WO2018107692A1 - 一种预防和治疗脂肪代谢紊乱及其相关病症的方法 - Google Patents

一种预防和治疗脂肪代谢紊乱及其相关病症的方法 Download PDF

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WO2018107692A1
WO2018107692A1 PCT/CN2017/089052 CN2017089052W WO2018107692A1 WO 2018107692 A1 WO2018107692 A1 WO 2018107692A1 CN 2017089052 W CN2017089052 W CN 2017089052W WO 2018107692 A1 WO2018107692 A1 WO 2018107692A1
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plasminogen
subject
drugs
drug
fat
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PCT/CN2017/089052
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English (en)
French (fr)
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李季男
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深圳瑞健生命科学研究院有限公司
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Priority to US16/470,174 priority Critical patent/US20190328850A1/en
Priority to CN201780078126.2A priority patent/CN110114082A/zh
Priority to JP2019532073A priority patent/JP7160351B2/ja
Priority to EP17881180.8A priority patent/EP3556387A4/en
Priority to CA3046664A priority patent/CA3046664C/en
Publication of WO2018107692A1 publication Critical patent/WO2018107692A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/484—Plasmin (3.4.21.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a method for preventing and/or treating a disorder of fat metabolism and a related disorder thereof, comprising administering an effective amount of plasminogen to a subject susceptible to or suffering from a disorder of fat metabolism and a related disorder thereof to reduce fat Abnormal deposition of tissues and organs in the body, thereby achieving the purpose of preventing and/or treating disorders of fat metabolism and related disorders and complications.
  • Fat metabolism disorder also known as lipodystrophy
  • lipids lipids
  • Fat metabolism disorder is one of the metabolic diseases, which is caused by primary or acquired factors, lipids (lipids) and their metabolic substances and amounts in blood and other tissues and organs. abnormal. Lipid metabolism involves digestion and absorption of lipids in the small intestine, entry into the blood circulation by the lymphatic system (transport by lipoproteins), transformation by the liver, storage in adipose tissue, and utilization by tissues when needed. The main function of lipids in the body is to oxidize energy. Adipose tissue is the energy store of the body. Fat can also protect the internal organs with skin, bones and muscles, prevent body temperature from spreading and help the absorption of fat-soluble vitamins in food.
  • Phospholipids are an important structural component of all cell membranes, and cholesterol is a precursor of bile acids and steroid hormones (adrenal cortical hormones and gonadal hormones). Lipid metabolism is regulated by genetics, neurohumoral fluids, hormones, enzymes, and tissues such as the liver. When these factors are abnormal, it can cause lipid metabolism disorder and pathophysiological changes of related organs, such as hyperlipoproteinemia and its clinical syndrome, obesity, fatty liver and so on.
  • Hyperlipoproteinemia is caused by excessive lipoproteins in the blood. Lipids in the blood such as triglyceride (TG), free cholesterol (FC), cholesterol (CE) and phospholipids are rarely soluble in water, and only a macromolecular complex (lipoprotein) is formed with apolipoprotein (APO). In order to dissolve, operate and metabolize in the blood. When the blood lipid is higher than the upper limit of normal people, it is hyperlipemia. Hyperlipidemia is also called hyperlipoproteinemia because blood lipids are transported in the form of lipoproteins in the blood. Generally, adult fasting blood triglycerides exceed 160mg/dl, cholesterol exceeds 260mg/dl, and children's cholesterol exceeds 160mg/dl as standard [1] .
  • Hyperlipoproteinemia hyperlipoproteinemia
  • type I mainly chylomicrons, serum.
  • turbidity is milky white, which contains a large amount of triglyceride (TG);
  • type II is divided into two subtypes IIa and IIb.
  • the former is mainly a low-density lipoprotein (LDL), and the latter is a very low-density lipoprotein (VLDL).
  • LDL low-density lipoprotein
  • VLDL very low-density lipoprotein
  • type III serum turbidity LDL and VLDL are increased, electrophoresis on the fusion
  • type IV mainly VLDL increase, serum or turbidity
  • V type chylomicrons and VLDL increase, serum opacity milky.
  • type II and type IV are the most common [1] .
  • Hyperlipidemia can be divided into primary and secondary categories according to the cause.
  • Primary is mostly caused by congenital defects (or genetic defects) in lipid and lipoprotein metabolism, as well as certain environmental factors (including diet, nutrition, and drugs) through unknown mechanisms.
  • Secondary is mainly secondary to certain diseases such as diabetes, liver disease, kidney disease, thyroid disease, as well as alcohol and obesity.
  • Environmental factors such as diet and lifestyle are also the cause of the disease.
  • diabetes often involves disorders of lipid metabolism, so diabetes is also known as "glycolipidosis" [2] .
  • the pathogenesis of diabetes is related to B cell function damage and insulin resistance, which is characterized by chronic hyperglycemia, and the disorder of glucose metabolism often involves disorder of lipid metabolism.
  • Diabetic lipid metabolism disorder has become an independent risk factor for cardiovascular disease, mainly characterized by hypertriglyceridemia, low levels of HDL, and increased LDL concentrations.
  • diabetes lipid metabolism disorder is still unclear, but there is a lot of evidence that insulin resistance is the central link. Recent studies have also found that intestinal insulin resistance is also involved. Animal models of diabetes and population studies have found that abnormal expression of certain genes associated with lipid metabolism further leads to insulin resistance. The occurrence of atherosclerosis in diabetic patients is associated with a variety of factors, but abnormalities in plasma lipid levels are the most important factors. Studies have shown that the incidence and mortality of cardiovascular disease in diabetic patients is significantly higher than non-diabetic patients, diabetes has become an independent risk factor for cardiovascular disease [3] .
  • nephropathy In recent years, the relationship between nephropathy and lipid metabolism disorders has become more and more conspicuous. In chronic progressive renal injury, abnormal lipid metabolism is often accompanied, and hyperlipidemia can promote and aggravate kidney damage, in addition to mediating glomerular injury. In addition, it also plays a role in tubulointerstitial damage. In 1913 Munk first described dyslipidemia in nephrotic syndrome. Some researchers have reported that 70%-10% of patients with nephrotic syndrome may have hyperlipidemia. It is mainly characterized by a significant increase in total cholesterol (TC) and a decrease in low-density lipoprotein cholesterol, a slight increase in triglyceride (TG), and an increase in low-density lipoprotein (LDL) and urine protein.
  • TC total cholesterol
  • TG triglyceride
  • LDL low-density lipoprotein
  • TC total cholesterol
  • TG triglyceride
  • LDLC low-density lipoprotein cholesterol
  • VLDLC very low-density lipoprotein cholesterol
  • Obesity is divided into two categories: simple and secondary. Simple obesity refers to obesity without obvious endocrine and metabolic diseases. It can be divided into two types: physical obesity and acquired obesity. There is a family history of constitutional obesity. The patient is rich in food intake from childhood, and the amount of excess is obese. The fat cells are hypertrophic and hypertrophic. Acquired obesity is mostly caused by overnutrition and/or reduced physical activity, such as improvement of living conditions after a middle age, adequate recovery of disease and rest, postpartum physical exercise or physical labor. The fat cells showed hypertrophic changes, no hyperplasia, and the treatment effect was better. Secondary obesity is mainly caused by neuroendocrine diseases.
  • the hypothalamus has a center of appetite regulation, central nervous system inflammation sequelae, trauma, tumors, etc. can cause hypothalamic dysfunction, causing an appetite and causing obesity.
  • 2 increased insulin secretion, such as early non-insulin-dependent diabetes mellitus injection of excessive insulin, resulting in hyperinsulinemia; islet B cell tumors secrete too much insulin, which increases fat synthesis, causing obesity.
  • Cortisol is often accompanied by central obesity.
  • 6 hypothyroidism due to low metabolic rate, fat accumulation, and mucus edema.
  • 7 hypogonads can also be obese, such as obesity reproductive incompetence (cerebral obesity, Florik's syndrome, trauma, encephalitis, pituitary tumor, craniopharyngioma, etc. caused by damage to the hypothalamus, manifested as central obesity , with diabetes insipidus and sexual growth retardation).
  • Lipid metabolism disorders often lead to fatty liver.
  • Fatty liver refers to a lesion of excessive accumulation of fat in liver cells due to various reasons.
  • the liver plays a particularly important role in lipid metabolism. It can synthesize lipoproteins, which is beneficial to lipid transport and is also the main site for fatty acid oxidation and ketone body formation.
  • the normal liver fat content is not much, about 4%, mainly phospholipids. If the liver cannot transport the fat out in time, the fat accumulates in the liver cells, which forms fatty liver.
  • Fatty liver can be an independent disease or can be caused by other causes, such as obesity fatty liver, alcoholic fatty liver, rapid weight loss fatty liver, malnutrition fatty liver, diabetic fatty liver, drug-induced fatty liver and so on.
  • Certain drugs or chemical poisons cause fatty livers by inhibiting the synthesis of proteins such as tetracycline, adrenocortical hormone, puromycin, cyclohexylamine, ipecaine, and arsenic, lead, silver, mercury, and the like.
  • Lipid lowering drugs can also form fatty liver by interfering with the metabolism of lipoproteins.
  • Atherosclerosis One of the dangers of fatty liver is that it promotes the formation of atherosclerosis.
  • One of the causes of atherosclerosis is that patients with fatty liver are often accompanied by hyperlipidemia, increased blood viscosity, and low-density lipoprotein (LDL). Because of its extremely small molecular weight, it easily passes through the intima of the arteries and settles on the vessel wall, which reduces the elasticity of the artery, narrows the diameter of the tube, and weakens the flexibility, eventually leading to blood circulation disorder.
  • the second risk of fatty liver is to induce or aggravate high blood pressure, coronary heart disease, and easily lead to myocardial infarction and sudden death.
  • the third risk of fatty liver is encephalopathy fatty liver syndrome (Reye syndrome).
  • the fourth risk of fatty liver is cirrhosis, liver failure, and liver cancer.
  • Fatty liver is a product of liver lipid metabolism disorder, and at the same time it is a causative factor that aggravates liver damage. This is a development of mutual causal and vicious circle.
  • the increase of lipid droplets in the liver cells causes the liver cells to be steatotic and swollen, and the nucleus is squeezed off-center.
  • the metabolism of fat should be carried out in the mitochondria.
  • the transport of fat to the extracellular cells mainly passes through the smooth endoplasmic reticulum.
  • the accumulation of fat in the hepatocytes further aggravates the burden of mitochondria and endoplasmic reticulum and reduces its function, thereby affecting other nutrients and hormones. Metabolism of vitamins.
  • Cirrhosis is associated with a higher incidence of hepatocellular carcinoma.
  • the fifth risk of fatty liver is acute pregnancy fatty liver with high mortality.
  • This disease also known as obstetric acute yellow liver atrophy, is a rare pregnancy with a prognosis. Most occur in the last three months of pregnancy, clinical manifestations are often similar to acute liver, acute liver failure, pancreatitis, renal failure, systemic coagulation abnormalities leading to rapid death, the majority of pregnant women in the first pregnancy.
  • the sixth risk of fatty liver is to induce or aggravate diabetes.
  • the blood glucose concentration exceeds the normal level, although it does not meet the diagnostic criteria for diabetes, it is generally considered to be pre-diabetes.
  • Fatty liver and diabetes often accompany each other and affect each other, which brings greater difficulties to clinical treatment.
  • plasminogen can prevent and/or reduce abnormal deposition of fat in tissues and organs of the body, for example, can prevent and reduce abnormal deposition of lipids in tissues of blood, blood vessel walls, internal organs and organs, and improve these.
  • the function of tissues and organs thus providing a new preventive and therapeutic solution for disorders of fat metabolism and related disorders, as well as its accompanying diseases or complications
  • the present invention relates to the prevention and/or treatment of disorders of fat metabolism and related disorders in a subject.
  • the invention relates to a method of preventing and/or treating a disorder of fat metabolism and a related disorder thereof, comprising administering to a subject a prophylactically and/or therapeutically effective amount of plasminogen, wherein said subject is susceptible Suffering from fat metabolism disorder, suffering from fat metabolism disorder or suffering from other diseases accompanied by disorders of fat metabolism.
  • the invention also relates to the use of plasminogen for the prevention and/or treatment of disorders of fat metabolism and related disorders in a subject.
  • the invention further relates to the use of plasminogen for the preparation of a medicament, pharmaceutical composition, preparation, kit for the prevention and/or treatment of a disorder of fat metabolism and a related disorder in a subject.
  • the present invention relates to plasminogen for preventing and/or treating a disorder of fat metabolism and a related condition thereof in a subject.
  • the present invention also relates to a plasminogen-containing drug, pharmaceutical composition, preparation, kit for use in preventing and/or treating a disorder of fat metabolism and a related condition thereof.
  • the disorder of fat metabolism is an endocrine disorder disease, a glucose metabolism disease, a liver disease, a kidney disease, a cardiovascular disease, an intestinal disease, a thyroid disease, a gallbladder or a biliary tract disease, an obesity, a drinking, or a medical treatment. Or accompanying disorders of fat metabolism.
  • the disorder of fat metabolism is hypertension, diabetes, chronic hepatitis, cirrhosis, kidney damage, chronic glomerulonephritis, chronic pyelonephritis, nephrotic syndrome, renal insufficiency, kidney transplantation, uremia, A disorder of fat metabolism caused by or associated with hypothyroidism, obstructive cholecystitis, obstructive cholangitis, drug or hormonal therapy.
  • the disorder of fat metabolism is hyperlipidemia, hyperlipoproteinemia, fatty liver, atherosclerosis, obesity, organ fat deposition.
  • the atherosclerosis comprises aortic atherosclerosis, coronary atherosclerosis, cerebral atherosclerosis, renal atherosclerosis, hepatic atherosclerosis, mesenteric atherosclerosis Lower extremity atherosclerosis.
  • the present invention relates to a method of preventing and/or reducing abnormal deposition of fat in a body tissue of a subject, comprising administering to the subject an effective amount of plasminogen.
  • the invention also relates to fibrinolysis
  • the zymogen is used to prevent and/or reduce the abnormal deposition of fat in the body tissues and organs of the subject.
  • the invention further relates to the use of plasminogen for the preparation of a medicament, a pharmaceutical composition, an article, a kit for preventing and/or reducing abnormal deposition of fat in a body tissue of a subject.
  • the present invention relates to plasminogen for preventing and/or reducing abnormal deposition of fat in a body tissue of a subject.
  • the present invention also relates to a medicament, a pharmaceutical composition, an article, a kit comprising plasminogen for preventing and/or reducing abnormal deposition of fat in a body tissue of a subject.
  • the present invention relates to a method of preventing and/or treating a condition caused by abnormal deposition of fat in a body tissue organ of a subject, comprising administering to the subject an effective amount of plasminogen.
  • the invention further relates to the use of plasminogen for preventing and/or treating a condition caused by abnormal deposition of fat in a body tissue or organ of a subject.
  • the invention further relates to the use of plasminogen for the preparation of a medicament, a pharmaceutical composition, an article, a kit for the prevention and/or treatment of a condition in which fat in a subject is abnormally deposited in tissues and organs of the body.
  • the present invention relates to a plasminogen-containing drug, pharmaceutical composition, preparation, kit for preventing and/or treating a condition caused by abnormal deposition of fat in a body tissue of a subject.
  • the abnormal deposition of the fat in the body tissue refers to the abnormal deposition of fat in the blood, subcutaneous tissue, blood vessel walls, internal organs.
  • the disorder caused by abnormal deposition of fat in body tissues and organs includes obesity, hyperlipidemia, hyperlipoproteinemia, fatty liver, atherosclerosis, lipid heart damage, lipidity Kidney damage, lipid islet damage.
  • the present invention relates to a method of preventing and/or treating a condition caused by a disorder of fat metabolism in a subject, comprising administering to the subject an effective amount of plasminogen.
  • the invention further relates to the use of plasminogen for the prevention and/or treatment of a condition caused by a disorder of fat metabolism in a subject.
  • the invention further relates to the use of plasminogen for the preparation of a medicament, a pharmaceutical composition, an article, a kit for the prevention and/or treatment of a disorder caused by a disorder of fat metabolism in a subject.
  • the present invention relates to plasminogen for preventing and/or treating a disorder caused by a disorder of fat metabolism in a subject.
  • the present invention also relates to a plasminogen-containing drug, pharmaceutical composition, preparation, kit for preventing and/or treating a disorder caused by a disorder of fat metabolism in a subject.
  • the condition comprises obesity, hyperlipidemia, hyperlipoproteinemia, fatty liver, atherosclerosis, lipid heart tissue damage, lipid renal injury.
  • the present invention relates to a method of treating a disease in a subject by reducing fat abnormal deposition comprising administering to the subject an effective amount of plasminogen.
  • the invention also relates to plasminogen for use in The use of reducing fat abnormal deposition to treat a subject's disease.
  • the invention further relates to the use of plasminogen for the preparation of a medicament, pharmaceutical composition, preparation, kit for treating a disease in a subject by reducing fat abnormal deposition.
  • the present invention also relates to plasminogen for treating a disease in a subject by reducing fat abnormal deposition.
  • the present invention also relates to a plasminogen-containing drug, pharmaceutical composition, preparation, kit for treating a disease in a subject by reducing fat abnormal deposition.
  • the disease comprises atherosclerosis, coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, fatty liver, cirrhosis, cerebral ischemia, cerebral infarction, renal insufficiency, nephrotic syndrome, renal insufficiency Obesity.
  • the present invention relates to a method of preventing and/or treating lipid damage in a tissue of a subject comprising administering to the subject an effective amount of plasminogen.
  • the invention further relates to the use of plasminogen for the prevention and/or treatment of lipid damage in tissues and organs of a subject.
  • the invention further relates to the use of plasminogen for the preparation of a medicament, pharmaceutical composition, preparation, kit for the prevention and/or treatment of lipid damage in tissues and organs of a subject.
  • the present invention relates to plasminogen for preventing and/or treating lipid damage in tissues and organs of a subject.
  • the present invention also relates to a plasminogen-containing drug, pharmaceutical composition, preparation, kit for preventing and/or treating lipid damage in tissues and organs of a subject.
  • the tissue organ comprises an arterial wall, a heart, a liver, a kidney, a pancreas.
  • the invention relates to a method of improving hyperlipidemia in a subject comprising administering to the subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for improving hyperlipidemia in a subject.
  • the invention further relates to the use of plasminogen for the preparation of a medicament, a pharmaceutical composition, an article, a kit for improving hyperlipidemia in a subject.
  • the present invention also relates to plasminogen for improving hyperlipidemia in a subject.
  • the present invention also relates to a medicament, a pharmaceutical composition, an article, a kit comprising plasminogen for improving hyperlipidemia in a subject.
  • the hyperlipidemia is selected from one or more of the group consisting of hypercholesterolemia, hypertriglyceridemia, mixed hyperlipidemia, and low high density lipoproteinemia.
  • the invention relates to a method of reducing the risk of atherosclerosis in a subject comprising administering to the subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for reducing the risk of atherosclerosis in a subject.
  • the invention further relates to the use of plasminogen in the manufacture of a medicament, pharmaceutical composition, article, kit for reducing the risk of atherosclerosis in a subject.
  • the present invention also relates to plasminogen for reducing the risk of atherosclerosis in a subject.
  • this invention also contemplated are plasminogen-containing drugs, pharmaceutical compositions, articles, kits for reducing the risk of atherosclerosis in a subject.
  • the subject has hypertension, obesity, diabetes, chronic hepatitis, cirrhosis, kidney injury, chronic glomerulonephritis, chronic pyelonephritis, nephrotic syndrome, renal insufficiency, kidney transplantation , uremia, hypothyroidism, obstructive cholecystitis or obstructive cholangitis, or the subject taking a drug or hormone that affects fat metabolism.
  • the plasminogen reduces the risk of atherosclerosis in a subject by one or more of the following: lowering total cholesterol levels in the blood, triglyceride levels, low density lipoprotein levels, Increase blood HDL levels.
  • the invention relates to a method of treating a disease by ameliorating hyperlipidemia in a subject comprising administering to the subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for the treatment of diseases by improving hyperlipidemia in a subject.
  • the invention further relates to the use of plasminogen for the preparation of a medicament, pharmaceutical composition, preparation, kit for the treatment of a disease by improving hyperlipidemia in a subject.
  • the present invention also relates to plasminogen for treating diseases by improving hyperlipidemia in a subject.
  • the present invention also relates to a medicament, a pharmaceutical composition, an article, a kit comprising plasminogen for treating a disease by improving hyperlipidemia in a subject.
  • the condition comprises diabetes, hypertension, atherosclerosis, coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, chronic hepatitis, fatty liver, cirrhosis, cerebral insufficiency, cerebral ischemia, cerebral infarction Chronic nephritis, chronic pyelonephritis, renal insufficiency, nephrotic syndrome, uremia, obesity.
  • the present invention relates to a method of preventing and/or treating a hyperlipidemia-related disorder in a subject comprising administering to the subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for the prevention and/or treatment of a hyperlipidemia-related disorder in a subject.
  • the invention further relates to the use of plasminogen for the preparation of a medicament, pharmaceutical composition, preparation, kit for the prevention and/or treatment of a hyperlipidemic disorder in a subject.
  • the present invention also relates to plasminogen for preventing and/or treating a hyperlipidemia-related disorder in a subject.
  • the present invention also relates to a plasminogen-containing drug, pharmaceutical composition, preparation, kit for use in preventing and/or treating a hyperlipidemia-related disorder in a subject.
  • the condition comprises diabetes, hypertension, atherosclerosis, coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, chronic hepatitis, fatty liver, cirrhosis, cerebral insufficiency, cerebral ischemia, cerebral infarction Chronic nephritis, chronic pyelonephritis, renal insufficiency, nephrotic syndrome, uremia, obesity.
  • the plasminogen can be administered in combination with one or more other drugs or methods of treatment.
  • the one or more other drugs include a therapeutic drug for hypertension, a drug for treating diabetes, a drug for treating atherosclerosis, a drug for treating chronic glomerulonephritis, a drug for treating chronic pyelonephritis, and a combination of nephropathy Drugs for treatment, drugs for treatment of renal insufficiency, drugs for uremia treatment, drugs for kidney transplantation, drugs for treatment of fatty liver, drugs for treatment of liver cirrhosis, drugs for treatment of obesity.
  • the other drug comprises: a hypolipidemic drug, an antiplatelet drug, a blood pressure lowering drug, a dilated vascular drug, a hypoglycemic drug, an anticoagulant drug, a thrombolytic drug, a hepatoprotective drug, an antiarrhythmic drug, Cardiotonic drugs, diuretic drugs, anti-infective drugs, antiviral drugs, immunomodulatory drugs, inflammatory regulating drugs, anti-tumor drugs, hormone drugs, thyroxine.
  • the medicament comprises a hypolipidemic drug: a statin; a fibrate; a niacin; a cholestyramine; clofibrate; an unsaturated fatty acid such as erosin, a blood lipid, and a heart pulse; alginic acid Sodium diester; antiplatelet drugs: aspirin; dipyridamole; clopidogrel; cilostazol; dilated vascular drugs: hydralazine; nitroglycerin and heartache; sodium nitroprusside; alpha nitrate receptor blockers such as prazosin ; alpha receptor blockers such as phentolamine; beta pull receptor stimulants such as salbutamol; captopril, enalapril; heart pain, thiazolone; lysine, long pressure , prostaglandins, atrial natriuretic peptide; thrombolytic drugs: urokinase and strept
  • the plasminogen may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97 with sequence 2, 6, 8, 10 or 12. %, 98% or 99% sequence identity and still have plasminogen activity.
  • the plasminogen is added, deleted, and/or substituted on the basis of sequence 2, 6, 8, 10, or 12, 1-100, 1-90, 1-80, 1-70 , 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1 -3, 1-2, 1 amino acid, and still a protein with plasminogen activity.
  • the plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
  • the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, delta-plasminogen, or their retention A variant of plasminogen activity.
  • the plasminogen is a native or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity.
  • the plasminogen is a human plasminogen ortholog from a primate or a rodent or a variant or fragment thereof that still retains plasminogen activity.
  • the amino acid of plasminogen is as shown in sequence 2, 6, 8, 10 or 12.
  • the plasminogen is human natural plasminogen.
  • the subject is a human. In some embodiments, the subject lacks or lacks plasminogen. In some embodiments, the deficiency or deficiency is innate, secondary, and/or local.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier and plasminogen for use in the foregoing methods.
  • the kit can be a prophylactic or therapeutic kit comprising: (i) plasminogen for use in the foregoing methods and (ii) for delivery of the plasminogen to The subject of the subject.
  • the member is a syringe or vial.
  • the kit further comprises a label or instructions for use, the label or instructions for use indicating administration of the plasminogen to the subject to perform any of the methods described above.
  • the article of manufacture comprises: a container comprising a label; and a pharmaceutical composition comprising (i) plasminogen or a plasminogen for use in the foregoing method, wherein the label indicates that the fiber is to be The lysogen or composition is administered to the subject to perform any of the methods described above.
  • the kit or article further comprises an additional one or more components or containers containing other drugs.
  • the other drug is selected from the group consisting of a hypolipidemic drug, an antiplatelet drug, a blood pressure lowering drug, a dilated vascular drug, a hypoglycemic drug, an anticoagulant drug, a thrombolytic drug, a hepatoprotective drug, and an anti-heart rhythm Abnormal drugs, cardiotonic drugs, diuretic drugs, anti-infective drugs, antiviral drugs, immunomodulatory drugs, inflammatory regulatory drugs, anti-tumor drugs, hormone drugs, thyroxine.
  • the plasminogen is administered systemically or locally, preferably by the following route: intravenous, intramuscular, subcutaneous administration of plasminogen for treatment.
  • the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer.
  • the plasminogen is 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg per day.
  • the present invention expressly covers all combinations of the technical features between the embodiments of the present invention, and these combined technical solutions are explicitly disclosed in the present application, just as the above technical solutions have been separately and explicitly disclosed.
  • the present invention also explicitly covers combinations between various embodiments and elements thereof, and the combined technical solutions are explicitly disclosed herein.
  • fat metabolism disorder is also called “abnormal fat metabolism” and "fat metabolism disorder”, and is a general term for clinical or pathological manifestations caused by abnormal, disorder or disorder of fat metabolism.
  • fat metabolism disorder “abnormal fat metabolism”, and “fat metabolic disorder” are used interchangeably.
  • fat metabolism “lipid metabolism”, and “lipid metabolism” are used interchangeably.
  • Fat metabolism disorder-related disorders is a general term for disorders associated with disorders of fat metabolism.
  • the correlation may be related to etiology, pathogenesis, pathological relevance, clinical symptom correlation, and/or treatment principles.
  • “Blood fat” is a general term for triglycerides, cholesterol and phospholipids.
  • Lipoprotein is a spherical macromolecular complex composed of apolipoprotein and blood lipid. Because lipoproteins contain different components of cholesterol and triglycerides, and the density is different. Divided into five categories: chylomicrons (CM) very low density lipoprotein (VLDL) medium density lipoprotein (IDL) low density lipoprotein (LDL) high density lipoprotein (HDL).
  • CM chylomicrons
  • VLDL very low density lipoprotein
  • IDL medium density lipoprotein
  • LDL low density lipoprotein
  • HDL high density lipoprotein
  • Secondary dyslipidemia is found in diabetes, hypothyroidism, nephrotic syndrome, kidney transplantation, severe liver disease, obstructive biliary tract disease, obesity, alcohol consumption, drug treatment, such as estrogen therapy, etc., if secondary dyslipidemia can be ruled out Consider primary dyslipidemia.
  • “Hyperlipidemia” refers to a pathological condition in which blood lipids such as cholesterol, triglyceride, phospholipids, and non-lipidated fatty acids are increased in plasma.
  • “Hyperlipid-related disorder” refers to a condition associated with high blood lipids, etiology, pathogenesis, pathological manifestations, clinical symptoms, and/or treatment principles.
  • the condition includes, but is not limited to, diabetes, hypertension, atherosclerosis, coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, chronic hepatitis, fatty liver, cirrhosis, cerebral insufficiency, cerebral ischemia, cerebral infarction, chronic Nephritis, chronic pyelonephritis, renal insufficiency, nephrotic syndrome, uremia, obesity.
  • hypolipidemia One or several lipid abnormalities in plasma are referred to as “hyperlipidemia”, “hyperlipemia” or “dyslipidemia” due to fat metabolism or abnormal function.
  • lipids are insoluble or slightly soluble in water, they must bind to proteins to form lipoproteins to function in the blood circulation. Therefore, hyperlipidemia is often a reflection of "high-lipoproteinemia.”
  • hyperlipidemia-related disorder of the present invention may also be referred to as “hyperlipidemic-related disorder” and “hyperlipoproteinemia-related disorder”.
  • Fat liver refers to a lesion of excessive accumulation of fat in liver cells due to various reasons, which may be an independent disease or may be caused by other causes, such as obese fatty liver, alcoholic fatty liver, and rapid weight loss. Fatty liver, malnutrition fatty liver, diabetic fatty liver, drug-induced fatty liver and so on.
  • the lipid droplets in the liver cells increase, causing the liver cells to be fatty degenerated, swollen, and the nucleus is squeezed off center.
  • the metabolism of fat should be carried out in the mitochondria.
  • the transport of fat to the extracellular cells mainly passes through the smooth endoplasmic reticulum.
  • the accumulation of fat in the hepatocytes further aggravates the burden of mitochondria and endoplasmic reticulum and reduces its function, thereby affecting other nutrients and hormones. Metabolism of vitamins. Long-term degeneration of hepatocytes leads to arrhythmia and necrosis of hepatocytes, which in turn leads to liver fibrosis and cirrhosis.
  • Atherosclerosis is a chronic, progressive arterial disease in which the fat deposited in the arteries partially or completely blocks blood flow. Atherosclerosis occurs when the smooth, firm arterial intima is roughened, thickened, and blocked by fat, fibrin, calcium, and cellular debris. Atherosclerosis is a gradual process. When the concentration of lipids in the blood is greatly increased, fat streaks are formed along the walls of the artery. These streaks cause fat and cholesterol deposits that attach to the originally smooth arterial intima, forming a nodule. These nodules then grow fibrotic scar tissue, resulting in calcium deposition. The deposited calcium gradually evolves into a chalky hard film (called an atheroma) that cannot be removed. This permanent film inside the artery blocks the normal expansion and contraction of the artery, slowing the blood flow velocity within the artery, which can easily form a blood clot that prevents or prevents blood from flowing through the artery.
  • an atheroma chalky hard film
  • Atherosclerosis The exact cause of atherosclerosis has not yet been determined, but important pathogenic factors have been identified: hyperlipidemia, hypertension, a history of smoking, and a family history of atherosclerosis (previous illness before age 60) Or diabetes.
  • Hyperlipidemia can promote the formation of fat streaks. Because hypertension exerts a certain constant force on the artery, it accelerates the process of arterial occlusion and hardening, thus increasing the prevalence of atherosclerosis.
  • Smoking can cause arterial contractions and restrict blood flow, thus creating conditions for arterial occlusion. Diabetes can also contribute to the development of atherosclerosis, especially for very small arteries.
  • Atherosclerosis As far as atherosclerosis is concerned, people do not feel any symptoms. The disease is only discovered when the artery connected to an important organ in the body is blocked. Symptoms caused by blocked arteries in the organ are more pronounced. For example, if the heart's blood supply artery is partially blocked, people may feel angina; but if it is completely blocked, it may lead to heart disease (heart tissue death from the blocked arteries). If atherosclerosis affects the brain arteries, people may feel dizziness, blurred vision and syncope, and may even cause a stroke (the brain tissue that is supplied by the blocked arteries dies, causing nerve damage, such as limbs controlled by dead brain tissue) ). Blockage of the arteries leading to the kidney may also lead to kidney failure. Blockage of blood vessels leading to the eye can lead to blindness. Arterial obstruction of the extremities may cause lesions in various limbs.
  • Atherosclerosis is the main cause of coronary heart disease, cerebral infarction, and peripheral vascular disease.
  • Lipid metabolism disorder is the basis of atherosclerosis, which is characterized by the involvement of the affected arterial lesions from the intima, usually the accumulation of lipids and complex carbohydrates, hemorrhage and thrombosis, and then fibrous tissue hyperplasia and calcium deposition, and The gradual metamorphosis and calcification of the middle layer of the artery leads to thickening and hardening of the arterial wall and narrowing of the vascular lumen.
  • the lesion often involves the large and medium muscular arteries, and once developed enough to block the arterial lumen, the tissue or organ supplied by the artery will be ischemic or necrotic.
  • Atherosclerosis is a systemic disease in which an organ angiogenic atherosclerotic lesion means that the same lesion may already exist in the blood vessels elsewhere; likewise, a vascular event in one organ means that a vascular event occurs elsewhere. The danger increases.
  • Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease that hydrolyzes several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycans [9] . In addition, plasmin activates some metalloproteinase precursors (pro-MMPs) to form active metalloproteinases (MMPs). Therefore, plasmin is considered to be an important upstream regulator of extracellular proteolysis [10,11] .
  • ECM extracellular matrix
  • MMPs active metalloproteinases
  • Plasmin is formed by proteolytic plasminogen by two physiological PAs: tissue plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). Due to the relatively high levels of plasminogen in plasma and other body fluids, it has been traditionally believed that the regulation of the PA system is primarily achieved by the synthesis and activity levels of PAs. The synthesis of components of the PA system is tightly regulated by various factors such as hormones, growth factors and cytokines. In addition, specific physiological inhibitors of plasmin and PAs are also present. The main inhibitor of plasmin is ⁇ 2-antiplasmin.
  • PAI-1 plasminogen activator inhibitor-1
  • PAI-2 lysogen activator inhibitor-2
  • Some cell surface has direct hydrolysis activity of uPA-specific cell surface receptors (uPAR) [12,13] .
  • Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa [14,15] . Plasminogen is mainly synthesized in the liver and is abundantly present in the extracellular fluid. The plasma plasminogen content is approximately 2 ⁇ M. Therefore, plasminogen is a huge potential source of proteolytic activity in tissues and body fluids [16,17] . Plasminogen exists in two molecular forms: glutamate-plasminogen and Lys-plasminogen. The naturally secreted and uncleaved forms of plasminogen have an amino terminal (N-terminal) glutamate and are therefore referred to as glutamate-plasminogen.
  • plasminogen in the presence of plasmin, glutamate-plasminogen is hydrolyzed to Lys-Lysinogen at Lys76-Lys77. Compared to glutamate-plasminogen, lysine-plasminogen has a higher affinity for fibrin and can be activated by PAs at a higher rate.
  • the Arg560-Val561 peptide bond of these two forms of plasminogen can be cleaved by uPA or tPA, resulting in the formation of a disulfide-linked double-chain protease plasmin [18] .
  • the amino terminal portion of plasminogen contains five homologous tricycles, the so-called kringles, which contain a protease domain.
  • Some kringles contain a lysine binding site that mediates the specific interaction of plasminogen with fibrin and its inhibitor alpha2-AP.
  • the main substrate for plasmin is fibrin, which is the key to preventing pathological thrombosis [19] .
  • Plasmin also has substrate specificity for several components of ECM, including laminin, fibronectin, proteoglycans and gelatin, suggesting that plasmin also plays an important role in ECM reconstruction [ 15, 20, 21] .
  • plasmin can also degrade other components of ECM, including MMP-1, MMP-2, MMP-3 and MMP-9, by converting certain protease precursors into active proteases. Therefore, it has been suggested that plasmin may be an important upstream regulator of extracellular proteolysis [22] .
  • plasmin has the ability to activate certain potential forms of growth factors [23-25] . In vitro, plasmin also hydrolyzes components of the complement system and releases chemotactic complement fragments.
  • Plasmid is a very important enzyme found in the blood that hydrolyzes fibrin clots into fibrin degradation products and D-dimers.
  • Plasmidogen is a zymogen form of plasmin, consisting of 810 amino acids calculated according to the sequence in swiss prot, based on the native human plasminogen amino acid sequence (sequence 4) containing the signal peptide, A glycoprotein having a molecular weight of about 90 kD, which is mainly synthesized in the liver and capable of circulating in the blood, and a cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO:3.
  • Full-length plasminogen contains seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAp) domain at the N-terminus, and five Kringle domains (Kringle 1-5).
  • the signal peptide includes the residue Met1-Gly19
  • PAp includes the residue Glu20-Val98
  • Kringle1 includes the residue Cys103-Cys181
  • Kringle2 includes the residue Glu184-Cys262
  • Kringle3 includes the residue Cys275-Cys352
  • Kringle4 Including the residue Cys377-Cys454
  • Kringle5 includes the residue Cys481-Cys560.
  • the serine protease domain includes the residues Val581-Arg804.
  • Glu-plasminogen is a natural full-length plasminogen consisting of 791 amino acids (not containing a 19 amino acid signal peptide), and the cDNA sequence encoding the sequence is shown in SEQ ID NO: 1, and its amino acid sequence is sequence 2. Shown. In vivo, there is also a Lys-plasminogen which is hydrolyzed from amino acids 76-77 of Glu-plasminogen, and as shown in SEQ ID NO: 6, the cDNA sequence encoding the amino acid sequence is as shown in SEQ ID NO: 5 Shown.
  • Delta-plasminogen is a fragment of full-length plasminogen deleted from the Kringle2-Kringle5 structure and contains only the Kringle1 and serine protease domains [26,27] .
  • the delta-plasminogen has been reported in the literature.
  • the amino acid sequence (SEQ ID NO: 8) [27] the cDNA sequence encoding the amino acid sequence is shown in Sequence 7.
  • Mini-plasminogen consists of Kringle5 and a serine protease domain, which has been reported in the literature to include the residue Val443-Asn791 (starting amino acid with a Glu residue of Glu-plasminogen sequence not containing a signal peptide) [28] , the amino acid sequence thereof is shown in SEQ ID NO: 10, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 9.
  • Micro-plasminogen contains only the serine protease domain, and its amino acid sequence has been reported to include the residue Ala543-Asn791 (from the Glu residue of the Glu-plasminogen sequence containing no signal peptide).
  • Plasin of the present invention is used interchangeably with “fibrinolytic enzyme” and “fibrinolytic enzyme”, and has the same meaning; “plasminogen” and “plasminogen”, “fibrinogenase” "Interchangeable use, meaning the same.
  • the term "deficiency" of plasminogen means that the content or activity of plasminogen in the subject is lower than that of a normal person, and is low enough to affect the normal physiological function of the subject;
  • the meaning of "deficient" of plasminogen is that the content or activity of plasminogen in the subject is significantly lower than that of normal people, and even the activity or expression is minimal, and only by external supply can maintain normal physiological functions.
  • plasminogen adopts a closed inactive conformation, but when bound to the surface of a thrombus or cell, it is converted to openness mediated by plasminogen activator (PA).
  • PA plasminogen activator
  • Conformational active plasmin The active plasmin further hydrolyzes the fibrin clot into a fibrin degradation product and a D-dimer, thereby dissolving the thrombus.
  • the PAp domain of plasminogen contains an important determinant that maintains plasminogen in an inactive blocking conformation, while the KR domain is capable of binding to lysine residues present on the receptor and substrate.
  • plasminogen activators include tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hag Mann factor) and so on.
  • a "plasminogen active fragment” refers to an active fragment that binds to a target sequence in a substrate and exerts a proteolytic function in a plasminogen protein.
  • the technical solution of the present invention relating to plasminogen covers the technical solution of replacing plasminogen with a plasminogen active fragment.
  • the plasminogen active fragment of the present invention is a protein comprising a serine protease domain of plasminogen.
  • the plasminogen active fragment of the present invention comprises the sequence 14, and the sequence 14 has at least 80%, 90.
  • the plasminogen of the present invention comprises a protein comprising the plasminogen active fragment and still retaining the plasminogen activity.
  • blood plasminogen and its activity assays include: detection of tissue plasminogen activator activity (t-PAA), detection of plasma tissue plasminogen activator antigen (t-PAAg), Detection of plasma tissue plasminogen activity (plgA), detection of plasma tissue plasminogen antigen (plgAg), detection of plasma tissue plasminogen activator inhibitor activity, inhibition of plasma tissue plasminogen activator Detection of antigens, plasma plasmin-anti-plasmin complex assay (PAP).
  • t-PAA tissue plasminogen activator activity
  • t-PAAg detection of plasma tissue plasminogen activator antigen
  • plgA Detection of plasma tissue plasminogen activity
  • plgAg detection of plasma tissue plasminogen antigen
  • PAP plasma plasmin-anti-plasmin complex assay
  • the most commonly used detection method is the chromogenic substrate method: adding streptokinase (SK) and chromogenic substrate to the plasma to be tested, and the PLG in the tested plasma is converted into PLM under the action of SK, and the latter acts on The chromogenic substrate is then measured spectrophotometrically and the increase in absorbance is directly proportional to the plasminogen activity.
  • plasminogen activity in blood can also be measured by immunochemical method, gel electrophoresis, immunoturbidimetry, or radioimmunoassay.
  • ortholog or ortholog refers to homologs between different species, including both protein homologs and DNA homologs, also known as orthologs, orthologs. It specifically refers to a protein or gene that has evolved from the same ancestral gene in different species.
  • the plasminogen of the present invention includes human natural plasminogen, and also includes plasminogen orthologs or orthologs of plasminogen activity derived from different species.
  • Constant substitution variant refers to a change in one of the given amino acid residues without altering the overall conformation and function of the protein or enzyme, including but not limited to similar properties (eg, acidic, basic, hydrophobic, etc.)
  • the amino acid replaces the amino acid in the amino acid sequence of the parent protein.
  • Amino acids having similar properties are well known. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and are interchangeable.
  • isoleucine is a hydrophobic amino acid that can be replaced by leucine, methionine or valine. Therefore, the similarity of two protein or amino acid sequences of similar function may be different.
  • Constant substitution variants also includes determining polypeptides or enzymes having more than 60% amino acid identity by BLAST or FASTA algorithm. If it is more than 75%, preferably more than 85%, or even more than 90%. Optimal and have the same or substantially similar properties or functions as the native or parent protein or enzyme.
  • Isolated plasminogen refers to a plasminogen protein that is isolated and/or recovered from its natural environment.
  • the plasminogen will purify (1) to a purity greater than 90%, greater than 95%, or greater than 98% by weight, as determined by the Lowry method, eg, over 99% (by weight), (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotating cup sequence analyzer, or (3) to homogeneity, which is by use Coomassie blue or silver staining was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions.
  • Isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering techniques and isolated by at least one purification step.
  • polypeptide peptide
  • protein protein
  • fusion proteins including, but not limited to, fusion proteins having a heterologous amino acid sequence, fusions having heterologous and homologous leader sequences (with or without an N-terminal methionine residue);
  • percent amino acid sequence identity with respect to a reference polypeptide sequence is defined as the introduction of a gap as necessary to achieve maximum percent sequence identity, and does not treat any conservative substitution as a sequence.
  • Comparisons for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art will be able to determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum contrast over the full length of the sequences being compared. However, for the purposes of the present invention, amino acid sequence identity percent values are generated using the sequence comparison computer program ALIGN-2.
  • amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or may be expressed as having or comprising relative to, and, or for a given amino acid sequence)
  • a given amino acid sequence A of a certain % amino acid sequence identity of B is calculated as follows:
  • X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 in the A and B alignments of the program, and wherein Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A relative to B will not be equal to the % amino acid sequence identity of B relative to A. All % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph, unless explicitly stated otherwise.
  • the terms “treating” and “treating” refer to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be to completely or partially prevent the disease or its symptoms, and/or to partially or completely cure the disease and/or its symptoms, and includes: (a) preventing the disease from occurring in the subject, the subject may have The cause of the disease, but not yet diagnosed as having a disease; (b) inhibiting the disease, ie, retarding its formation; and (c) reducing the disease and/or its symptoms, ie causing the disease and/or its symptoms to subside.
  • the terms "individual”, “subject” and “patient” are used interchangeably herein to refer to a mammal, including but not limited to a mouse (rat, mouse), a non-human primate, a human, a dog, a cat. Hoofed animals (such as horses, cattle, sheep, pigs, goats).
  • “Therapeutically effective amount” or “effective amount” refers to an amount of plasminogen sufficient to effect such prevention and/or treatment of a disease when administered to a mammal or other subject to treat the disease.
  • the “therapeutically effective amount” will vary depending on the plasminogen used, the severity of the disease and/or its symptoms of the subject to be treated, and the age, weight, and the like.
  • Plasminogen can be isolated and purified from nature for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When the polypeptide is chemically synthesized, it can be synthesized in a liquid phase or a solid phase.
  • Solid phase polypeptide synthesis SPPS
  • Fmoc and Boc Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize plasminogen.
  • the attached solid phase free N-terminal amine is coupled to a single N-protected amino acid unit. This unit is then deprotected to reveal a new N-terminal amine that can be attached to other amino acids.
  • the peptide remains immobilized on the solid phase and then cut off.
  • the plasminogen of the present invention can be produced using standard recombinant methods.
  • a nucleic acid encoding plasminogen is inserted into an expression vector operably linked to a regulatory sequence in an expression vector.
  • Expression control sequences include, but are not limited to, promoters (eg, naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • Expression regulation can be a eukaryotic promoter system in a vector that is capable of transforming or transfecting eukaryotic host cells (eg, COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of plasminogen.
  • Suitable expression vectors are typically replicated as an episome in the host organism or as an integral part of the host chromosomal DNA.
  • expression vectors typically contain a selection marker (eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate transformation of the desired DNA sequence with foreign sources. Those cells are tested.
  • a selection marker eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance
  • Escherichia coli is an example of a prokaryotic host cell that can be used to clone a subject antibody-encoding polynucleotide.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Genus (Pseudomonas) kind.
  • expression vectors can also be generated which will typically contain expression control sequences (e.g., origins of replication) that are compatible with the host cell.
  • promoters such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from phage lambda. Promoters typically control expression, optionally in the context of manipulating a gene sequence, and have a ribosome binding site sequence, etc., to initiate and complete transcription and translation.
  • yeast can also be used for expression.
  • Yeast e.g., S. cerevisiae
  • Pichia are examples of suitable yeast host cells in which a suitable vector has expression control sequences (e.g., a promoter), an origin of replication, a termination sequence, and the like, as desired.
  • a typical promoter comprises 3-phosphoglycerate kinase and other saccharolytic enzymes.
  • Inducible yeast is initiated by a promoter specifically comprising an alcohol dehydrogenase, an isocytochrome C, and an enzyme responsible for the utilization of maltose and galactose.
  • mammalian cells e.g., mammalian cells cultured in in vitro cell culture
  • an anti-Tau antibody of the invention e.g., a polynucleotide encoding a subject anti-Tau antibody.
  • Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for these cells may contain expression control sequences such as origins of replication, promoters and enhancers (Queen et al, Immunol. Rev.
  • RNA splice sites sites that are ribosome binding.
  • RNA splice sites sites that are ribosome binding.
  • polyadenylation sites sites that are ribosome binding sites.
  • transcription terminator sequences sites that are ribosome binding sites.
  • suitable expression control sequences are promoters derived from the white immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al, J. Immunol. 148: 1149 (1992).
  • the invention may be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, and the like.
  • Plasminogen is substantially pure, such as at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure. Or more pure, for example, free of contaminants, such as cellular debris, macromolecules other than the subject antibody, and the like.
  • the jelly can be formed by mixing plasminogen of the desired purity with an optional pharmaceutical carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980)).
  • the therapeutic formulation is prepared as a dry formulation or as an aqueous solution.
  • Acceptable carriers, excipients, and stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as Octadecyldimethylbenzylammonium chloride; chlorinated hexane diamine; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl p-hydroxybenzoic acid Esters such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol; low molecular weight polypeptide (less than about 10 residues) Protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, his
  • the formulations of the invention may also contain more than one active compound as required for the particular condition being treated, preferably those having complementary activities and no side effects to each other.
  • active compound for example, antihypertensive drugs, antiarrhythmic drugs, drugs for treating diabetes, and the like.
  • the plasminogen of the present invention may be encapsulated in microcapsules prepared by, for example, coacervation techniques or interfacial polymerization, for example, may be placed in a glial drug delivery system (eg, liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules are placed in hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a macroemulsion.
  • glial drug delivery system eg, liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules are placed in hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a macroemulsion.
  • the plasminogen of the invention for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filter before or after lyophilization and reconstitution.
  • the plasminogen of the present invention can prepare a sustained release preparation.
  • sustained release formulations include solid hydrophobic polymeric semi-permeable matrices having a shape and containing glycoproteins, such as films or microcapsules.
  • sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) (Langer et al, J. Biomed. Mater. Res., 15: 167-277 (1981); Langer, Chem .Tech., 12:98-105 (1982)) or poly(vinyl alcohol), polylactide (U.S.
  • Patent 3,739,919, EP 58,481 L-glutamic acid and ⁇ -ethyl-L-glutamic acid Copolymer (Sidman, et al, Biopolymers 22: 547 (1983)), not Degraded ethylene-vinyl acetate (Langer, et al., supra), or degradable lactic acid-glycolic acid copolymer such as Lupron DepotTM (from lactic acid-glycolic acid copolymer and leucine hydrazide) Injectable microspheres composed of leuprolide acetate, and poly D-(-)-3-hydroxybutyric acid.
  • Lupron DepotTM from lactic acid-glycolic acid copolymer and leucine hydrazide
  • Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can sustain release molecules for more than 100 days, while some hydrogels release proteins for shorter periods of time.
  • a rational strategy for stabilizing proteins can be designed based on relevant mechanisms. For example, if the mechanism of aggregation is found to be an intermolecular SS bond by thiodisulfide bond exchange, it can be modified by modifying the thiol residue, lyophilizing from an acidic solution, controlling humidity, using suitable additives, and developing specific The polymer matrix composition is used to achieve stability.
  • compositions of this invention may be effected intramuscularly in different ways, such as by intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery).
  • Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffering media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils.
  • Intravenous vehicles contain liquid and nutritional supplements, electrolyte supplements, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, and the like.
  • the medical staff will determine the dosage regimen based on various clinical factors. As is well known in the medical arts, the dosage of any patient depends on a variety of factors, including the patient's size, body surface area, age, specific compound to be administered, sex, number and route of administration, overall health, and other medications administered simultaneously. .
  • the pharmaceutical composition of the present invention comprising plasminogen may be, for example, in the range of about 0.0001 to 2000 mg/kg per day, or about 0.001 to 500 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 10 mg/kg, 50 mg/kg, etc.) Subject weight.
  • the dose can be 1 mg/kg body weight or 50 mg/kg body weight or in the range of 1-50 mg/kg, or at least 1 mg/kg. Dosages above or below this exemplary range are also contemplated, particularly in view of the above factors. Intermediate doses in the above ranges are also included in the scope of the present invention.
  • the subject can administer such doses daily, every other day, every week, or according to any other schedule determined by empirical analysis.
  • An exemplary dosage schedule includes 1-10 mg/kg for several days. The therapeutic effect and safety need to be evaluated in real time during the administration of the drug of the present invention.
  • One embodiment of the invention relates to an article or kit comprising a plasminogen or plasmin of the invention useful for treating a cardiovascular disease caused by diabetes and a related disorder thereof.
  • the article preferably includes a container, label or package insert. Suitable containers are bottles, vials, syringes, and the like.
  • the container can be made of various materials such as glass or plastic.
  • the container contains a composition that is effective to treat a disease or condition of the invention and has a sterile access port (eg, the container can be an intravenous solution or vial containing a stopper that can be penetrated by a hypodermic needle) of).
  • At least one active agent in the composition is plasminogen/plasmin.
  • the label on or attached to the container indicates that the composition is used to treat the cardiovascular disease caused by diabetes and its related conditions of the present invention.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
  • the article comprises a package insert with instructions for use, including, for example, a user instructing the composition to administer the plasminogen composition and other drugs to treat the accompanying disease.
  • FIG. 1 Serum high-density lipoprotein cholesterol test results after 10 days and 20 days of plasminogen administration in 3% cholesterol hyperlipidemia model mice. The results showed that the serum HDL-C concentration in the plasminogen group was significantly higher than that in the vehicle-treated PBS group after administration of plasminogen, and the high-density lipoprotein concentrations were statistically different after 10 and 20 days of administration. Extremely significant (** means P ⁇ 0.01). It is indicated that plasminogen can effectively increase the content of high-density lipoprotein cholesterol in serum of hyperlipidemia model mice and improve dyslipidemia in hyperlipidemia model mice.
  • Figure 2 Results of serum total cholesterol after 20 days of plasminogen administration in 3% cholesterol hyperlipidemia model mice. The results showed that the total cholesterol concentration in the plasminogen group was significantly lower than that in the vehicle PBS control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can reduce the total cholesterol content in the serum of hyperlipidemia model mice and has the function of lowering blood lipids.
  • Figure 3 Results of serum low-density lipoprotein cholesterol after 20 days of administration of plasminogen in 3% cholesterol hyperlipidemia model mice. The results showed that the concentration of LDL-C in the plasminogen group was significantly lower than that in the vehicle-treated PBS control group, and the statistical difference was significant (* indicates P ⁇ 0.05). Explain that plasminogen can decrease The content of low-density lipoprotein cholesterol in the serum of mice with hypolipidemia has the function of improving hyperlipemia.
  • Figure 4 shows the results of liver oil red O staining after administration of plasminogen for 30 days in a 16-week hyperlipidemia model mouse.
  • A is the control vehicle PBS control group
  • B is the plasminogen group
  • C is the quantitative analysis result.
  • the results showed that the liver fat deposition in the plasminogen group was significantly less than that in the vehicle control group, and the statistical analysis was statistically significant (* indicates P ⁇ 0.05). This indicates that plasminogen can improve the deposition of fat in the liver of hyperlipidemia model mice.
  • Fig. 5 Observation results of aortic sinus oil red O staining after administration of plasminogen for 30 days in a 16-week hyperlipidemia model mouse.
  • a and C are the control group for the vehicle PBS
  • B and D are for the plasminogen group
  • E is the quantitative analysis result.
  • the results showed that the aortic sinus fat deposition in the plasminogen group was significantly less than that in the vehicle control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can improve the deposition of fat in the aortic sinus of hyperlipidemia model mice.
  • FIG. 6 Representative images of HE staining of aortic sinus after administration of plasminogen for 30 weeks in a 16-week hyperlipidemia model mouse.
  • a and C were given to the vehicle PBS control group, and B and D were given to the plasminogen group.
  • the results showed that foam cell deposition (marked by the arrow) was observed in the aortic wall of the vehicle control group, and plaque deposition was severe; only mild foam cell deposition was observed in the aortic wall of the plasminogen group, and under the intima No obvious atheroma deposits were found, and the aorta in the plasminogen group was less damaged. This indicates that plasminogen can ameliorate the damage caused by lipid deposition in the aortic sinus wall of mice with hyperlipidemia.
  • FIG. 7 Immunofibrotic staining of cardiac fibrin 30 days after administration of plasminogen in a 16-week hyperlipidemia model mouse.
  • A is the control vehicle PBS control group
  • B is the plasminogen group
  • C is the quantitative analysis result.
  • the results showed that the positive expression of cardiac fibrin in the plasminogen group was significantly less than that in the vehicle control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can reduce heart damage caused by hyperlipidemia.
  • FIG. 8 Representative pictures of cardiac IgM immunostaining 30 days after administration of plasminogen in a 16-week hyperlipidemia model mouse.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that the positive expression of IgM in the plasminogen group was significantly less than that in the vehicle PBS control group, indicating that plasminogen can alleviate the heart damage caused by hyperlipidemia.
  • Figure 9 Representative pictures of Sirius red staining after 30 days of plasminogen administration in a 16-week hyperlipidemia model mouse.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that the deposition of collagen in the plasminogen group was significantly less than that in the vehicle PBS control group, indicating that plasminogen can alleviate cardiac fibrosis in hyperlipidemia model mice.
  • FIG. 10 Serum troponin assay results after 30 days of administration of plasminogen in a 16-week hyperlipidemia model mouse. The results showed that the concentration of serum cardiac troponin in the control group was significantly higher than that in the plasminogen group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can significantly repair the damage of hyperlipidemic heart.
  • FIG 11 Results of serum atherosclerosis index after 20 days of administration of plasminogen in 3% cholesterol hyperlipidemia model mice. The results showed that the atherosclerosis index of the plasminogen group was significantly lower than that of the vehicle PBS control group, and the statistical difference was extremely significant (** means P ⁇ 0.01). This indicates that plasminogen can effectively reduce the risk of atherosclerosis in mice with hyperlipidemia.
  • FIG. 12 Serum cardiac risk risk index results after 20 days of plasminogen administration in 3% cholesterol hyperlipidemia model mice. The results showed that the CRI for the plasminogen group was significantly smaller than that of the vehicle control group, and the statistical difference was extremely significant (** indicates P ⁇ 0.01). This indicates that plasminogen can effectively reduce the risk of heart disease in mice with hyperlipidemia.
  • Figure 13 is a picture of liver oil red O staining after 35 days of plasminogen administration in 24-25 week diabetic mice. The results showed that the lipid deposition area of the liver of the plasminogen group was significantly smaller than that of the vehicle PBS control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can reduce the deposition of fat in the liver of diabetic mice.
  • Figure 14 is a picture of aortic HE staining after administration of plasminogen for 23 days in 24-25 week old diabetic mice.
  • a and C were given to the vehicle PBS control group, and B and D were given to the plasminogen group.
  • the results showed that there were foam cell deposition (arrow mark) on the vascular wall of the control vehicle PBS control group, the middle elastic membrane was disorderly arranged, the blood vessel wall was thickened, and the tube wall was uneven; the structure of the middle elastic membrane of the plasminogen group was regular.
  • the shape of the wave is uniform and the thickness of the vessel wall is uniform. It indicates that injection of plasminogen has a certain repairing effect on aortic injury caused by diabetes.
  • FIG. 15 Representative pictures of ventricular oil red O staining after 26 days of administration of plasminogen in 26-week-old diabetic mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that ventricular lipid deposition (arrow markers) was significantly less in the plasminogen group than in the vehicle PBS control group. This indicates that plasminogen can reduce ventricular lipid deposition in diabetic mice and promote the repair of ventricular injury.
  • Figure 16 Results of detection of high-density lipoprotein cholesterol in serum after administration of plasminogen for 35 days in 26-week-old diabetic mice. The results showed that after 35 days of continuous injection of human plasmin source in diabetic mice, the serum level of HDL-C in the plasminogen group was higher than that in the vehicle control group, and the statistical difference was significant (* indicates P ⁇ 0.05). It indicated that injection of plasminogen could promote the increase of serum high-density lipoprotein cholesterol and improve the dyslipidemia in diabetic mice.
  • FIG 17 Results of detection of serum low-density lipoprotein cholesterol (LDL-C) in rabbits aged 24-25 weeks of age after administration of plasminogen for 31 days.
  • FIG. 18 Results of serum total cholesterol test after 30 days of administration of plasminogen in ApoE atherosclerotic model mice. The results showed that the total cholesterol concentration in the plasminogen group was significantly lower than that in the vehicle PBS control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can reduce the total cholesterol in the serum of ApoE atherosclerotic model mice and improve the dyslipidemia in atherosclerotic model mice.
  • Figure 19 Results of serum triglyceride test after 30 days of administration of plasminogen in ApoE atherosclerotic model mice. The results showed that the concentration of triglyceride in the plasminogen group was significantly lower than that in the vehicle PBS control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can reduce the serum triglyceride content of ApoE atherosclerosis model mice and improve dyslipidemia in atherosclerotic model mice.
  • Figure 20 shows the results of serum low density lipoprotein cholesterol assay after 30 days of plasminogen administration in ApoE atherosclerotic model mice.
  • the results showed that the concentration of LDL-C in the plasminogen group was significantly lower than that in the vehicle-treated PBS control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can reduce the content of low-density lipoprotein cholesterol in serum of ApoE atherosclerotic model mice and improve dyslipidemia in atherosclerotic model mice.
  • Figure 21 is a representative picture of liver oil red O staining after 30 days of plasminogen administration in ApoE atherosclerotic model mice.
  • A is the control vehicle PBS control group
  • B is the plasminogen group
  • C is the quantitative analysis result.
  • the results showed that the liver fat deposition in the plasminogen group was significantly less than that in the vehicle control group, and the statistical analysis was statistically significant (* indicates P ⁇ 0.05). This indicates that plasminogen can reduce the deposition of fat in the liver of atherosclerotic model mice.
  • Figure 22 is a representative picture of aortic sinus oil red O staining after 30 days of plasminogen administration in ApoE atherosclerotic model mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that the aortic sinus fat deposition in the plasminogen group was significantly less than that in the vehicle PBS control group. This indicates that plasminogen can improve the deposition of fat in the aortic sinus of atherosclerotic model mice.
  • FIG 23 Representative pictures of aortic sinus simulone red staining after administration of plasminogen for 16 weeks in a 16-week-old hyperlipidemia model mouse.
  • a and C were given to the vehicle PBS control group, and B and D were given to the plasminogen group.
  • the results showed that the area of collagen deposition (arrow mark) in the vascular sinus of the plasminogen group was significantly smaller than that of the vehicle PBS control group, indicating that plasminogen can attenuate the aortic sinus fibrosis in hyperlipidemia model mice. Level.
  • Figure 24 is a graph showing cardiac coefficient statistics after 30 days of plasminogen administration in ApoE atherosclerotic model mice. The results showed that the cardiac organ coefficient of the plasminogen group was significantly lower than that of the vehicle PBS control group. This indicates that plasminogen can ameliorate cardiac compensatory hypertrophy caused by cardiac injury in ApoE atherosclerotic model mice.
  • Figure 25 shows the results of Sirius red staining of 3% cholesterol hyperlipidemia model mice after 30 days of administration of plasminogen.
  • A is a blank control group
  • B is a vehicle control group
  • C is a plasminogen group
  • D is a quantitative analysis result.
  • the results showed that the collagen deposition (arrow mark) in the plasminogen group was significantly less than that in the vehicle control group, and the statistical difference was significant; the fibrosis in the plasminogen group was basically restored to normal levels. This indicates that plasminogen can effectively reduce renal fibrosis in 3% cholesterol hyperlipidemia model mice.
  • Figure 26 shows the results of observation of renal oil red O in mice of 3% cholesterol hyperlipidemia model 30 days after administration of plasminogen.
  • A is a blank control group
  • B is a vehicle control group
  • C is a plasminogen group
  • D is a quantitative analysis result.
  • the results showed that the renal fat deposition (arrow mark) in the plasminogen group was significantly less than that in the vehicle control group, and the quantitative analysis was statistically significant.
  • the lipid deposition level in the plasminogen group was compared with the blank control group. The mice are similar. This indicates that plasminogen can reduce the deposition of fat in the kidney of hyperlipidemia model mice, thereby reducing kidney damage caused by fat deposition.
  • mice Sixteen-week-old male C57 mice were fed with 3% cholesterol and high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia [30 , 31] .
  • This model was designated as a model of 3% cholesterol hyperlipidemia.
  • the modeled mice continued to be fed a 3% cholesterol high fat diet.
  • 50 ⁇ L of blood was taken from each mouse three days before the administration, and total cholesterol was measured, and was randomly divided into two groups according to the total cholesterol concentration and body weight, with 8 in each group. The first dose was recorded as the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 20 days.
  • mice On the 10th and 20th day, the mice were fasted for 16 hours. On the 11th and 21st day, 50 ⁇ l of blood was taken from the iliac venous plexus, and the supernatant was centrifuged to detect serum high-density lipoprotein cholesterol (HDL-C). The high-density lipoprotein cholesterol content in this paper was detected by the method described in the test kit (Nanjing Institute of Bioengineering, item number A112-1).
  • High-density lipoprotein is an anti-atherosclerotic plasma lipoprotein, a protective factor for coronary heart disease, commonly known as “vascular scavenger.”
  • mice Sixteen-week-old male C57 mice were fed with 3% cholesterol and high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia [30 , 31] .
  • This model was designated as a model of 3% cholesterol hyperlipidemia.
  • the modeled mice continued to be fed a 3% cholesterol high fat diet.
  • 50 ⁇ L of blood was taken from each mouse three days before the administration, and total cholesterol was measured, and was randomly divided into two groups according to the total cholesterol concentration and body weight, with 8 in each group. The first dose was recorded as the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 20 days.
  • mice On the 20th day, the mice were fasted for 16 hours. On the 21st day, 50 ⁇ L of blood was taken from the iliac venous plexus, and the supernatant was obtained by centrifugation. Total cholesterol test was performed using the total cholesterol test kit (Nanjing Institute of Bioengineering, article number A111-1). .
  • the test results showed that the total cholesterol concentration in the plasminogen group was significantly lower than that in the vehicle PBS control group, and the statistical difference was significant (Fig. 2). This indicates that plasminogen can reduce the total cholesterol in the serum of hyperlipidemia model mice.
  • mice Sixteen-week-old male C57 mice were fed with 3% cholesterol and high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia [30 , 31] .
  • This model was designated as a model of 3% cholesterol hyperlipidemia.
  • the modeled mice continued to be fed a 3% cholesterol high fat diet.
  • 50 ⁇ l of blood was taken from each mouse three days before the administration, and total cholesterol was measured, and was randomly divided into two groups according to the total cholesterol concentration and body weight, with 8 in each group. The first dose was recorded as the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 20 days.
  • mice On the 20th day, the mice were fasted for 16 hours. On the 21st day, 50 ⁇ L of blood was taken from the iliac venous plexus, and the supernatant was obtained by centrifugation.
  • the low-density lipoprotein cholesterol test kit (Nanjing Institute of Bioengineering, item number A113-1) was used. Low density lipoprotein cholesterol (LDL-C) detection.
  • Low-density lipoprotein is a lipoprotein particle that carries cholesterol into peripheral tissue cells and can be oxidized to oxidized low-density lipoprotein when low-density lipoproteins, especially oxidized low-density lipoprotein (OX-LDL), are excessive.
  • OX-LDL oxidized low-density lipoprotein
  • the cholesterol it carries will accumulate on the arterial wall and cause arteriosclerosis. Therefore, low density lipoprotein cholesterol is called "bad cholesterol.”
  • plasminogen can reduce the content of low-density lipoprotein cholesterol in serum of hyperlipidemia model mice and improve dyslipidemia in hyperlipidemia mice.
  • Example 4 Degradation of plasminogen in the liver of a 16-week hyperlipidemia model mouse
  • mice 11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model [30,31] .
  • This model was assigned to 16-week hyperlipidemia.
  • Mice after modeling continue to feed high cholesterol feed. 50 ⁇ l of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. .
  • the start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the mice were sacrificed for 30 days, and the mice were sacrificed on the 31st day.
  • the liver was fixed in 4% paraformaldehyde for 24-48 hours, respectively, in a 15%, 30% sucrose solution at 4 ° C overnight, embedded in OCT, and frozen section thickness. 8 ⁇ m, oil red O staining for 15min, 75% alcohol differentiation for 5 seconds, hematoxylin staining for 30s, glycerin gelatin sealing. Sections were observed under a 200x optical microscope.
  • Oil red O staining can show lipid deposition, reflecting the extent of lipid deposition [32] .
  • the results showed that the liver fat deposition in the plasminogen group (Fig. 4B) was significantly less than that in the vehicle PBS control group (Fig. 4A), and the statistical analysis was statistically significant (Fig. 4C). This indicates that plasminogen can reduce the deposition of fat in the liver of hyperlipidemia model mice.
  • Example 5 Plasminogen-reducing lipid deposition in aortic sinus of a 16-week hyperlipidemia model mouse
  • mice 11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model [30,31] .
  • This model was assigned to 16-week hyperlipidemia.
  • Mice after modeling continue to feed high cholesterol feed. 50 ⁇ l of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. .
  • the start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the mice were sacrificed for 30 days, and the mice were sacrificed on the 31st day.
  • the heart tissue was fixed in 4% paraformaldehyde for 24-48 hours, respectively, in a 15%, 30% sucrose overnight at 4 ° C, OCT embedding, aorta
  • the sinus frozen section thickness was 8 ⁇ m, oil red O staining for 15 min, 75% alcohol differentiation for 5 seconds, hematoxylin staining for 30 s, and glycerin gelatin for sealing. Sections were observed under 40 (Fig. 5A, 5B), 200 times (Fig. 5C, 5D) magnification optical microscope.
  • mice 11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model [30,31] .
  • This model was assigned to 16-week hyperlipidemia.
  • Mice after modeling continue to feed high cholesterol feed. 50 ⁇ l of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. .
  • the start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the mice were administered for 30 days, and the mice were sacrificed on the 31st day, and the heart tissue was fixed in 4% paraformaldehyde for 24-48 hours.
  • the fixed tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene.
  • the fixed tissue samples were dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the aortic sinus tissue section was 3 ⁇ m.
  • the sections were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining). After 1% hydrochloric acid alcohol differentiation, the ammonia water returned to the blue and dehydrated with an alcohol gradient.
  • the slices were cut at 40 (Fig. 6A, B). 400 times (Fig. 6C, D) observed under an optical microscope.
  • mice 11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model [30,31] .
  • This model was assigned to 16-week hyperlipidemia.
  • Mice after modeling continue to feed high cholesterol feed. 50 ⁇ l of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. .
  • the start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the mice were administered for 30 days, and the mice were sacrificed on the 31st day, and the heart tissue was fixed in 4% paraformaldehyde for 24-48 hours.
  • the fixed tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time.
  • the color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes.
  • the gradient alcohol was dehydrated, the xylene was transparent and the neutral gum was sealed, and the sections were observed under a 200-fold optical microscope.
  • Fibrinogen is a precursor of fibrin.
  • fibrinogen is hydrolyzed into fibrin deposits at the site of injury [33,34] . Therefore, the level of damaged local fibrin can be used as a marker of the degree of damage.
  • Example 8 Plasminogen effectively protects myocardial injury in a 16-week hyperlipidemia model mouse
  • mice 11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model [30,31] .
  • This model was assigned to 16-week hyperlipidemia.
  • Mice after modeling continue to feed high cholesterol feed. 50 ⁇ l of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. .
  • the start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the mice were administered for 30 days, and the mice were sacrificed on the 31st day, and the heart tissue was fixed in 4% paraformaldehyde for 24-48 hours.
  • the fixed tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate with 3% hydrogen peroxide for 15 minutes and wash twice with water for 5 minutes each time.
  • IgM antibodies play an important role in the clearance of apoptotic and necrotic cells, and the level of local IgM antibodies in damaged tissues is positively correlated with the degree of injury [35,36] . Therefore, detecting the level of local IgM antibodies in tissues and organs can reflect the degree of damage of the tissues and organs.
  • Example 9 plasminogen attenuates cardiac fibrosis in a 16-week hyperlipidemia model mouse
  • mice 11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model [30,31] .
  • This model was assigned to 16-week hyperlipidemia.
  • Mice after modeling continue to feed high cholesterol feed. 50 ⁇ l of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. .
  • the start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the mice were administered for 30 days, and the mice were sacrificed on the 31st day, and the heart tissue was fixed in 4% paraformaldehyde for 24-48 hours.
  • the fixed tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene. The thickness of the tissue section was 3 ⁇ m.
  • the sections were dewaxed and rehydrated, washed once with water, stained with 0.1% Sirius red saturated picric acid for 30 minutes, rinsed with running water for 2 min, stained with hematoxylin for 1 minute, rinsed with water, differentiated with 1% hydrochloric acid, and returned to blue with ammonia. Rinse with running water, dry and seal with neutral gum, and observe under a 200x optical microscope.
  • Sirius red staining can make collagen staining for a long time. As a special staining method for pathological sections, Sirius red staining can specifically display collagen tissue.
  • mice 11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model [30,31] .
  • This model was assigned to 16-week hyperlipidemia.
  • Mice after modeling continue to feed high cholesterol feed. 50 ⁇ l of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. .
  • the start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the mice began to fast after the 30th day of administration, fasting for 16 hours.
  • the eyeballs were taken for blood collection, and the supernatant was obtained by centrifugation.
  • Cardiac troponin I (CTNI) detection kit was used. (Nanjing built) to measure the concentration of troponin in serum.
  • Cardiac troponin I is an important marker of myocardial injury, and its serum concentration can reflect the extent of myocardial damage [37] .
  • mice Sixteen-week-old male C57 mice were fed with 3% cholesterol and high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia [30 , 31] .
  • This model was designated as a model of 3% cholesterol hyperlipidemia.
  • the modeled mice continued to be fed a 3% cholesterol high fat diet.
  • 50 ⁇ l of blood was taken from each mouse three days before administration, total cholesterol (T-CHO) was detected, and two groups were randomly divided into two groups according to the total cholesterol concentration and body weight.
  • the start of administration was recorded as the first day, and the plasminogen group mice were injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • mice After the 20th day of administration, the mice began to fast, fasted for 16 hours, and on the 21st day, 50 ⁇ L of blood was taken from the iliac venous plexus, and the supernatant was obtained by centrifugation.
  • the total cholesterol content was determined by the total cholesterol test kit (Nanjing Jianshe Bioengineering Research Institute). , Item No. A111-1) was tested; high-density lipoprotein cholesterol (HDL-C) content was detected using a high-density lipoprotein cholesterol test kit (Nanjing Institute of Bioengineering, item number A112-1).
  • the atherosclerosis index is a comprehensive indicator of clinical prediction of atherosclerosis. It is considered to be clinically more meaningful than the individual cholesterol, triglyceride, high-density lipoprotein and low-density lipids in estimating the degree of risk of coronary heart disease.
  • the protein is larger [38] .
  • Atherosclerosis index (T-CHO-HDL-C) / HDL-C.
  • mice Sixteen-week-old male C57 mice were fed with 3% cholesterol and high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia [30 , 31] .
  • This model was designated as a model of 3% cholesterol hyperlipidemia.
  • the modeled mice continued to be fed a 3% cholesterol high fat diet.
  • 50 ⁇ l of blood was taken from each mouse three days before administration, and total cholesterol (T-CHO) was measured, and randomly divided into two groups according to the total cholesterol concentration, 8 in each group. The start of administration was recorded as the first day, and the plasminogen group mice were injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • mice After the 20th day of administration, the mice began to fast, fasted for 16 hours, and on the 21st day, 50 ⁇ L of blood was taken from the iliac venous plexus, and the supernatant was obtained by centrifugation.
  • the total cholesterol content was determined by the total cholesterol test kit (Nanjing Jianshe Bioengineering Research Institute). , Item No. A111-1) was tested; high-density lipoprotein cholesterol (HDL-C) content was detected using a high-density lipoprotein cholesterol test kit (Nanjing Institute of Bioengineering, item number A112-1).
  • Cardiac risk index T-CHO/HDL-C.
  • Cardiac risk index (CRI) is used to assess the risk of dyslipidemia-induced heart disease [38] .
  • mice Ten male db/db mice aged 24-25 weeks were randomly divided into two groups, and the vehicle PBS control group and the plasminogen group were each given 5 rats. On the day of the start of the experiment, the day 0 was recorded and the group was weighed, and the first day was given to plasminogen or PBS. The plasminogen group was injected with plasminogen at a dose of 2 mg/0.2 ml/day/day, and the same volume of PBS was administered to the vehicle PBS control group for 30 days. On the 36th day, the mice were sacrificed and the liver tissue was fixed with 4% paraformaldehyde for 24-48 hours.
  • mice Ten male db/db mice aged 24-25 weeks were randomly divided into two groups, and the vehicle PBS control group and the plasminogen group were each given 5 rats. On the day of the start of the experiment, the group was weighed on the 0th day, and the PBS or plasminogen was administered on the first day for 31 days. The plasminogen group was injected with plasminogen at a dose of 2 mg/0.2 ml/day/day, and the same volume of PBS was administered to the vehicle PBS control group by tail vein injection. Mice were sacrificed on day 32 and the aorta was fixed in 10% neutral formalin fixative for 24 hours. The fixed aorta was paraffin-embedded by alcohol gradient dehydration and xylene transparency.
  • the thickness of the tissue section was 5 ⁇ m, the sections were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), and after 1% hydrochloric acid alcohol was differentiated, the ammonia water returned to the blue and dehydrated with an alcohol gradient to seal the pieces, and the sections were 400 times (Fig. 14A, B). And 1000 times (Fig. 14C, D) observed under the oil microscope.
  • HE staining hematoxylin and eosin
  • Diabetes with hyperlipidemia is a common complication of diabetes and an important risk factor for diabetic macroangiopathy [39] .
  • the staining results showed that there was foam cell deposition (arrow mark) on the vessel wall of the vehicle PBS control group (Fig. 14A, C), the middle layer elastic membrane was disordered, the vessel wall was thickened, and the tube wall was uneven; the plasminogen group was given.
  • FIG. 14B, D The structure of the middle elastic membrane is regular, wavy, and the thickness of the vessel wall is uniform. It indicates that injection of plasminogen can alleviate the lipid deposition in the aortic wall of diabetic mice, and has a certain protective effect on the damage caused by lipid deposition in arterial wall.
  • mice at 26 weeks of age were randomly assigned to 4 rats in the plasminogen group and 5 to the vehicle PBS control group.
  • the plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group for 35 days.
  • the mice were sacrificed on the 36th day, and the hearts were fixed in 4% paraformaldehyde for 24-48 hours, respectively, in 15%, 30% sucrose at 4 ° C overnight, embedded in OCT, frozen section thickness 8 ⁇ m, oil red O staining 15 min, 75% alcohol differentiation for 5 seconds, hematoxylin staining for 30s, glycerin gelatin seal. Sections were observed under a 400x optical microscope.
  • ventricular lipid deposition (arrow mark) was significantly less in the plasminogen group (Fig. 15B) than in the vehicle PBS control group (Fig. 15A). This indicates that plasminogen can reduce the deposition of fat in the ventricle of diabetic mice and promote the repair of ventricular damage.
  • mice Twenty male 26-week-old db/db mice were randomly divided into groups of 11 in the plasminogen group and 9 in the vehicle PBS control group. On the day of the start of the experiment, the day 0 was recorded and the group was weighed, and on the first day, plasminogen or PBS was administered for 35 days. The plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail vein of the control group.
  • mice were harvested from the eyeballs for whole blood, centrifuged at 3500C/min for 10 minutes at 4°C, and the supernatant was taken and high-density in serum was detected using the High Density Lipoprotein Test Kit (Nanjing Institute of Bioengineering, Cat. No. A112-1). Lipoprotein cholesterol (HDL-C) concentration.
  • HDL-C Lipoprotein cholesterol
  • mice of 24-25 weeks old were randomly divided into groups, 5 rats in the plasminogen group and the vehicle control group, and 3 db/m were used as the normal control group.
  • the day of the experiment was recorded as the 0th day weighing group, and the first day was given to plasminogen or PBS for 31 days.
  • the plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail vein of the PBS control group.
  • the normal control mice were not treated.
  • mice were harvested from the eyeballs for whole blood, centrifuged at 3500C/min for 10 minutes at 4°C, and the supernatant was taken and tested in serum using the Low Density Lipoprotein Cholesterol Detection Kit (Nanjing Institute of Bioengineering, Cat. No. A113-1). Low density lipoprotein cholesterol (LDL-C) concentration.
  • LDL-C Low density lipoprotein cholesterol
  • Example 18 plasminogen reduces serum total cholesterol in ApoE atherosclerotic mice
  • mice at 6 weeks of age were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce an atherosclerosis model [40,41] .
  • Mice after modeling continue to feed high-fat, high-cholesterol feed.
  • 50 ⁇ l of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 7 rats in the vehicle PBS control group, and 6 cells in the plasminogen group. .
  • the first dose was started on the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 30 days. On the 30th day, the mice were fasted for 16 hours. On the 31st day, the eyeballs were removed and blood was taken. The supernatant was centrifuged to obtain a total cholesterol test using a total cholesterol test kit (Nanjing Institute of Bioengineering, item number A111-1).
  • mice at 6 weeks of age were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce an atherosclerosis model [40,41] .
  • Mice after modeling continue to feed high-fat, high-cholesterol feed.
  • 50 ⁇ l of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 7 rats in the vehicle PBS control group, and 6 cells in the plasminogen group. .
  • the first dose was recorded as the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 30 days. On the 30th day, the mice were fasted for 16 hours. On the 31st day, the eyeballs were taken for blood collection, and the supernatant was centrifuged to obtain a triglyceride detection kit (Nanjing Institute of Bioengineering, article number A110-1) for triglyceride detection.
  • mice at 6 weeks of age were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce an atherosclerosis model [40,41] .
  • Mice after modeling continue to feed high-fat, high-cholesterol feed.
  • 50 ⁇ l of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 7 rats in the vehicle PBS control group, and 6 cells in the plasminogen group. .
  • the first dose was recorded as the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 30 days. On the 30th day, the mice were fasted for 16 hours. On the 31st day, the eyeballs were taken for blood collection, and the supernatant was centrifuged to obtain a low-density lipoprotein cholesterol (LDL-C) test kit (Nanjing Institute of Bioengineering, item number A113-1). LDL-C detection.
  • LDL-C low-density lipoprotein cholesterol
  • mice at 6 weeks of age were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce an atherosclerosis model [40,41] .
  • Mice after modeling continue to feed high-fat, high-cholesterol feed.
  • 50 ⁇ l of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 7 rats in the vehicle PBS control group, and 6 cells in the plasminogen group. .
  • the first dose was recorded as the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 30 days.
  • the mice were sacrificed on the 31st day, and the liver tissues were fixed in 4% paraformaldehyde for 24-48 hours, respectively, in a 15%, 30% sucrose overnight at 4 ° C, embedded in OCT, frozen section thickness 8 ⁇ m, oil red O Dyeing for 15 min, 75% alcohol differentiation for 5 seconds, hematoxylin staining for 30s, glycerin gelatin for sealing. Sections were observed under a 400x optical microscope.
  • Example 22 Plasminogen improves lipid deposition in aortic sinus of ApoE atherosclerotic mice
  • mice at 6 weeks of age were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce an atherosclerosis model [40,41] .
  • Mice after modeling continue to feed high-fat, high-cholesterol feed.
  • 50 ⁇ l of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 7 rats in the vehicle PBS control group, and 6 cells in the plasminogen group. .
  • the first dose was recorded as the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 30 days.
  • the mice were sacrificed on the 31st day, and the heart tissue was fixed in 4% paraformaldehyde for 24-48 hours.
  • the cells were sedimented in 15% and 30% sucrose at 4 °C overnight, embedded in OCT, and the aortic sinus frozen section was 8 ⁇ m thick. Oil red O staining for 15 min, 75% alcohol differentiation for 5 seconds, hematoxylin staining for 30s, glycerin gelatin for sealing. Sections were observed under a 40x optical microscope.
  • mice 11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model [30,31] .
  • This model was assigned to 16-week hyperlipidemia.
  • Mice after modeling continue to feed high cholesterol feed. 50 ⁇ l of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. .
  • the start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the mice were sacrificed on the 31st day, and the hearts were fixed in 4% paraformaldehyde for 24-48 hours.
  • the fixed tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene.
  • the thickness of the aortic sinus slice was 3 ⁇ m. The slice was dewaxed and rehydrated and washed once.
  • mice at 6 weeks of age were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce an atherosclerosis model [40,41] .
  • the mice after the modeling were taken 50 ⁇ l of blood for three days before the administration to detect the total cholesterol (T-CHO) content, and were randomly divided into two groups according to the T-CHO content, and the vehicle PBS control group was given 7 cells. 6 lysogen group.
  • the first dose was started on the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • Cardiac coefficient (%) heart weight / body weight ⁇ 100.
  • mice of 9 weeks old were fed with 3% cholesterol and high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia [30 , 31] .
  • This model was classified as a model of 3% cholesterol hyperlipidemia.
  • the modeled mice continued to be fed a 3% cholesterol high fat diet.
  • Another 5 male C57 mice of the same age were used as a blank control group, and normal maintenance feed was fed during the experiment.
  • the model mice were randomly divided into two groups according to the total cholesterol concentration and body weight, and given to the plasminogen group and the vehicle PBS control group, each group was 8 only.
  • mice were injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • Mice were dosed for 30 days after the 30th day of administration, and the mice were sacrificed on the 31st day, and the kidneys were fixed in 4% paraformaldehyde for 24-48 hours.
  • the fixed tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene.
  • the thickness of the slice was 3 ⁇ m, the slice was dewaxed and rehydrated, washed once with water, stained with 0.1% Sirius red saturated picric acid for 30 minutes, rinsed with running water for 2 min, stained with hematoxylin for 1 minute, rinsed with water, differentiated with 1% hydrochloric acid alcohol, and returned to blue with ammonia. Rinse with running water, dry and seal with neutral gum, and observe under a 200x optical microscope.
  • kidney collagen deposition (arrow mark) was significantly less in the plasminogen group (Fig. 25C) than in the vehicle PBS control group (Fig. 25B), and the statistical difference was significant (Fig. 25D); plasminogen group fibers were given. Basically returned to normal levels ( Figure 25A). This indicates that plasminogen can effectively reduce renal fibrosis in 3% cholesterol hyperlipidemia model mice.
  • mice of 9 weeks old were fed with 3% cholesterol and high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia [30 , 31] .
  • This model was classified as a model of 3% cholesterol hyperlipidemia.
  • the modeled mice continued to be fed a 3% cholesterol high fat diet.
  • Another 5 male C57 mice of the same age were used as a blank control group, and normal maintenance feed was fed during the experiment.
  • the model mice were randomly divided into two groups according to the total cholesterol concentration and body weight, and given to the plasminogen group and the vehicle PBS control group, each group was 8 only. The first dose was recorded as the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. 30 days. On the 31st day, the mice were sacrificed and the kidneys were fixed in 4% paraformaldehyde for 24-48 hours. They were sedimented in 15% and 30% sucrose at 4 °C overnight, embedded in OCT, frozen sections were 8 ⁇ m thick, and oil red O stained for 15 min. 75% alcohol differentiation for 5 seconds, hematoxylin staining for 30 seconds, glycerin gelatin seal. Sections were observed under a 400x optical microscope.
  • kidney fat deposition (arrow mark) in the plasminogen group (Fig. 26C) was significantly less than that in the vehicle PBS control group (Fig. 26B), and the quantitative analysis was statistically significant (Fig. 26D); Lipid deposition levels in the lysogen group were similar to those in the blank control group (Fig. 26A). It is indicated that plasminogen can reduce the deposition of fat in the kidney of hyperlipidemia model mice, thereby reducing the kidney damage caused by fat deposition.
  • Mooradian AD Cardiovascular disease in type 2 diabetes mellitus: Current management guidelines [J] . Arch Intern Med. 2003, 163: 33-40.
  • Grone HJ Glomerular lipids in non-hereditary forms of glomerulopathy/glomerulo nephritis [J] . Nephrol. Dial Transplant. 1999, 14: 1595-1598.

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Abstract

提供一种预防和/或治疗脂肪代谢紊乱及其相关病症的方法，包括给药易患或患有脂肪代谢紊乱及其相关病症的受试者有效量的纤溶酶原，以消减脂肪在身体各部位的异常沉积，从而实现预防和/或治疗脂肪代谢紊乱、其相关病症或并发症的目的。

Description

一种预防和治疗脂肪代谢紊乱及其相关病症的方法 技术领域
本发明涉及一种预防和/或治疗脂肪代谢紊乱及其相关病症的方法，包括给药易患或患有脂肪代谢紊乱及其相关病症的受试者有效量的纤溶酶原，以消减脂肪在身体组织器官的异常沉积，从而实现预防和/或治疗脂肪代谢紊乱及其相关病症、并发症的目的。
背景技术
脂肪代谢紊乱又称为脂肪代谢障碍，是代谢性疾病中的一种，是由原发性或获得性因素造成的血液及其他组织器官中脂质(脂类)及其代谢产物质和量的异常。脂质的代谢包括脂类在小肠内消化、吸收，由淋巴***进入血循环(通过脂蛋白转运)，经肝脏转化，储存于脂肪组织，需要时被组织利用。脂质在体内的主要功用是氧化供能，脂肪组织是机体的能量仓库，脂肪也能协同皮肤、骨骼、肌肉保护内脏，防止体温散发和帮助食物中脂溶性维生素的吸收。磷脂是所有细胞膜的重要结构成分，胆固醇是胆酸和类固醇激素(肾上腺皮质激素和性腺激素)的前体。脂类代谢受遗传、神经体液、激素、酶以及肝脏等组织器官的调节。当这些因素有异常时，可造成脂代谢紊乱和有关器官的病理生理变化，如高脂蛋白血症及其造成的临床综合征、肥胖症、脂肪肝等。
高脂蛋白血症(Hyperlipoproteinemia)由血液中的脂蛋白过高所致。血液中的脂质如甘油三脂(TG)、游离胆固醇(FC)、胆固醇脂(CE)和磷脂等很少溶于水，只有与载脂蛋白(APO)组成巨分子复合物(脂蛋白)，才能在血中溶解、运转和代谢。当血脂高于正常人上限即为高脂血症(Hyperlipemia)。由于血脂在血中以脂蛋白形式运输，故高脂血症也称为高脂蛋白血症。一般以成人空腹血甘油三脂超过160mg/dl，胆固醇超过260mg/dl，儿童胆固醇超过160mg/dl为标准[1]。
高脂蛋白血症(高脂血症)是动脉粥样硬化病变的重要原因之一，是体内脂质代谢异常的表现。由于血脂或脂蛋白的种类不同，超出正常范围的血脂或脂蛋白种类也可不同，所以世界卫生组织(WHO)将高脂蛋白血症分为五型：I型，主要是乳糜微粒增加，血清混浊呈乳白色，其中含大量 甘油三酯(TG)；II型，又分成II a型和IIb两亚型，前者主要为低密度脂蛋(LDL)明显增加，后者极低密度脂蛋白(VLDL)也有增加；III型，血清常混浊LDL和VLDL皆有增加，电泳上两者融合；IV型，主要为VLDL增加，血清或有混浊；V型，乳糜微粒及VLDL皆有增加，血清混浊呈乳白色。其中以II型和IV型最常见[1]。
高脂血症根据病因可分为原发性和继发性两大类。原发性多由于脂质和脂蛋白代谢先天性缺陷(或遗传性缺陷)以及某些环境因素(包括饮食、营养和药物等)通过未知的机理而引起。继发性主要继发于某种疾病，如糖尿病、肝脏疾病、肾脏疾病、甲状腺疾病，以及饮酒、肥胖。饮食与生活方式等环境因素也是该病的病因。
糖尿病常合并脂质代谢紊乱因此糖尿病又称为“糖脂病”[2]。糖尿病的发病机制与B细胞功能损伤及胰岛素抵抗相关，表现为慢性高血糖，而糖代谢的紊乱又常合并脂质代谢的紊乱。糖尿病脂代谢紊乱已经成为心血管疾病独立的危险因素，其主要表现为高甘油三脂血症、低水平的HDL、以及LDL浓度增加。
糖尿病脂质代谢紊乱的发生机制尚不清楚，但众多证据表明胰岛素抵抗是其发生的中心环节。近年来的研究还发现肠胰岛素抵抗也参与其中。对糖尿病动物模型和人群研究发现与脂代谢相关的某些基因表达异常进一步导致胰岛素抵抗。糖尿病患者动脉粥样硬化的发生与多种因素相关，但血浆脂质水平的异常是最主要的因素。研究表明，糖尿病患者心血管疾病的发病率和死亡率明显高于非糖尿病患者，糖尿病已经成为心血管疾病的独立危险因素[3]。
近年来，肾病与脂代谢紊乱的关系愈来愈引人注目，在慢性进行性肾损伤时常伴随脂代谢异常，而高脂血症又可促进并加重肾脏的损害，除了介导肾小球损伤外，在小管间质损伤中也起作用。1913年Munk首先描述了肾病综合征的血脂异常。有学者报道，70％-10％的肾病综合征患者可出现高脂血症。其主要表现为血总胆固醇(TC)明显增加，且以低密度脂蛋白胆固醇升高为主，甘油三酯(TG)的轻度增加，其中低密度脂蛋白(LDL)的升高与尿蛋白有一定的相关性[4]。慢性肾功能不全患者以中度甘油三酯血症为主，血浆总胆固醇水平一般正常，VLDLC、中等密度脂蛋白胆固醇(IDLC)中胆固醇增加。高密度脂蛋白胆固醇(HDLC)减少，各种脂蛋 白中的甘油三酯含量均增加。其根本原因就是***环境对甘油三酯的合成及分解代谢的不利影响以及对胆固醇逆向转运的抑制作用[5]。
随着肾移植疗法的普遍推广以及各种新型免疫抑制剂(特别是CsA、强的松)的广泛应用，慢性肾功能衰竭(CRF)病人的生存期显著延长，然而肾移植后高脂血症的发生率非常高。肾移植术后高脂血症主要表现为血浆总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDLC)、极低密度脂蛋白胆固醇(VLDLC)水平增高[6]。
临床研究证实，脂质代谢紊乱与糖尿病肾病之间有一定的相关性。糖尿病患者脂代谢紊乱，升高的脂类沉积在肾小球基底膜，刺激基底膜细胞增殖和细胞外间质生成。早在1936年，Kimmelstiel和Wilson便在糖尿病肾病患者的肾小动脉、肾小球和肾小管内发现有大量的脂质沉积[7]。脂代谢异常使肾小球和肾小管间质纤维化是导致肾功能进行性损害的重要原因之一[8]。
脂代谢紊乱也可导致肥胖症(肥胖综合症)的发生。肥胖症分单纯性和继发性两类。单纯性肥胖指无明显内分泌代谢疾病的肥胖。又可分为体质性肥胖及获得性肥胖两种。体质性肥胖有家族遗传史，患者自幼进食丰富，入量过剩，从小肥胖，脂肪细胞呈增生肥大。获得性肥胖大多由于营养过度和/或体力活动减少所致，如人到中年后生活物质条件的改善、疾病恢复和休养充分、产后停止体育锻炼或体力劳动等。脂肪细胞呈肥大变化，没有增生现象，治疗效果较好。继发性肥胖主要为神经内分泌疾病所致。神经内分泌对代谢有重要调节作用：①下丘脑有调节食欲的中枢，中枢神经***炎症后遗症、创伤、肿瘤等均可引起下丘脑功能异常，使食欲旺盛而造成肥胖。②胰岛素分泌增多，如早期非胰岛素依赖型糖尿病患者注射过多胰岛素，致高胰岛素血症；胰岛B细胞瘤分泌过多的胰岛素，这都使脂肪合成增加，引起肥胖。③垂体功能低减，特别是***及促甲状腺激素减少引起性腺及甲状腺功能低下时，可发生肥胖症。④经产妇或口服女性避孕药者易发生肥胖，这提示***有促进脂肪合成的作用。⑤皮质醇增多症常伴有向心性肥胖。⑥甲状腺功能减退，由于代谢率低下，脂肪堆积，且伴粘液水肿。⑦性腺低下也可肥胖，如肥胖性生殖无能症(脑性肥胖症，弗洛利克氏综合征，外伤、脑炎、垂体瘤、颅咽管瘤等损伤下丘脑所致，表现为向心性肥胖，伴尿崩症及性发育迟缓)。
脂代谢紊乱常导致脂肪肝。脂肪肝是指由于各种原因引起的肝细胞内脂肪堆积过多的病变。肝脏在脂质代谢中起着特别重要的作用，它能合成脂蛋白，有利于脂质运输，也是脂肪酸氧化和酮体形成的主要场所。正常时肝含脂质量不多，约为4％，其中主要是磷脂。若肝脏不能及时将脂肪运出，脂肪在肝细胞中堆积，即形成脂肪肝。
脂肪肝可以是一个独立的疾病也可以由其它原因所致，例如肥胖性脂肪肝、酒精性脂肪肝、快速减肥性脂肪肝、营养不良性脂肪肝、糖尿病脂肪肝、药物性脂肪肝等。
某些药物或化学毒物通过抑制蛋白质的合成而致脂肪肝，如四环素、肾上腺皮质激素、嘌呤霉素、环已胺、吐根碱以及砷、铅、银、汞等。降脂药也可通过干扰脂蛋白的代谢而形成脂肪肝。
脂肪肝的危害之一是其促进动脉粥样硬化的形成，导致动脉粥样硬化的原因之一是脂肪肝患者常伴有高血脂症，血液粘稠度增加，其中的低密度脂蛋白(LDL)因其分子量极小，很容易穿过动脉血管内膜在血管壁沉着，使动脉弹性降低，管径变窄，柔韧性减弱，最终导致血液循环障碍。脂肪肝的危害之二是诱发或加重高血压、冠心病，容易导致心肌梗塞而猝死。脂肪肝的危害之三是脑病脂肪肝综合征(Reye综合征)。脂肪肝的危害之四是导致肝硬化、肝功能衰竭、肝癌。
脂肪肝是肝脏脂代谢失调的产物，同时又是加重肝脏损伤的致病因素，这是一种互为因果、恶性循环的发展。肝细胞中脂滴增多，使肝细胞脂肪变性、肿大，细胞核被挤压偏离中心。脂肪的代谢工要在线粒体中进行，脂肪向细胞外运输主要通过光面内质网，脂肪在肝细胞内的堆积进一步加重线粒体和内质网的负担降低其功能，进而影响其他营养素、激素、维生素的代谢。长期的肝细胞变性会导致肝细胞的再生障碍和坏死，进而形成肝纤维化、肝硬化。肝硬化继发肝细胞癌的机率较高。
脂肪肝的危害之五是急性妊娠性脂肪肝，病死率高。此病又称产科急性黄色肝萎缩，是一种较少见、预后凶险的妊娠合并症。多发生在怀孕的最后三个月，临床表现常与急重肝相似，可出现急性肝功能衰竭、胰腺炎、肾功能衰竭、全身凝血异常而导致快速死亡，首次妊娠的孕妇居多。
脂肪肝的危害之六是诱发或加重糖尿病。肥胖性脂肪肝患者若血糖浓度超过正常水平，虽未达到糖尿病的诊断标准，一般认为是糖尿病前期。 脂肪肝与糖尿病常常相伴而生，而且互相影响，这给临床治疗带来了更大的困难。
本发明研究发现纤溶酶原可以预防和/或消减脂肪在身体组织器官的异常沉积，例如可以预防和消减脂质在血液、血管壁、内脏器官以及器官间的组织内的异常沉积，改善这些组织器官的功能，从而为脂肪代谢紊乱及其相关病症、以及其伴随的疾病或并发症提供了全新的预防和治疗方案
发明概述
本发明涉及预防和/或治疗受试者脂肪代谢紊乱及其相关病症。
一方面，本发明涉及预防和/或治疗受试者脂肪代谢紊乱及其相关病症的方法，包括给药受试者预防和/或治疗有效量的纤溶酶原，其中所述受试者易患脂肪代谢紊乱、患有脂肪代谢紊乱或罹患其它疾病并伴有脂肪代谢紊乱。本发明还涉及纤溶酶原用于预防和/或治疗受试者脂肪代谢紊乱及其相关病症的用途。本发明还涉及纤溶酶原在制备用于预防和/或治疗受试者脂肪代谢紊乱及其相关病症的药物、药物组合物、制品、试剂盒中的用途。进一步地，本发明还涉及用于预防和/或治疗受试者脂肪代谢紊乱及其相关病症的纤溶酶原。本发明还涉及用于预防和/或治疗受试者脂肪代谢紊乱及其相关病症的包含纤溶酶原的药物、药物组合物、制品、试剂盒。
在一些实施方案中，所述脂肪代谢紊乱为内分泌紊乱疾病、糖代谢疾病、肝脏疾病、肾脏疾病、心血管疾病、肠道疾病、甲状腺疾病、胆囊或胆道疾病、肥胖症、饮酒、药物治疗引发或伴随的脂肪代谢紊乱。在一些实施方案中，所述脂肪代谢紊乱为高血压、糖尿病、慢性肝炎、肝硬化、肾损伤、慢性肾小球肾炎、慢性肾盂肾炎、肾病综合征、肾功能不全、肾移植、***、甲状腺功能低下、阻塞性胆囊炎、阻塞性胆管炎、药物或激素治疗引发或伴随的脂肪代谢紊乱。在一些实施方案中，所述脂肪代谢紊乱为高脂血症、高脂蛋白血症、脂肪肝、动脉粥样硬化、肥胖症、脏器脂肪沉积。在又一些实施方案中，所述动脉粥样硬化包括包括主动脉粥样硬化、冠状动脉粥样硬化、脑动脉粥样硬化、肾动脉粥样硬化、肝动脉粥样硬化、肠系膜动脉粥样硬化、下肢动脉粥样硬化。
在又一个方面，本发明涉及预防和/或消减受试者脂肪在身体组织器官异常沉积的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶 酶原用于预防和/或消减受试者脂肪在身体组织器官异常沉积的用途。本发明还涉及纤溶酶原在制备用于预防和/或消减受试者脂肪在身体组织器官异常沉积的药物、药物组合物、制品、试剂盒中的用途。进一步地，本发明还涉及用于预防和/或消减受试者脂肪在身体组织器官异常沉积的纤溶酶原。本发明还涉及用于预防和/或消减受试者脂肪在身体组织器官异常沉积的包含纤溶酶原的药物、药物组合物、制品、试剂盒。
在又一个方面，本发明涉及预防和/或治疗受试者脂肪在身体组织器官异常沉积导致的病症的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于预防和/或治疗受试者脂肪在身体组织器官异常沉积导致的病症的用途。本发明还涉及纤溶酶原在制备用于预防和/或治疗受试者脂肪在身体组织器官异常沉积导致的病症的药物、药物组合物、制品、试剂盒中的用途。进一步地，本发明还涉及用于预防和/或治疗受试者脂肪在身体组织器官异常沉积导致的病症的包含纤溶酶原的药物、药物组合物、制品、试剂盒。
在一些实施方案中，所述脂肪在身体组织器官异常沉积是指脂肪在血液，皮下组织、血管壁、内脏器官的异常沉积。在一些实施方案中，所述脂肪在身体组织器官异常沉积导致的病症包括肥胖症，高脂血症、高脂蛋白血症、脂肪肝、动脉粥样硬化、脂质性心脏损害、脂质性肾损害，脂质性胰岛损害。
在又一个方面，本发明涉及预防和/或治疗受试者脂肪代谢紊乱所致病症的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于预防和/或治疗受试者脂肪代谢紊乱所致病症的用途。本发明还涉及纤溶酶原在制备用于预防和/或治疗受试者脂肪代谢紊乱所致病症的药物、药物组合物、制品、试剂盒中的用途。进一步地，本发明还涉及用于预防和/或治疗受试者脂肪代谢紊乱所致病症的纤溶酶原。本发明还涉及用于预防和/或治疗受试者脂肪代谢紊乱所致病症的包含纤溶酶原的药物、药物组合物、制品、试剂盒。在一些实施方案中，所述病症包括肥胖症，高脂血症、高脂蛋白血症、脂肪肝、动脉粥样硬化、脂质性心脏组织损伤，脂质性肾损伤。
在又一个方面，本发明涉及通过减少脂肪异常沉积治疗受试者疾病的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于通 过减少脂肪异常沉积治疗受试者疾病的用途。本发明还涉及纤溶酶原在制备用于通过减少脂肪异常沉积治疗受试者疾病的药物、药物组合物、制品、试剂盒中的用途。进一步地，本发明还涉及用于通过减少脂肪异常沉积治疗受试者疾病的纤溶酶原。本发明还涉及通过减少脂肪异常沉积治疗受试者疾病的包含纤溶酶原的药物、药物组合物、制品、试剂盒。
在一些实施方案中，所述疾病包括动脉粥样硬化、冠心病、心绞痛、心肌梗死、心律失常、脂肪肝、肝硬化，脑缺血、脑梗死、肾功能不全、肾病综合征、肾功能不全、肥胖症。
在又一个方面，本发明涉及预防和/或治疗受试者组织器官脂质性损伤的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于预防和/或治疗受试者组织器官脂质性损伤的用途。本发明还涉及纤溶酶原在制备用于预防和/或治疗受试者组织器官脂质性损伤的药物、药物组合物、制品、试剂盒中的用途。进一步地，本发明还涉及用于预防和/或治疗受试者组织器官脂质性损伤的纤溶酶原。本发明还涉及用于预防和/或治疗受试者组织器官脂质性损伤的包含纤溶酶原的药物、药物组合物、制品、试剂盒。
在一些实施方案中，所述组织器官包括动脉管壁、心脏、肝脏、肾脏、胰腺。
在又一个方面，本发明涉及改善受试者高脂血症的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于改善受试者高脂血症的用途。本发明还涉及纤溶酶原在制备用于改善受试者高脂血症的药物、药物组合物、制品、试剂盒中的用途。进一步地，本发明还涉及用于改善受试者高脂血症的纤溶酶原。本发明还涉及用于改善受试者高脂血症的包含纤溶酶原的药物、药物组合物、制品、试剂盒。
在一些实施方案中，所述高脂血症选自如下的一项或多项：高胆固醇血症、高甘油三酯血症、混合型高脂血症和低高密度脂蛋白血症。
在又一个方面，本发明涉及降低受试者动脉粥样硬化风险的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于降低受试者动脉粥样硬化风险的用途。本发明还涉及纤溶酶原在制备用于降低受试者动脉粥样硬化风险的药物、药物组合物、制品、试剂盒中的用途。进一步地，本发明还涉及用于降低受试者动脉粥样硬化风险的纤溶酶原。本发明 还涉及用于降低受试者动脉粥样硬化风险的包含纤溶酶原的药物、药物组合物、制品、试剂盒。
在一些实施方案中，所述受试者患有高血压、肥胖症、糖尿病、慢性肝炎、肝硬化、肾损伤、慢性肾小球肾炎、慢性肾盂肾炎、肾病综合征、肾功能不全、肾移植、***、甲状腺功能低下、阻塞性胆囊炎或阻塞性胆管炎，或所述受试者服用影响脂肪代谢的药物或激素。在一些实施方案中，所述纤溶酶原通过选自如下的一项或多项降低受试者动脉粥样硬化风险：降低血中总胆固醇水平、甘油三酯水平、低密度脂蛋白水平、提高血中高密度脂蛋白水平。
在又一个方面，本发明涉及通过改善受试者高脂血症治疗疾病的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于通过改善受试者高脂血症治疗疾病的用途。本发明还涉及纤溶酶原在制备用于通过改善受试者高脂血症治疗疾病的药物、药物组合物、制品、试剂盒中的用途。进一步地，本发明还涉及用于通过改善受试者高脂血症治疗疾病的纤溶酶原。本发明还涉及用于通过改善受试者高脂血症治疗疾病的包含纤溶酶原的药物、药物组合物、制品、试剂盒。
在一些实施方案中，所述病症包括糖尿病、高血压、动脉粥样硬化、冠心病、心绞痛、心肌梗死、心律失常、慢性肝炎、脂肪肝、肝硬化，脑供血不足、脑缺血、脑梗死、慢性肾炎、慢性肾盂肾炎、肾功能不全、肾病综合征、***、肥胖症。
在又一个方面，本发明涉及预防和/或治疗受试者高血脂相关病症的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于预防和/或治疗受试者高血脂相关病症的用途。本发明还涉及纤溶酶原在制备用于预防和/或治疗受试者高血脂相关病症的药物、药物组合物、制品、试剂盒中的用途。进一步地，本发明还涉及用于预防和/或治疗受试者高血脂相关病症的纤溶酶原。本发明还涉及用于预防和/或治疗受试者高血脂相关病症的包含纤溶酶原的药物、药物组合物、制品、试剂盒。在一些实施方案中，所述病症包括糖尿病、高血压、动脉粥样硬化、冠心病、心绞痛、心肌梗死、心律失常、慢性肝炎、脂肪肝、肝硬化，脑供血不足、脑缺血、脑梗死、慢性肾炎、慢性肾盂肾炎、肾功能不全、肾病综合征、***、肥胖症。
在本发明的上述任一实施方案中，所述纤溶酶原可与一种或多种其它药物或治疗方法联合施用。在一些实施方案中，所述一种或多种其它药物包括高血压治疗药物、糖尿病治疗用药物、动脉粥样硬化治疗用药物、慢性肾小球肾炎治疗药物、慢性肾盂肾炎治疗药物、肾病综合征治疗用药物、肾功能不全治疗用药物、***治疗用药物、肾移植治疗用药物、脂肪肝治疗用药物、肝硬化治疗用药物、肥胖症治疗用药物。在一些实施方案中，所述其它药物包括：降血脂药物、抗血小板药物、降血压药物、扩张血管药物、降血糖药物、抗凝血药物、溶血栓药物，保肝药物，抗心律失常药物，强心药物，利尿药物，抗感染药物、抗病毒药物、免疫调节药物、炎症调节类药物、抗肿瘤药物、激素类药物、甲状腺素。在一些进一步的实施方案中，所述药物包括降血脂药物：他汀类；贝特类；烟酸；消胆胺；安妥明；不饱和脂肪酸如益寿宁、血脂平及心脉乐；藻酸双酯钠；抗血小板药物：阿司匹林；潘生丁；氯吡格雷；西洛他；扩张血管药物：肼苯哒嗪；***和消心痛；硝普钠；α硝受体阻断剂如哌唑嗪；α受体阻断剂如酚妥拉明；β拉受体***如舒喘灵；卡托普利、依那普利；心痛定、硫氮卓酮；柳丁氨酸、长压定、***素、心钠素；溶血栓药物：尿激酶和链激酶；组织型纤溶酶原激活剂；单链尿激酶型纤溶酶原激活剂；TNK-组织型纤溶酶原激活剂；抗凝血药物：肝素；依诺肝素；那曲肝素；比伐卢定。
在本发明的上述任一实施方案中，所述纤溶酶原可与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。在一些实施方案中，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。
在一些实施方案中，所述纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。在一些实施方案中，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。在一些实施方案中，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的变体或片段。在 一些实施方案中，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的变体或片段。在一些实施方案中，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。在一些实施方案中，所述纤溶酶原是人天然纤溶酶原。
在一些实施方案中，所述受试者是人。在一些实施方案中，所述受试者缺乏或缺失纤溶酶原。在一些实施方案中，所述缺乏或缺失是先天的、继发的和/或局部的。
在一些实施方案中，所述药物组合物包含药学上可接受的载剂和用于前述方法的纤溶酶原。在一些实施方案中，所述试剂盒可以是预防性或治疗性试剂盒，其包含：(i)用于前述方法的纤溶酶原和(ii)用于递送所述纤溶酶原至所述受试者的构件(means)。在一些实施方案中，所述构件为注射器或小瓶。在一些实施方案中，所述试剂盒还包含标签或使用说明书，该标签或使用说明书指示将所述纤溶酶原投予所述受试者以实施前述任一方法。
在一些实施方案中，所述制品包含：含有标签的容器；和包含(i)用于前述方法的纤溶酶原或包含纤溶酶原的药物组合物，其中所述标签指示将所述纤溶酶原或组合物投予所述受试者以实施前述任一方法。
在一些实施方案中，所述试剂盒或制品还包含另外的一个或多个构件或容器，该构件或容器中含有其他药物。在一些实施方案中，所述其他药物选自下组：降血脂药物、抗血小板药物、降血压药物、扩张血管药物、降血糖药物、抗凝血药物、溶血栓药物，保肝药物，抗心律失常药物，强心药物，利尿药物，抗感染药物、抗病毒药物、免疫调节药物、炎症调节类药物、抗肿瘤药物、激素类药物、甲状腺素。
在前述方法的一些实施方案中，所述纤溶酶原通过全身或局部给药，优选通过以下途径施用：静脉内、肌内、皮下给予纤溶酶原来进行治疗。在前述方法的一些实施方案中，所述纤溶酶原与适当的多肽载体或稳定剂组合施用。在前述方法的一些实施方案中，所述纤溶酶原以每天0.0001-2000mg/kg、0.001-800mg/kg、0.01-600mg/kg、0.1-400mg/kg、1-200mg/kg、1-100mg/kg、10-100mg/kg(以每公斤体重计算)或0.0001-2000mg/cm2、0.001-800mg/cm2、0.01-600mg/cm2、0.1-400mg/cm2、1-200mg/cm2、1-100mg/cm2、10-100mg/cm2(以每平方厘米体表面积计算)的剂量施用，优选至少重复一次，优选至少每天施用。
本发明明确涵盖了属于本发明实施方案之间的技术特征的所有组合，并且这些组合后的技术方案在本申请中已经明确公开，就像上述技术方案已经单独且明确公开一样。另外，本发明还明确涵盖各个实施方案及其要素的之间的组合，该组合后的技术方案在本文中明确公开。
定义
本发明所述的“脂肪代谢紊乱”又称“脂肪代谢异常”、“脂肪代谢障碍”，为脂肪代谢发生异常、紊乱或障碍所引发的临床或病理表现的总称。在本文中，“脂肪代谢紊乱”、“脂肪代谢异常”、“脂肪代谢障碍”可互换使用。本发明中“脂肪代谢”、“脂代谢”、“脂质代谢”可互换使用。
“脂肪代谢紊乱相关病症”是与脂肪代谢紊乱相关的病症的总称。所述的相关，可以是病因相关、发病机理相关、病理表现相关、临床症状相关和/或治疗原则相关。
“血脂”是甘油三酯、胆固醇和磷脂等的总称，脂蛋白是由载脂蛋白和血脂组成的球型大分子复合体，由于脂蛋白包含胆固醇、甘油三酯的成分不同及密度大小不同被分为5大类：乳糜微粒(CM)极低密度脂蛋白(VLDL)中密度脂蛋白(IDL)低密度脂蛋白(LDL)高密度脂蛋白(HDL)。依据血脂危险水平，临床最常见的异常脂蛋白血症类型：高胆固醇血症、高甘油三酯血症、混合型高脂血症和低高密度脂蛋白血症。继发性血脂异常见于糖尿病、甲状腺功能低下、肾病综合症、肾移植、严重肝病、阻塞性胆道疾病、肥胖症、饮酒、药物治疗，例如***治疗等，如能排除继发性血脂异常可考虑原发性血脂异常。
“高血脂”是指血浆中的胆固醇、甘油三脂、磷脂和未脂化的脂酸等血脂成分增高的病理状况。
“高血脂相关病症”是指病因、发病机理、病理表现、临床症状和/或治疗原则与高血脂相关的病症。优选所述病症包括但不限于糖尿病、高血压、动脉粥样硬化、冠心病、心绞痛、心肌梗死、心律失常、慢性肝炎、脂肪肝、肝硬化，脑供血不足、脑缺血、脑梗死、慢性肾炎、慢性肾盂肾炎、肾功能不全、肾病综合征、***、肥胖症。
由于脂肪代谢或运转异常使血浆中一种或几种脂质异常称为“高脂血症”、“高血脂症”或“血脂异常”(dyslipidemia)。
由于脂质不溶或微溶于水，必须与蛋白质结合成脂蛋白才能在血液循环中运转，因此高脂血症常为“高脂蛋白血症”的反映。
本发明的“高血脂相关病症”也可称为“高脂血症相关病症”、“高脂蛋白血症相关病症”。
“脂肪肝”是指由于各种原因引起的肝细胞内脂肪堆积过多的病变，其可以是一个独立的疾病也可以由其它原因所致，例如肥胖性脂肪肝、酒精性脂肪肝、快速减肥性脂肪肝、营养不良性脂肪肝、糖尿病脂肪肝、药物性脂肪肝等。
在脂肪肝情况下，肝细胞中脂滴增多，使肝细胞脂肪变性、肿大，细胞核被挤压偏离中心。脂肪的代谢工要在线粒体中进行，脂肪向细胞外运输主要通过光面内质网，脂肪在肝细胞内的堆积进一步加重线粒体和内质网的负担降低其功能，进而影响其他营养素、激素、维生素的代谢。长期的肝细胞变性会导致肝细胞的再生障碍和坏死，进而形成肝纤维化、肝硬化。
“动脉粥样硬化”是一种慢性的、渐进性动脉疾病，发病时动脉中沉积的脂肪部分或全部堵塞血流。当原本光滑、坚实的动脉内膜***糙、增厚，并被脂肪、纤维蛋白、钙和细胞碎屑堵塞时，便出现动脉粥样硬化。动脉粥样硬化是个渐进的过程。当血液中的脂类浓度大大增加时，便会沿着动脉壁形成脂肪条纹。这些条纹会导致脂肪和胆固醇沉积，这些沉淀依附在原本平滑的动脉内膜上，从而形成小结。这些小结下面继而长出纤维化的瘢痕组织，导致钙沉积。沉积的钙逐渐演变为无法除去的白垩状坚硬薄膜(称为动脉粥样斑)。动脉内部的这层永久薄膜会阻碍动脉的正常扩张和收缩，从而减缓了动脉内的血流速度，从而很容易形成血块，妨碍或阻止血液流经动脉。
人们尚未确定动脉粥样硬化的确切原因，但是人们已经发现了重要的致病因素：高脂血症、高血压、有吸烟史、有动脉粥样硬化家族史(60岁以前患上该病)或糖尿病。高脂血症能促进脂肪条纹的形成。因高血压对动脉施加一定的恒力，加速了动脉阻塞和硬化过程，因此能增加动脉粥样硬化的患病率。抽烟可以引致动脉收缩，限制血液流动，因而为动脉阻塞创造了条件。糖尿病也能促使动脉粥样硬化的发生，特别是对于很小的动脉。
仅就动脉粥样硬化症而言，人们感觉不到任何症状。仅当与体内的某个重要器官相连的动脉被堵塞后，才会发现此病。因该器官中的动脉受阻而引起的症状较为明显。例如，如果心脏供血动脉部分受阻，人们就可能感到心绞痛；但是如果完全被阻塞，就可能导致心脏病(由受阻动脉供血的心脏组织死亡)。如果动脉粥样硬化影响到脑部动脉，人们会感觉眩晕、视线模糊和晕厥，甚至可能导致中风(由受阻动脉供血的脑组织死亡，从而引起神经损伤，如受死亡脑组织控制的肢体出现瘫痪)。通向肾部的动脉受阻还可能导致肾衰竭。通向眼部的血管受阻可能导致失明。四肢动脉阻塞可能导致各肢体的病变。
动脉粥样硬化是冠心病、脑梗死、外周血管病的主要原因。脂质代谢障碍为动脉粥样硬化的病变基础，其特点是受累动脉病变从内膜开始，一般先有脂质和复合糖类积聚、出血及血栓形成，进而纤维组织增生及钙质沉着，并有动脉中层的逐渐蜕变和钙化，导致动脉壁增厚***、血管腔狭窄。病变常累及大中肌性动脉，一旦发展到足以阻塞动脉腔，则该动脉所供应的组织或器官将缺血或坏死。
动脉粥样硬化是一种全身性疾病，一个器官血管发生动脉粥样硬化病变，意味着其他地方的血管也可能已经存在同样的病变；同样，一个器官发生血管事件，意味着其他地方发生血管事件的危险性增加。
发明详述
纤溶酶是纤溶酶原激活***(PA***)的关键组分。它是一种广谱的蛋白酶，能够水解细胞外基质(ECM)的几个组分，包括纤维蛋白、明胶、纤连蛋白、层粘连蛋白和蛋白聚糖[9]。此外，纤溶酶能将一些金属蛋白酶前体(pro-MMPs)激活形成具有活性的金属蛋白酶(MMPs)。因此纤溶酶被认为是胞外蛋白水解作用的一个重要的上游调节物[10，11]。纤溶酶是由纤溶酶原通过两种生理性的PAs：组织型纤溶酶原激活剂(tPA)或尿激酶型纤溶酶原激活剂(uPA)蛋白水解形成的。由于纤溶酶原在血浆和其他体液中相对水平较高，传统上认为PA***的调节主要通过PAs的合成和活性水平实现。PA***组分的合成受不同因素严格调节，如激素、生长因子和细胞因子。此外，还存在纤溶酶和PAs的特定生理抑制剂。纤溶酶的主要抑制剂是α2-抗纤溶酶(α2-antiplasmin)。PAs的活性同时被uPA和tPA的纤 溶酶原激活剂抑制剂-1(PAI-1)抑制以及主要抑制uPA的溶酶原激活剂抑制剂-2(PAI-2)调节。某些细胞表面具有直接水解活性的uPA特异性细胞表面受体(uPAR)[12，13]。
纤溶酶原是一个单链糖蛋白，由791个氨基酸组成，分子量约为92kDa[14，15]。纤溶酶原主要在肝脏合成，大量存在于胞外液中。血浆中纤溶酶原含量约为2μM。因此纤溶酶原是组织和体液中蛋白质水解活性的一个巨大的潜在来源[16，17]。纤溶酶原存在两种分子形式：谷氨酸-纤溶酶原(Glu-plasminogen)和赖氨酸-纤溶酶原(Lys-plasminogen)。天然分泌和未裂解形式的纤溶酶原具有一个氨基末端(N-末端)谷氨酸，因此被称为谷氨酸-纤溶酶原。然而，在纤溶酶存在时，谷氨酸-纤溶酶原在Lys76-Lys77处水解成为赖氨酸-纤溶酶原。与谷氨酸-纤溶酶原相比，赖氨酸-纤溶酶原与纤维蛋白具有更高的亲和力，并可以更高的速率被PAs激活。这两种形式的纤溶酶原的Arg560-Val561肽键可被uPA或tPA切割，导致二硫键连接的双链蛋白酶纤溶酶的形成[18]。纤溶酶原的氨基末端部分包含五个同源三环，即所谓的kringles，羧基末端部分包含蛋白酶结构域。一些kringles含有介导纤溶酶原与纤维蛋白及其抑制剂α2-AP特异性相互作用的赖氨酸结合位点。最新发现一个纤溶酶原为38kDa的片段，其中包括kringles1-4，是血管生成的有效抑制剂。这个片段被命名为血管抑素，可通过几个蛋白酶水解纤溶酶原产生。
纤溶酶的主要底物是纤维蛋白，纤维蛋白的溶解是预防病理性血栓形成的关键[19]。纤溶酶还具有对ECM几个组分的底物特异性，包括层粘连蛋白、纤连蛋白、蛋白聚糖和明胶，表明纤溶酶在ECM重建中也起着重要作用[15，20，21]。间接地，纤溶酶还可以通过转化某些蛋白酶前体为活性蛋白酶来降解ECM的其他组分，包括MMP-1，MMP-2，MMP-3和MMP-9。因此，有人提出，纤溶酶可能是细胞外蛋白水解的一个重要的上游调节器[22]。此外，纤溶酶具有激活某些潜在形式的生长因子的能力[23-25]。在体外，纤溶酶还能水解补体***的组分并释放趋化补体片段。
“纤溶酶”是存在于血液中的一种非常重要的酶，能将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体。
“纤溶酶原”是纤溶酶的酶原形式，根据swiss prot中的序列，按含有信号肽的天然人源纤溶酶原氨基酸序列(序列4)计算由810个氨基酸组成， 分子量约为90kD，主要在肝脏中合成并能够在血液中循环的糖蛋白，编码该氨基酸序列的cDNA序列如序列3所示。全长的纤溶酶原包含七个结构域：位于C末端的丝氨酸蛋白酶结构域、N末端的Pan Apple(PAp)结构域以及5个Kringle结构域(Kringle1-5)。参照swiss prot中的序列，其信号肽包括残基Met1-Gly19，PAp包括残基Glu20-Val98，Kringle1包括残基Cys103-Cys181，Kringle2包括残基Glu184-Cys262，Kringle3包括残基Cys275-Cys352，Kringle4包括残基Cys377-Cys454，Kringle5包括残基Cys481-Cys560。根据NCBI数据，丝氨酸蛋白酶域包括残基Val581-Arg804。
Glu-纤溶酶原是天然全长的纤溶酶原，由791个氨基酸组成(不含有19个氨基酸的信号肽)，编码该序列的cDNA序列如序列1所示，其氨基酸序列如序列2所示。在体内，还存在一种是从Glu-纤溶酶原的第76-77位氨基酸处水解从而形成的Lys-纤溶酶原，如序列6所示，编码该氨基酸序列的cDNA序列如序列5所示。Delta-纤溶酶原(δ-plasminogen)是全长纤溶酶原缺失了Kringle2-Kringle5结构的片段，仅含有Kringle1和丝氨酸蛋白酶域[26，27]，有文献报道了delta-纤溶酶原的氨基酸序列(序列8)[27]，编码该氨基酸序列的cDNA序列如序列7。小纤溶酶原(Mini-plasminogen)由Kringle5和丝氨酸蛋白酶域组成，有文献报道其包括残基Val443-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)[28]，其氨基酸序列如序列10所示，编码该氨基酸序列的cDNA序列如序列9所示。而微纤溶酶原(Micro-plasminogen)仅含有丝氨酸蛋白酶结构域，有文献报道其氨基酸序列包括残基Ala543-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)[29]，也有专利文献CN102154253A报道其序列包括残基Lys531-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，本专利序列参考专利文献CN102154253A，其氨基酸序列如序列12所示，编码该氨基酸序列的cDNA序列如序列11所示。
本发明的“纤溶酶”与“纤维蛋白溶酶”、“纤维蛋白溶解酶”可互换使用，含义相同；“纤溶酶原”与“纤溶酶原”、“纤维蛋白溶解酶原”可互换使用，含义相同。
在本申请中，所述纤溶酶原“缺乏”的含义为受试者体内纤溶酶原的含量或活性比正常人低，低至足以影响所述受试者的正常生理功能；所述 纤溶酶原“缺失”的含义为受试者体内纤溶酶原的含量或活性显著低于正常人，甚至活性或表达极微，只有通过外源提供才能维持正常生理功能。
本领域技术人员可以理解，本发明纤溶酶原的所有技术方案适用于纤溶酶，因此，本发明描述的技术方案涵盖了纤溶酶原和纤溶酶。
在循环过程中，纤溶酶原采用封闭的非活性构象，但当结合至血栓或细胞表面时，在纤溶酶原激活剂(plasminogen activator，PA)的介导下，其转变为呈开放性构象的活性纤溶酶。具有活性的纤溶酶可进一步将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体，进而溶解血栓。其中纤溶酶原的PAp结构域包含维持纤溶酶原处于非活性封闭构象的重要决定簇，而KR结构域则能够与存在于受体和底物上的赖氨酸残基结合。已知多种能够作为纤溶酶原激活剂的酶，包括：组织纤溶酶原激活剂(tPA)、尿激酶纤溶酶原激活剂(uPA)、激肽释放酶和凝血因子XII(哈格曼因子)等。
“纤溶酶原活性片段”是指在纤溶酶原蛋白中，能够与底物中的靶序列结合并发挥蛋白水解功能的活性片段。本发明涉及纤溶酶原的技术方案涵盖了用纤溶酶原活性片段代替纤溶酶原的技术方案。本发明所述的纤溶酶原活性片段为包含纤溶酶原的丝氨酸蛋白酶域的蛋白质，优选，本发明所述的纤溶酶原活性片段包含序列14、与序列14具有至少80％、90％、95％、96％、97％、98％、99％同源性的氨基酸序列的蛋白质。因此，本发明所述的纤溶酶原包括含有该纤溶酶原活性片段、并且仍然保持该纤溶酶原活性的蛋白。
目前，对于血液中纤溶酶原及其活性测定方法包括：对组织纤溶酶原激活剂活性的检测(t-PAA)、血浆组织纤溶酶原激活剂抗原的检测(t-PAAg)、对血浆组织纤溶酶原活性的检测(plgA)、血浆组织纤溶酶原抗原的检测(plgAg)、血浆组织纤溶酶原激活剂抑制物活性的检测、血浆组织纤溶酶原激活剂抑制物抗原的检测、血浆纤维蛋白溶酶-抗纤维蛋白溶酶复合物检测(PAP)。其中最常用的检测方法为发色底物法：向受检血浆中加链激酶(SK)和发色底物，受检血浆中的PLG在SK的作用下，转变成PLM，后者作用于发色底物，随后用分光光度计测定，吸光度增加与纤溶酶原活性成正比。此外也可采用免疫化学法、凝胶电泳、免疫比浊法、放射免疫扩散法等对血液中的纤溶酶原活性进行测定。
“直系同源物或直系同系物(ortholog)”指不同物种之间的同源物，既包括蛋白同源物也包括DNA同源物，也称为直向同源物、垂直同源物。其具体指不同物种中由同一祖先基因进化而来的蛋白或基因。本发明的纤溶酶原包括人的天然纤溶酶原，还包括来源于不同物种的、具有纤溶酶原活性的纤溶酶原直系同源物或直系同系物。
“保守取代变体”是指其中一个给定的氨基酸残基改变但不改变蛋白质或酶的整体构象和功能，这包括但不限于以相似特性(如酸性，碱性，疏水性，等)的氨基酸取代亲本蛋白质中氨基酸序列中的氨基酸。具有类似性质的氨基酸是众所周知的。例如，精氨酸、组氨酸和赖氨酸是亲水性的碱性氨基酸并可以互换。同样，异亮氨酸是疏水氨基酸，则可被亮氨酸，蛋氨酸或缬氨酸替换。因此，相似功能的两个蛋白或氨基酸序列的相似性可能会不同。例如，基于MEGALIGN算法的70％至99％的相似度(同一性)。“保守取代变体”还包括通过BLAST或FASTA算法确定具有60％以上的氨基酸同一性的多肽或酶，若能达75％以上更好，最好能达85％以上，甚至达90％以上为最佳，并且与天然或亲本蛋白质或酶相比具有相同或基本相似的性质或功能。
“分离的”纤溶酶原是指从其天然环境分离和/或回收的纤溶酶原蛋白。在一些实施方案中，所述纤溶酶原会纯化(1)至大于90％、大于95％、或大于98％的纯度(按重量计)，如通过Lowry法所确定的，例如超过99％(按重量计)，(2)至足以通过使用旋转杯序列分析仪获得N端或内部氨基酸序列的至少15个残基的程度，或(3)至同质性，该同质性是通过使用考马斯蓝或银染在还原性或非还原性条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确定的。分离的纤溶酶原也包括通过生物工程技术从重组细胞制备，并通过至少一个纯化步骤分离的纤溶酶原。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用，指任何长度的氨基酸的聚合形式，其可以包括遗传编码的和非遗传编码的氨基酸，化学或生物化学修饰的或衍生化的氨基酸，和具有经修饰的肽主链的多肽。该术语包括融合蛋白，包括但不限于具有异源氨基酸序列的融合蛋白，具有异源和同源前导序列(具有或没有N端甲硫氨酸残基)的融合物；等等。
关于参照多肽序列的“氨基酸序列同一性百分数(％)”定义为在必要时引入缺口以实现最大百分比序列同一性后，且不将任何保守替代视为序列 同一性的一部分时，候选序列中与参照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式实现，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员能决定用于比对序列的适宜参数，包括对所比较序列全长实现最大对比需要的任何算法。然而，为了本发明的目的，氨基酸序列同一性百分数值是使用序列比较计算机程序ALIGN-2产生的。
在采用ALIGN-2来比较氨基酸序列的情况中，给定氨基酸序列A相对于给定氨基酸序列B的％氨基酸序列同一性(或者可表述为具有或包含相对于、与、或针对给定氨基酸序列B的某一％氨基酸序列同一性的给定氨基酸序列A)如下计算：
分数X/Y乘100
其中X是由序列比对程序ALIGN-2在该程序的A和B比对中评分为相同匹配的氨基酸残基的数目，且其中Y是B中的氨基酸残基的总数。应当领会，在氨基酸序列A的长度与氨基酸序列B的长度不相等的情况下，A相对于B的％氨基酸序列同一性会不等于B相对于A的％氨基酸序列同一性。除非另有明确说明，本文中使用的所有％氨基酸序列同一性值都是依照上一段所述，使用ALIGN-2计算机程序获得的。
如本文中使用的，术语“治疗”和“处理”指获得期望的药理和/或生理效果。所述效果可以是完全或部分预防疾病或其症状，和/或部分或完全治愈疾病和/或其症状，并且包括：(a)预防疾病在受试者体内发生，所述受试者可以具有疾病的素因，但是尚未诊断为具有疾病；(b)抑制疾病，即阻滞其形成；和(c)减轻疾病和/或其症状，即引起疾病和/或其症状消退。
术语“个体”、“受试者”和“患者”在本文中可互换使用，指哺乳动物，包括但不限于鼠(大鼠、小鼠)、非人灵长类、人、犬、猫、有蹄动物(例如马、牛、绵羊、猪、山羊)等。
“治疗有效量”或“有效量”指在对哺乳动物或其它受试者施用以治疗疾病时足以实现对疾病的所述预防和/或治疗的纤溶酶原的量。“治疗有效量”会根据所使用的纤溶酶原、要治疗的受试者的疾病和/或其症状的严重程度以及年龄、体重等而变化。
本发明纤溶酶原的制备
纤溶酶原可以从自然界分离并纯化用于进一步的治疗用途，也可以通过标准的化学肽合成技术来合成。当通过化学合成多肽时，可以经液相或固相进行合成。固相多肽合成(SPPS)(其中将序列的C末端氨基酸附接于不溶性支持物，接着序贯添加序列中剩余的氨基酸)是适合纤溶酶原化学合成的方法。各种形式的SPPS，诸如Fmoc和Boc可用于合成纤溶酶原。用于固相合成的技术描述于Barany和Solid-Phase Peptide Synthesis；第3-284页于The Peptides：Analysis，Synthesis，Biology.第2卷：Special Methods in Peptide Synthesis，Part A.，Merrifield，等J.Am.Chem.Soc.，85：2149-2156(1963)；Stewart等，Solid Phase Peptide Synthesis，2nd ed.Pierce Chem.Co.，Rockford，Ill.(1984)；和Ganesan A.2006Mini Rev.Med Chem.6：3-10和Camarero JA等2005Protein Pept Lett.12：723-8中。简言之，用其上构建有肽链的功能性单元处理小的不溶性多孔珠。在偶联/去保护的重复循环后，将附接的固相游离N末端胺与单个受N保护的氨基酸单元偶联。然后，将此单元去保护，露出可以与别的氨基酸附接的新的N末端胺。肽保持固定在固相上，之后将其切掉。
可以使用标准重组方法来生产本发明的纤溶酶原。例如，将编码纤溶酶原的核酸***表达载体中，使其与表达载体中的调控序列可操作连接。表达调控序列包括但不限于启动子(例如天然关联的或异源的启动子)、信号序列、增强子元件、和转录终止序列。表达调控可以是载体中的真核启动子***，所述载体能够转化或转染真核宿主细胞(例如COS或CHO细胞)。一旦将载体掺入合适的宿主中，在适合于核苷酸序列的高水平表达及纤溶酶原的收集和纯化的条件下维持宿主。
合适的表达载体通常在宿主生物体中作为附加体或作为宿主染色体DNA的整合部分复制。通常，表达载体含有选择标志物(例如氨苄青霉素抗性、潮霉素抗性、四环素抗性、卡那霉素抗性或新霉素抗性)以有助于对外源用期望的DNA序列转化的那些细胞进行检测。
大肠杆菌(Escherichia coli)是可以用于克隆主题抗体编码多核苷酸的原核宿主细胞的例子。适合于使用的其它微生物宿主包括杆菌，诸如枯草芽孢杆菌(Bacillus subtilis)和其他肠杆菌科(enterobacteriaceae)，诸如沙门氏菌属(Salmonella)、沙雷氏菌属(Serratia)、和各种假单胞菌属(Pseudomonas)物 种。在这些原核宿主中，也可以生成表达载体，其通常会含有与宿主细胞相容的表达控制序列(例如复制起点)。另外，会存在许多公知的启动子，诸如乳糖启动子***，色氨酸(trp)启动子***，beta-内酰胺酶启动子***，或来自噬菌体λ的启动子***。启动子通常会控制表达，任选在操纵基因序列的情况中，并且具有核糖体结合位点序列等，以启动并完成转录和翻译。
其他微生物，诸如酵母也可用于表达。酵母(例如酿酒酵母(S.cerevisiae))和毕赤酵母(Pichia)是合适的酵母宿主细胞的例子，其中合适的载体根据需要具有表达控制序列(例如启动子)、复制起点、终止序列等。典型的启动子包含3-磷酸甘油酸激酶和其它糖分解酶。诱导型酵母启动于特别包括来自醇脱氢酶、异细胞色素C、和负责麦芽糖和半乳糖利用的酶的启动子。
在微生物外，哺乳动物细胞(例如在体外细胞培养物中培养的哺乳动物细胞)也可以用于表达并生成本发明的抗-Tau抗体(例如编码主题抗-Tau抗体的多核苷酸)。参见Winnacker，From Genes to Clones，VCH Publishers，N.Y.，N.Y.(1987)。合适的哺乳动物宿主细胞包括CHO细胞系、各种Cos细胞系、HeLa细胞、骨髓瘤细胞系、和经转化的B细胞或杂交瘤。用于这些细胞的表达载体可以包含表达控制序列，如复制起点，启动子和增强子(Queen等，Immunol.Rev.89：49(1986))，以及必需的加工信息位点，诸如核糖体结合位点，RNA剪接位点，多聚腺苷酸化位点，和转录终止子序列。合适的表达控制序列的例子是白免疫球蛋白基因、SV40、腺病毒、牛***瘤病毒、巨细胞病毒等衍生的启动子。参见Co等，J.Immunol.148：1149(1992)。
一旦合成(化学或重组方式)，可以依照本领域的标准规程，包括硫酸铵沉淀，亲和柱，柱层析，高效液相层析(HPLC)，凝胶电泳等来纯化本发明所述的纤溶酶原。该纤溶酶原是基本上纯的，例如至少约80％至85％纯的，至少约85％至90％纯的，至少约90％至95％纯的，或98％至99％纯的或更纯的，例如不含污染物，所述污染物如细胞碎片，除主题抗体以外的大分子，等等。
药物配制剂
可以通过将具有所需纯度的纤溶酶原与可选的药用载体，赋形剂，或稳定剂(Remington′s Pharmaceutical Sciences，16版，Osol，A.ed.(1980))混合形成冻干制剂或水溶液制备治疗配制剂。可接受的载体、赋形剂、稳定剂在所用剂量及浓度下对受者无毒性，并包括缓冲剂例如磷酸盐，柠檬酸盐及其它有机酸；抗氧化剂包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；氯化己烷双胺；氯化苄烷铵(benzalkonium chloride)，苯索氯铵；酚、丁醇或苯甲醇；烷基对羟基苯甲酸酯如甲基或丙基对羟基苯甲酸酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；间甲酚)；低分子量多肽(少于约10个残基)；蛋白质如血清白蛋白，明胶或免疫球蛋白；亲水聚合物如聚乙烯吡咯烷酮；氨基酸如甘氨酸，谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖，二糖及其它碳水化合物包括葡萄糖、甘露糖、或糊精；螯合剂如EDTA；糖类如蔗糖、甘露醇、岩藻糖或山梨醇；成盐反离子如钠；金属复合物(例如锌-蛋白复合物)；和/或非离子表面活性剂，例如TWEENTM，PLURONICSTM或聚乙二醇(PEG)。优选冻干的抗-VEGF抗体配制剂在WO 97/04801中描述，其包含在本文中作为参考。
本发明的配制剂也可含有需治疗的具体病症所需的一种以上的活性化合物，优选活性互补并且相互之间没有副作用的那些。例如，抗高血压的药物，抗心律失常的药物，治疗糖尿病的药物等。
本发明的纤溶酶原可包裹在通过诸如凝聚技术或界面聚合而制备的微胶囊中，例如，可置入在胶质药物传送***(例如，脂质体，白蛋白微球，微乳剂，纳米颗粒和纳米胶囊)中或置入粗滴乳状液中的羟甲基纤维素或凝胶-微胶囊和聚-(甲基丙烯酸甲酯)微胶囊中。这些技术公开于Remington′s Pharmaceutical Sciences 16th edition，Osol，A.Ed.(1980)。
用于体内给药的本发明的纤溶酶原必需是无菌的。这可以通过在冷冻干燥和重新配制之前或之后通过除菌滤膜过滤而轻易实现。
本发明的纤溶酶原可制备缓释制剂。缓释制剂的适当实例包括具有一定形状且含有糖蛋白的固体疏水聚合物半通透基质，例如膜或微胶囊。缓释基质实例包括聚酯、水凝胶(如聚(2-羟基乙基-异丁烯酸酯)(Langer等，J.Biomed.Mater.Res.，15：167-277(1981)；Langer，Chem.Tech.，12：98-105(1982))或聚(乙烯醇)，聚交酯(美国专利3773919，EP 58，481)，L-谷氨酸与γ乙基-L-谷氨酸的共聚物(Sidman，等，Biopolymers 22：547(1983))，不可 降解的乙烯-乙烯乙酸酯(ethylene-vinyl acetate)(Langer，等，出处同上)，或可降解的乳酸-羟基乙酸共聚物如Lupron DepotTM(由乳酸-羟基乙酸共聚物和亮氨酰脯氨酸(leuprolide)乙酸酯组成的可注射的微球体)，以及聚D-(-)-3-羟丁酸。聚合物如乙烯-乙酸乙烯酯和乳酸-羟基乙酸能持续释放分子100天以上，而一些水凝胶释放蛋白的时间却较短。可以根据相关机理来设计使蛋白稳定的合理策略。例如，如果发现凝聚的机理是通过硫代二硫键互换而形成分子间S-S键，则可通过修饰巯基残基、从酸性溶液中冻干、控制湿度、采用合适的添加剂、和开发特定的聚合物基质组合物来实现稳定。
给药和剂量
可以通过不同方式，例如通过静脉内，腹膜内，皮下，颅内，鞘内，动脉内(例如经由颈动脉)，肌内来实现本发明药物组合物的施用。
用于胃肠外施用的制备物包括无菌水性或非水性溶液、悬浮液和乳剂。非水性溶剂的例子是丙二醇、聚乙二醇、植物油如橄榄油，和可注射有机酯，如油酸乙酯。水性载体包括水、醇性/水性溶液、乳剂或悬浮液，包括盐水和缓冲介质。胃肠外媒介物包含氯化钠溶液、林格氏右旋糖、右旋糖和氯化钠、或固定油。静脉内媒介物包含液体和营养补充物、电解质补充物，等等。也可以存在防腐剂和其他添加剂，诸如例如，抗微生物剂、抗氧化剂、螯合剂、和惰性气体，等等。
医务人员会基于各种临床因素确定剂量方案。如医学领域中公知的，任一患者的剂量取决于多种因素，包括患者的体型、体表面积、年龄、要施用的具体化合物、性别、施用次数和路径、总体健康、和同时施用的其它药物。本发明包含纤溶酶原的药物组合物的剂量范围可以例如为例如每天约0.0001至2000mg/kg，或约0.001至500mg/kg(例如0.02mg/kg，0.25mg/kg，0.5mg/kg，0.75mg/kg，10mg/kg，50mg/kg等等)受试者体重。例如，剂量可以是1mg/kg体重或50mg/kg体重或在1-50mg/kg的范围，或至少1mg/kg。高于或低于此例示性范围的剂量也涵盖在内，特别是考虑到上述的因素。上述范围中的中间剂量也包含在本发明的范围内。受试者可以每天、隔天、每周或根据通过经验分析确定的任何其它日程表施用此类剂量。例示性的剂量日程表包括连续几天1-10mg/kg。在本发明的药物施用过程中需要实时评估治疗效果和安全性。
制品或药盒
本发明的一个实施方案涉及一种制品或药盒，其包含可用于治疗由糖尿病引起的心血管病及其相关病症的本发明纤溶酶原或纤溶酶。所述制品优选包括一个容器，标签或包装插页。适当的容器有瓶子，小瓶，注射器等。容器可由各种材料如玻璃或塑料制成。所述容器含有组合物，所述组合物可有效治疗本发明的疾病或病症并具有无菌入口(例如所述容器可为静脉内溶液包或小瓶，其含有可被皮下注射针穿透的塞子的)。所述组合物中至少一种活性剂为纤溶酶原/纤溶酶。所述容器上或所附的标签说明所述组合物用于治疗本发明所述由糖尿病引起的心血管病及其相关病症。所述制品可进一步包含含有可药用缓冲液的第二容器，诸如磷酸盐缓冲的盐水，林格氏溶液以及葡萄糖溶液。其可进一步包含从商业和使用者角度来看所需的其它物质，包括其它缓冲液，稀释剂，过滤物，针和注射器。此外，所述制品包含带有使用说明的包装插页，包括例如指示所述组合物的使用者将纤溶酶原组合物以及治疗伴随的疾病的其它药物给药患者。
附图简述
图1 3％胆固醇高脂血症模型小鼠给予纤溶酶原10天和20天后血清高密度脂蛋白胆固醇检测结果。结果显示，给予纤溶酶原后给纤溶酶原组小鼠血清HDL-C浓度明显高于给溶媒PBS对照组，且二者在给药10天和20天后高密度脂蛋白浓度均统计差异极显著(**表示P＜0.01)。说明纤溶酶原能有效提高高脂血症模型小鼠血清中高密度脂蛋白胆固醇的含量，改善高脂血症模型小鼠血脂紊乱。
图2 3％胆固醇高脂血症模型小鼠给予纤溶酶原20天后血清总胆固醇检测结果。结果显示，给纤溶酶原组小鼠总胆固醇浓度明显低于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05)。说明纤溶酶原能降低高脂血症模型小鼠血清中总胆固醇的含量，具有降低血脂的功能。
图3 3％胆固醇高脂血症模型小鼠给予纤溶酶原20天后血清低密度脂蛋白胆固醇检测结果。结果显示，给纤溶酶原组小鼠LDL-C浓度明显低于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05)。说明纤溶酶原能降 低高脂血症模型小鼠血清中低密度脂蛋白胆固醇的含量，具有改善高血脂的功能。
图4 16周高脂血症模型小鼠给予纤溶酶原30天后肝脏油红O染色观察结果。A为给溶媒PBS对照组，B为给纤溶酶原组，C为定量分析结果。结果显示，给纤溶酶原组小鼠肝脏脂肪沉积明显少于给溶媒PBS对照组，且定量分析统计差异显著(*表示P＜0.05)。说明纤溶酶原能改善脂肪在高脂血症模型小鼠肝脏中的沉积。
图5 16周高脂血症模型小鼠给予纤溶酶原30天后主动脉窦油红O染色观察结果。A、C为给溶媒PBS对照组，B、D为给纤溶酶原组，E为定量分析结果。结果显示，给纤溶酶原组小鼠主动脉窦脂肪沉积明显少于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05)。说明纤溶酶原能改善脂肪在高脂血症模型小鼠主动脉窦中的沉积。
图6 16周高脂血症模型小鼠给予纤溶酶原30天后主动脉窦HE染色代表性图片。A、C为给溶媒PBS对照组，B、D为给纤溶酶原组。结果显示，给溶媒PBS对照组主动脉管壁可见泡沫细胞沉积(箭头所指)，斑块沉积严重；给纤溶酶原组主动脉管壁仅可见轻度的泡沫细胞沉积，且内膜下未见明显的粥样斑块沉积，给纤溶酶原组主动脉损伤较轻。说明纤溶酶原能改善高脂血症模型小鼠主动脉窦内壁由于脂质沉积所导致的损伤。
图7 16周高脂血症模型小鼠给予纤溶酶原30天后心脏纤维蛋白免疫组化染色图片。A为给溶媒PBS对照组，B为给纤溶酶原组，C为定量分析结果。结果显示，给纤溶酶原组小鼠心脏纤维蛋白的阳性表达明显少于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05)。说明纤溶酶原能减少高血脂所致的心脏损伤。
图8 16周高脂血症模型小鼠给予纤溶酶原30天后心脏IgM免疫染色代表性图片。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给纤溶酶原组小鼠心脏IgM的阳性表达明显少于给溶媒PBS对照组，说明纤溶酶原能减轻高血脂所致心脏损伤。
图9 16周高脂血症模型小鼠给予纤溶酶原30天后心脏天狼星红染色代表性图片。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给纤溶酶原组胶原的沉积明显少于给溶媒PBS对照组，说明纤溶酶原能减轻高脂血症模型小鼠心脏纤维化。
图10 16周高脂血症模型小鼠给予纤溶酶原30天后血清肌钙蛋白检测结果。结果显示，给溶媒PBS对照组血清心肌肌钙蛋白浓度明显高于给纤溶酶原组，且统计差异显著(*表示P＜0.05)。说明纤溶酶原能显著修复高血脂心脏的损伤。
图11 3％胆固醇高脂血症模型小鼠给予纤溶酶原20天后血清动脉粥样硬化指数检测结果。结果显示，给纤溶酶原组小鼠动脉粥样硬化指数明显低于给溶媒PBS对照组，且统计差异极显著(**表示P＜0.01)。说明纤溶酶原能有效降低高脂血症模型小鼠发生动脉粥样硬化的风险。
图12 3％胆固醇高脂血症模型小鼠给予纤溶酶原20天后血清心脏风险险指数结果。结果显示，给纤溶酶原组CRI明显小于给溶媒PBS对照组，且统计差异极其显著(**表示P＜0.01)。说明纤溶酶原能有效的降低高脂血症模型小鼠发生心脏疾病的风险。
图13 24-25周糖尿病小鼠给予纤溶酶原35天后肝脏油红O染色图片。结果显示，给纤溶酶原组小鼠肝脏的脂质沉积面积要显著小于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05)。说明纤溶酶原能减少脂肪在糖尿病小鼠肝脏中的沉积。
图14 24-25周龄糖尿病小鼠给予纤溶酶原31天后主动脉HE染色图片。A、C为给溶媒PBS对照组，B、D为给纤溶酶原组。结果显示，给溶媒PBS对照组血管管壁有泡沫细胞沉积(箭头标识)，中层弹性膜排列紊乱，血管壁增厚，管壁凸凹不均；给纤溶酶原组中层弹性膜结构规则，呈波浪形，血管管壁厚度均匀。表明注射纤溶酶原对糖尿病所致的主动脉损伤具有一定的修复作用。
图15 26周龄糖尿病小鼠给予纤溶酶原35天后心室油红O染色代表性图片。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给纤溶酶原组小鼠心室脂质沉积(箭头标识)明显少于给溶媒PBS对照组。说明纤溶酶原能减少糖尿病小鼠心室脂质沉积，促进心室损伤的修复。
图16 26周龄糖尿病小鼠给予纤溶酶原35天后血清中高密度脂蛋白胆固醇的含量检测结果。结果显示，在对糖尿病小鼠连续注射人源纤溶酶源35天后，给纤溶酶原组小鼠血清中HDL-C的含量高于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05)。说明注射纤溶酶原能促进血清高密度脂蛋白胆固醇的含量升高，改善糖尿病小鼠血脂紊乱。
图17 24-25周龄糖尿病小鼠给予纤溶酶原31天后血清中低密度脂蛋白胆固醇(LDL-C)的含量检测结果。结果显示，糖尿病模型小鼠连续注射人源纤溶酶源31天后，给纤溶酶原组小鼠血清中的LDL-C含量低于给溶媒PBS对照组，统计差异接近显著(P＝0.1)。说明纤溶酶原能降低糖尿病小鼠血清中低密度脂蛋白胆固醇的含量。
图18ApoE动脉粥样硬化模型小鼠给予纤溶酶原30天后血清总胆固醇检测结果。结果显示，给纤溶酶原组小鼠总胆固醇浓度明显低于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05)。说明纤溶酶原能降低ApoE动脉粥样硬化模型小鼠血清中总胆固醇的含量，改善动脉粥样硬化模型小鼠血脂紊乱。
图19ApoE动脉粥样硬化模型小鼠给予纤溶酶原30天后血清甘油三酯检测结果。结果显示，给纤溶酶原组小鼠甘油三酯浓度明显低于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05)。说明纤溶酶原能够降低ApoE动脉粥样硬化模型小鼠血清中甘油三酯的含量，改善动脉粥样硬化模型小鼠血脂紊乱。
图20ApoE动脉粥样硬化模型小鼠给予纤溶酶原30天后血清低密度脂蛋白胆固醇检测结果。结果显示，给纤溶酶原组小鼠LDL-C浓度明显低于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05)。说明纤溶酶原能降低ApoE动脉粥样硬化模型小鼠血清中低密度脂蛋白胆固醇的含量，改善动脉粥样硬化模型小鼠血脂紊乱。
图21ApoE动脉粥样硬化模型小鼠给予纤溶酶原30天后肝脏油红O染色代表性图片。A为给溶媒PBS对照组，B为给纤溶酶原组，C为定量分析结果。结果显示，给纤溶酶原组小鼠肝脏脂肪沉积明显少于给溶媒PBS对照组，且定量分析统计差异显著(*表示P＜0.05)。说明纤溶酶原能减少脂肪在动脉粥样硬化模型小鼠肝脏中的沉积。
图22ApoE动脉粥样硬化模型小鼠给予纤溶酶原30天后主动脉窦油红O染色代表性图片。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给纤溶酶原组小鼠主动脉窦脂肪沉积明显少于给溶媒PBS对照组。说明纤溶酶原能改善脂肪在动脉粥样硬化模型小鼠主动脉窦中的沉积。
图23 16周龄高脂血症模型小鼠给予纤溶酶原30天后主动脉窦天狼星红染色代表性图片。A、C为给溶媒PBS对照组，B、D为给纤溶酶原组。 结果显示，给纤溶酶原组主动脉窦血管内壁胶原蛋白沉积(箭头标识)的面积明显小于给溶媒PBS对照组，说明纤溶酶原能消减高脂血症模型小鼠主动脉窦纤维化水平。
图24ApoE动脉粥样硬化模型小鼠给予纤溶酶原30天后心脏系数统计结果。结果显示，给纤溶酶原组小鼠心脏脏器系数明显低于给溶媒PBS对照组。说明纤溶酶原能改善ApoE动脉粥样硬化模型小鼠心脏损伤所致的心脏代偿性肥大。
图25给予纤溶酶原30天后3％胆固醇高脂血症模型小鼠肾脏天狼星红染色观察结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为定量分析结果。结果显示，给纤溶酶原组肾脏胶原蛋白沉积(箭头标识)明显少于给溶媒PBS对照组，且统计差异显著；给纤溶酶原组纤维化基本恢复到正常水平。说明纤溶酶原能有效减少3％胆固醇高脂血症模型小鼠肾脏纤维化。
图26给予纤溶酶原30天后3％胆固醇高脂血症模型小鼠肾脏油红O观察结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为定量分析结果。结果显示，给纤溶酶原组小鼠肾脏脂肪沉积(箭头标识)明显少于给溶媒PBS对照组，且定量分析统计差异显著；此外，给纤溶酶原组脂质沉积水平与空白对照组小鼠相似。说明纤溶酶原能消减脂肪在高脂血症模型小鼠肾脏中的沉积，从而减少脂肪沉积所致的肾脏损伤。
实施例
实施例1纤溶酶原提高3％胆固醇高脂血症模型小鼠血清高密度脂蛋白胆固醇浓度
9周龄雄性C57小鼠16只饲喂3％胆固醇高脂饲料(南通特洛菲)4周，诱导高脂血症[30，31]，此模型定为3％胆固醇高脂血症模型。成模后的小鼠继续饲喂3％胆固醇高脂饲料。在给药前三天每只小鼠取血50μL，检测总胆固醇，并根据总胆固醇浓度和体重随机分为两组，每组各8只。开始给药记为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，给药20天。在第10、20天小鼠禁食16小时，第11、21天扎眼眶静脉丛取血50μl，离心获得上清， 用以检测血清高密度脂蛋白胆固醇(HDL-C)。本文中高密度脂蛋白胆固醇含量通过检测试剂盒(南京建成生物工程研究所，货号A112-1)所述方法检测。
高密度脂蛋白是一种抗动脉粥样硬化的血浆脂蛋白，是冠心病的保护因子，俗称“血管清道夫”。
检测结果显示，给纤溶酶原组小鼠血清HDL-C浓度明显高于给溶媒PBS对照组，且二者在给药10、20天后HDL-C浓度均有统计学差异(图1)。说明纤溶酶原能提高高脂血症模型小鼠血清中高密度脂蛋白胆固醇的含量，改善高脂血症小鼠血脂紊乱。
实施例2纤溶酶原降低3％胆固醇高脂血症模型小鼠血清总胆固醇水平
9周龄雄性C57小鼠16只饲喂3％胆固醇高脂饲料(南通特洛菲)4周，诱导高脂血症[30，31]，此模型定为3％胆固醇高脂血症模型。成模后的小鼠继续饲喂3％胆固醇高脂饲料。在给药前三天每只小鼠取血50μL，检测总胆固醇，并根据总胆固醇浓度和体重随机分为两组，每组各8只。开始给药记为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，给药20天。在第20天小鼠禁食16小时，第21天扎眼眶静脉丛取血50μL，离心获得上清，采用总胆固醇检测试剂盒(南京建成生物工程研究所，货号A111-1)进行总胆固醇检测。
检测结果显示，给纤溶酶原组小鼠总胆固醇浓度明显低于给溶媒PBS对照组，且统计差异显著(图2)。说明纤溶酶原能降低高脂血症模型小鼠血清中总胆固醇的含量。
实施例3纤溶酶原降低3％胆固醇高脂血症模型小鼠血清低密度脂蛋白胆固醇水平
9周龄雄性C57小鼠16只饲喂3％胆固醇高脂饲料(南通特洛菲)4周，诱导高脂血症[30，31]，此模型定为3％胆固醇高脂血症模型。成模后的小鼠继续饲喂3％胆固醇高脂饲料。在给药前三天每只小鼠取血50μl，检测总胆固醇，并根据总胆固醇浓度和体重随机分为两组，每组各8只。开始给药记为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，给药20天。在第20天小鼠 禁食16小时，第21天扎眼眶静脉丛取血50μL，离心获得上清，采用低密度脂蛋白胆固醇检测试剂盒(南京建成生物工程研究所，货号A113-1)进行低密度脂蛋白胆固醇(LDL-C)检测。
低密度脂蛋白是一种运载胆固醇进入外周组织细胞的脂蛋白颗粒，可被氧化成氧化低密度脂蛋白，当低密度脂蛋白，尤其是氧化修饰的低密度脂蛋白(OX-LDL)过量时，它携带的胆固醇便积存在动脉壁上，引发动脉硬化。因此低密度脂蛋白胆固醇被称为“坏的胆固醇”。
结果显示，给纤溶酶原组小鼠(LDL-C)浓度明显低于给溶媒PBS对照组，且统计差异显著(图3)。说明纤溶酶原能降低高脂血症模型小鼠血清中低密度脂蛋白胆固醇的含量，改善高脂血症小鼠血脂紊乱。
实施例4纤溶酶原消减脂肪在16周高脂血症模型小鼠肝脏中的沉积
6周龄雄性C57小鼠11只饲喂高脂高胆固醇饲料(南通特洛菲，货号TP2031)16周以诱导高脂血症模型[30，31]，此模型定为16周高脂血症模型。成模后的小鼠继续饲喂高胆固醇饲料。在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS对照组6只，给纤溶酶原组5只。开始给药记为第1天，给纤溶酶原组尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。给药30天，并于第31天处死小鼠，取肝脏于4％多聚甲醛固定24-48小时，分别于15％、30％蔗糖中4℃过夜沉底，OCT包埋，冰冻切片厚度8μm，油红O染色15min，75％酒精分化5秒，苏木素染核30s，甘油明胶封片。切片在200倍光学显微镜下观察。
油红O染色可显示脂质沉积，反映脂质沉积的程度[32]。结果显示，给纤溶酶原组(图4B)小鼠肝脏脂肪沉积明显少于给溶媒PBS对照组(图4A)，且定量分析统计差异显著(图4C)。说明纤溶酶原能消减脂肪在高脂血症模型小鼠肝脏中的沉积。
实施例5纤溶酶原减少脂质在16周高脂血症模型小鼠主动脉窦中的沉积
6周龄雄性C57小鼠11只饲喂高脂高胆固醇饲料(南通特洛菲，货号TP2031)16周以诱导高脂血症模型[30，31]，此模型定为16周高脂血症模型。成模后的小鼠继续饲喂高胆固醇饲料。在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS 对照组6只，给纤溶酶原组5只。开始给药记为第1天，给纤溶酶原组尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。给药30天，并于第31天处死小鼠，取心脏组织于4％多聚甲醛固定24-48小时，分别于15％、30％蔗糖中4℃过夜沉底，OCT包埋，主动脉窦冰冻切片厚度8μm，油红O染色15min，75％酒精分化5秒，苏木素染核30s，甘油明胶封片。切片在40(图5A、5B)、200倍(图5C、5D)倍光学显微镜下观察。
结果显示，给纤溶酶原组(图5B、5D)小鼠主动脉窦脂肪沉积明显少于给溶媒PBS对照组(图5A、5C)，且统计差异显著(图5E)。说明纤溶酶原能减少脂质在高脂血症模型小鼠主动脉窦中的沉积。
实施例6纤溶酶原改善16周高脂血症模型小鼠主动脉窦损伤
6周龄雄性C57小鼠11只饲喂高脂高胆固醇饲料(南通特洛菲，货号TP2031)16周以诱导高脂血症模型[30，31]，此模型定为16周高脂血症模型。成模后的小鼠继续饲喂高胆固醇饲料。在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS对照组6只，给纤溶酶原组5只。开始给药记为第1天，给纤溶酶原组尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。给药30天，并于第31天处死小鼠，取心脏组织于4％多聚甲醛固定24-48小时。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。固定后的组织样本经酒精梯度脱水和二甲苯透明后进行石蜡包埋。主动脉窦组织切片厚度为3μm，切片脱蜡复水并用苏木素和伊红染色(HE染色)，1％盐酸酒精分化后氨水返蓝并酒精梯度脱水封片，切片在40(图6A、B)、400倍(图6C、D)光学显微镜下观察。
结果显示，给溶媒PBS对照组(图6A、C)主动脉窦内壁泡沫细胞沉积(箭头所指)，斑块沉积重；给纤溶酶原组(图6B、D)主动脉窦内壁仅可见轻度的泡沫细胞沉积，且内膜下未见明显的粥样斑块沉积，给纤溶酶原组主动脉窦内壁损伤较轻。说明纤溶酶原能改善高脂血症模型小鼠动脉窦内壁损伤。
实施例7纤溶酶原降低16周高脂血症模型小鼠心脏纤维蛋白的表达
6周龄雄性C57小鼠11只饲喂高脂高胆固醇饲料(南通特洛菲，货号TP2031)16周以诱导高脂血症模型[30，31]，此模型定为16周高脂血症模型。 成模后的小鼠继续饲喂高胆固醇饲料。在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS对照组6只，给纤溶酶原组5只。开始给药记为第1天，给纤溶酶原组尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。给药30天，并于第31天处死小鼠，取心脏组织于4％多聚甲醛固定24-48小时。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。以3％双氧水孵育15分钟，水洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，用PAP笔圈出组织。以3％双氧水孵育15分钟，水洗2次，每次5分钟。兔抗小鼠纤维蛋白抗体(Abcam)4℃孵育过夜，0.01MPBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水、二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
纤维蛋白原是纤维蛋白的前体，在组织存在损伤的情况下，作为机体对损伤的一种应激反应，纤维蛋白原水解成纤维蛋白沉积在损伤部位[33，34]。因此，可将损伤局部纤维蛋白水平作为损伤程度的一个标志。
免疫组化染色结果显示，给纤溶酶原组小鼠(图7B)心脏纤维蛋白的阳性表达明显少于给溶媒PBS对照组(图7A)，且统计差异显著(图7C)，说明纤溶酶原能减少高血脂所致的心肌损伤。
实施例8纤溶酶原有效保护16周高脂血症模型小鼠心肌损伤
6周龄雄性C57小鼠11只饲喂高脂高胆固醇饲料(南通特洛菲，货号TP2031)16周以诱导高脂血症模型[30，31]，此模型定为16周高脂血症模型。成模后的小鼠继续饲喂高胆固醇饲料。在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS对照组6只，给纤溶酶原组5只。开始给药记为第1天，给纤溶酶原组尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。给药30天，并于第31天处死小鼠，取心脏组织于4％多聚甲醛固定24-48小时。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。以3％双氧水孵育 15分钟，水洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，用PAP笔圈出组织。以3％双氧水孵育15分钟，水洗2次，每次5分钟。山羊抗鼠IgM(HRP)抗体(Abcam)室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素染核30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明，中性树胶封片，切片在200倍光学显微镜下观察。
IgM抗体在清除凋亡和坏死细胞过程中发挥着重要作用，损伤的组织器官局部IgM抗体的水平与损伤程度呈正相关[35，36]。因此，检测组织器官局部IgM抗体的水平能够反映该组织器官的损伤程度。
免疫染色结果显示，给纤溶酶原组小鼠(图8B)心脏IgM的阳性表达明显少于给溶媒PBS对照组(图8A)，说明纤溶酶原能减少高脂血症模型动物心脏的损伤。
实施例9纤溶酶原减轻16周高脂血症模型小鼠心脏纤维化
6周龄雄性C57小鼠11只饲喂高脂高胆固醇饲料(南通特洛菲，货号TP2031)16周以诱导高脂血症模型[30，31]，此模型定为16周高脂血症模型。成模后的小鼠继续饲喂高胆固醇饲料。在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS对照组6只，给纤溶酶原组5只。开始给药记为第1天，给纤溶酶原组尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。给药30天，并于第31天处死小鼠，取心脏组织于4％多聚甲醛固定24-48小时。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2min，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
天狼星红染色可使胶原持久染色，作为病理切片特殊染色方法，天狼星红染色可以特异显示胶原组织。
染色结果显示，给纤溶酶原组(图9B)胶原的沉积明显少于给溶媒PBS对照组(图9A)，说明纤溶酶原能减轻高脂血症模型小鼠心脏组织胶原蛋白的沉积，减轻心肌纤维化。
实施例10纤溶酶原修复16周高脂血症模型小鼠心肌损伤
6周龄雄性C57小鼠11只饲喂高脂高胆固醇饲料(南通特洛菲，货号TP2031)16周以诱导高脂血症模型[30，31]，此模型定为16周高脂血症模型。成模后的小鼠继续饲喂高胆固醇饲料。在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS对照组6只，给纤溶酶原组5只。开始给药记为第1天，给纤溶酶原组尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。给药30天，第30天给药后小鼠开始禁食，禁食16小时，第31天摘眼球取血，离心获得上清，采用心肌肌钙蛋白(Cardiac troponin I，CTNI)检测试剂盒(南京建成)检测血清中肌钙蛋白的浓度。
心肌肌钙蛋白I是心肌损伤的重要标志物，其血清浓度能够反映心肌损伤的程度[37]。
检测结果显示，给溶媒PBS对照组血清心肌肌钙蛋白浓度明显高于给纤溶酶原组，且统计差异显著(图10)。说明纤溶酶原能显著改善高脂血症模型小鼠心脏损伤。
实施例11纤溶酶原降低3％胆固醇高脂血症模型小鼠动脉粥样硬化形成的风险
9周龄雄性C57小鼠16只饲喂3％胆固醇高脂饲料(南通特洛菲)4周，诱导高脂血症[30，31]，此模型定为3％胆固醇高脂血症模型。成模后的小鼠继续饲喂3％胆固醇高脂饲料。在给药前三天每只小鼠取血50μl，检测总胆固醇(T-CHO)，并根据总胆固醇浓度和体重随机分为两组，每组各8只。开始给药记为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。在第20天给药后小鼠开始禁食，禁食16小时，第21天扎眼眶静脉丛取血50μL，离心获得上清，总胆固醇含量采用总胆固醇检测试剂盒(南京建成生物工程研究所，货号A111-1)进行检测；高密度脂蛋白胆固醇(HDL-C)含量采用高密度脂蛋白胆固醇检测试剂盒(南京建成生物工程研究所，货号A112-1)进行检测。
动脉粥样硬化指数，是临床上预测动脉粥样硬化的综合指标，认为它在用作对冠心病风险程度的估计方面临床意义比单项的总胆固醇、甘油三 酯、高密度脂蛋白和低密度脂蛋白更大[38]。动脉粥样硬化指数＝(T-CHO-HDL-C)/HDL-C。
计算结果显示，给纤溶酶原组小鼠动脉粥样硬化指数明显低于给溶媒PBS对照组，且统计差异显著(图11)。说明纤溶酶原能降低高脂血症模型小鼠发生动脉粥样硬化的风险。
实施例12纤溶酶原降低3％胆固醇高脂血症模型小鼠心脏发病风险
9周龄雄性C57小鼠16只饲喂3％胆固醇高脂饲料(南通特洛菲)4周，诱导高脂血症[30，31]，此模型定为3％胆固醇高脂血症模型。成模后的小鼠继续饲喂3％胆固醇高脂饲料。在给药前三天每只小鼠取血50μl，检测总胆固醇(T-CHO)，并根据总胆固醇浓度随机分为两组，每组各8只。开始给药记为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。在第20天给药后小鼠开始禁食，禁食16小时，第21天扎眼眶静脉丛取血50μL，离心获得上清，总胆固醇含量采用总胆固醇检测试剂盒(南京建成生物工程研究所，货号A111-1)进行检测；高密度脂蛋白胆固醇(HDL-C)含量采用高密度脂蛋白胆固醇检测试剂盒(南京建成生物工程研究所，货号A112-1)进行检测。心脏风险指数＝T-CHO/HDL-C。
心脏风险指数(cardiac risk index，CRI)用以评估血脂紊乱诱发心脏疾病的风险[38]。
结果显示，给纤溶酶原组CRI明显小于给溶媒PBS对照组，且统计差异极其显著(图12)。说明纤溶酶原能有效的降低高脂血症模型小鼠发生心脏疾病的风险。
实施例13纤溶酶原改善糖尿病小鼠肝脏脂质沉积
24-25周龄雄性db/db小鼠10只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组各5只。实验开始当天记为第0天并称重分组，第1天开始给纤溶酶原或PBS。给纤溶酶原组小鼠按2mg/0.2ml/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组尾静脉注射给予相同体积的PBS，连续给药35天。在第36天处死小鼠取肝脏组织4％多聚甲醛固定24-48小时，分别于15％、30％蔗糖中4℃过夜沉底，OCT包埋，冰冻切片厚度8μm，油红O染色15min，75％酒精分化5秒，苏木素染核30s，甘油明胶封片。切片在200倍光学显微镜下观察。
染色结果显示，给纤溶酶原组(图13B)小鼠肝脏的脂质沉积面积显著小于给溶媒PBS对照组(图13A)，且统计差异显著(P＝0.02)(图13C)。说明纤溶酶原能减少脂肪在糖尿病小鼠肝脏中的沉积。
实施例14纤溶酶原减轻糖尿病小鼠主动脉管壁的损伤
24-25周龄雄性db/db小鼠10只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组各5只。实验开始当天记为第0天称重分组，第1天开始给PBS或纤溶酶原，连续给药31天。给纤溶酶原组小鼠按2mg/0.2ml/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组尾静脉注射给予相同体积的PBS。在第32天处死小鼠并取主动脉在10％中性***固定液中固定24小时。固定后的主动脉经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为5μm，切片脱蜡复水并用苏木素和伊红染色(HE染色)，1％盐酸酒精分化后氨水返蓝并酒精梯度脱水封片，切片在400倍(图14A、B)光学显微镜以及1000倍(图14C、D)油镜下观察。
糖尿病合并高血脂是糖尿病较为常见的并发症，是糖尿病大血管病变的重要危险因子[39]。
染色结果显示，给溶媒PBS对照组(图14A、C)血管管壁有泡沫细胞沉积(箭头标识)，中层弹性膜排列紊乱，血管壁增厚，管壁凸凹不均；给纤溶酶原组(图14B、D)中层弹性膜结构规则，呈波浪形，血管管壁厚度均匀。表明注射纤溶酶原可减轻糖尿病小鼠主动脉管壁的脂质沉积，对动脉管壁脂质沉积导致的损伤具有一定的保护作用。
实施例15纤溶酶原降低糖尿病小鼠心室脂质沉积
26周龄雄性db/db小鼠9只随机分组，给纤溶酶原组4只，给溶媒PBS对照组5只。给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，给药35天。于第36天处死小鼠，取心脏于4％多聚甲醛固定24-48小时，分别于15％、30％蔗糖中4℃过夜沉底，OCT包埋，冰冻切片厚度8μm，油红O染色15min，75％酒精分化5秒，苏木素染核30s，甘油明胶封片。切片在400倍光学显微镜下观察。
结果显示，给纤溶酶原组小鼠(图15B)心室脂质沉积(箭头标识)明显少于给溶媒PBS对照组(图15A)。说明纤溶酶原能减少脂肪在糖尿病小鼠心室沉积，促进心室损伤的修复。
实施例16纤溶酶原提高糖尿病小鼠血清中的高密度脂蛋白胆固醇水平
26周龄雄性db/db小鼠20只随机分组，给纤溶酶原组11只，给溶媒PBS对照组各9只。实验开始当天记为第0天并称重分组，第1天开始给纤溶酶原或PBS，连续给药35天。给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，对照组尾静脉注射同体积的PBS。第36天小鼠摘眼球采全血，4℃3500r/min离心10分钟，取上清液并采用高密度脂蛋白检测试剂盒(南京建成生物工程研究所，货号A112-1)检测血清中高密度脂蛋白胆固醇(HDL-C)浓度。
检测结果显示，给纤溶酶原组小鼠血清中HDL-C的含量高于给溶媒PBS对照组，统计差异显著(图16)。说明注射纤溶酶原能促进血清高密度脂蛋白胆固醇的含量升高，改善糖尿病血脂紊乱。
实施例17纤溶酶原降低糖尿病小鼠血清中低密度脂蛋白胆固醇
24-25周龄雄性db/db小鼠10只随机分组，给纤溶酶原组和给溶媒PBS对照组各5只，并取3只db/m作为正常对照组。实验开始当天记为第0天称重分组，第1天开始给纤溶酶原或PBS，连续给药31天。给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，PBS对照组尾静脉注射同体积的PBS，正常对照组小鼠不作任何处理。第32天小鼠摘眼球采全血，4℃3500r/min离心10分钟，取上清液并采用低密度脂蛋白胆固醇检测试剂盒(南京建成生物工程研究所，货号A113-1)检测血清中低密度脂蛋白胆固醇(LDL-C)浓度。
结果显示，糖尿病模型小鼠连续注射人源纤溶酶源31天后，给纤溶酶原组小鼠血清中的LDL-C含量低于给溶媒PBS对照组，且统计差异接近显著(P＝0.1)(图17)。说明纤溶酶原能降低血清中LDL-C的含量。
实施例18纤溶酶原降低ApoE动脉粥样硬化小鼠血清总胆固醇的含量
6周龄雄性ApoE小鼠13只饲喂高脂高胆固醇饲料(南通特洛菲，TP2031)16周以诱导动脉粥样硬化模型[40，41]。成模后的小鼠继续饲喂高脂高胆固醇饲料。在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS对照组7只，给纤溶酶原组6只。开始给药定为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，给药 30天。在第30天小鼠禁食16小时，第31天摘除眼球取血，离心获得上清采用总胆固醇检测试剂盒(南京建成生物工程研究所，货号A111-1)进行总胆固醇检测。
检测结果显示，给纤溶酶原组小鼠总胆固醇浓度明显低于给溶媒PBS对照组，且统计差异显著(P＝0.014)(图18)。说明纤溶酶原能降低ApoE动脉粥样硬化模型小鼠血清中总胆固醇的含量，改善动脉粥样硬化的血脂紊乱。
实施例19纤溶酶原降低ApoE动脉粥样硬化小鼠血清甘油三酯的含量
6周龄雄性ApoE小鼠13只饲喂高脂高胆固醇饲料(南通特洛菲，TP2031)16周以诱导动脉粥样硬化模型[40，41]。成模后的小鼠继续饲喂高脂高胆固醇饲料。在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS对照组7只，给纤溶酶原组6只。开始给药记为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，给药30天。在第30天小鼠禁食16小时，第31天摘眼球取血，离心获得上清采用甘油三酯检测试剂盒(南京建成生物工程研究所，货号A110-1)进行甘油三酯检测。
检测结果显示，给纤溶酶原组小鼠甘油三酯浓度明显低于给溶媒PBS对照组，且统计差异显著(P＝0.013)(图19)。说明纤溶酶原能降低ApoE动脉粥样硬化模型小鼠血清中甘油三酯的含量，改善动脉粥样硬化血脂紊乱。
实施例20纤溶酶原降低ApoE动脉粥样硬化小鼠血清低密度脂蛋白胆固醇的含量
6周龄雄性ApoE小鼠13只饲喂高脂高胆固醇饲料(南通特洛菲，TP2031)16周以诱导动脉粥样硬化模型[40，41]。成模后的小鼠继续饲喂高脂高胆固醇饲料。在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS对照组7只，给纤溶酶原组6只。开始给药记为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，给药30天。第30天小鼠禁食16小时，第31天摘眼球取血，离心获得上清采用 低密度脂蛋白胆固醇(LDL-C)检测试剂盒(南京建成生物工程研究所，货号A113-1)进行LDL-C检测。
结果显示，给纤溶酶原组小鼠LDL-C浓度明显低于给溶媒PBS对照组，且统计差异显著(P＝0.017)(图20)。说明纤溶酶原能降低ApoE动脉粥样硬化模型小鼠血清中低密度脂蛋白胆固醇含量，改善动脉粥样硬化模型小鼠血脂紊乱。
实施例21纤溶酶原改善ApoE动脉粥样硬化小鼠肝脏脂质沉积
6周龄雄性ApoE小鼠13只饲喂高脂高胆固醇饲料(南通特洛菲，TP2031)16周以诱导动脉粥样硬化模型[40，41]。成模后的小鼠继续饲喂高脂高胆固醇饲料。在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS对照组7只，给纤溶酶原组6只。开始给药记为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，给药30天。于第31天处死小鼠，取材肝脏组织于4％多聚甲醛固定24-48小时，分别于15％、30％蔗糖中4℃过夜沉底，OCT包埋，冰冻切片厚度8μm，油红O染色15min，75％酒精分化5秒，苏木素染核30s，甘油明胶封片。切片在400倍光学显微镜下观察。
染色结果显示，给纤溶酶原组(图21B)小鼠肝脏脂肪沉积明显少于给溶媒PBS对照组(图21A)，且定量分析统计差异显著(P＝0.02)(图21C)。说明纤溶酶原能减少脂肪在动脉粥样硬化模型小鼠肝脏中的沉积。
实施例22纤溶酶原改善脂质在ApoE动脉粥样硬化小鼠主动脉窦中的沉积
6周龄雄性ApoE小鼠13只饲喂高脂高胆固醇饲料(南通特洛菲，TP2031)16周以诱导动脉粥样硬化模型[40，41]。成模后的小鼠继续饲喂高脂高胆固醇饲料。在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS对照组7只，给纤溶酶原组6只。开始给药记为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，给药30天。于第31天处死小鼠，取心脏组织于4％多聚甲醛固定24-48小时，分别于15％、30％蔗糖中4℃过夜沉底，OCT包埋，主动脉窦冰冻切片厚 度8μm，油红O染色15min，75％酒精分化5秒，苏木素染核30s，甘油明胶封片。切片在40倍光学显微镜下观察。
染色结果显示，给纤溶酶原组(图22B)小鼠主动脉窦脂肪沉积明显少于给溶媒PBS对照组(图22A)。说明纤溶酶原能减少脂质在动脉粥样硬化模型小鼠主动脉窦中的沉积。
实施例23纤溶酶原降低16周高脂血症模型小鼠主动脉窦纤维化
6周龄雄性C57小鼠11只饲喂高脂高胆固醇饲料(南通特洛菲，货号TP2031)16周以诱导高脂血症模型[30，31]，此模型定为16周高脂血症模型。成模后的小鼠继续饲喂高胆固醇饲料。在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS对照组6只，给纤溶酶原组5只。开始给药记为第1天，给纤溶酶原组尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。给药30天，于第31天处死小鼠，取心脏于4％多聚甲醛固定24-48小时。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。主动脉窦切片厚度为3μm，切片脱蜡复水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2min，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在40(图23A、23B)、200倍(图23C、23D)光学显微镜下观察。
结果显示，给纤溶酶原组(图23B、23D)主动脉窦血管内壁胶原蛋白沉积(箭头标识)的面积明显小于给溶媒PBS对照组(图23A、23C)，说明纤溶酶原能够消减高脂血症模型小鼠主动脉窦纤维化水平。
实施例24纤溶酶原改善ApoE动脉粥样硬化小鼠心脏代偿性肥大
6周龄雄性ApoE小鼠13只饲喂高脂高胆固醇饲料(南通特洛菲，TP2031)16周以诱导动脉粥样硬化模型[40，41]。成模后的小鼠在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS对照组7只，给纤溶酶原组6只。开始给药定为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。给药30天，给药期间继续饲喂高脂高胆固醇饲料。于给药的第31天称重后处死小鼠，取心脏称重，并计算心脏系数。心脏系数(％)＝心脏重量/体重×100。
结果显示，给纤溶酶原组小鼠心脏系数明显低于给溶媒PBS对照组(图24)。说明纤溶酶原能减轻ApoE动脉粥样硬化模型小鼠由于心脏损伤所致的心脏代偿性肥大。
实施例25纤溶酶原降低3％胆固醇高脂血症模型小鼠肾脏纤维化
9周龄雄性C57小鼠16只饲喂3％胆固醇高脂饲料(南通特洛菲)4周，诱导高脂血症[30，31]，此模型定为3％胆固醇高脂血症模型，成模后的小鼠继续饲喂3％胆固醇高脂饲料。另取相同周龄的雄性C57小鼠5只作为空白对照组，实验期间饲喂普通维持饲料。在给药前三天每只小鼠取血50μL，检测总胆固醇，模型小鼠根据总胆固醇浓度和体重随机分为两组，给纤溶酶原组和给溶媒PBS对照组，每组各8只。开始给药记为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。在第30天给药后小鼠给药30天，于第31天处死小鼠，取肾脏于4％多聚甲醛固定24-48小时。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。切片厚度为3μm，切片脱蜡复水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2min，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
结果显示，给纤溶酶原组(图25C)肾脏胶原蛋白沉积(箭头标识)明显少于给溶媒PBS对照组(图25B)，且统计差异显著(图25D)；给纤溶酶原组纤维化基本恢复到正常水平(图25A)。说明纤溶酶原能有效的减少3％胆固醇高脂血症模型小鼠肾脏纤维化。
实施例26纤溶酶原降低3％胆固醇高脂血症模型小鼠肾脏脂肪沉积
9周龄雄性C57小鼠16只饲喂3％胆固醇高脂饲料(南通特洛菲)4周，诱导高脂血症[30，31]，此模型定为3％胆固醇高脂血症模型，成模后的小鼠继续饲喂3％胆固醇高脂饲料。另取相同周龄的雄性C57小鼠5只作为空白对照组，实验期间饲喂普通维持饲料。在给药前三天每只小鼠取血50μL，检测总胆固醇，模型小鼠根据总胆固醇浓度和体重随机分为两组，给纤溶酶原组和给溶媒PBS对照组，每组各8只。开始给药记为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，给药30天。第31天处死小鼠，取肾脏于4％多聚甲醛固定24-48小时，分别于15％、30％蔗糖中4℃过夜沉底，OCT包埋， 冰冻切片厚度8μm，油红O染色15min，75％酒精分化5秒，苏木素染核30秒，甘油明胶封片。切片在400倍光学显微镜下观察。
结果显示，给纤溶酶原组(图26C)小鼠肾脏脂肪沉积(箭头标识)明显少于给溶媒PBS对照组(图26B)，且定量分析统计差异显著(图26D)；此外，给纤溶酶原组脂质沉积水平与空白对照组小鼠(图26A)相似。说明纤溶酶原能消减脂肪在高脂血症模型小鼠肾脏中的沉积，从而减少脂肪沉积所致肾脏的损伤。
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Claims (46)

  1. 一种预防和/或治疗受试者脂肪代谢紊乱及其相关病症的方法，包括给药受试者预防和/或治疗有效量的纤溶酶原，其中所述受试者易患脂肪代谢紊乱、患有脂肪代谢紊乱或罹患其它疾病并伴有脂肪代谢紊乱。
  2. 权利要求1的方法，其中所述脂肪代谢紊乱为内分泌紊乱疾病、糖代谢疾病、肝脏疾病、肾脏疾病、心血管疾病、肠道疾病、甲状腺疾病、胆囊或胆道疾病、肥胖症、饮酒、药物治疗引发或伴随的脂肪代谢紊乱。
  3. 权利要求2的方法，其中所述脂肪代谢紊乱为高血压、糖尿病、慢性肝炎、肝硬化、肾损伤、慢性肾小球肾炎、慢性肾盂肾炎、肾病综合征、肾功能不全、肾移植、***、甲状腺功能低下、阻塞性胆囊炎、阻塞性胆管炎、药物或激素治疗引发或伴随的脂肪代谢紊乱。
  4. 权利要求1-3任一项的方法，其中所述脂肪代谢紊乱为高脂血症、高脂蛋白血症、脂肪肝、动脉粥样硬化、肥胖症、脏器脂肪沉积。
  5. 权利要求4的方法，其中所述动脉粥样硬化包括包括主动脉粥样硬化、冠状动脉粥样硬化、脑动脉粥样硬化、肾动脉粥样硬化、肝动脉粥样硬化、肠系膜动脉粥样硬化、下肢动脉粥样硬化。
  6. 一种预防和/或消减受试者脂肪在身体组织器官异常沉积的方法，包括给药受试者有效量的纤溶酶原。
  7. 一种预防和/或治疗受试者脂肪在身体组织器官异常沉积导致的病症的方法，包括给药受试者有效量的纤溶酶原。
  8. 权利要求6或7的方法，其中所述脂肪在身体组织器官异常沉积是指脂肪在血液，皮下组织、血管壁、内脏器官的异常沉积。
  9. 权利要求8的方法，其中所述脂肪在身体组织器官异常沉积导致的病症包括肥胖症，高脂血症、高脂蛋白血症、脂肪肝、动脉粥样硬化、脂质性心脏损害、脂质性肾损害，脂质性胰岛损害。
  10. 一种预防和/或治疗受试者脂肪代谢紊乱所致病症的方法，包括给药受试者有效量的纤溶酶原。
  11. 权利要求10的方法，所述病症包括肥胖症，高脂血症、高脂蛋白血症、脂肪肝、动脉粥样硬化、脂质性心脏组织损伤，脂质性肾损伤。
  12. 一种通过减少脂肪异常沉积治疗受试者疾病的方法，包括给药受试者有效量的纤溶酶原。
  13. 权利要求12的方法，其中所述疾病包括动脉粥样硬化、冠心病、心绞痛、心肌梗死、心律失常、脂肪肝、肝硬化，脑缺血、脑梗死、肾功能不全、肾病综合征、肾功能不全、肥胖症。
  14. 一种预防和/或治疗受试者组织器官脂质性损伤的方法，包括给药受试者有效量的纤溶酶原。
  15. 权利要求14的方法，其中所述组织器官包括动脉管壁、心脏、肝脏、肾脏、胰腺。
  16. 一种改善受试者高脂血症的方法，包括给药受试者有效量的纤溶酶原。
  17. 权利要求16的方法，其中所述高脂血症选自如下的一项或多项：高胆固醇血症、高甘油三酯血症、混合型高脂血症和低高密度脂蛋白血症。
  18. 一种降低受试者动脉粥样硬化风险的方法，包括给药受试者有效量的纤溶酶原。
  19. 权利要求18的方法，其中所述受试者患有高血压、肥胖症、糖尿病、慢性肝炎、肝硬化、肾损伤、慢性肾小球肾炎、慢性肾盂肾炎、肾病综合征、肾功能不全、肾移植、***、甲状腺功能低下、阻塞性胆囊炎或阻塞性胆管炎，或所述受试者服用影响脂肪代谢的药物或激素。
  20. 权利要求19的方法，其中所述纤溶酶原通过选自如下的一项或多项降低受试者动脉粥样硬化风险：降低血中总胆固醇水平、甘油三酯水平、低密度脂蛋白水平、提高血中高密度脂蛋白水平。
  21. 一种通过改善受试者高脂血症治疗疾病的方法，包括给药受试者有效量的纤溶酶原。
  22. 权利要求21的方法，其中所述病症包括糖尿病、高血压、动脉粥样硬化、冠心病、心绞痛、心肌梗死、心律失常、慢性肝炎、脂肪肝、肝硬化，脑供血不足、脑缺血、脑梗死、慢性肾炎、慢性肾盂肾炎、肾功能不全、肾病综合征、***、肥胖症。
  23. 一种预防和/或治疗受试者高血脂相关病症的方法，包括给药受试者有效量的纤溶酶原，其中所述病症包括糖尿病、高血压、动脉粥样硬化、冠心病、心绞痛、心肌梗死、心律失常、慢性肝炎、脂肪肝、肝硬化，脑 供血不足、脑缺血、脑梗死、慢性肾炎、慢性肾盂肾炎、肾功能不全、肾病综合征、***、肥胖症。
  24. 根据权利要求1-23任一项的方法，其中所述纤溶酶原可与一种或多种其它药物或治疗方法联合施用。
  25. 权利要求24的方法，其中所述一种或多种其它药物包括高血压治疗药物、糖尿病治疗用药物、动脉粥样硬化治疗用药物、慢性肾小球肾炎治疗药物、慢性肾盂肾炎治疗药物、肾病综合征治疗用药物、肾功能不全治疗用药物、***治疗用药物、肾移植治疗用药物、脂肪肝治疗用药物、肝硬化治疗用药物、肥胖症治疗用药物。
  26. 根据权利要求25的方法，其中所述其它药物包括：降血脂药物、抗血小板药物、降血压药物、扩张血管药物、降血糖药物、抗凝血药物、溶血栓药物，保肝药物，抗心律失常药物，强心药物，利尿药物，抗感染药物、抗病毒药物、免疫调节药物、炎症调节类药物、抗肿瘤药物、激素类药物、甲状腺素。
  27. 权利要求26的方法，其中所述药物包括降血脂药物：他汀类；贝特类；烟酸；消胆胺；安妥明；不饱和脂肪酸如益寿宁、血脂平及心脉乐；藻酸双酯钠；抗血小板药物：阿司匹林；潘生丁；氯吡格雷；西洛他；扩张血管药物：肼苯哒嗪；***和消心痛；硝普钠；α硝受体阻断剂如哌唑嗪；α受体阻断剂如酚妥拉明；β拉受体***如舒喘灵；卡托普利、依那普利；心痛定、硫氮卓酮；柳丁氨酸、长压定、***素、心钠素；溶血栓药物：尿激酶和链激酶；组织型纤溶酶原激活剂；单链尿激酶型纤溶酶原激活剂；TNK-组织型纤溶酶原激活剂；抗凝血药物：肝素；依诺肝素；那曲肝素；比伐卢定。
  28. 权利要求1-27任一项的方法，其中所述纤溶酶原与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。
  29. 权利要求1-28任一项的方法，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。
  30. 权利要求1-29任一项的方法，所述纤溶酶原是包含纤溶酶原活性片 段、并且仍然具有纤溶酶原活性的蛋白质。
  31. 权利要求1-30任一项的方法，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。
  32. 权利要求1-31任一项的方法，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的变体或片段。
  33. 权利要求1-31任一项的方法，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的变体或片段。
  34. 权利要求1-33任一项的方法，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。
  35. 权利要求1-34任一项的方法，其中所述纤溶酶原是人天然纤溶酶原。
  36. 权利要求1-35任一项的方法，其中所述受试者是人。
  37. 权利要求1-36任一项的方法，其中所述受试者缺乏或缺失纤溶酶原。
  38. 权利要求37的方法，其中所述缺乏或缺失是先天的、继发的和/或局部的。
  39. 一种用于权利要求1-38任一项的方法的纤溶酶原。
  40. 一种药物组合物，其包含药学上可接受的载剂和用于权利要求1-38中任一项所述方法的纤溶酶原。
  41. 一种预防性或治疗性试剂盒，其包含：(i)用于权利要求1-38中任一项所述方法的纤溶酶原和(ii)用于递送所述纤溶酶原至所述受试者的构件(means)。
  42. 根据权利要求41所述的试剂盒，其中所述构件为注射器或小瓶。
  43. 权利要求41或42的试剂盒，其还包含标签或使用说明书，该标签或使用说明书指示将所述纤溶酶原投予所述受试者以实施权利要求1-38中任一项所述方法。
  44. 一种制品，其包含：
    含有标签的容器；和
    包含(i)用于权利要求1-38中任一项所述方法的纤溶酶原或包含纤溶酶原的药物组合物，其中所述标签指示将所述纤溶酶原或组合物投予所述受试者以实施权利要求1-38中任一项所述方法。
  45. 权利要求41-43中任一项的试剂盒或权利要求44的制品，还包含另外的一个或多个构件或容器，该构件或容器中含有其他药物。
  46. 权利要求45的试剂盒或制品，其中所述其他药物选自下组：降血脂药物、抗血小板药物、降血压药物、扩张血管药物、降血糖药物、抗凝血药物、溶血栓药物，保肝药物，抗心律失常药物，强心药物，利尿药物，抗感染药物、抗病毒药物、免疫调节药物、炎症调节类药物、抗肿瘤药物、激素类药物、甲状腺素。
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