CN106890318A - A kind of new method for preventing and treating diabetic cardiomyopathy - Google Patents

Abstract

Effect the present invention relates to plasminogen in terms for the treatment of and/or eliminating diabetic cardiomyopathy, and then provide brand-new therapeutic strategy to treat different types of diabetic cardiomyopathy and its associated conditions.

Description

A kind of new method for preventing and treating diabetic cardiomyopathy

Technical field

Effect the present invention relates to plasminogen in terms for the treatment of and/or eliminating diabetic cardiomyopathy, and then be treatment Different types of diabetic cardiomyopathy and its associated conditions provide brand-new therapeutic strategy.

Background

Diabetes are one group causes insulin secretion to reduce or insulin work by various h and E factor collective effects Cause endocrine, the metabolic syndrome of internal sugar, protein, fat, water and metabolic disturbance of electrolyte with defect.It is with chronic Blood glucose level rises are high to be characterized, and prolonged illness can cause the chronic complicating diseases of multiple system organs, and wherein diabetic cardiomyopathy is sugar Urinate the major complications of disease.

" diabetic cardiomyopathy " refers to the histology of the heart caused by diabetes and the lesion of changes of function, is most common One of diabetic complication.Diabetic cardiomyopathy this diabetic complication harm is big, clinical visible electrocardiographic abnormality, the heart Dirty expansion, arrhythmia cordis, angina pectoris, painless myocardial infarction, heart failure.According to statistics, the patient of diabetes of about 70%-80% Person finally dies from cardiac complication, and diabetes merge cardiopathic patient in short term or long-term prognosis are significantly worse than non-diabetic Patient.

Fibrinolysin is the key component of plasminogen activating system system (PA systems).It is a kind of protease of wide spectrum, can Several components of hydrolyzed cellular epimatrix (ECM), including fibrin, gelatin, fibronectin, laminin and albumen are poly- Sugar^[1].Additionally, the activation of some metalloprotein enzyme precursors (pro-MMP) can be formed active metalloproteinases by fibrinolysin (MMP).Therefore fibrinolysin is considered as an important upstream regulation thing of extracellular proteolysis effect^[2,3].Fibrinolysin be by Plasminogen is by two kinds of PA of physiological：Tissue-type plasminogen activator (tPA) or urokinase type plasminogen activator (uPA) proteolysis are formed.Due to plasminogen, relative level is higher in blood plasma and other body fluid, conventionally PA systems The regulation of system is main to be realized by the synthesis of PA and activity level.The synthesis of PA system components is strictly adjusted by different factors, such as Hormone, growth factor and cell factor.Additionally, also there is the specific physiological inhibitor of fibrinolysin and PA.The main suppression of fibrinolysin Preparation is α 2- antiplasmins (α 2-antiplasmin).Some cell surfaces have the uPA specific cells of direct hydrolysis activity Surface receptor (uPAR)^[4,5]。

Plasminogen (plasminogen, plg) is a single chain glycoprotein, is made up of 791 amino acid, and molecular weight is about It is 92kD^[6,7].Plasminogen is main in liver synthesis, is largely present in extracellular fluid.Content of plasminogen is about 2 μ in blood plasma M.Therefore plasminogen is a huge potential source of its proteolytic activity in tissue and body fluid^[8,9].Plasminogen is deposited In two kinds of molecular forms：Glutamic acid-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys- plasminogen).The plasminogen of natural secretion and uncracked form has amino terminal (N- ends) glutamic acid, because This is referred to as glutamic acid-plasminogen.However, in the presence of fibrinolysin, glutamic acid-plasminogen water at Lys76-Lys77 Solution turns into lysine-plasminogen.Compared with glutamic acid-plasminogen, lysine-plasminogen has higher with fibrin Affinity, it is possible to speed higher is activated by PA.The Arg560-Val561 peptide bonds of the plasminogen of both forms can quilt UPA or tPA cuts, and causes the formation of the dichain proteins enzyme fibrinolysin of disulfide bond^[10].The amino terminus portion of plasminogen Comprising five homologous three rings, i.e., so-called kringle, carboxy-terminal sections include protease domain.Some kringle contain The lysine-binding site that mediation plasminogen interacts with fibrin and its inhibitor α 2-AP specificity.Latest find One is the plasminogen fragment of 38kD, is effective inhibitor of angiogenesis including kringle1-4.This Fragment is named as angiostatin, can be produced by several protease hydrolytic plasminogens.

The main substrate of fibrinolysin is fibrin, and fibrinous dissolving is the pass for preventing pathologic thrombus to be formed Key^[11].Fibrinolysin also has the substrate specificity to the several components of ECM, including laminin, fibronectin, proteoglycans And gelatin, show that fibrinolysin also plays an important role in ECM reconstructions^[7,12,13].Indirectly, fibrinolysin can also by by certain A little protease precursors are converted into active protease come the other components of the ECM that degrades, including MMP-1, MMP-2, MMP-3 and MMP-9. It is thus proposed that, fibrinolysin is probably an important upstream regulator of extracellular proteolysis^[14].Additionally, fibrinolysin Ability with the growth factor for activating some potential forms^[15-17].In vitro, fibrinolysin can also hydrolyze the component of complement system And discharge chemotactic complement fragment.

At present, the method for the treatment of diabetic cardiomyopathy complication is mainly control diabetes and controls to the ill cardiopathic Treat etc..

Our research is surprised to find that plasminogen has obvious reparation to make to the damage to cardiac tissue that diabetes are caused With, and the dissolving of microthrombus can be promoted, repair internal organs, such as damage of kidney, liver, retinal tissue caused by diabetes Wound, recovers the inducing function of injured nerve, is diabetic complication, and such as diabetic cardiomyopathy opens one and brand-new controls Treatment approach.

Detailed description of the invention

The present invention relates to one kind prevention and/or the treatment cardiopathic method of sub-ject's property, including administration subject The plasminogen or fibrinolysin of effective dose.The invention further relates to be organized caused by one kind prevention and/or treatment sub-ject The method of microthrombus, prevention and/or the method for tissue cell insult caused by treatment sub-ject, including administration subject Plasminogen or fibrinolysin.In one embodiment, tissue microthrombus includes heart tissue, liver caused by the diabetes Tissue, nerve fiber, renal tissue and retinal tissue microthrombus；Tissue cell insult includes heart caused by the diabetes Tissue, liver organization, nerve fiber, renal tissue and retinal tissue are damaged.

In one embodiment, the diabetic cardiomyopathy include cardiomegaly, cardiac insufficiency, arrhythmia cordis, Coronary heart diseases and angina pectoris, painless myocardial infarction, heart failure.In the above-described embodiment, the subject is mammal, It is preferred that being people.

In one embodiment, subject's fibrinolysin or plasminogen are low.Specifically, it is described low Under be inborn, secondary and/or part.

In one embodiment, plasminogen and sequence 2,6,8,10 or 12 have at least 80%, 85%, 90%th, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is plasminogen activity. In one embodiment, plasminogen is addition, deletion and/or substitution 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- 4th, 1-3,1-2,1 amino acid, and still there is the protein of plasminogen activity.In one embodiment, Plasminogen is comprising activities of endothelial tissue plasminogen fragment and still with the protein of plasminogen activity.At one In embodiment, plasminogen is selected from Glu- plasminogens, Lys- plasminogens, small fibrinolysin Former, fibrillin lyase original, δ-plasminogen or its any combination.In one embodiment, plasminogen is selected from such as Under conservative substitution variant：Glu- plasminogens, Lys- plasminogens, small plasminogen, δ-fibre Lyase is former or fibrillin lyase is former.In one embodiment, plasminogen behaviour natural fiber plasmin Original, such as plasminogen shown in sequence 2 it is straight to homologue, for example, the fiber from primate or rodent Plasmin is former straight to homologue, such as the plasminogen from gorilla, rhesus macaque, mouse, ox, horse, dog is straight to same It is thing.Most preferably, the amino acid sequence of plasminogen of the invention is as shown in sequence 2,6,8,10 or 12.

In one embodiment, the plasminogen is treated by administered either systemically or locally, preferably by with Lower approach is applied：Intravenous, intramuscular, subcutaneous, suction give plasminogen.

In one embodiment, the plasminogen is applied with appropriate peptide carrier or combination of stabilizers.At one In embodiment, the plasminogen with daily 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 (being calculated with per kilogram of body weight) or 0.0001- 2000mg/cm²、0.001-800mg/cm²、0.01-600mg/cm²、0.1-400mg/cm²、1-200mg/cm²、1-100mg/ cm²、10-100mg/cm²The dosage of (being calculated with body surface area every square centimeter) is applied, and is preferably at least repeated once, preferably at least Apply daily.In the case of local application, above-mentioned dosage according to circumstances can also be adjusted further.

Stating plasminogen can be administered alone, it is also possible to be used in combination with other medicines, the other medicines include but not It is limited to：Cardiovascular disease resistant medicine, antidiabetic medicine, antithrombotic reagent, anti-infectives, antiarrhythmic drug, reducing blood lipid Medicine etc..

Preventing and/or treating the cardiopathic medicine of sub-ject's property the invention further relates to plasminogen or fibrinolysin In purposes.It is used to preventing and/or treat the invention further relates to plasminogen or fibrinolysin and is organized caused by sub-ject The purposes of microthrombus, prevention and/or the purposes of tissue cell insult caused by treatment sub-ject, including administration subject Plasminogen or fibrinolysin.In one embodiment, tissue microthrombus includes heart tissue, liver caused by the diabetes Tissue, nerve fiber, renal tissue and retinal tissue microthrombus；Tissue cell insult includes heart caused by the diabetes Tissue, liver organization, nerve fiber, renal tissue and retinal tissue are damaged.In one embodiment, the diabetic keratopathy Heart disease includes diabetic cardiomyopathy, including cardiomegaly, cardiac insufficiency, arrhythmia cordis, coronary heart diseases and angina pectoris, painless Property myocardial infarction, heart failure.

In the above-mentioned technical solutions, the subject is mammal, is preferably people.

In one embodiment, subject's fibrinolysin or plasminogen are low.Specifically, it is described low Under be inborn, secondary and/or part.

In one embodiment, the plasminogen is preferably applied by administered either systemically or locally by following approach： Intravenous, intramuscular, subcutaneous, suction give fibrinolysin and were treated originally.In one embodiment, the plasminogen leads to Cross it is administered either systemically or locally treated, preferably applied by following approach：Intravenous, intramuscular, subcutaneous, suction give fibrinolytic Proenzyme.

Above-mentioned plasminogen can be administered alone, it is also possible to be used in combination with other medicines, the other medicines include but It is not limited to：Cardiovascular disease resistant medicine, antidiabetic medicine, antithrombotic reagent, anti-infectives, antiarrhythmic drug, drop blood Fat medicine etc..

In one embodiment, subject's fibrinolysin or plasminogen are low.Specifically, it is described low Under be inborn, secondary and/or part.

The invention further relates to preventing and/or treating the cardiopathic plasminogen of sub-ject's property or fibrinolysin, and Sub-ject's property is cardiopathic, pharmaceutical composition comprising plasminogen or fibrinolysin for prevention and/or treatment.The present invention is also It is related to organize the plasminogen or fibrinolysin of microthrombus caused by sub-ject or comprising the fibre for preventing and/or treat The pharmaceutical composition of lyase original or fibrinolysin；The fibrinolytic of tissue cell insult caused by prevention and/or treatment sub-ject Proenzyme or fibrinolysin or the pharmaceutical composition comprising the plasminogen or fibrinolysin.In one embodiment, the diabetes Caused tissue microthrombus includes heart tissue, liver organization, nerve fiber, renal tissue and retinal tissue microthrombus；Institute Tissue cell insult includes heart tissue, liver organization, nerve fiber, renal tissue and retinal tissue caused by stating diabetes Damage.In one embodiment, the diabetic cardiomyopathy includes diabetic cardiomyopathy, including cardiomegaly, heart work( Can not entirely, arrhythmia cordis, coronary heart diseases and angina pectoris, painless myocardial infarction, heart failure.

In one embodiment, plasminogen and sequence 2,6,8,10 or 12 have at least 80%, 85%, 90%th, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is plasminogen activity. In one embodiment, plasminogen is addition, deletion and/or substitution 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- 4th, 1-3,1-2,1 amino acid, and still there is the protein of plasminogen activity.In one embodiment, Plasminogen is comprising activities of endothelial tissue plasminogen fragment and still with the protein of plasminogen activity.At one In embodiment, plasminogen is selected from Glu- plasminogens, Lys- plasminogens, small fibrinolysin Former, fibrillin lyase original, δ-plasminogen or its any combination.In one embodiment, plasminogen is selected from such as Under conservative substitution variant：Glu- plasminogens, Lys- plasminogens, small plasminogen, δ-fibre Lyase is former or fibrillin lyase is former.In one embodiment, plasminogen behaviour natural fiber plasmin Original, such as plasminogen shown in sequence 2 it is straight to homologue, for example, the fiber from primate or rodent Plasmin is former straight to homologue, such as, from gorilla, rhesus macaque, mouse, ox, horse, the plasminogen of dog are straight to same It is thing.Most preferably, the amino acid sequence of plasminogen of the invention is as shown in sequence 2,6,8,10 or 12.

In one embodiment, the plasminogen is preferably applied by administered either systemically or locally by following approach： Intravenous, intramuscular, subcutaneous, suction give fibrinolysin and were treated originally.

In one embodiment, the plasminogen is applied with appropriate peptide carrier or combination of stabilizers.At one In embodiment, the plasminogen with daily 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 (being calculated with per kilogram of body weight) or 0.0001- 2000mg/cm²、0.001-800mg/cm²、0.01-600mg/cm²、0.1-400mg/cm²、1-200mg/cm²、1-100mg/ cm²、10-100mg/cm²The dosage of (being calculated with body surface area every square centimeter) is applied, and is preferably at least repeated once, preferably at least Apply daily.In the case of local application, above-mentioned dosage according to circumstances can also be adjusted further.

In one embodiment, subject's fibrinolysin or plasminogen are low.Specifically, it is described low Under be inborn, secondary and/or part.

The invention further relates to prevent and/or treat sub-ject's property it is cardiopathic, comprising plasminogen or fibrinolysin Product or medicine box.The invention further relates to be used to preventing and/or treat organize caused by sub-ject microthrombus, include The product or medicine box of plasminogen or fibrinolysin；Tissue cell insult, bag caused by prevention and/or treatment sub-ject Product or medicine box containing plasminogen or fibrinolysin.In one embodiment, tissue microthrombus bag caused by the diabetes Include heart tissue, liver organization, nerve fiber, renal tissue and retinal tissue microthrombus；Tissue caused by the diabetes Cellular damage includes that heart tissue, liver organization, nerve fiber, renal tissue and retinal tissue are damaged.In an embodiment party In case, the diabetic cardiomyopathy includes diabetic cardiomyopathy, including cardiomegaly, cardiac insufficiency, arrhythmia cordis, hat Worry, angina pectoris, painless myocardial infarction, heart failure.In one embodiment, the product or medicine box are included and are divided in Plasminogen/the fibrinolysin of the effective dose in container.It is preferred that, the medicine box also includes one or more other medicine, packing In other containers of the medicine box.The medicine box can also include operation instructions, illustrate that the plasminogen can be used for treating institute State diabetic cardiomyopathy, and can further illustrate, the plasminogen can other medicines administration before, meanwhile, And/or apply afterwards.In one embodiment, the other medicines can be included but is not limited to：It is cardiovascular disease resistant medicine, anti- Diabetes medicament, antithrombotic reagent, anti-infectives, antiarrhythmic drug, blood lipid-lowering medicine etc..In an embodiment In, plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% with sequence 2,6,8,10 or 12 Or 99% sequence identity, and still have plasminogen activity.In one embodiment, plasmin Proenzyme be on the basis of sequence 2,6,8,10 or 12, addition, delete and/or substitution 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 So with the protein of plasminogen activity.In one embodiment, plasminogen is comprising activities of endothelial tissue plasminogen Fragment and still have plasminogen activity protein.In one embodiment, plasminogen is selected from Glu- Plasminogen, Lys- plasminogens, small plasminogen, fibrillin lyase are former, δ-fibrinolysin Former or its any combination.In one embodiment, plasminogen is selected from following conservative substitution variant：Glu- fibrins Lyase original, Lys- plasminogens, small plasminogen, δ-plasminogen or fibrillin lyase are former.One In individual embodiment, plasminogen behaviour natural fiber plasmin is former, and for example the plasminogen shown in sequence 2 is straight To homologue, for example, the plasminogen from primate or rodent is straight to homologue, for example, come arrogant Orangutan, rhesus macaque, mouse, ox, horse, the plasminogen of dog is straight to homologue.Most preferably, fibrinolysin of the invention Former amino acid sequence is as shown in sequence 2,6,8,10 or 12.

In one embodiment, the plasminogen is preferably applied by administered either systemically or locally by following approach： Intravenous, intramuscular, subcutaneous, suction give fibrinolysin and were treated originally.

In one embodiment, the plasminogen is applied with appropriate peptide carrier or combination of stabilizers.At one In embodiment, the plasminogen with daily 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 (being calculated with per kilogram of body weight) or 0.0001- 2000mg/cm²、0.001-800mg/cm²、0.01-600mg/cm²、0.1-400mg/cm²、1-200mg/cm²、1-100mg/ cm²、10-100mg/cm²The dosage of (being calculated with body surface area every square centimeter) is applied, and is preferably at least repeated once, preferably at least Apply daily.In the case of local application, above-mentioned dosage according to circumstances can also be adjusted further.

In one embodiment, subject's fibrinolysin or plasminogen are low.Specifically, it is described low Under be inborn, secondary and/or part.

On the one hand, the diabetic keratopathy of prevention and/or treatment subject is being prepared the present invention relates to plasminogen or fibrinolysin The purposes that tissue and internal organs are damaged in medicine, product, the medicine box of (infringement).In one embodiment, it is described tissue and Internal organs damage (infringement) including to heart, liver, kidney, nerve, retinal tissue organ damage (infringement).One side Face, medicine, product, the medicine box of the diabetic complication for preventing and/or treating subject are being prepared the present invention relates to plasminogen In purposes.In one embodiment, the diabetic complication is diabetic keratopathy cerebral diseased, the glycosuria that diabetes trigger Characteristic of disease heart change, diabetic keratopathy hepatic disease, diabetic nephropathy change, diabetic keratopathy lung lesion, diabetic nerve Lesion, BDR.

On the one hand, the present invention relates to a kind of pharmaceutical methods, including by plasminogen or fibrinolysin and pharmaceutically acceptable load Body is prepared into diabetic keratopathy body tissue for preventing and/or treating subject and internal organs damage (infringement) medicine, Product, medicine box.In one embodiment, it is described tissue and internal organs damage (infringement) including to heart, liver, kidney, Nerve, the damage (infringement) of retinal tissue organ.On the one hand, the present invention relates to a kind of pharmaceutical methods, including by plasminogen Or fibrinolysin and pharmaceutical acceptable carrier be prepared into the medicine of the diabetic complication of prevention and/or treatment subject, product or Medicine box.In one embodiment, the diabetic complication is diabetic keratopathy cerebral diseased, the diabetic keratopathy that diabetes trigger Heart change, diabetic keratopathy hepatic disease, diabetic nephropathy change, diabetic neuropathy, diabetic retinopathy Become.

On the one hand, damaged the present invention relates to be used to prevent and/or treat the diabetic keratopathy tissue and internal organs of subject The plasminogen or fibrinolysin of (infringement), the pharmaceutical composition comprising plasminogen or fibrinolysin, product, medicine box.In a reality Apply in scheme, the tissue and internal organs damage (infringement) and include to heart, liver, kidney, nerve, retinal tissue organ Damage (infringement).On the one hand, the fibrinolysin of the diabetic complication the present invention relates to be used to preventing and/or treating subject Original, the pharmaceutical composition comprising plasminogen, product or medicine box.In one embodiment, the diabetic complication is sugar Diabetic keratopathy cerebral diseased, diabetic cardiomyopathy change, diabetic keratopathy hepatic disease, diabetic keratopathy lung lesion that urine disease triggers Diabetic nephropathy change, diabetic neuropathy, BDR.

On the one hand, damaged the present invention relates to a kind of diabetic keratopathy tissue and internal organs for preventing and/or treating subject The method of (infringement), including administration subject's plasminogen or fibrinolysin, the drug regimen comprising plasminogen or fibrinolysin Thing, product, medicine box.The invention further relates to plasminogen or fibrinolysin, the pharmaceutical composition comprising plasminogen or fibrinolysin, system Product, medicine box are used to prevent and/or treat the purposes of the diabetic keratopathy body tissue and internal organs damage (infringement) of subject. In one embodiment, the tissue and internal organs damage (infringement) and include to heart, liver, kidney, nerve, retina group Knit the damage (infringement) of organ.On the one hand, the present invention relates to a kind of side of the diabetic complication for preventing and/or treating subject Method, including administration subject's plasminogen or fibrinolysin, the pharmaceutical composition comprising plasminogen or fibrinolysin, product or medicine Box.Used present invention additionally comprises plasminogen or fibrinolysin, the pharmaceutical composition comprising plasminogen or fibrinolysin, product or medicine box In prevention and/or the purposes of the diabetic complication for the treatment of subject.In one embodiment, the diabetic complication is Diabetic keratopathy cerebral diseased, diabetic cardiomyopathy change, diabetic keratopathy hepatic disease, diabetic keratopathy lungs disease that diabetes trigger Change, diabetic nephropathy change, diabetic neuropathy, BDR.

In one embodiment, subject's fibrinolysin or plasminogen are low.Specifically, it is described low Under be inborn, secondary and/or part.

In one embodiment, plasminogen and sequence 2,6,8,10,12 have at least 80%, 85%, 90%th, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is plasminogen activity. In one embodiment, plasminogen is addition, deletion and/or substitution 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- 4th, 1-3,1-2,1 amino acid, and still there is the protein of plasminogen activity.In one embodiment, Plasminogen is comprising activities of endothelial tissue plasminogen fragment and still with the protein of plasminogen activity.At one In embodiment, plasminogen is selected from Glu- plasminogens, Lys- plasminogens, small fibrinolysin Former, fibrillin lyase original, δ-plasminogen or its any combination.In one embodiment, plasminogen is selected from such as Under conservative substitution variant：Glu- plasminogens, Lys- plasminogens, small plasminogen, δ-fibre Lyase is former or fibrillin lyase is former.In one embodiment, plasminogen behaviour natural fiber plasmin Original, such as plasminogen shown in sequence 2 it is straight to homologue, for example, the fiber from primate or rodent Plasmin is former straight to homologue, such as, from gorilla, rhesus macaque, mouse, ox, horse, the plasminogen of dog are straight to same It is thing.Most preferably, the amino acid sequence of plasminogen of the invention is as shown in sequence 2,6,8,10 or 12.

In one embodiment, the plasminogen passes through administered either systemically or locally, preferably by following way Apply in footpath：Surface, intravenous, intramuscular, subcutaneous, suction, intraspinal tube, local injection, intra-articular injection or by rectum.At one In embodiment, the local administration by contain the dressing and/or conduit of plasminogen in thrombus area application come Carry out.

In one embodiment, the plasminogen is applied with appropriate peptide carrier or combination of stabilizers.At one In embodiment, the plasminogen with daily 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 (being calculated with per kilogram of body weight) or 0.0001- 2000mg/cm²、0.001-800mg/cm²、0.01-600mg/cm²、0.1-400mg/cm²、1-200mg/cm²、1-100mg/ cm²、10-100mg/cm²The dosage of (being calculated with body surface area every square centimeter) is applied, and is preferably at least repeated once, preferably at least Apply daily.In the case of local application, above-mentioned dosage according to circumstances can also be adjusted further.

Above-mentioned plasminogen can be administered alone, it is also possible to be used in combination with other medicines, the other medicines, For example, treating cardiovascular disease medicine, treating irregular heart pulse medicine, Remedies for diabetes etc., with treatment and pathologic thrombus There are associated Other diseases.

In one embodiment, subject's fibrinolysin or plasminogen are low.Specifically, it is described low Under be inborn, secondary and/or part.

The present invention clearly covers all combinations for belonging to the technical characteristic between embodiment of the present invention, and these groups Technical scheme after conjunction is clearly disclosed in this application, just as above-mentioned technical proposal is individually and clearly disclosing. In addition, the present invention also clearly covers all sub-combinations of each embodiment and its key element, and it is disclosed herein, just as every One such sub-combination is independent and clearly disclosed herein the same.

Detailed description of the invention

" diabetes " are by inherent cause, immunologic function disorder, microorganism infection and its toxin, free radical toxin, spirit The various virulence factors of factor etc. act on the sugar that body causes hypoinsulinism, insulin resistance etc. and trigger, protein, A series of metabolic disorder syndromes such as fat, water and electrolyte, clinically with hyperglycaemia as main feature.

" diabetic complication " is by bad caused other organ or tissues of body of glycemic control in diabetes mellitus Infringement or dysfunction, including liver, kidney, heart, retina, the infringement of nervous system or dysfunction etc..According to generation Boundary's health organization statistics, diabetic complication is up to kind more than 100, is to be currently known a kind of most disease of complication.

" diabetic microangiopathies " refer to the different degrees of exception of each organ or tissue's microcirculation of diabetic's body Caused microangiopathies.The process that microangiopathy is deformed into is substantially：Microcirculation function changes；Vascular damaged, such as it is interior Skin damaged is hindered, basal membrane thickening；Blood viscosity increases, erythrocyte aggregation, platelet adhesion reaction and aggregation, finally result in microvascular corrosion cast and/ Or microvascular occlusion, thrombosis, cell hypoxia, form blood clot, thrombus and inflammation, and further the tissue on influence periphery and Organ dysfunction, and then cause diabetic complication.

" diabetic cardiomyopathy " refers to the histology of the cardiovascular system caused by diabetes and the lesion of changes of function, its Be one of most common diabetic complication, wherein, patient clinical can behave as electrocardiographic abnormality, Heart enlargement, arrhythmia cordis, Angina pectoris, painless myocardial infarction, heart failure.According to statistics, the diabetic of about 70%-80% finally dies from heart simultaneously Hair disease.The incidence of diabetic's atherosclerosis, hypertension, acute myocardial infarction AMI, chronic heart failure and sudden death It is significantly raised.At present, diabetes have been the pathogenetic high risk factor of heart and prediction index, and diabetes merge cardiopathic disease People is in short term or long-term prognosis are significantly worse than the patient of non-diabetic.

" fibrinolysin " is a kind of very important enzyme being present in blood, fibrin clot can be hydrolyzed into fiber egg White catabolite and DDi.

" plasminogen " is the zymogen forms of fibrinolysin, according to the sequence in swiss prot, by the day containing signal peptide Right people source plasminogen amino acid sequence (sequence 4) calculates and is made up of 810 amino acid, and molecular weight is about 92kD, mainly in liver Dirty middle synthesis and the glycoprotein that can circulate in blood, encode the cDNA sequence of the amino acid sequence as shown in sequence 3.Total length Plasminogen include seven domains：The Pan Apple (PAp) of serine protease domain, N-terminal positioned at C-terminal Domain and 5 Kringle domains (Kringle1-5).With reference to the sequence in swiss prot, its signal peptide includes residual Base Met1-Gly19, PAp include that residue Glu20-Val98, Kringle1 include residue Cys103-Cys181, Kringle2 bag Including residue Glu184-Cys262, Kringle3 includes that residue Cys275-Cys352, Kringle4 include residue Cys377- Cys454, Kringle5 include residue Cys481-Cys560.According to NCBI data, serine protease domain includes residue Val581-Arg804。

Glu- plasminogens are the plasminogens of Native full-length, are made up of 791 amino acid and (do not contain 19 amino acid Signal peptide), the cDNA sequence of the sequence is encoded as shown in sequence 1, its amino acid sequence is as shown in sequence 2.In vivo, also exist A kind of is that the Lys- plasminogens so as to be formed are hydrolyzed from the 76-77 amino acids of Glu- plasminogens, such as the institute of sequence 6 Show, encode the cDNA sequence of the amino acid sequence as shown in sequence 5.δ-plasminogen (δ-plasminogen) is total length fibrinolytic Proenzyme has lacked the fragment of Kringle2-Kringle5 structures, only contains Kringle1 and serine protease domain^[18,19], have The document report amino acid sequence (sequence 8) of δ-plasminogen^[19], encode the cDNA sequence such as sequence 7 of the amino acid sequence. Miniplasminogen (Mini-plasminogen) is made up of Kringle5 and serine protease domain, have document report it include it is residual Base Val443-Asn791 (being initial amino acid with the Glu residues for not containing the Glu- plasminogen sequences of signal peptide)^[20], its Amino acid sequence encodes the cDNA sequence of the amino acid sequence as shown in sequence 9 as shown in sequence 10.And Microplasminogen (Micro-plasminogen) only contain serine protease domain, there is its amino acid sequence of document report to include residue Ala543-Asn791 (being initial amino acid with the Glu residues for not containing the Glu- plasminogen sequences of signal peptide)^[21], also have Patent document CN102154253A reports that its sequence includes residue Lys531-Asn791 (not contain the Glu- fibrinolytics of signal peptide The Glu residues of proenzyme sequence are initial amino acid), this patent sequence reference patent document CN102154253A, its amino acid sequence Row encode the cDNA sequence of the amino acid sequence as shown in sequence 11 as shown in sequence 12.

" thrombus " is the product of formation in coagulation process.Coagulation process is that body keeps closing high-pressure recycle system integrality Defense mechanism.Under normal circumstances, the process should keep its non-activated state, but when tissue sustains damage, it is necessary to open immediately The mechanism is moved to reduce blood extravasation.When vascular injury, the fibrinogen in being dissolved in blood plasma in the presence of fibrin ferment (fibrinogen) will finally be changed into water insoluble fibrin (fibrin) polymer, and be interlaced with one another into net, by blood Including cell is enlisted the services of, blood clot is formed, complete coagulation process.In this process, the size of blood clot and injury is to closing weight Will.Therefore initial blood clot is formed molecule (fibrin, fibrin ferment) and molecule (fibrinolysin, the fibrinolysin of dissolved blood clot Activator etc.) between should exist balance.But in pathologic process, the destruction of the balance will cause excessive blood clot formation point Son, and then thrombus (thrombus) is formed, the thrombus is " pathologic thrombus ".

In human body, thrombus can occur in any position with blood flow, and two major classes are mainly classified as at present：Venous blood Bolt and arterial thrombus.Phlebothrombosis is caused by the blood clot produced in vein.Most common phlebothrombosis type is：Deep venou Thrombus (DVT), it generally influences limbs vein such as femoral vein, causes the pain of affected area and redness；Portal Vein Thrombosis, It can influence vena portae hepatica, and then cause pancreatitis, cirrhosis, diverticulitis or cholangiocarcinoma；Renal vein thrombosis, cause renal embolism； Jugular vein thrombus, it can cause the multiple complications such as systemic sepsis, pulmonary embolism；Cerebral venous thrombosis, cause patient to present The symptoms such as headache, paropsia apoplexy.Arterial thrombus may then cause the infarct of substantially any organ in vivo, its disease for triggering Disease includes but is not limited to：It is cerebral infarction, myocardial infarction, embolic stroke, atherosclerosis disease, unstable angina, stupid Solidity angina pectoris, transient ischemic attack, pulmonary embolism etc..

Under diabetic condition, arterial wall endothelial cell damage, hemodynamic responses make blood mechanically rush for a long time Blood vessel endothelium is hit, causes endothelial injuries, and then cause blood platelet, fibrin etc. to form thrombus in damage location adhesion and aggregation. Present invention experiment finds that plasminogen can make the DDi in diabetic mice serum significantly raised, heart, liver, kidney The local fibrin of dirty, nerve fiber is significantly reduced relative to control group, illustrates that plasminogen can promote diabetic mice The dissolving of microthrombus.The present invention covers treatment of the plasminogen to diabetes microthrombus.

" fibrinolysin " of the invention is used interchangeably with " fibrinolysin ", " fibrinoclase ", and implication is identical； " plasminogen " is used interchangeably with " plasminogen ", " plasminogen ", and implication is identical.

It will be understood by those skilled in the art that all technical schemes of plasminogen of the present invention are applied to fibrinolysin, therefore, The technical scheme of present invention description covers plasminogen and fibrinolysin.

Thrombus of the present invention includes fresh thrombus and outmoded thrombus." fresh thrombus " of the invention and " acute thrombus " Can be with used interchangeably；" outmoded thrombus " and " Chronic Thrombotic " can be with used interchangeablies.

In cyclic process, plasminogen uses the nonactive conformation of closing, but when thrombus or cell surface is bound to, Under the mediation of plasminogen activator (plasminogen activator, PA), it is changed into the activity in open conformation Fibrinolysin.Fibrin clot further can be hydrolyzed to fibrin degradation product (FDP) and D- dimerization by active fibrinolysin Body, and then thrombus.Wherein the PAp domains of plasminogen include the weight for maintaining plasminogen to be in nonactive closing conformation Determinant is wanted, and KR domains can then be combined with the lysine residue being present on acceptor and substrate.It is known various to make It is the enzyme of plasminogen activator, including：Tissue plasminogen activator (tPA), uPA (uPA), Kallikrein and Hageman factor (Hageman factor (HF)) etc..

" activities of endothelial tissue plasminogen fragment " refers to that in plasminogen protein, can be combined and played with the target sequence in substrate The active fragment of proteolysis function.Technical scheme the present invention relates to plasminogen is covered and uses activities of endothelial tissue plasminogen fragment Instead of the technical scheme of plasminogen.Activities of endothelial tissue plasminogen fragment of the present invention is the serine stretch protein comprising plasminogen The protein in enzyme domain, it is preferable that activities of endothelial tissue plasminogen fragment of the present invention has at least comprising sequence 14 and sequence 14 80%th, the protein of the amino acid sequence of 90%, 95%, 96%, 97%, 98%, 99% homology.Therefore, it is of the present invention Plasminogen include containing the activities of endothelial tissue plasminogen fragment and remain in that the albumen of the activities of endothelial tissue plasminogen.

At present, include for plasminogen in blood and its activity determination method：To tissue plasminogen Activator activity detection (t-PAA), the detection (t-PAAg) of plasma tissue PLA antigen, to blood Starch detection (plgA), the detection (plgAg) of plasma tissue plasminogen antigen, the plasma tissue fiber of tissue plasminogen activity The detection of Plasminogen Activators Inhibitor Activity, the inspection of plasma tissue Plasminogen activator inhibitor antigen Survey, plasma fibrin lyase-antiplasmin complex detects (PAP).The detection method of most common of which is color development Substrate method：Add streptokinase (SK) and chromophoric substrate to by inspection blood plasma, by the PLG in inspection blood plasma in the presence of SK, be transformed into PLM, the latter acts on chromophoric substrate, then with spectrophotometric determination, absorbance increase with plasminogen activity into Direct ratio.In addition also can be using immuno-chemical method, gel electrophoresis, immunoturbidimetry, radioimmunodiffusion etc. to the fibre in blood The molten zymogen activity of fibrillarin is measured.

" ortholog thing or lineal homologue (ortholog) " refers to the homologue between different plant species, both same including albumen Source thing also includes DNA homology thing, also referred to as straight homologues, Paralog thing.It is referred specifically in different plant species by same ancestors Gene evolution and come albumen or gene.Plasminogen of the invention includes the natural plasminogen of people, also including from not Plasminogen ortholog thing infraspecific, with activities of endothelial tissue plasminogen or lineal homologue.

" conservative substitution variant " refer to one of them given amino acid residue change but do not change protein or enzyme it is whole Body conformation and function, this is included but is not limited to the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor parent of similar characteristic (such as acid, alkalescence, hydrophobicity, etc.) Amino acid in Amino Acids in Proteins sequence.Amino acid with similarity is well-known.For example, arginine, group Propylhomoserin and lysine are hydrophilic basic amino acids and can exchange.Equally, isoleucine is hydrophobic amino acid, then can be bright Propylhomoserin, methionine or valine are replaced.Therefore, two albumen of identity function or the similitude of amino acid sequence may not Together.For example, based on MEGALIGN algorithms 70% to 99% similarity (homogeneity)." conservative substitution variant " also includes passing through BLAST or fasta algorithm determine the polypeptide or enzyme of the amino acid identities with more than 60%, if can be more preferable up to more than 75%, Preferably up to more than 85%, or even it is optimal up to more than 90%, and has compared with natural or parent protein or enzyme identical Or similar property or function substantially.

" separation " plasminogen refers to the plasminogen protein for separating and/or reclaiming from its natural surroundings.In some realities Apply in scheme, the plasminogen can purify (1) extremely more than the 90%, purity (by weight) more than 95% or more than 98%, As by determined by Lowry methods, such as, more than 99% (by weight), (2) are to being enough to by using rotating cup sequence analysis Instrument obtains at least 15 degree of residue of N-terminal or internal amino acid sequence, or (3), to homogeney, the homogeney is by making With the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Coomassie blue or silver staining under reproducibility or non-reducing conditions (SDS-PAGE) determine.The plasminogen of separation also includes being prepared from recombinant cell by biotechnology, and by extremely The plasminogen that a few purification step is separate.

Term " polypeptide ", " peptide " and " protein " is used interchangeably herein, and refers to the polymerization of the amino acid of any length It is form, its amino acid that can include genetic coding and non-genetic coding, chemistry or biochemical modification or derivatization Amino acid, and the polypeptide with modified peptide backbone.The term includes fusion protein, including but not limited to heterologous amino The fusion protein of acid sequence, with heterologous and homologous leader sequences (with and without N-terminal methionine residues) fusions；Deng Deng.

It is defined as introducing breach when necessary on " amino acid sequence identity percentage (%) " with reference to polypeptide sequence After realizing largest percentage sequence identity, and when any conservative replacement not being considered as into a part for sequence identity, candidate With the percentage with reference to the amino acid residue identical amino acid residue in polypeptide sequence in sequence.To determine percent amino acid The contrast of sequence identity purpose can be realized with the various ways in the range of art technology, such as using publicly available Computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can be certainly Surely it is used for the suitable parameter of aligned sequences, including any algorithm that maximum contrast needs is realized to institute's comparative sequences total length.However, For the purposes of the present invention, amino acid sequence identity percent value is to compare computer program ALIGN-2 using sequence to produce 's.

In the case of using ALIGN-2 come comparing amino acid sequence, amino acid sequence A is given relative to given amino acid The % amino acid sequence identities of sequence B (or can be expressed as having or comprising relative to or for given amino acid sequence Arrange the given amino acid sequence A of a certain % amino acid sequence identities of B) it is calculated as below：

Fraction X/Y multiplies 100

Wherein X is that scoring is the amino of identical match in the A and B of the program are compared by alignment programs ALIGN-2 The number of sour residue, and wherein Y is the sum of the amino acid residue in B.It will be appreciated that in the length and ammonia of amino acid sequence A In the case of the length of base acid sequence B is unequal, % amino acid sequence identities of the A relative to B can be not equal to B relative to A % amino acid sequence identities.Unless expressly stated otherwise, all % amino acid sequence identities values used herein are all It is, according to described in the preceding paragraph, to be obtained using ALIGN-2 computer programs.

As used in this article, term " treatment " and " treatment " refers to the desired pharmacology of acquisition and/or physiologic effect.The effect Fruit can be prevention disease or its symptom wholly or in part, and/or partially or completely cure diseases and/or its symptom, and wrap Include：A () prevention disease occurs in subject, the subject can have the procatarxis of disease, but not yet be diagnosed as tool There is disease；B () suppresses disease, that is, block its formation；(c) mitigates disease and/or its symptom, that is, cause disease and/or its disease Shape disappears.

Term " individuality ", " subject " and " patient " is used interchangeably herein, and refers to mammal, including but not limited to Mouse (rat, mouse), non-human primates, people, dog, cat, ungulate (such as horse, ox, sheep, pig, goat) etc..

" therapeutically effective amount " or " effective dose " refers to and is enough to when applying mammal or other subjects to treat disease Realize the amount to the prevention of disease and/or the plasminogen for the treatment of." therapeutically effective amount " can be according to the fibrinolysin for being used The former, disease of subject to be treated and/or the order of severity of its symptom and age, body weight etc. and change.

The preparation of plasminogen of the present invention

Plasminogen can be separated and purified for further treatment purposes from nature, it is also possible to by the change of standard Peptide symthesis technology is learned to synthesize.When by chemically synthesized polypeptide, can be synthesized through liquid phase or solid phase.Solid phase peptide synthssis (SPPS) (the C-terminal amino acid of sequence is wherein attached into insoluble holder, then remaining amino in sequential addition sequence Acid) it is the method for being adapted to plasminogen chemical synthesis.Various forms of SPPS, such as Fmoc and Boc can be used to synthesize fibrinolysin It is former.Technology for synthesis in solid state is described in Barany and Solid-Phase Peptide Synthesis；The 3-284 pages in The Peptides:Analysis, Synthesis, Biology. volume 2：Special Methods in Peptide Synthesis, Part A., Merrifield, wait J.Am.Chem.Soc., 85:2149-2156(1963)；Stewart etc., Solid Phase Peptide Synthesis,2nd ed.Pierce Chem.Co.,Rockford,Ill.(1984)；With Ganesan A.2006Mini Rev.Med Chem.6:The 2005Protein such as 3-10 and Camarero JA Pept Lett.12:In 723-8.In short, processing small insoluble porous bead with the functional element for being built with peptide chain thereon.In idol After connection/de-protected repetitive cycling, the free N-terminal amine of solid phase that will be attached is coupled with the single Amino Acid Unit protected by N.So Afterwards, this element is deprotected, exposes the new N-terminal amine that can be attached with other amino acid.Peptide is remained fixed in solid phase, it Cut away afterwards.

Plasminogen of the invention can be produced using Standard recombinant methods.For example, by the nucleic acid of encoding plasminogen In insertion expression vector, it is set to be operatively connected with the regulating and controlling sequence in expression vector.Expression regulation sequence is included but is not limited to Promoter (such as naturally associated or heterologous promoter), signal sequence, enhancer element and transcription terminator.Expression Regulation and control can be eukaryotic promoter system in carrier, the carrier can convert or transfect eukaryotic host cell (such as COS or Chinese hamster ovary celI).Once during carrier mixed into suitable host, it is being suitable for the high level expression and plasminogen of nucleotide sequence Collection and purifying under conditions of maintain host.

Suitable expression vector is generally in host organisms as episome or the integration portion as host chromosome DNA Divide and replicate.Generally, expression vector contain selection marker thing (for example amicillin resistance, hygromycin resistance, tetracyclin resistance, Kalamycin resistance or neomycin resistance) contributing to those cells converted with desired DNA sequence dna to external source to detect.

Escherichia coli (Escherichia coli) can be used for cloning the protokaryon place of theme antibody coding polynucleotides The example of chief cell.Other microbial hosts being adapted for use with include bacillus, such as bacillus subtilis (Bacillus Subtilis) and other enterobacteriaceaes (Enterobacteriaceae), such as Salmonella (Salmonella), sand Lei Shi Pseudomonas (Serratia) and various pseudomonas (Pseudomonas) species.In these prokaryotic hosts, it is also possible to generate Expression vector, it would generally contain the expression control sequenc (such as replication orgin) compatible with host cell.In addition, can exist being permitted Many known promoters, such as lactose promoter system, tryptophan (trp) promoter systems, beta-lactamase promoter systems, Or the promoter systems from phageλ.Promoter would generally control table reach, optionally in the case of operator sequence, and And with ribosome bind site sequence etc., to start and complete transcription and translation.

Other microorganisms, such as yeast can also be used for expression.Yeast (such as saccharomyces cerevisiae (S.cerevisiae)) and finish Red yeast (Pichia) is the example of suitable yeast host cell, wherein suitable carrier has expression control sequence as needed Row (such as promoter), replication orgin, terminator sequence etc..Typical promoter includes glycerol 3-phosphate acid kinase and other sugars Solution enzyme.Induction type yeast starts in particularly including from alcohol dehydrogenase, different cell pigment C and responsible maltose and galactolipin profit The promoter of enzyme.

Outside microorganism, mammalian cell (mammalian cell for example cultivated in cell culture in vitro) also may be used For expressing and generate plasminogen of the invention (polynucleotides of the anti-Tau antibody of such as encoding schemes).Referring to Winnacker,From Genes to Clones,VCH Publishers,N.Y.,N.Y.(1987).Suitable mammal Host cell includes Chinese hamster ovary celI system, various Cos cell lines, HeLa cells, myeloma cell line, and inverted B cell or miscellaneous Hand over knurl.Expression vector for these cells can include expression control sequenc, such as replication orgin, promoter and enhancer (Queen etc., Immunol.Rev.89:49 (1986)), and required machining information site, such as ribosome bind site, RNA splice sites, polyadenylation site, and transcription terminator sequences.The example of suitable expression control sequenc is white exempting from The derivative promoter such as epidemic disease globulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus.Referring to Co etc., J.Immunol.148:1149(1992)。

Once synthesis (chemistry or recombination form), can be according to the standard schedule of this area including ammonium sulfate precipitation, affine Post, column chromatography, high performance liquid chroma- tography (HPLC), gel electrophoresis etc. purify plasminogen of the present invention.The plasminogen It is substantially pure, for example, at least about 80% to 85% is pure, at least about 85% to 90% is pure, at least about 90% to 95% is pure , or 98% to 99% is pure or purer, such as without pollutant, the pollutant such as cell fragment, except theme antibody with Outer macromolecular, etc..

Pharmaceutical formulation

Can by will have needed for purity plasminogen and optional pharmaceutical carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16 editions, Osol, A.ed. (1980)) be mixed to form lyophilized formulations or The aqueous solution prepares treatment preparaton.Acceptable carrier, excipient, stabilizer are nontoxic to receptor under dosage used and concentration Property, and including buffer such as phosphate, citrate and other organic acids；Antioxidant includes ascorbic acid and methionine； Preservative (such as stearyl dimethyl benzyl ammonium chloride；Hexamethonium chloride；Benzalkonium chloride (benzalkonium Chloride), benzethonium chloride；Phenol, butanol or phenmethylol；Alkyl parabens such as methyl or propyl p-hydroxybenzoate Ester；Catechol；Resorcinol；Cyclohexanol；3- amylalcohols；Metacresol)；Low molecular weight polypeptide (less than about 10 residues)；Albumen Matter such as seralbumin, gelatin or immunoglobulin；Hydrophilic polymer such as polyvinylpyrrolidone；Amino acid such as glycine, paddy Glutamine, asparagine, histidine, arginine or lysine；Monose, disaccharides and other carbohydrate include glucose, sweet Dew sugar or dextrin；Chelating agent such as EDTA；Carbohydrate such as sucrose, mannitol, fucose or sorbierite；Into salt counter ion such as sodium；Metal Compound (such as zinc-albumen composition)；And/or nonionic surfactant, such as TWEENTM, PLURONICSTM or poly- second Glycol (PEG).It is preferred that lyophilized anti-VEGF antibodies preparaton is described in WO 97/04801, it is included in herein as ginseng Examine.

Preparaton of the invention can also contain the specific illness that need to treat needed for more than one reactive compound, preferably Complementary activities and be free from side effects each other those.For example, antihypertensive medicine, antiarrhythmic medicine, controlling Treat medicine of diabetes etc..

Plasminogen of the invention can be wrapped in the microcapsules prepared by such as condensation technique or interfacial polymerization, example Such as, colloidal drug delivery systems (for example, liposome, albumin microsphere, microemulsion, nano particle and Nano capsule) can be embedded in In or the hydroxymethyl cellulose inserted in macro emulsion or gelatin-microcapsule and poly- (methyl methacrylate) microcapsules in. These technologies are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)。

Plasminogen of the invention for vivo medicine-feeding is necessarily aseptic.This can be by freeze-drying and again Realized easily by degerming membrane filtration before or after preparation.

Plasminogen of the invention can prepare sustained release preparation.The appropriate example of sustained release preparation includes having definite shape and contain There are the penetrating matrix of solid hydrophobic polymers half of glycoprotein, such as film or microcapsules.Sustained-release matrix example includes polyester, hydrogel (such as poly- (2- hydroxyethyl-methacrylates) (Langer, J.Biomed.Mater.Res., 15:167-277(1981)； Langer,Chem.Tech.,12:98-105 (1982)) or poly- (vinyl alcohol), polyactide (United States Patent (USP) 3773919, EP 58, 481), the copolymer (Sidman, etc. Biopolymers 22 of Pidolidone and γ ethyl-L-glutamates:547 (1983)), no Degradable ethylene vinyl acetate (ethylene-vinyl acetate) (Langer, etc. ibid), or it is degradable Poly lactic coglycolic acid such as Lupron DepotTM (by poly lactic coglycolic acid and leucyl proline (leuprolide) microsphere of the injectable of acetic acid esters composition), and poly- D- (-) -3- hydroxybutyric acids.Polymer such as ethene-second Vinyl acetate and lactic-co-glycolic acid energy sustained release molecule more than 100 days, and the time of some hydrogels release proteins compared with It is short.Can be designed according to Related Mechanism makes protein stabilized rational strategy.For example, if it find that the mechanism of cohesion is by sulphur Intermolecular S -- S is formed for Disulfide interchange, then can by modify sulfhydryl residue, from acid solution freeze, control humidity, Stabilization is realized using suitable additive and the specific polymer matrix composition of exploitation.

Administration and dosage

Can by different modes, for example by intravenous, intraperitoneal, subcutaneous, encephalic, intrathecal, intra-arterial (for example via Arteria carotis), intramuscular, intranasal, surface or intradermal administration or spinal cord or brain deliver to realize the administration of pharmaceutical composition of the present invention.Gas Aqueous or other solution and preservative and isotonic agent of purifying of the sol preparation such as nose spray preparation comprising activating agent.By such system Agent is adjusted to the pH and isotonic state compatible with schneiderian membrane.

Prepared product for parenteral administration includes sterile aqueous or non-aqueous solution, suspension and emulsion.It is non-aqueous molten The example of agent is propane diols, polyethylene glycol, vegetable oil such as olive oil, and injectable organic ester, such as ethyl oleate.Aqueous carrier bag Include water, alcohol/aqueous solution, emulsion or suspension, including salt solution and buffer medium.Parenteral medium is molten comprising sodium chloride Liquid, woods grignard dextrose, dextrose and sodium chloride or fixing oil.Intravenous vehicles include liquid and nutritional supplement, electrolysis Matter supplement, etc..Can also there are preservative and other additives, such as, antimicrobial, antioxidant, chelating Agent and inert gas, etc..

Medical worker can determine dosage based on various clinical factors.It is such as known in medical domain, any patient's Dosage depend on many factors, including patient build, body surface area, age, the particular compound to be applied, sex, administration Number of times and path, general health and the other medicines being administered simultaneously.The dosage of pharmaceutical composition of the present invention comprising plasminogen Scope can be, for example, for example daily about 0.0001 to 2000mg/kg, or about 0.001 to 500mg/kg (such as 0.02mg/kg, 0.25mg/kg, 0.5mg/kg, 0.75mg/kg, 10mg/kg, 50mg/kg etc.) subject's body weight.For example, dosage can be 1mg/kg body weight or 50mg/kg body weight or the scope in 1-50mg/kg, or at least 1mg/kg.Higher or lower than this exemplary model Including the dosage for enclosing is also covered by, above-mentioned factor is especially considering that.Middle dosage in above range is also contained in the present invention In the range of.Subject can apply this daily, every other day, weekly or according to any other schedule determined by empirical analysis Class dosage.Exemplary dosage schedule includes continuous several days 1-10mg/kg.Reality is needed during medicament administration of the invention When assessment, the therapeutic effect and security of periodical evaluation diabetic cardiomyopathy and its associated conditions.

Treatment effect and treatment safetyProperty

After one embodiment of the invention is directed to use with plasminogen treatment subject, to treatment effect and treatment safety The judgement of property.Blood pressure to subject, electrocardiogram, blood wherein are included but is not limited to the determination methods of the treatment effect normal Rule, routine urinalysis, blood fat, blood sugar, haemodynamics are measured.Specifically, including to subject following inspection is implemented：1) heart Vascular Ultrasonography, comprehensively to diagnose, detect each chamber size of heart, ventricular wall motion, VPV and cardiac function aspect；2) heart Flesh marker detection, such as c reactive protein (CRP), myoglobins (Mb), CK-MB (CK-MB), BNP (BNP), isoconcentration is detected that these marks are the important diagnostic mark of acute myocardial infarction AMI, it is contemplated that subject is receiving Above-mentioned detection recovers to range of normal value or is improved after plasminogen treatment of the invention, and such as Mb is extensive in male subject Again to 19~92 μ g/L, women is 12~76 μ g/L；3) dynamic ecg monitoring.Moreover, it relates to use fibrinolysin Original is carried out in therapeutic process and after treatment to subject, and various bad things are monitored in the judgement to the therapeutic scheme security Part.

Product or medicine box

One embodiment of the invention is related to a kind of product or medicine box, and it includes and can be used to treat diabetic cardiomyopathy And its plasminogen of the present invention or fibrinolysin of associated conditions.The product preferably includes a container, label or package insert. Appropriate container has bottle, bottle, syringe etc..Container can be made up of various materials such as glass or plastics.The container contains Composition, the composition can effectively treat disease of the invention or illness and have sterile access port (such as described container can be Intravenous solution bag or bottle, it contains the stopper that can be penetrated by hypodermic needle).At least one of composition activity Agent is plasminogen/fibrinolysin.On the container or appended label illustrates that the composition is used to treat sugar of the present invention Urine characteristic of disease heart disease and its associated conditions.The product can further include the second container containing pharmaceutically acceptable buffer solution, such as The salt solution of phosphate-buffered, Ringer's mixture and glucose solution.It can further include and comes from business and user's angle See required other materials, including other buffer solutions, diluent, filtrate, pin and syringe.Additionally, the product includes band There is the package insert of operation instruction, including for example indicate the user of the composition by plasminogen composition and treat companion With disease other medicines administered patient.

Brief description of the drawings

The db/db mouse of Fig. 1 display 24-25 week old changes of weight after continuous 31 days administration plasminogens.To fibrinolysin Former group and to solvent PBS control group in the 0th, 4,7,11,16,21,26,31 days body weight without significant difference.

The db/db mouse of Fig. 2 display 24-25 week old heart HE coloration results after continuous 31 days administration plasminogens.

The cardiac fibers proteinogen dyeing after continuous 31 days administration plasminogens of the db/db mouse of Fig. 3 display 24-25 week old As a result.

The db/db mouse of Fig. 4 display 24-25 week old were in continuous 31 days administration plasminogen aorta posterior bow HE dyeing knots Really.

Fig. 5 display 24-25 weeks diabetes later stage mouse give PBS or plasminogen 31 days serum cardiac troponin concentration Measurement result.

DDi contains the db/db mouse of Fig. 6 display 24-25 week old in serum after the continuous 15 days administration plasminogens Amount testing result.

The retina PAS dyeing observations after continuous 31 days administration plasminogens of the db/db mouse of Fig. 7 display 24-25 week old As a result.

The db/db mouse of Fig. 8 24-25 week old kidney fibrin immunostaining after continuous 31 days administration plasminogens Observation result.

The kidney Bcl2 immunostainings observation after continuous 31 days administration plasminogens of the db/db mouse of Fig. 9 24-25 week old As a result.

The hepatic fibrosis protein immunization dyeing after continuous 31 days administration plasminogens of the db/db mouse of Figure 10 24-25 week old Observation result.

The liver F4/80 immunostainings observation after continuous 31 days administration plasminogens of the db/db mouse of Figure 11 24-25 week old As a result.

The db/db mouse of Figure 12 display 24-25 week old the 0th, 4,7,11,16 days detection machinery after plasminogen is administered is touched Induce the result of pain sensing capability.The pain threshold of plasminogen group 50% is found when detecting within the 16th day and to solvent PBS control Group is compared and occurs in that pole significant difference.

The db/db mouse of Figure 13 display 24-25 week old the 0th, 4,7,11,16 days detection cold stimulations after plasminogen is administered The result of sensing capability.

15 days os hypogastroidale nerve fiber proteins of Figure 14 24-25 week diabetes later stage neurotrosis mouse plasminogen are immunized Histochemical staining observes result.

Figure 15 24-25 week diabetic mices give plasminogen 31 days kidney IgM immunostainings observation results.

Figure 16 24-25 week diabetic mices give plasminogen serum glutamic pyruvic transminase (ALT) testing result after 31 days.

Embodiment

Influence of the plasminogen of embodiment 1 to advanced diabetes Mouse Weight

24-25 week old db/db hero mouse 20, are randomly divided into two groups, to solvent PBS control group and to plasminogen group each 10 Only.Experiment starts the same day and is designated as packet of weighing in the 0th day, and experiment starts to plasminogen or PBS and is designated as the 1st day, company for second day Continuous administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS control is given Group gives the PBS of same volume.In the 0th, 4,7,11,16,21,26,31 natural gift another name body weight.

Result shows, to plasminogen group and to solvent PBS control group in the 0th, 4,7,11,16,21,26,31 days body weight Without significant difference (Fig. 1).Illustrate that plasminogen is little on the weight of animals influence, administration treatment does not have significant shadow to the weight of animals Ring.

The plasminogen of embodiment 2 is acted on the injury repair of advanced diabetes mouse cardiac muscle

24-25 week old db/db hero mouse 10, are randomly divided into two groups, to solvent PBS control group and to plasminogen group each 5 Only.Experiment starts the same day and is designated as packet of weighing in the 0th day, and experiment starts to plasminogen or PBS and is designated as the 1st day, company for second day Continuous administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS control is given Group gives the PBS of same volume.Mouse was put to death at the 32nd day and is cored and dirty fix 24 in 10% neutral formalin fixer Hour.Heart after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Histotomy thickness is 5 μm, Section dewaxing rehydration and with haematoxylin and eosin stains (HE dyeing), 1% hydrochloride alcohol breaks up, and ammoniacal liquor returns indigo plant, and alcohol gradient Dehydration mounting, section is observed under 400 times under the microscope.

Result shows, gives solvent PBS control group cardiac myocyte hypertrophy, the loose nucleus of even visible spindle shape, slight fat Fat is denatured, in empty balloon-shaped, in the visible slight cell infiltration of vessel boundary or myocyte gap, and the broadening (figure in muscle fibre gap 2A)；To plasminogen group cardiac muscle cell circle or fusiformis, loose cell is few compared with control group, and muscle fibre gap causes compared with control group Close, cell infiltration and steatosis mitigate (Fig. 2 B) with to solvent PBS control group than obvious.Illustrate that injection plasminogen makes The damage of diabetic mice cardiac muscle is significantly repaired.

The plasminogen of embodiment 3 promotes the hydrolysis of advanced diabetes mouse heart fibrin

24-25 week old db/db hero mouse 10, are randomly divided into two groups, to solvent PBS control group and to plasminogen group each 5 Only.Experiment starts the same day and is designated as packet of weighing in the 0th day, and experiment starts to plasminogen or PBS and is designated as the 1st day, company for second day Continuous administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS control is given Group gives the PBS of same volume.Mouse was put to death at the 32nd day and is cored and dirty fix 24 in 10% neutral formalin fixer Hour.Heart tissue after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Histotomy thickness is 5 μm, washed 1 time after section dewaxing rehydration, it is incubated 15 minutes with 3% hydrogen peroxide, wash 2 times, every time 5 minutes.10% normal sheep Serum solution (Vectorlaboratories, Inc., USA) is closed 1 hour；Reject sheep blood serum liquid, group is irised out with PAP afterwards Knit.Rabbit anti-mouse fibrin (original) antibody (Abcam) 4 DEG C of overnight incubations, TBS is washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and TBS is washed 2 times, every time 5 minutes.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water is rinsed 5 minutes.Serial dehydration is transparent And mounting, section observed under 400 times under the microscope.

Fibrinogen is fibrinous precursor, is deposited in the case of a fracture in tissue, as body to the one of damage Plant stress reaction, fibrinogen hydrolysis fibroblast cells^[22-24], therefore can be using fibrin level as the one of degree of injury Individual mark.Fibrin is also thrombosed main component after tissue damage, therefore, also can be using fibrin level as blood One mark of bolt.

Result shows, compared with to solvent PBS control group (Fig. 3 A), to the mouse heart tissue of plasminogen group (Fig. 3 B) The positive coloring of fibrin is shallower, illustrates to be reduced to the fibrin of plasminogen group heart tissue deposition, reflects plasminogen Damage to cardiac tissue reparation caused by diabetes can be promoted, also illustrate that plasminogen can promote the dissolving of heart tissue thrombus.

The repair that the plasminogen of embodiment 4 is damaged to advanced diabetes rat aorta wall

24-25 week old db/db hero mouse 10, are randomly divided into two groups, to solvent PBS control group and to plasminogen group each 5 Only.Experiment starts the same day and is designated as packet of weighing in the 0th day, and experiment starts to plasminogen or PBS and is designated as the 1st day, company for second day Continuous administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS control is given Group gives the PBS of same volume.Mouse being put to death at the 32nd day and taking the arch of aorta consolidate in 10% neutral formalin fixer It is fixed 24 hours.Heart after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Histotomy thickness is 5 μm, section dewaxing rehydration simultaneously uses haematoxylin and eosin stains (HE dyeing), and 1% hydrochloride alcohol breaks up, and ammoniacal liquor returns indigo plant, and alcohol is terraced Degree dehydration mounting, section is observed under 400 times under the microscope.

Result shows have foam cells to deposit to solvent PBS control group vascular wall, middle elastic film arrangement disorder, blood Tube wall thickening, tube wall convex-concave inequality (Fig. 4 A)；Give plasminogen group middle elastic membrane structure rule, undulate, vascular wall Thickness is uniform (Fig. 4 B).Show that injection plasminogen has repair to the sustainer vessel wall damage caused by diabetes.

The plasminogen of embodiment 5 significantly mitigates the damage of advanced diabetes cardiac muscle

24-25 week old db/db hero mouse 28, are randomly divided into two groups, to solvent PBS control group 12, give plasminogen group 16.Experiment starts the same day and is designated as packet of weighing in the 0th day, tests and starts within second day to plasminogen or PBS and be designated as the 1st day, Successive administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, to solvent PBS pairs The PBS of same volume is given according to group.Extract eyeball within 32nd day and take blood, be centrifuged 15-20 minutes with 3500r/min, and take supernatant, examine Survey the concentration of serum Myocardial Troponin I.

Cardiac muscle troponin I (cardiac troponin, CTNI) is the important symbol thing of myocardial damage, its serum-concentration It is capable of the degree of reflecting myocardium damage^[25].Result shows, to the concentration of plasminogen group cardiac muscle troponin I be substantially less than to Solvent PBS control group, and with extremely notable significant difference (Fig. 5).Illustrate that plasminogen can significantly mitigate advanced diabetes The damage of cardiac muscle.

The dissolving of the microthrombus that the diabetes that the fibrinolysin raw sugar of embodiment 6 promote are caused

24-25 week old db/db hero mouse 10, are randomly divided into two groups, to solvent PBS control group and to plasminogen group each 5 Only.Experiment starts the same day and is designated as packet of weighing in the 0th day, and experiment starts to plasminogen or PBS and is designated as the 1st day, company for second day Continuous administration 15 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS control is given Group gives the PBS of same volume.Last time administration is plucked eyeball and takes blood after 24 hours, serum is used to detect blood after whole blood stands Middle DDi (D-dimer) content.

Result shows that after 15 days, the content of DDi significantly rises (Fig. 6) to administration plasminogen in serum, illustrates fibre Lyase proper energy remarkably promotes the microthrombus dissolving that diabetes are caused.

The plasminogen of embodiment 7 promotes the reparation that diabetic mice retina blood capillary is damaged

24-25 week old db/db hero mouse 20, are randomly divided into two groups, to solvent PBS control group and to plasminogen group each 10 Only.Experiment starts the same day and is designated as packet of weighing in the 0th day, and experiment starts to plasminogen or PBS and is designated as the 1st day, company for second day Continuous administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS control is given Group gives the PBS of same volume.Mouse was put to death at the 32nd day and left side eyeball is taken 24 hours are fixed in paraformaldehyde fixer. After eyeball after fixation separates retina, it is placed in the EP pipes of the pancreatin of 1mL 3% (Solarbio), 37 DEG C of concussions in shaking table Digestion 2-3h.Careful move into retina is equipped with the EP pipes of distilled water after the phenomenon for softening, coming off occurs in retina, 37 DEG C of concussion 2-3h, make tissue loss unnecessary on retina in shaking table.Soft piping and druming retina, makes its surplus vascular lamina The tile on slide, natural air drying afterwards.Retina dyes (PAS dyeing) in SchiffShi liquid, the differentiation of 1% hydrochloride alcohol, ammoniacal liquor Indigo plant, and the transparent rear mounting of alcohol serial dehydration dimethylbenzene are returned, is observed under 400 times under the microscope.

From experimental result as can be seen that compared with plasminogen group (Fig. 7 B), giving solvent PBS control group (Fig. 7 A) db/db Mouse capillary caliber thickness differs, and vascular wall thickens deep dye, and vascular endothelial cell (Δ) hyperplasia, pericyte (↓) substantially subtracts It is few；Quantitative analysis finds that acellular vascular length (Fig. 7 C) is substantially reduced compared with to solvent PBS control group to plasminogen group, And results of statistical analysis shows significant difference.Illustrate that plasminogen can remarkably promote diabetes later stage Mouse Retina hair The reparation of thin injury of blood vessel, so as to promote the reparation that diabetic retina is damaged.

The plasminogen of embodiment 8 reduces diabetes later stage mouse kidney fibrin deposition

24-25 week old db/db hero mouse 20, are randomly divided into two groups, to solvent PBS control group and to plasminogen group each 10 Only.Experiment starts the same day and is designated as packet of weighing in the 0th day, and experiment starts to plasminogen or PBS and is designated as the 1st day, company for second day Continuous administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS control is given Group gives the PBS of same volume.Mouse was put to death at the 32nd day and kidney is taken and fixes 24 in 10% neutral formalin fixer Hour.Renal tissue after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Histotomy thickness is 5 μm, washed 1 time after section dewaxing rehydration.It is incubated 15 minutes with 3% hydrogen peroxide, is washed 2 times, every time 5 minutes.10% normal sheep Serum solution (Vector laboratories, Inc., USA) is closed 1 hour；After time arrives, reject sheep blood serum liquid, with PAP circle Go out tissue.Rabbit anti-mouse fibrin (original) antibody (Abcam) 4 DEG C of overnight incubations, TBS is washed 2 times, every time 5 minutes.Goat antirabbit IgG (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and TBS is washed 2 times, every time 5 minutes.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water is rinsed 5 minutes.Serial dehydration is transparent And mounting, section observed under 200 times under the microscope.

Fibrinogen is fibrinous precursor, is deposited in the case of a fracture in tissue, as body to the one of damage Plant stress reaction, fibrinogen hydrolysis fibroblast cells^[22-24], therefore can be using fibrin level as the one of degree of injury Individual mark.Fibrin is also thrombosed main component after tissue damage, therefore, also can be using fibrin level as blood One mark of bolt.

Result shows, to plasminogen group (Fig. 8 B) than giving solvent PBS control group (Fig. 8 A) Antigen positive hybridomas coloring of fibrin It is shallow.Illustrate that injection plasminogen can significantly reduce diabetic mice kidney fibrous proteinosis, reflect plasminogen to sugar The sick mouse kidney of urine is damaged significant repair, also illustrates that plasminogen can promote the dissolving of renal tissue thrombus.

The plasminogen of embodiment 9 promotes diabetic mice kidney IAP Bcl-2 expression

24-25 week old db/db hero mouse 20, are randomly divided into two groups, to solvent PBS control group and to plasminogen group each 10 Only.Experiment starts the same day and is designated as packet of weighing in the 0th day, and experiment starts to plasminogen or PBS and is designated as the 1st day, company for second day Continuous administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS control is given Group gives the PBS of same volume.Mouse was put to death at the 32nd day and kidney is taken and fixes 24 in 10% neutral formalin fixer Hour.Renal tissue after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Histotomy thickness is 5 μm, washed 1 time after section dewaxing rehydration.It is incubated 15 minutes with 3% hydrogen peroxide, is washed 2 times, every time 5 minutes.10% normal sheep Serum solution (Vector laboratories, Inc., USA) is closed 1 hour；After time arrives, reject sheep blood serum liquid, with PAP circle Go out tissue.4 DEG C of overnight incubations of rabbit anti-mouse Bcl2 antibody (Abcam), TBS is washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) Antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and TBS is washed 2 times, every time 5 minutes.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water is rinsed 5 minutes.Serial dehydration is transparent And mounting, section observed under 200 times under the microscope.

Bcl-2 is iap protein, and expression can be lowered under the effect of the apoptotic stimulus factor^[26,27].Bcl-2 is immunized Groupization result shows, to be substantially deeper than to the coloring of plasminogen group (Fig. 9 B) renal cells positive expression and give solvent PBS Control group (Fig. 9 A), and the former color range it is wider.Quantitative analysis results are consistent with observation result, and with significant difference (Fig. 9 C).This shows that plasminogen can promote the expression of diabetic mice kidney cell Apoptosis inhibitor molecule Bcl-2, so as to suppress The apoptosis of diabetic mice renal tissue cell.

The plasminogen of embodiment 10 reduces advanced diabetes hepatic tissue fibrin level

24-25 week old db/db hero mouse 10, are randomly divided into two groups, to solvent PBS control group and to plasminogen group each 5 Only.Experiment starts the same day and is designated as packet of weighing in the 0th day, and experiment starts to plasminogen or PBS and is designated as the 1st day, company for second day Continuous administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS control is given Group gives the PBS of same volume.Mouse being put to death at the 32nd day and taking liver organization consolidate in 10% neutral formalin fixer It is fixed 24 hours.Liver organization after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Histotomy is thick It is 5 μm to spend, and is washed 1 time after section dewaxing rehydration.It is incubated 15 minutes with 3% hydrogen peroxide, is washed 2 times, every time 5 minutes.10% Normal sheep serum liquid (Vector laboratories, Inc., USA) is closed 1 hour；After time arrives, reject sheep blood serum liquid is used PAP is irised out tissue.Rabbit anti-mouse fibrin (original) antibody (Abcam) 4 DEG C of overnight incubations, TBS is washed 2 times, every time 5 minutes. Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and TBS is washed 2 times, every time 5 minutes.By DAB kits (Vector laboratories, Inc., USA) is developed the color, and haematoxylin is redyed 30 seconds after washing 3 times, and flowing water is rinsed 5 minutes.Gradient Transparent and mounting is dehydrated, section is observed under 200 times under the microscope.

Fibrinogen is fibrinous precursor, is deposited in the case of a fracture in tissue, as body to the one of damage Plant stress reaction, fibrinogen hydrolysis fibroblast cells^[22-24], therefore can be using fibrin level as the one of degree of injury Individual mark.Fibrin is also thrombosed main component after tissue damage, therefore, also can be using fibrin level as blood One mark of bolt.

Research discovery, compared with to solvent PBS control group (Figure 10 A), gives the mouse of plasminogen group (Figure 10 B) its liver The positive coloring of tissue fibrin is shallow, illustrates that injection plasminogen can significantly reduce diabetic mice hepatic fibrosis albumen and sink Product, reflects that plasminogen has notable repair to diabetic mice hepar damnification, also illustrates that plasminogen can promote liver The dissolving of dirty tissue thrombus.

The plasminogen of embodiment 11 promotes the inflammation reparation of advanced diabetes liver organization

24-25 week old db/db hero mouse 10, are randomly divided into two groups, to solvent PBS control group and to plasminogen group each 5 Only.Experiment starts the same day and is designated as packet of weighing in the 0th day, and experiment starts to plasminogen or PBS and is designated as the 1st day, company for second day Continuous administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS control is given Group gives the PBS of same volume.To putting to death mouse after plasminogen 31 days and to take liver organization solid in 10% neutral formalin Determine to fix 24 hours in liquid.Liver organization after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Group Slice thickness is knitted for 5 μm, is washed 1 time after section dewaxing rehydration.It is incubated 15 minutes with 3% hydrogen peroxide, is washed 2 times, every time 5 points Clock.10% normal sheep serum (Vector laboratories, Inc., USA) is closed 1 hour, and the time gets rid of serum deprivation after, is used PAP is irised out tissue.For 4 DEG C of overnight incubations of rabbit polyclonal antibody (Abcam) of F4/80, TBS is washed 2 times, every time 5 minutes.Mountain Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and TBS is washed 2 times.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water is rinsed 5 minutes.Serial dehydration is transparent And mounting, section observed under 200 times under the microscope.

F4/80 is macrophage marker.Macrophage is responsible for removing body as the main phagocyte of inflammation phase Necrotic Debris and pathogen of injury region tissue and cell etc., therefore, the amount of local macrophage can represent inflammatory reaction Degree and the stage.Experimental result shows, to plasminogen group (Figure 11 B) compared with to solvent PBS control group (Figure 11 A), gives The F4/80 Positive Levels of plasminogen group mouse are substantially reduced, and illustrate that to plasminogen liver organization inflammation can be mitigated.Figure 11C is F4/80 SABC positive expression number quantitative analysis results, is substantially reduced to plasminogen group F4/80 expression quantity, and tool There is significant difference, illustrate that injecting plasminogen can remarkably promote the reparation of diabetic mice inflammation.

The plasminogen of embodiment 12 promotes diabetes later stage neurotrosis mouse to repair mechanical allodynia sensing capability It is multiple

24-25 week old db/db hero mouse 10, are randomly divided into two groups, to solvent PBS control group and to plasminogen group each 5 Only.Experiment starts the same day and is designated as weighing for the 0th day and is grouped and starts to do Physiological Experiment, experiment start within second day to plasminogen or PBS is simultaneously designated as the 1st day, successive administration 15 days.2mg/0.2mL/ pcs/day of tail vein injection fibrinolysin is pressed to plasminogen group mouse Original, the PBS of same volume is given to solvent PBS control group.After to plasminogen the 0th, 4,7,11,16 day it is fine with Von-Frey Sensitivity of dimension silk (Stoelting, USA) the detection animal to mechanical injuries.With 2.0g power as initial force, first detect that it is left Pin.If stimulating to have 2 times and thering is paw withdrawal to be the positive for 5 times, if positive stimulated its right crus of diaphragm again with the power of small one-level； If negative, then its right crus of diaphragm is stimulated with the power of big one-level, such left and right pin alternately stimulates, stimulus intervals is 5 minutes, always Costimulation 6 times, then according to S.R.Chaplan et al. (1994)^[28]The method of introduction calculates the threshold value of its 50% paw withdrawal.

Research finds that compared with to solvent PBS control group, the diabetic mice mechanical allodynia to plasminogen group is anti- Answering homogeneity increases, and finds to occur in that pole significant difference (Figure 12) compared with to solvent PBS control group when detecting within the 16th day, Illustrate that plasminogen can repair diabetes later stage neurotrosis mouse to mechanical allodynia (mechanical Allodynia sensing capability).

The reparation that the plasminogen of embodiment 13 senses to diabetes later stage neurotrosis mouse cold stimulation

24-25 week old db/db hero mouse 10, are randomly divided into two groups, to solvent PBS control group and to plasminogen group each 5 Only.Experiment starts the same day and is designated as weighing for the 0th day and is grouped and starts to do Physiological Experiment, experiment start within second day to plasminogen or PBS is simultaneously designated as the 1st day, successive administration 15 days.2mg/0.2mL/ pcs/day of tail vein injection fibrinolysin is pressed to plasminogen group mouse Original, the PBS of same volume is given to solvent PBS control group.Needle applicator extrusion is spent within the 0th, 4,7,11,16 day upon administration One drop acetone solution pearl simultaneously touches db/db mouse vola, makes its whole vola of covering.Since left foot, it was stimulated in turn every 3 minutes Left and right pin, costimulation 10 times, and count paw withdrawal number of times.Percent reaction=paw withdrawal number of times/stimulation number of times meter 100%.

Experimental result shows, at the 0th and the 4th day, acetone is stimulated simultaneously to plasminogen group and to solvent PBS control group Without significant difference, and significant difference is initially observed within the 7th day, pole significant difference, P values were then observed at the 16th day<0.0001 (figure 13) after, illustrating administration 15 days, diabetic mice has almost recovered the reaction to cold stimulation completely, and indicating plasminogen can Repair sensing capability of the diabetes later stage neurotrosis mouse to cold stimulation.

The plasminogen of embodiment 14 reduces diabetes later stage neurotrosis mouse Nerve tissue fibrin level

24-25 week old db/db hero mouse 10, are randomly divided into two groups, to solvent PBS control group and give plasminogen group, often Group is each 5.Experiment starts the same day and is designated as packet of weighing in the 0th day, tests and starts within second day to plasminogen or PBS and be designated as the 1st My god, successive administration 15 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS is given Control group gives the PBS of same volume.Mouse was put to death at the 16th day and sciatic nerve is taken in 10% neutral formalin fixer In fix 24 hours.Sciatic nerve after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out FFPE.Tissue is cut Piece thickness is 5 μm, is washed 1 time after section dewaxing rehydration, then irises out tissue with PAP.Incubated with the hydrogen peroxide that 3%TBS dilutes Educate 15 minutes, wash 3 times.10% normal sheep serum (Vector laboratories, Inc., USA) is closed 1 hour, is siphoned away many Remaining serum.Rabbit anti-mouse fibrin (original) antibody (Abcam) is incubated at room temperature 1 hour or 4 DEG C of overnight incubations, and TBS is washed 3 times.Mountain Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and TBS is washed 3 times.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water is rinsed 5 minutes.Serial dehydration is transparent And mounting, section observed under 400 times under the microscope.

Fibrinogen is fibrinous precursor, is deposited in the case of a fracture in tissue, as body to the one of damage Plant stress reaction, fibrinogen hydrolysis fibroblast cells^[22-24], therefore can be using fibrin level as the one of degree of injury Individual mark.Fibrin is also thrombosed main component after tissue damage, therefore, also can be using fibrin level as blood One mark of bolt.

Research discovery, compared with to solvent PBS control group (Figure 14 A), gives the mouse of plasminogen group (Figure 14 B) its ischium The level reduction of nerve fiber protein, illustrates that plasminogen has the function of fibrin degradation level, and damage obtains certain journey The reparation of degree, also illustrates that plasminogen can promote the dissolving of thrombus around nerve fiber.

The plasminogen of embodiment 15 mitigates the damage of advanced diabetes mouse kidney

24-25 week old db/db hero mouse 8, are randomly divided into two groups, to solvent PBS control group and give plasminogen group, every group Each 4.Experiment starts the same day and is designated as packet of weighing in the 0th day, tests and starts within second day to plasminogen or PBS and be designated as the 1st day, Successive administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, to solvent PBS pairs The PBS of same volume is given according to group.Terminate in the 32nd day detection physical signs, put to death mouse and take kidney in 10% neutrality Fu Er 24 hours are fixed in Malin's fixer.Renal tissue after fixation through alcohol serial dehydration and dimethylbenzene it is transparent after carry out paraffin bag Bury.Histotomy thickness is 5 μm, is washed 1 time after section dewaxing rehydration.It is incubated 15 minutes with 3% hydrogen peroxide, washing 2 times, every time 5 minutes.Mountain sheep anti mouse IgM (HRP) antibody (Abcam) is incubated at room temperature 1 hour, and TBS is washed 2 times, every time 5 minutes.By DAB kits (Vector laboratories, Inc., USA) is developed the color, and haematoxylin is redyed 30 seconds after washing 3 times, and flowing water is rinsed 5 minutes.Gradient Transparent and mounting is dehydrated, section is observed under 400 times under the microscope.

IgM antibody is played an important role during apoptosis and non-viable non-apoptotic cell is removed, the apoptosis and non-viable non-apoptotic cell of cell More, local I gM antibody levels are higher^[29-31].Therefore, local I gM antibody levels can reflect the degree of impairment of histoorgan.

Result shows, is shallower than to the positive coloring of plasminogen group (Figure 15 B) murine glomerular IgM and gives solvent PBS control Group (Figure 15 A), and scope is also small compared with control group, and statistic analysis result is consistent (Figure 15 C) with observation result, and this shows that injection is fine The damage of glomerulus is obviously improved after lyase original, reflect plasminogen have to the body injury of diabetic mice significantly protection and Repair.

The plasminogen of embodiment 16 promotes the reparation of diabetic mice hepar damnification

25-28 week old db/db hero mouse 9, are randomly divided into two groups, to solvent PBS control group 3, to plasminogen group 6 Only.Experiment starts the same day and is designated as packet of weighing in the 0th day, and experiment starts to plasminogen or PBS and is designated as the 1st day, company for second day Continuous administration 31 days.2mg/0.2mL/ pcs/day of tail vein injection plasminogen is pressed to plasminogen group mouse, solvent PBS control is given Group gives the PBS of same volume.Eyeball is plucked after 31 days to plasminogen and adopts whole blood, after 4 DEG C of 3500r/min centrifugations after serum precipitation 10 minutes, take supernatant and detected.Using glutamic-pyruvic transaminase detection kit, (bio-engineering research is built up in Nanjing for this experiment Institute, article No. C009-2), glutamic-pyruvic transaminase (ALT's) contains in Lai Shi colorimetric methods (Reitman-Frankel) detection serum Amount.

Glutamic-pyruvic transaminase is an important indicator of liver health state^[32,33], the normal reference value area of glutamic-pyruvic transaminase Between be 9~50U/L.Testing result shows that the content to solvent PBS control group serum alt is significantly higher than normal physiological index, And then have been restored to internal normal level to plasminogen group, and to plasminogen group ALT content be substantially less than to Solvent PBS control group, and with significant difference (Figure 16).Illustrate in advanced diabetes model mice, inject plasminogen Hepatic injury can substantially be repaired.

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[20]Sottrup-Jensen L,Claeys H,Zajdel M,et al.The primary structure of human plasminogen:Isolation of two lysine-binding fragments and one“mini”- plasminogen(MW,38,000)by elastase-catalyzed-specific limited proteolysis[J] .Progress in chemical fibrinolysis and thrombolysis,1978,3:191-209.

[21]Nagai N,Demarsin E,Van Hoef B,et al.Recombinant human microplasmin:production and potential therapeutic properties[J].Journal of Thrombosis and Haemostasis,2003,1(2):307-313.

[22]Jae Kyu Ryu,Mark A.Petersen,Sara G.Murray et al.Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation.NATURE COMMUNICATIONS,2015,6:8164.

[23]Dimitrios Davalos,Katerina Akassoglou.Fibrinogen as a key regulator of inflammation in disease.Seminars in Immunopathology,2012.34(1): 43-62.

[24]Valvi D,Mannino DM,Mullerova H,et al.Fibrinogen,chronic obstructive pulmonary disease(COPD)and outcomes in two United States cohorts.Int J Chron Obstruct Pulmon Dis 2012；7:173–82.

[25]R.Langhorn and J.L.Willesen.Cardiac Troponins in Dogs and Cats.J Vet Intern Med2016；30:36–50.

[26]Moungjaroen J,Nimmannit U,Callery PS,Wang L,Azad N,Lipipun V, Chanvorachote P,Rojanasakul Y(2006).Reactive oxygen species mediate caspase activation and apoptosis induced by lipoic acid in human lung epithelial cancer cells through Bcl-2 downregulation.J Pharmacol Exp Ther 319,1062–1069.

[27]Wang L,Chanvorachote P,Toledo D,Stehlik C,Mercer RR,Castranova V, Rojanasakul Y(2008).Peroxide is a key mediator of Bcl-2 down-regulation and apoptosis induction by cisplatinin human lung cancer cells.Mol Pharmacol 73, 119–127.

[28]S.R.Chaplan et al.Quantitative assessment of tactile allodynia in the rat paw,Journal of Neuroscience Methods 53(1994)55-63.

[29]Zwart B,Ciurana C,Rensink I,Manoe R,Hack CE,et al.(2004) Complement activation by apoptotic cells occurs predominantly via IgM and is limited to late apoptotic(secondary necrotic)cells.Autoimmunity 37:95–102.

[30]Zhang M,Takahashi K,Alicot EM,Vorup-Jensen T,Kessler B,et al. (2006)Activation of the lectin pathway by natural IgM in a model of ischemia/ reperfusion injury.J Immunol 177:4727–4734.

[31]Kim SJ,Gershov D,Ma X,Brot N,Elkon KB(2002)I-PLA2 Activation during Apoptosis Promotes the Exposure of Membrane Lysophosphatidylcholine Leading to Binding by Natural Immunoglobulin M Antibodies and Complement Activation.The Journal of Experimental Medicine 196:655–665.

[32]Karmen A,Wroblewski F,Ladue JS(Jan 1955).Transaminase activity in human blood.The Journal of Clinical Investigation.34(1):126–31.

[33]Wang CS,Chang TT,Yao WJ,Wang ST,Chou P(Apr 2012).Impact of increasing alanine aminotransferase levels within normal range on incident Diabetes.Journal of the Formosan Medical Association=Taiwan Yi Zhi.111 (4): 201–8.

Sequence table

<110>Shenzhen Co., Ltd of Rui Jian life sciences institute

<120>A kind of new method for preventing and treating diabetic cardiomyopathy

<130> PCK02775

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 2376

<212> DNA

<213>Natural plasminogen (Glu-PLG, Glu- plasminogen) nucleotide sequence of signal peptide is not contained

<400> 1

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggaatgtat gcattgcagt ggagaaaact atgacggcaa aatttccaag 540

accatgtctg gactggaatg ccaggcctgg gactctcaga gcccacacgc tcatggatac 600

attccttcca aatttccaaa caagaacctg aagaagaatt actgtcgtaa ccccgatagg 660

gagctgcggc cttggtgttt caccaccgac cccaacaagc gctgggaact ttgtgacatc 720

ccccgctgca caacacctcc accatcttct ggtcccacct accagtgtct gaagggaaca 780

ggtgaaaact atcgcgggaa tgtggctgtt accgtgtccg ggcacacctg tcagcactgg 840

agtgcacaga cccctcacac acataacagg acaccagaaa acttcccctg caaaaatttg 900

gatgaaaact actgccgcaa tcctgacgga aaaagggccc catggtgcca tacaaccaac 960

agccaagtgc ggtgggagta ctgtaagata ccgtcctgtg actcctcccc agtatccacg 1020

gaacaattgg ctcccacagc accacctgag ctaacccctg tggtccagga ctgctaccat 1080

ggtgatggac agagctaccg aggcacatcc tccaccacca ccacaggaaa gaagtgtcag 1140

tcttggtcat ctatgacacc acaccggcac cagaagaccc cagaaaacta cccaaatgct 1200

ggcctgacaa tgaactactg caggaatcca gatgccgata aaggcccctg gtgttttacc 1260

acagacccca gcgtcaggtg ggagtactgc aacctgaaaa aatgctcagg aacagaagcg 1320

agtgttgtag cacctccgcc tgttgtcctg cttccagatg tagagactcc ttccgaagaa 1380

gactgtatgt ttgggaatgg gaaaggatac cgaggcaaga gggcgaccac tgttactggg 1440

acgccatgcc aggactgggc tgcccaggag ccccatagac acagcatttt cactccagag 1500

acaaatccac gggcgggtct ggaaaaaaat tactgccgta accctgatgg tgatgtaggt 1560

ggtccctggt gctacacgac aaatccaaga aaactttacg actactgtga tgtccctcag 1620

tgtgcggccc cttcatttga ttgtgggaag cctcaagtgg agccgaagaa atgtcctgga 1680

agggttgtag gggggtgtgt ggcccaccca cattcctggc cctggcaagt cagtcttaga 1740

acaaggtttg gaatgcactt ctgtggaggc accttgatat ccccagagtg ggtgttgact 1800

gctgcccact gcttggagaa gtccccaagg ccttcatcct acaaggtcat cctgggtgca 1860

caccaagaag tgaatctcga accgcatgtt caggaaatag aagtgtctag gctgttcttg 1920

gagcccacac gaaaagatat tgccttgcta aagctaagca gtcctgccgt catcactgac 1980

aaagtaatcc cagcttgtct gccatcccca aattatgtgg tcgctgaccg gaccgaatgt 2040

ttcatcactg gctggggaga aacccaaggt acttttggag ctggccttct caaggaagcc 2100

cagctccctg tgattgagaa taaagtgtgc aatcgctatg agtttctgaa tggaagagtc 2160

caatccaccg aactctgtgc tgggcatttg gccggaggca ctgacagttg ccagggtgac 2220

agtggaggtc ctctggtttg cttcgagaag gacaaataca ttttacaagg agtcacttct 2280

tggggtcttg gctgtgcacg ccccaataag cctggtgtct atgttcgtgt ttcaaggttt 2340

gttacttgga ttgagggagt gatgagaaat aattaa 2376

<210> 2

<211> 791

<212> PRT

<213>Natural plasminogen (Glu-PLG, Glu- plasminogen) amino acid sequence of signal peptide is not contained

<400> 2

Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser

1 5 10 15

Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala

20 25 30

Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His

35 40 45

Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser

50 55 60

Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr

65 70 75 80

Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met

85 90 95

Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser

100 105 110

Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu

115 120 125

Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp

130 135 140

Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu

145 150 155 160

Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly

165 170 175

Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser

180 185 190

Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys

195 200 205

Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro

210 215 220

Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile

225 230 235 240

Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys

245 250 255

Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val

260 265 270

Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His

275 280 285

Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr

290 295 300

Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn

305 310 315 320

Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser

325 330 335

Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr

340 345 350

Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly

355 360 365

Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser

370 375 380

Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala

385 390 395 400

Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro

405 410 415

Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu

420 425 430

Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val

435 440 445

Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe

450 455 460

Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly

465 470 475 480

Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile

485 490 495

Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys

500 505 510

Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn

515 520 525

Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro

530 535 540

Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly

545 550 555 560

Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln

565 570 575

Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu

580 585 590

Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser

595 600 605

Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val

610 615 620

Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu

625 630 635 640

Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala

645 650 655

Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr

660 665 670

Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr

675 680 685

Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val

690 695 700

Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val

705 710 715 720

Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser

725 730 735

Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys

740 745 750

Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro

755 760 765

Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile

770 775 780

Glu Gly Val Met Arg Asn Asn

785 790

<210> 3

<211> 2433

<212> DNA

<213>Natural plasminogen containing signal peptide（From swiss prot）Nucleotide sequence

<400> 3

atggaacata aggaagtggt tcttctactt cttttatttc tgaaatcagg tcaaggagag 60

cctctggatg actatgtgaa tacccagggg gcttcactgt tcagtgtcac taagaagcag 120

ctgggagcag gaagtataga agaatgtgca gcaaaatgtg aggaggacga agaattcacc 180

tgcagggcat tccaatatca cagtaaagag caacaatgtg tgataatggc tgaaaacagg 240

aagtcctcca taatcattag gatgagagat gtagttttat ttgaaaagaa agtgtatctc 300

tcagagtgca agactgggaa tggaaagaac tacagaggga cgatgtccaa aacaaaaaat 360

ggcatcacct gtcaaaaatg gagttccact tctccccaca gacctagatt ctcacctgct 420

acacacccct cagagggact ggaggagaac tactgcagga atccagacaa cgatccgcag 480

gggccctggt gctatactac tgatccagaa aagagatatg actactgcga cattcttgag 540

tgtgaagagg aatgtatgca ttgcagtgga gaaaactatg acggcaaaat ttccaagacc 600

atgtctggac tggaatgcca ggcctgggac tctcagagcc cacacgctca tggatacatt 660

ccttccaaat ttccaaacaa gaacctgaag aagaattact gtcgtaaccc cgatagggag 720

ctgcggcctt ggtgtttcac caccgacccc aacaagcgct gggaactttg tgacatcccc 780

cgctgcacaa cacctccacc atcttctggt cccacctacc agtgtctgaa gggaacaggt 840

gaaaactatc gcgggaatgt ggctgttacc gtgtccgggc acacctgtca gcactggagt 900

gcacagaccc ctcacacaca taacaggaca ccagaaaact tcccctgcaa aaatttggat 960

gaaaactact gccgcaatcc tgacggaaaa agggccccat ggtgccatac aaccaacagc 1020

caagtgcggt gggagtactg taagataccg tcctgtgact cctccccagt atccacggaa 1080

caattggctc ccacagcacc acctgagcta acccctgtgg tccaggactg ctaccatggt 1140

gatggacaga gctaccgagg cacatcctcc accaccacca caggaaagaa gtgtcagtct 1200

tggtcatcta tgacaccaca ccggcaccag aagaccccag aaaactaccc aaatgctggc 1260

ctgacaatga actactgcag gaatccagat gccgataaag gcccctggtg ttttaccaca 1320

gaccccagcg tcaggtggga gtactgcaac ctgaaaaaat gctcaggaac agaagcgagt 1380

gttgtagcac ctccgcctgt tgtcctgctt ccagatgtag agactccttc cgaagaagac 1440

tgtatgtttg ggaatgggaa aggataccga ggcaagaggg cgaccactgt tactgggacg 1500

ccatgccagg actgggctgc ccaggagccc catagacaca gcattttcac tccagagaca 1560

aatccacggg cgggtctgga aaaaaattac tgccgtaacc ctgatggtga tgtaggtggt 1620

ccctggtgct acacgacaaa tccaagaaaa ctttacgact actgtgatgt ccctcagtgt 1680

gcggcccctt catttgattg tgggaagcct caagtggagc cgaagaaatg tcctggaagg 1740

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 1800

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 1860

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 1920

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 1980

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 2040

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 2100

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 2160

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 2220

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 2280

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 2340

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 2400

acttggattg agggagtgat gagaaataat taa 2433

<210> 4

<211> 810

<212> PRT

<213>Natural plasminogen containing signal peptide（From swiss prot）Amino acid sequence

<400> 4

Met Glu His Lys Glu Val Val Leu Leu Leu Leu Leu Phe Leu Lys Ser

1 5 10 15

Gly Gln Gly Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser

20 25 30

Leu Phe Ser Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu

35 40 45

Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe

50 55 60

Gln Tyr His Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg

65 70 75 80

Lys Ser Ser Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys

85 90 95

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg

100 105 110

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser

115 120 125

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser

130 135 140

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln

145 150 155 160

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys

165 170 175

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn

180 185 190

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala

195 200 205

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe

210 215 220

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu

225 230 235 240

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu

245 250 255

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr

260 265 270

Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala

275 280 285

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro

290 295 300

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp

305 310 315 320

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His

325 330 335

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys

340 345 350

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro

355 360 365

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser

370 375 380

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser

385 390 395 400

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr

405 410 415

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp

420 425 430

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr

435 440 445

Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro

450 455 460

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp

465 470 475 480

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr

485 490 495

Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg

500 505 510

His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys

515 520 525

Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr

530 535 540

Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

545 550 555 560

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

565 570 575

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

580 585 590

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

595 600 605

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

610 615 620

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

625 630 635 640

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

645 650 655

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

660 665 670

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

675 680 685

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

690 695 700

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

705 710 715 720

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

725 730 735

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

740 745 750

Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu

755 760 765

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

770 775 780

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

785 790 795 800

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

805 810

<210> 5

<211> 2145

<212> DNA

<213> LYS77-PLG（Lys- plasminogens）Nucleotide sequence

<400> 5

aaagtgtatc tctcagagtg caagactggg aatggaaaga actacagagg gacgatgtcc 60

aaaacaaaaa atggcatcac ctgtcaaaaa tggagttcca cttctcccca cagacctaga 120

ttctcacctg ctacacaccc ctcagaggga ctggaggaga actactgcag gaatccagac 180

aacgatccgc aggggccctg gtgctatact actgatccag aaaagagata tgactactgc 240

gacattcttg agtgtgaaga ggaatgtatg cattgcagtg gagaaaacta tgacggcaaa 300

atttccaaga ccatgtctgg actggaatgc caggcctggg actctcagag cccacacgct 360

catggataca ttccttccaa atttccaaac aagaacctga agaagaatta ctgtcgtaac 420

cccgataggg agctgcggcc ttggtgtttc accaccgacc ccaacaagcg ctgggaactt 480

tgtgacatcc cccgctgcac aacacctcca ccatcttctg gtcccaccta ccagtgtctg 540

aagggaacag gtgaaaacta tcgcgggaat gtggctgtta ccgtgtccgg gcacacctgt 600

cagcactgga gtgcacagac ccctcacaca cataacagga caccagaaaa cttcccctgc 660

aaaaatttgg atgaaaacta ctgccgcaat cctgacggaa aaagggcccc atggtgccat 720

acaaccaaca gccaagtgcg gtgggagtac tgtaagatac cgtcctgtga ctcctcccca 780

gtatccacgg aacaattggc tcccacagca ccacctgagc taacccctgt ggtccaggac 840

tgctaccatg gtgatggaca gagctaccga ggcacatcct ccaccaccac cacaggaaag 900

aagtgtcagt cttggtcatc tatgacacca caccggcacc agaagacccc agaaaactac 960

ccaaatgctg gcctgacaat gaactactgc aggaatccag atgccgataa aggcccctgg 1020

tgttttacca cagaccccag cgtcaggtgg gagtactgca acctgaaaaa atgctcagga 1080

acagaagcga gtgttgtagc acctccgcct gttgtcctgc ttccagatgt agagactcct 1140

tccgaagaag actgtatgtt tgggaatggg aaaggatacc gaggcaagag ggcgaccact 1200

gttactggga cgccatgcca ggactgggct gcccaggagc cccatagaca cagcattttc 1260

actccagaga caaatccacg ggcgggtctg gaaaaaaatt actgccgtaa ccctgatggt 1320

gatgtaggtg gtccctggtg ctacacgaca aatccaagaa aactttacga ctactgtgat 1380

gtccctcagt gtgcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 1440

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 1500

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 1560

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 1620

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 1680

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 1740

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 1800

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 1860

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1920

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1980

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 2040

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 2100

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 2145

<210> 6

<211> 714

<212> PRT

<213> LYS77-PLG（Lys- plasminogens）Amino acid sequence

<400> 6

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg

1 5 10 15

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser

20 25 30

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser

35 40 45

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln

50 55 60

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys

65 70 75 80

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn

85 90 95

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala

100 105 110

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe

115 120 125

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu

130 135 140

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu

145 150 155 160

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr

165 170 175

Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala

180 185 190

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro

195 200 205

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp

210 215 220

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His

225 230 235 240

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys

245 250 255

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro

260 265 270

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser

275 280 285

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser

290 295 300

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr

305 310 315 320

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp

325 330 335

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr

340 345 350

Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro

355 360 365

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp

370 375 380

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr

385 390 395 400

Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg

405 410 415

His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys

420 425 430

Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr

435 440 445

Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

450 455 460

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

465 470 475 480

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

485 490 495

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

500 505 510

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

515 520 525

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

530 535 540

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

545 550 555 560

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

565 570 575

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

580 585 590

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

595 600 605

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

610 615 620

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

625 630 635 640

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

645 650 655

Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu

660 665 670

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

675 680 685

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

690 695 700

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

705 710

<210> 7

<211> 1245

<212> DNA

<213> delta-plg（Delta- plasminogens）Nucleotide sequence

<400> 7

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 540

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 600

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 660

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 720

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 780

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 840

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 900

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 960

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1020

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1080

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 1140

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 1200

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 1245

<210> 8

<211> 414

<212> PRT

<213> delta-plg（Delta- plasminogens）Amino acid sequence

<400> 8

Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser

1 5 10 15

Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala

20 25 30

Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His

35 40 45

Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser

50 55 60

Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr

65 70 75 80

Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met

85 90 95

Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser

100 105 110

Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu

115 120 125

Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp

130 135 140

Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu

145 150 155 160

Glu Cys Glu Glu Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val

165 170 175

Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His

180 185 190

Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met

195 200 205

His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala

210 215 220

Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile

225 230 235 240

Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile

245 250 255

Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu

260 265 270

Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala

275 280 285

Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe

290 295 300

Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu

305 310 315 320

Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr

325 330 335

Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His

340 345 350

Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu

355 360 365

Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp

370 375 380

Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val

385 390 395 400

Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

405 410

<210> 9

<211> 1104

<212> DNA

<213> Mini-plg（Small plasminogen）Nucleotide sequence

<400> 9

gtcaggtggg agtactgcaa cctgaaaaaa tgctcaggaa cagaagcgag tgttgtagca 60

cctccgcctg ttgtcctgct tccagatgta gagactcctt ccgaagaaga ctgtatgttt 120

gggaatggga aaggataccg aggcaagagg gcgaccactg ttactgggac gccatgccag 180

gactgggctg cccaggagcc ccatagacac agcattttca ctccagagac aaatccacgg 240

gcgggtctgg aaaaaaatta ctgccgtaac cctgatggtg atgtaggtgg tccctggtgc 300

tacacgacaa atccaagaaa actttacgac tactgtgatg tccctcagtg tgcggcccct 360

tcatttgatt gtgggaagcc tcaagtggag ccgaagaaat gtcctggaag ggttgtaggg 420

gggtgtgtgg cccacccaca ttcctggccc tggcaagtca gtcttagaac aaggtttgga 480

atgcacttct gtggaggcac cttgatatcc ccagagtggg tgttgactgc tgcccactgc 540

ttggagaagt ccccaaggcc ttcatcctac aaggtcatcc tgggtgcaca ccaagaagtg 600

aatctcgaac cgcatgttca ggaaatagaa gtgtctaggc tgttcttgga gcccacacga 660

aaagatattg ccttgctaaa gctaagcagt cctgccgtca tcactgacaa agtaatccca 720

gcttgtctgc catccccaaa ttatgtggtc gctgaccgga ccgaatgttt catcactggc 780

tggggagaaa cccaaggtac ttttggagct ggccttctca aggaagccca gctccctgtg 840

attgagaata aagtgtgcaa tcgctatgag tttctgaatg gaagagtcca atccaccgaa 900

ctctgtgctg ggcatttggc cggaggcact gacagttgcc agggtgacag tggaggtcct 960

ctggtttgct tcgagaagga caaatacatt ttacaaggag tcacttcttg gggtcttggc 1020

tgtgcacgcc ccaataagcc tggtgtctat gttcgtgttt caaggtttgt tacttggatt 1080

gagggagtga tgagaaataa ttaa 1104

<210> 10

<211> 367

<212> PRT

<213> Mini-plg（Small plasminogen）Amino acid sequence

<400> 10

Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala

1 5 10 15

Ser Val Val Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr

20 25 30

Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly

35 40 45

Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala

50 55 60

Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg

65 70 75 80

Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly

85 90 95

Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys

100 105 110

Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln

115 120 125

Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala

130 135 140

His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly

145 150 155 160

Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr

165 170 175

Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val

180 185 190

Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu

195 200 205

Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala

210 215 220

Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro

225 230 235 240

Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys

245 250 255

Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu

260 265 270

Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg

275 280 285

Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly

290 295 300

His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro

305 310 315 320

Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser

325 330 335

Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg

340 345 350

Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

355 360 365

<210> 11

<211> 750

<212> DNA

<213> Micro-plg（Fibrillin lyase is former）Nucleotide sequence

<400> 11

gccccttcat ttgattgtgg gaagcctcaa gtggagccga agaaatgtcc tggaagggtt 60

gtaggggggt gtgtggccca cccacattcc tggccctggc aagtcagtct tagaacaagg 120

tttggaatgc acttctgtgg aggcaccttg atatccccag agtgggtgtt gactgctgcc 180

cactgcttgg agaagtcccc aaggccttca tcctacaagg tcatcctggg tgcacaccaa 240

gaagtgaatc tcgaaccgca tgttcaggaa atagaagtgt ctaggctgtt cttggagccc 300

acacgaaaag atattgcctt gctaaagcta agcagtcctg ccgtcatcac tgacaaagta 360

atcccagctt gtctgccatc cccaaattat gtggtcgctg accggaccga atgtttcatc 420

actggctggg gagaaaccca aggtactttt ggagctggcc ttctcaagga agcccagctc 480

cctgtgattg agaataaagt gtgcaatcgc tatgagtttc tgaatggaag agtccaatcc 540

accgaactct gtgctgggca tttggccgga ggcactgaca gttgccaggg tgacagtgga 600

ggtcctctgg tttgcttcga gaaggacaaa tacattttac aaggagtcac ttcttggggt 660

cttggctgtg cacgccccaa taagcctggt gtctatgttc gtgtttcaag gtttgttact 720

tggattgagg gagtgatgag aaataattaa 750

<210> 12

<211> 249

<212> PRT

<213> Micro-plg（Fibrillin lyase is former）Amino acid sequence

<400> 12

Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys

1 5 10 15

Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro

20 25 30

Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly

35 40 45

Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu

50 55 60

Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln

65 70 75 80

Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu

85 90 95

Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser

100 105 110

Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro

115 120 125

Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly

130 135 140

Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu

145 150 155 160

Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly

165 170 175

Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr

180 185 190

Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys

195 200 205

Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala

210 215 220

Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr

225 230 235 240

Trp Ile Glu Gly Val Met Arg Asn Asn

245

<210> 13

<211> 684

<212> DNA

<213>Serine protease（Structure）The nucleotide sequence in domain

<400> 13

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 60

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 120

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 180

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 240

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 300

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 360

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 420

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 480

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 540

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 600

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 660

acttggattg agggagtgat gaga 684

<210> 14

<211> 228

<212> PRT

<213>Serine protease（Structure）The amino acid sequence in domain

<400> 14

Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val

1 5 10 15

Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile

20 25 30

Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro

35 40 45

Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn

50 55 60

Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu

65 70 75 80

Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val

85 90 95

Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val

100 105 110

Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln

115 120 125

Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile

130 135 140

Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln

145 150 155 160

Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys

165 170 175

Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr

180 185 190

Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn

195 200 205

Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu

210 215 220

Gly Val Met Arg

225

Claims (10)

1. plasminogen is preparing the purposes in preventing and/or treating the cardiopathic medicine of sub-ject's property.

2. the purposes of claim 1, wherein the diabetic cardiomyopathy include cardiomegaly, cardiac insufficiency, arrhythmia cordis, Coronary heart diseases and angina pectoris, painless myocardial infarction, heart failure.

3. according to the purposes of claim 1 or 2, wherein the diabetic cardiomyopathy is the complication of diabetes.

4. according to the purposes of claim any one of 1-3, wherein the plasminogen be with sequence 2 have at least 80%, 85%, 90%th, the protein of 95%, 96%, 97%, 98% or 99% sequence identity.

5. according to the purposes of claim any one of 1-4, wherein the plasminogen can combine with one or more other medicines Using.

6. purposes according to claim 5, wherein the other medicines include：Antianginal drug, antihyperlipidemic, anti-hypertension Medicine, anti-inflammatory agent, anti-infectious agent, aldosterone antagonists, blood sugar regulator, insulin, antithrombotic.

7. the purposes in the medicine that plasminogen organizes microthrombus caused by preparation prevention and/or treatment sub-ject.

8. plasminogen prepare prevention and/or treatment sub-ject caused by tissue cell insult medicine in use On the way.

9. purposes according to claim 7, wherein organizes microthrombus to include heart tissue, liver organization, god caused by diabetes Through tissue, renal tissue and retinal tissue microthrombus.

10. purposes according to claim 8, wherein tissue cell insult includes heart tissue, liver caused by the diabetes Tissue, nerve fiber, renal tissue and retinal tissue are damaged.