WO2020010958A1 - Application of metrnl protein or gene in blocked blood vessel disease - Google Patents

Application of metrnl protein or gene in blocked blood vessel disease Download PDF

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WO2020010958A1
WO2020010958A1 PCT/CN2019/089441 CN2019089441W WO2020010958A1 WO 2020010958 A1 WO2020010958 A1 WO 2020010958A1 CN 2019089441 W CN2019089441 W CN 2019089441W WO 2020010958 A1 WO2020010958 A1 WO 2020010958A1
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metrnl
mice
atherosclerosis
protein
apoe
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PCT/CN2019/089441
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French (fr)
Chinese (zh)
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缪朝玉
郑斯莉
缪玉恩
胡文君
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上海风劲生物医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention relates to the technical field of medicine, in particular to the application of Metrnl protein or gene in vascular obstructive diseases.
  • Vascular occlusion refers to the occlusion of blood vessels due to slow blood flow, changes in blood composition, or increase in blood viscosity due to age, diet, and other factors.
  • Vascular occlusive diseases include: thrombosis of arteries on the basis of sclerosis, arterial embolism after cardiac thrombolysis, venous thrombosis, impeded blood return after varicose veins, etc. The more common include cerebrovascular occlusion, coronary coronary occlusion, etc. Atherosclerosis, diabetes, hyperlipidemia, and hypertension can accelerate their development. The goal of treatment of vascular obstructive diseases is recanalization of blood vessels.
  • SK streptokinase
  • APSAC acyl plasminogen streptokinase activator complex
  • UK urokinase
  • t-PA tissue-type plasminogen.
  • the existing thrombolytic drugs can cause adverse reactions caused by the reduction of fibrinogen and other coagulation factors, and the clinical manifestations are local bleeding, subcutaneous sheet bleeding, and even intracranial bleeding.
  • AS atherosclerosis
  • Metrnl also known as Subfatin, Cometin, Interleukin39
  • CN102164611A disclose the application of Metrnl in reducing hearing loss in the inner ear.
  • Chinese Patent Publication Nos. CN103536903A, CN103536904A and International Patent Publication No. WO2015062167A8 disclose the application of Metrnl in lowering blood lipids and blood glucose.
  • Chinese Patent Publication No. CN104808004A discloses the application of Metrnl in the diagnosis of colon cancer. However, the use of Metrnl protein or gene in vascular obstructive diseases has not been reported yet.
  • the first object of the present invention is to provide a new therapeutic use of Metrnl protein or gene in view of the shortcomings in the prior art.
  • Metrnl proteins or genes or their synergists in the preparation of anti-atherosclerotic drugs is provided.
  • Metrnl protein or gene in a drug for treating atherosclerosis is provided.
  • a Metrnl protein or gene or their synergist in the preparation of a medicament, the medicament being used for:
  • the medicament can be made into various dosage forms by using conventional methods in the medical field, using Metrnl protein as an active ingredient, and pharmaceutically acceptable excipients.
  • Metrnl protein as an active ingredient
  • pharmaceutically acceptable excipients When used for oral administration, it can be prepared into conventional solid preparations such as tablets, powders, or capsules; for injection, it can be prepared into injection solutions.
  • the active ingredient has a weight content of 0.1 to 99.9%, and a preferred weight content is 0.5 to 90%.
  • the medicament can be applied to individuals in need of treatment through different routes such as intraperitoneal injection, subcutaneous injection, intravenous injection, intramuscular injection, intra-lymph node injection, and mucosal medication.
  • the individual can be a human or an animal.
  • the dosage is generally 0.01 to 1000 mg / kg of body weight / day, which can be specifically changed according to the age and condition of the individual.
  • a second object of the present invention is to provide a method for treating atherosclerosis.
  • the technical solution adopted by the present invention is: a method for treating atherosclerosis, which comprises administering an effective amount of Metrnl protein or a synergist to an individual in need of treatment, Self-agonist, up-regulator or stabilizer.
  • a third object of the present invention is to provide a new diagnostic application of Metrnl protein.
  • Metrnl protein or a specific antibody thereof is provided for preparing a diagnostic reagent or a kit for detecting atherosclerosis.
  • Metrnl protein or a specific antibody thereof is provided for preparing a diagnostic reagent or a kit for detecting the degree of atherosclerotic disease.
  • Metrnl protein is provided as a diagnostic marker for detecting atherosclerosis.
  • a fourth object of the present invention is to provide a method for detecting atherosclerosis.
  • the technical solution adopted by the present invention is: a method for detecting atherosclerosis, the method is to detect the content of Metrnl protein in a serum sample, so as to determine whether the subject derived from the serum sample is suffering Atherosclerosis.
  • the test is a serum test.
  • the serum detection is an ELISA method.
  • the detection is to prepare an antibody against Metrnl protein by a conventional method, and establish a qualitative or quantitative method for detecting Metrnl protein.
  • detecting the content of Metrnl protein in the serum sample it is determined whether the subject from which the serum sample originates has an artery. Atherosclerosis.
  • the specifics are as follows: first, measure the content of Metrnl protein in the serum sample to be tested, and then compare it with the content of Metrnl protein in the normal human serum sample to determine whether the content of Metrnl protein in the serum sample to be tested is reduced, and judge the test based on the comparison result Whether the subject of the serum sample has atherosclerosis.
  • a fifth object of the present invention is to provide a method for detecting the degree of disease in patients with atherosclerosis.
  • the technical solution adopted by the present invention is: a method for detecting the degree of disease in atherosclerosis patients, the method is to detect the content of Metrnl protein in a patient's serum sample, thereby determining the origin of the serum sample The degree of atherosclerotic disease in the subject.
  • the detection is to prepare an antibody against Metrnl protein by a conventional method, establish a qualitative or quantitative method for detecting Metrnl protein, and continuously measure the content of Metrnl protein in serum samples of atherosclerotic patients at different times, Based on this, the degree of atherosclerotic disease in the subject from which the serum sample was derived was determined.
  • the details are as follows: The Metrnl protein content in serum samples of patients with atherosclerosis is measured for multiple consecutive days and compared. If the Metrnl protein content (concentration) in the patient's serum sample decreases, it indicates that the disease is aggravated. An increase in the content (concentration) of Metrnl protein indicates a reduction in disease severity.
  • the present invention found for the first time that Metrnl can be used to diagnose and treat atherosclerosis.
  • the present invention is constructed Metrnl - / - ApoE - / - double knockout mouse model of atherosclerosis and arterial EC-Metrnl - / - ApoE - / - mouse model of atherosclerotic arteries
  • experiments show: Metrnl - / - ApoE - / - double knockout mice aorta compared with the control mice had more white plaque, EC-Metrnl - / - ApoE - / - mice than in control mice aorta having more white atheroma Block
  • Metrnl -/- ApoE -/- double-knock mice have larger aortic plaque area than control mice, EC-Metrnl -/- ApoE -/- mice have aortic plaque area smaller than control Mice; atherosclerotic mice, the aortic root vessels have significant pathological thickening and narrowing of the lumen, and the thickness of the
  • Metrnl can be secreted by vascular endothelial cells, which is achieved through the classic secretion pathway; the level of vascular endothelial and blood Metrnl decreased during atherosclerosis, and the level of Metrnl decreased with the increase of disease. It is suggested that Metrnl is not only useful for anti-atherosclerosis, but also can be used as a diagnostic indicator of disease.
  • the anti-thrombotic effect of Metrnl in mice was found in the early stage.
  • the present invention has found that there is no species difference in the anti-thrombotic effect of Metrnl recombinant protein.
  • the mouse Metrnl recombinant protein can inhibit rat and human platelet clot retraction, and human Metrnl recombinant protein. Can also inhibit mouse and rat platelet clot contraction response.
  • Metrnl recombinant protein on the clot contraction reaction showed a dose-effect relationship, and both human and mouse Metrnl protein showed inhibitory effect at low concentration (0.1 ⁇ g / ml), and the inhibitory effect increased with increasing concentration, of which Metrnl When the protein was 30 ⁇ g / ml, the initiation time of clot contraction was more than doubled than normal. Metrnl recombinant protein also inhibited the clot contraction response of platelets in Metrnl gene knockout mice. Metrnl protein can inhibit platelet activation, which is manifested by a decrease in the level of platelet activation marker CD62p.
  • Figure 1 Metrnl -/- mouse breeding process and genotype identification.
  • Figure 2 Metrnl -/- ApoE -/- double-knock breeding process and genotype identification.
  • Figure 3 EC-Metrnl -/- mouse breeding process and genotype identification.
  • Figure 4 The cultivation process and genotype identification of EC-Metrnl -/- ApoE -/- mice.
  • Figure 5 In vivo morphology of the aorta at 2.5 months of HFD in Metrnl -/- ApoE -/- double knock mice and their control mice.
  • the black arrow in the figure shows the location of the atheromatous plaque.
  • Figure 6 The aortic morphology of EC-Metrnl -/- ApoE -/- and its control mice at 5.5 months of HFD.
  • the white arrow in the picture shows the location of the atheromatous plaque.
  • Figure 7 Results of oil red O staining in longitudinal section of aorta at 2.5 months of HFD in Metrnl -/- ApoE -/- double knock mice and their control mice. ** P ⁇ 0.01 vs control mice.
  • Figure 8 Results of oil red O staining in longitudinal sections of the aorta at 5.5 months of HFD in EC-Metrnl -/- ApoE -/- and its control mice. Panel A shows the results of male mice, and Panel B shows the results of female mice. * P ⁇ 0.05 vs control mice.
  • Figure 9 Representative images of HE staining of aortic roots at 2.5 months of HFD in C57 wild-type mice and Metrnl -/- ApoE -/- double knock mice and their control mice.
  • Figure 10 Representative diagram of aortic root HE staining in EC-Metrnl -/- ApoE -/- and its control mice at 5.5 months of HFD.
  • Figure 11 Results of oil red O staining on frozen sections of aortic roots at 2.5 months of HFD in C57 wild-type mice and Metrnl -/- ApoE -/- double knock mice and their control mice. * P ⁇ 0.05 vs control mice.
  • Figure 12 Results of oil red O staining of frozen sections of aortic roots at 5.5 months of HFD in EC-Metrnl -/- ApoE -/- and its control mice. * P ⁇ 0.05 vs control mice.
  • Figure 13 Representative diagram of oil red O staining of frozen sections of aorta at 5.5 months of HFD in EC-Metrnl -/- ApoE -/- and its control mice.
  • Figure 14 Representative diagram of immunohistochemistry of frozen section F4 / 80 of aortic root in EC-Metrnl -/- ApoE -/- and its control mice at 5.5 months of HFD.
  • Figure 15 Representative map of ⁇ -SMA immunohistochemistry of frozen sections of aortic roots in EC-Metrnl -/- ApoE -/- mice and their control mice at 5.5 months of HFD.
  • Figure 16 Quantitative detection results of aortic inflammatory factor mRNA in EC-Metrnl -/- ApoE -/- and its control mice at 5.5 months of HFD. * P ⁇ 0.05, ** P ⁇ 0.01 vs control mice.
  • Figure 17 Metrnl concentration in mouse primary endothelial cell incubation solution and its concentrate.
  • Figure 18 Concentration of Metrnl in human umbilical vein endothelial cells treated with brefeldin A or eeyarestatin1. ** P ⁇ 0.01 vs Vehicle Group.
  • Figure 20 Aortic and carotid vascular morphology of mice with different degrees of AS disease.
  • Figure 22 Metrnl and CD31 immunofluorescence staining in AS plaque area and non-AS plaque area of mouse aorta.
  • FIG. 23 Blood Metrnl levels in patients with atherosclerosis complicated with myocardial infarction (AS + MI) are significantly lower than those in the control group. There were 11 patients in the control group (8 males and 3 females), and 21 patients in the AS + MI group (17 males and 4 females). *** P ⁇ 0.001 vs control patients.
  • Figure 24 There is no species difference in the effect of Metrnl in inhibiting platelet clot contraction.
  • Figure 25 Dose-effect relationship of Metrnl recombinant protein inhibiting platelet clot shrinkage response.
  • Figure 26 Inhibition effect of Metrnl recombinant protein on platelet clot shrinkage response in Metrnl knockout mice.
  • Figure 28 Comparison of anticoagulant characteristics of human Metrnl recombinant protein and t-PA recombinant protein.
  • Metrnl protein used may be naturally occurring, for example, it may be isolated and purified from mammals.
  • the Metrnl protein may also be artificially prepared, for example, according to conventional genetic engineering techniques. Any suitable Metrnl protein may be suitable for use in the present invention.
  • the Metrnl protein includes a full-length Metrnl protein or a biologically active fragment thereof.
  • the Metrnl protein may be a human Metrnl protein with a size of about 30 kDa, and the amino acid sequence is shown in NP_001004431.1. Metrnl proteins such as rats and mice may also be used.
  • the amino acid sequence of Metrnl protein formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention.
  • the Metrnl protein or a biologically active fragment thereof includes a part of a conservative amino acid substitution sequence which does not affect its activity or retains a part of its activity.
  • Proper substitution of amino acids is a well-known technique in the art that can be easily implemented and ensures that the biological activity of known molecules is not altered. These techniques have taught those skilled in the art that, in general, changing a single amino acid in a non-essential amino acid region of a polypeptide does not alter biological activity.
  • any biologically active fragment of Metrnl protein can be used in the present invention.
  • the meaning of the biologically active fragment of the Metrnl protein refers to a polypeptide that can still retain all or part of the function of the full-length Metrnl protein.
  • the biologically active fragment retains at least 50%, 60% to 99%, or 100% of the activity of the full-length Metrnl protein.
  • the present invention can also use modified or improved all or part of the amino acid Metrnl protein, for example, modified or improved Metrnl protein to promote half-life, effectiveness, metabolism and / or protein efficacy.
  • the modified or modified Metrnl protein may be a conjugate of a Metrnl protein, or a substituted or artificial amino acid thereof.
  • the modified or improved Metrnl protein or gene may have certain differences from the natural Metrnl protein or gene, but it also has the functions described in the present invention and does not cause other adverse reactions or toxicity. That is, any change that does not affect the biological activity of the Metrnl protein or the biological function of the gene can be used in the present invention.
  • Metal synergist includes agonists, up-regulators, stabilizers, etc., and refers to any substance that can increase the activity of Metrnl, increase the stability of Metrnl, increase the expression of Metrnl, and increase the effective action time of Metrnl.
  • Substances can be used in the present invention. They can be compounds, small chemical molecules, biomolecules, and the like.
  • the biomolecule may be at a nucleic acid level (including DNA, RNA), at a protein level, or may be a virus product that up-regulates the expression of Metrnl.
  • Example 1 Construction of a atherosclerotic model of Metrnl genetically engineered mice
  • Metrnl systemic knockout mice (Metrnl -/- for short) were obtained using the Cre-LoxP gene knockout system (Diabetes2015; 64: 4011-4022) according to the breeding process of FIG. 1A.
  • Metrnl loxP / loxP came from this unit, Ella-Cre mice were purchased from Shanghai N forcing Model Biotechnology Development Co., Ltd., and C57BL / 6J mice were purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.
  • Genotype identification method Ella-Cre gene and Metrnl gene need to be identified. The mouse tail genotype was identified using upstream and downstream primers of the corresponding genes.
  • Ella-Cre gene was positive with a 100bp band; the amplified product of the Metrnl knockout sequence was 289bp, and the amplified product of the Metrnl wild sequence was 150bp.
  • Metrnl +/- Ella-Cre mice showed three bands of 100bp, 150bp, and 289bp (lane 1); Metrnl +/- appeared two bands of 150bp and 289bp (lane 2); Metrnl -/- full knock The mouse showed a band of 289 bp (lane 4); the wild control mouse showed a band of 150 bp (lane 3).
  • Metrnl -/- ApoE -/- double knock mice Metrnl -/- mice and ApoE -/- mice were obtained by mating and breeding according to the incubation process of Fig. 2A, of which ApoE -/- mice were purchased from Shanghai N searching Biotechnology Development Co., Ltd. Genotype identification method: Metrnl gene and ApoE gene need to be identified. The mouse tail genotype was identified using upstream and downstream primers of the corresponding genes.
  • the amplified product of Metrnl knockout sequence was 289bp
  • the amplified product of Metrnl wild sequence was 150bp
  • the amplified product of ApoE knockout sequence was 245bp
  • the amplified product of ApoE wild sequence was 155bp.
  • three bands of 289bp, 150bp, 245bp, and 155bp appeared in Metrnl +/- ApoE +/- mice (lane 3); two bands of 289bp and 245bp appeared in Metrnl -/- ApoE -/- double-click mice (Lane 6); Wild control mice with double knock mice showed two bands of 150 bp and 245 bp (lane 2).
  • Western high-fat diet feed was purchased from Shanghai Pluton Biotechnology Co., Ltd.
  • the feed formula was 100g of feed: 60g of common breeding feed, 16.4g of lard, 10g of sucrose, 8.5g of casein, 1.3g of cholesterol, and 0.3g of bile salt.
  • HFD high-fat diet
  • Metrnl endothelial cell-specific knockout mice ie, Metrnl loxP / loxP Tek-Cre mice, referred to as EC-Metrnl -/-
  • EC-Metrnl -/- Metrnl endothelial cell-specific knockout mice
  • a 100bp band was positive for the Tek-Cre gene; the amplified product of the Metrnl-flox sequence was 243bp, and the corresponding amplified product of the wild sequence was 131bp.
  • 100 bp, 243 bp, and 131 bp appeared in Metrnl loxP / wt Tek-Cre mice (lane 1); 100 bp and 243 bp appeared in EC-Metrnl -/- mice (lane 2); the control of special knockout mice was small The mouse appeared 243 bp (lane 3).
  • EC-Metrnl -/- ApoE -/- double knock mice were obtained by mating and breeding according to the incubation flow of Figure 4A using EC-Metrnl -/- mice and ApoE -/- mice.
  • Genotype identification method Tek-Cre needs to be identified Gene, Metrnl-flox gene and ApoE gene.
  • a 100bp band was positive for the Tek-Cre gene; the Metrnl-flox sequence amplification product was 243bp, and the corresponding wild sequence amplification product was 131bp; the ApoE knockout sequence amplification product It was 245bp, and the amplified ApoE wild sequence product was 155bp.
  • EC-Metrnl -/- ApoE -/- double knock mice showed 100 bp, 243 bp, and 245 bp (lane 2), and control mice showed two bands of 243 bp and 245 bp (lane 3).
  • mice were intraperitoneally injected with 1% sodium pentobarbital (100mg / kg) for anesthesia.
  • the alcohol and cotton balls were used to moisten their thorax and abdomen.
  • the abdominal cavity and thorax were cut in sequence from below the sternal handle to fully expose the heart and its bottom veins.
  • Blood was taken from the vena cava with a syringe, then a small cut was made in the right atrium of the heart.
  • a 10 ml syringe was filled with pre-chilled 1 ⁇ PBS buffer.
  • the lungs, liver, stomach, spleen, pancreas, intestine, fat and genital organs were cut off, leaving the heart, aorta and kidneys, and the aorta was separated under a microscope.
  • the aorta was separated under a microscope.
  • the heart was immersed in 4% paraformaldehyde and fixed; the aorta was fixed in 4% paraformaldehyde or immediately frozen with liquid nitrogen to extract mRNA.
  • Oil red O dye solution preparation method First use oil isopropyl alcohol to mix oil red O powder (Sigma) into 0.5% oil red O stock solution, filter with filter paper, and use oil red O stock solution and ddH 2 O according to 3: 2 Mix by volume. Staining method: When the aorta is immersed and fixed in 4% paraformaldehyde for 24 hours, it is transferred to a silicone dish filled with 1 ⁇ PBS buffer solution, the aorta is dissected longitudinally, and it is fixed flatly on a silicone surface with a needle.
  • the F4 / 80 antibody was purchased from abcam company, and the ⁇ -SMA antibody and SABC kit were purchased from Wuhan Baoshide Biological Engineering Co., Ltd.
  • Immunohistochemical method Take frozen sections from -20 ° C, bake at 37 ° C for 1h, and draw circles around the tissue of each piece with an immunohistochemical pen.
  • the kit "RNeasy Micro Kit” for extracting mouse aortic mRNA was purchased from QIAGEN.
  • the method for obtaining and cryopreserving the mouse aorta was the same as the experimental method "(1) Mouse heart and aorta separation”.
  • the primer sequences used are as follows:
  • IL-6 CCACTTCACAAGTCGGAGGCTTA GCAAGTGCATCATCGTTGTTCATAC IL-1 ⁇ TCCAGGATGAGGACATGAGCAC GAACGTCACACACCAGCAGGTTA F4 / 80 GAGATTGTGGAAGCATCCGAGAC GATGACTGTACCCACATGGCTGA CD68 CATCAGAGCCCGAGTACAGTCTACC AATTCTGCGCCATGAATGTCC IL-10 CTTACTGACTGGCATGAGGATCA GCAGCTCTAGGAGCATGTGG Fizz1 TTGCAACTGCCTGTGCTTAC CAAGAAGCAGGGTAAATGGG Ym1 TTTGATGGCCTCAACCTGGA AGTGAGTAGCAGCCTTGGAATGTC GAPDH GTATGACTCCACTCACGGCAAA GGTCTCGCTCCTGGAAGATG
  • Figure 5 shows the morphology of aorta in the body of Metrnl -/- ApoE -/- double knock mice and their control mice at 2.5 months of HFD. The heart, aorta and muscle tissue can be seen. The results show that Metrnl- / -The aorta of ApoE -/- double knock mice has more white atheromatous plaques (shown by the black arrows in the figure).
  • Figure 6 shows the aortic morphology of EC-Metrnl -/- ApoE -/- and its control mice at 5.5 months of HFD. The heart and aorta can be seen. The results show that EC-Metrnl -/- The aorta of ApoE -/- mice has more white atheromatous plaques (shown by yellow arrows in the figure).
  • Oil red O is a fat-soluble bright red dye. After oil red O staining, the lipid deposition site is stained red, and the non-lipid deposition site is not colored, so it can reflect the aortic atheromatous plaque. Severity. As shown in Figure 7, in the 2.5-month HFD of Metrnl -/- ApoE -/- double-click mice, the proportion of the area of the red aortic plaque in the longitudinal section of the entire aorta was significantly higher than that in the control group.
  • FIG. 9 shows the results of HE staining of the aortic roots of C57, Metrnl -/- ApoE -/- double knock mice and their control mice at 2.5 months of HFD. It was found that the vascular wall of the aortic root of C57 wild-type mice without atherosclerosis was thin, while Metrnl -/- ApoE -/- double-knocking mice and their control mice with atherosclerosis The aortic root vessels had a significant pathological thickening and narrowed lumen, and the thickness of the tube walls of Metrnl -/- ApoE -/- double-knocked mice was more severe than that of the control group.
  • Figure 10 shows the results of HE staining of aortic roots in EC-Metrnl -/- ApoE -/- and its control mice at 5.5 months of HFD. Similarly, EC-Metrnl -/- ApoE -/- mice The tube wall thickened more severely than the control mice.
  • Oil red O staining of frozen sections of mouse aortic roots reflects the formation of atherosclerotic plaques in the aortic roots.
  • C57 wild-type mice without atherosclerosis did not have lipid deposits in their aortic roots, but Metrnl -/- ApoE -/- double knock mice with atherosclerosis and their In control mice, aortic roots had obvious lipid plaques, which caused the old old lumen to narrow to form new ones.
  • the ratio of the area of the red atheroma in the lumen to the area of the old lumen was used to indicate the involvement of the aortic root.
  • Atherosclerosis is a chronic inflammatory disease.
  • the F4 / 80 molecule is used as a marker of macrophages and can be used to observe the inflammatory response of atherosclerosis.
  • the expression of F4 / 80 molecules in the aortic roots of EC-Metrnl -/- ApoE -/- mice at 5.5 months of HFD was significantly higher than that of control mice.
  • ⁇ -SMA smooth muscle cells migrate from the tunica media to the intima and proliferate.
  • ⁇ -SMA can reflect the severity of atherosclerosis.
  • FIG. 15 the expression of ⁇ -SMA molecules in the aortic root of EC-Metrnl -/- ApoE -/- mice at 5.5 months of HFD was significantly higher than that of control mice.
  • Atherosclerosis as a chronic inflammatory disease, changes in the expression levels of many inflammatory factors during its development.
  • EC-Metrnl -/- ApoE -/- mice had pro-inflammatory factors VCAM-1, ICAM-1, TNF ⁇ , IL-6 and macrophages in aortic tissues at 5.5 months of HFD Cell marker molecules F4 / 80 and CD68 were all up-regulated, IL-1 ⁇ was up-regulated but not statistically significant, and the expression levels of anti-inflammatory factors IL-10, Fizz and Ym1 were not significantly changed.
  • Example 3 Endothelial cells can secrete Metrnl, which is achieved through the classical secretion pathway
  • the cells When the cells grow to about 90% confluence, the cells are gently rinsed 3 times with 1 ⁇ PBS buffer, serum-free endothelial medium is added, and the cells are cultured in a cell incubator for 12 hours.
  • the cell incubation solution was aspirated into a 15 ml centrifuge tube, and centrifuged: room temperature ⁇ 1200 rpm ⁇ 5 min, and the obtained supernatant was the cell incubation solution.
  • Serum-free medium was used as a control treatment group. Store separately at -80 ° C.
  • Serum-free medium containing drugs (brefeldin A or eeyarestatin1, each with a final concentration of 5 ⁇ g / ml) was added and placed in a cell culture incubator for 24 h. Pipet the incubation solution into a 15 mL centrifuge tube, centrifuge: room temperature ⁇ 1200 rpm ⁇ 5 min, and suck the supernatant. Serum-free medium was used as a control treatment group. Store separately at -80 ° C.
  • ELISA kit (# DY6679, R & D system) was used to measure Metrnl levels in mouse primary endothelial cell incubation solution; ELISA kit (# DY7867-05, R & D system) was used to determine human umbilical vein endothelial cell incubation solution Metrnl level. The experiments were performed with double duplicate wells, and the operation steps were strictly performed according to the instructions provided by the kit. The standard curves were highly fit (R 2 > 0.99).
  • Metrnl was successfully detected in the mouse primary endothelial cell incubation stock solution and its concentrated solution, and the Metrnl concentration in the stock solution was about 1/3 of the concentration in the concentrated solution, which was consistent with the concentration step (2ml The stock solution was concentrated to about 0.7 ml of concentrate). In addition, the concentration of Metrnl in the blank serum-free medium was below the detection limit. This result demonstrates that endothelial cells can indeed secrete Metrnl.
  • Brefeldin A is an inhibitor of Golgi transport function
  • eeyarestatin 1 is an inhibitor of endoplasmic reticulum transport channels.
  • Metrnl was hardly detected in the culture medium, which indicates that the secretion process of endothelial cells Metrnl can be successfully inhibited by brefeldin A and eeyarestatin1, reflecting that the secretion pathway of endothelial cells Metrnl belongs Classical ER-Golgi protein secretion pathway.
  • Example 4 Endothelial and blood Metrnl levels decrease during atherosclerosis, and Metrnl levels decrease with increasing disease
  • ApoE -/- mice susceptible to atherosclerosis were purchased from Shanghai Nsweeping Model Biotechnology Development Co., Ltd. Preparation of mouse models with different AS levels: Under the same feeding environment, ApoE -/- mice were fed with ordinary feed to 7 months of age and randomly divided into two groups. One group continued to be fed with ordinary feed, and the other group was fed with ordinary feed. Feed was changed to Western high-fat diet feed. After 22 weeks, the atherosclerosis of mice (12 months old) was examined.
  • Isolation of mouse aorta was the same as before. Isolation of the mouse carotid artery was performed after the aortic separation was completed. The mice were supine, cut along the front of the neck, and separated until the common, internal and external carotid arteries were exposed. The carotid arteries near the bifurcation of the blood vessels were taken. .
  • the mouse thoracic aorta was isolated and cut into 2-3mm sections, immersed in 4% paraformaldehyde solution and fixed for at least 48 hours, and frozen sections were made and stored at -20 ° C.
  • the frozen sections were dried at 37 ° C for 2 hours, and then the tablets were rinsed twice with 1 ⁇ PBS buffer; the tablets were antigen-recovered using sodium citrate antigen repair solution and microwave heating; immunohistochemistry was used.
  • the pen draws a circle around the tissue; rinses the film 3 times; drops the blocking serum into the circle, and blocks the wet box at room temperature for 2h; shakes off the blocking serum, and drops the diluted Metrnl primary antibody (Abcam, # ab121775) into the circle.
  • ApoE -/- mice are widely used in preclinical studies of AS diseases. They can spontaneously AS under normal dietary conditions, and HFD can accelerate the occurrence and development of AS. As shown in Fig. 20, C57 normal mice without AS disease had clean blood vessels without foreign bodies; ApoE -/- mice (12 months old) with spontaneous AS under normal diet had both aorta and carotid arteries. White AS plaques were compared; in contrast, after 22 weeks of HFD in ApoE -/- mice (12 months of age), the AS condition was more severe, showing a large number of AS plaques in the aorta and carotid arteries.
  • CD31 molecule is a very abundant endothelial cell-to-cell junction protein, often used as a molecular marker of endothelial cells. Take C57 mice and ApoE -/- mice with spontaneous AS under normal diet (as shown in Figure 20), and perform immunofluorescence staining on frozen sections of their thoracic aorta (as shown in Figure 22).
  • CD31 molecules (green (Fluorescence) showed very obvious continuous expression in the vascular wall of normal C57 mice, overlapping with Metrnl staining (red fluorescence); CD31 molecules in non-plaque areas of ApoE -/- mice also linearly expressed in the vascular wall, suggesting non-plaque There are relatively complete endothelial cells in the region, and at the same time, the weakened expression of Metrnl in the vascular wall can also be detected. In contrast, the expression of CD31 and Metrnl in the plaque area are very low, almost lacking, indicating that the endothelial cells in the plaque area are damaged.
  • Metrnl is a secreted protein, and this result is not only consistent with the characteristics of exacerbation of vascular endothelium during the development of AS, but also consistent with the above-mentioned decrease in serum Metrnl levels with the increase in AS disease severity.
  • Metrnl can be secreted by vascular endothelial cells, which is achieved through the classic secretion pathway; the level of vascular endothelial and blood Metrnl decreases during atherosclerosis, and the level of Metrnl decreases with the increase of disease. It is suggested that Metrnl is not only useful for anti-atherosclerosis, but also can be used as a diagnostic indicator of disease.
  • Example 5 Metrnl recombinant protein inhibits platelet clot contraction response without species differences
  • mice intraperitoneally inject 10% chloral hydrate (300mg / kg) to anesthetize the mice, take a 1ml syringe and wet the inner wall of the syringe with 2% sodium citrate anticoagulant, and reserve 100 ⁇ l of anticoagulant in Inside the syringe. After the mouse was numbed, its limbs were fixed, and its abdomen was opened along the midline of the abdomen (to avoid damage to the blood vessels). Carefully poke out the internal organs to expose the abdominal aorta.
  • 10% chloral hydrate 300mg / kg
  • the two platelet layer solutions were mixed together and centrifuged (2750 rpm ⁇ 3min, room temperature) to obtain platelet precipitation.
  • the upper transparent PPP (Poor Platelet Plasma) solution was first transferred to a new 1.5 ml centrifuge tube and incubated at 37 ° C for subsequent experiments. Then continue to add an appropriate amount of 37 ° C TB (Tyrode's Buffer) solution (containing 0.04U / ml Apyrase) to a 15ml centrifuge tube, and carefully resuspend the platelets by pipetting to obtain a platelet suspension for subsequent experiments.
  • 37 ° C TB Tee's Buffer
  • rat platelet suspension intraperitoneally inject 10% chloral hydrate (350mg / kg) to anaesthetize the rats, take a 10ml syringe and wet the inner wall of the syringe with 3.8% sodium citrate anticoagulant, and reserve 1ml of anticoagulant in Inside the syringe. After the rat is anesthetized, place it in the supine position, open its abdomen along the midline of the abdomen (to avoid damaging blood vessels), carefully open the internal organs, and expose the abdominal aorta.
  • 10% chloral hydrate 350mg / kg
  • Centrifuge (1750rpm ⁇ 10min, room temperature), draw the platelet layer to a new 15ml centrifuge tube, centrifuge (2750rpm ⁇ 5min, room temperature) to obtain platelet precipitation, first transfer the upper transparent PPP solution to a new 1.5ml centrifuge tube at 37 ° C Incubate for subsequent experiments, and then continue to add an appropriate amount of 37 ° C TB solution (containing 0.04U / ml Apyrase) to a 15ml centrifuge tube, and carefully resuspend the platelets to obtain platelet suspensions for subsequent experiments.
  • 37 ° C TB solution containing 0.04U / ml Apyrase
  • Human platelet suspension preparation Volunteer elbow vein blood was collected by a hospital nurse, collected into a vacuum blood collection tube with ACD anticoagulant, and the blood was immediately mixed with the anticoagulant and transferred to a 15 ml centrifuge tube. Centrifuge (1750 rpm ⁇ 4min, room temperature), aspirate the platelet layer to a new 15ml centrifuge tube, centrifuge (2550rpm ⁇ 2.5min, room temperature) to obtain platelet precipitation, first transfer the upper transparent PPP solution to a new 1.5ml centrifuge tube at 37 Incubate at °C for subsequent experiments, and then continue to add an appropriate amount of 37 ° C TB solution (containing 0.04U / ml Apyrase) to a 15ml centrifuge tube, and carefully resuspend the platelets by pipetting to obtain platelet suspensions for subsequent experiments.
  • 37 ° C TB solution containing 0.04U / ml Apyrase
  • Metrnl recombinant protein solution or an equal volume of a control solvent was added to the platelet suspension (250 ⁇ l), and the mixture was incubated at 37 ° C. for 15 minutes for experiments.
  • Both human and mouse Metrnl recombinant proteins were purchased from R & D Systems, and the final concentration after adding to platelet suspension was 1.923 ⁇ g / ml.
  • mouse Metrnl recombinant protein can significantly inhibit platelet clot contraction speed in rats (Figure 24A) and human ( Figure 24B); similarly, human Metrnl recombinant protein can also significantly inhibit mice ( Figure 24C) and Platelet clot contraction rate in rats ( Figure 24D). This shows that there is no species difference in the inhibitory effect of Metrnl recombinant protein on platelet clot shrinkage response. The experiment was repeated three times.
  • mice Metrnl recombinant protein can inhibit the platelet clot shrinkage response, and this inhibitory effect increases with the concentration of Metrnl (0.1-10 ⁇ g / ml) And enhanced. Continue to increase the concentration of Metrnl, and this inhibitory effect continues to increase. Among them, platelets can be observed at 30 ⁇ g / ml without obvious clot contraction response at 25min. The response time of this group is later than that of the 10 ⁇ g / ml group, and is shorter than that of the Vehicle group. The start-up time is delayed more than 1 time, and the contraction response speed is reduced. In addition, human Metrnl recombinant protein also showed a similar dose-effect relationship. The experiment was repeated twice each.
  • Example 7 Metrnl recombinant protein has an inhibitory effect on platelet clot shrinkage in Metrnl knockout mice
  • Metrnl recombinant protein solution or an equal volume of a control solvent was added to the platelet suspension (250 ⁇ l), and the mixture was incubated at 37 ° C. for 15 minutes for experiments.
  • Both human and mouse Metrnl recombinant proteins were purchased from R & D Systems, and the final concentration after adding to platelet suspension was 1.923 ⁇ g / ml.
  • the platelet clot contraction response speed of Metrnl fully knocked mice was significantly faster than that of wild control mice (WT), which was consistent with the previous observation results.
  • the platelet contraction speed of KO mice was significantly reduced, which was comparable to that of the wild control group.
  • the platelet clot contraction response speed of Metrnl endothelial cell-specific knockout mice is also faster than that of control mice (WT).
  • mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg / kg).
  • a 1 ml syringe was used to wet the inner wall of the syringe with 2% sodium citrate anticoagulant, while leaving 100 ⁇ l of the anticoagulant in the syringe.
  • place it in the supine position fully open the chest cavity, expose the heart and remove the pericardium.
  • Use a 1ml syringe to slowly draw blood (about 1ml) from the confluence of veins at the bottom of the heart.
  • the blood and anticoagulant were mixed and transferred to a 15 ml centrifuge tube. Centrifuge (2000 rpm x 8 min, room temperature), pipette the upper PRP solution into a new 1.5 ml centrifuge tube for subsequent experiments.
  • PRP is divided into 4 equal volumes. Add equal volumes of human Metrnl recombinant protein or control solvent at different concentrations, mix and incubate at 37 ° C for 15-20 minutes, and then add 1.5 ⁇ l 1U / ml thrombin, mix, and mix. Continue to incubate at 37 ° C for 5 min for subsequent experiments.
  • the final Metrnl concentrations after addition to PRP were 10 ⁇ g / ml, 30 ⁇ g / ml, and 100 ⁇ g / ml, respectively.
  • the ELISA kit (Shanghai Haring Biotechnology Co., Ltd., # Hl1006) was used to detect the content of CD62p protein in mouse PRP. The experiments were performed in duplicate wells. The operation steps were strictly performed according to the instructions provided in the kit. The standard curves all have a high degree of fit (R 2 > 0.98).
  • human Metrnl recombinant protein can inhibit platelet activation, and it is shown that under the stimulation condition of thrombin, Metrnl recombinant protein can reduce the concentration of CD62p protein, a platelet activation index in PRP.
  • This experiment consists of 3 dose groups, with 3 cases in each dose group, and the experiment was repeated twice.
  • Example 9 Comparison of anticoagulant characteristics between Metrnl recombinant protein and t-PA
  • the inhibitory effects of the human Metrnl recombinant protein and the t-PA recombinant protein on the platelet clot shrinkage response in mice were different.
  • Metrnl recombinant protein can inhibit the speed of platelet clot shrinkage reaction; while the platelet suspension under t-PA does not shrink into a clot but flocculent, that is, no obvious clot shrinkage reaction.
  • the experiment was repeated three times.
  • mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (300mg / kg). After the mice were anesthetized, they were placed in the supine position, the chest cavity was fully opened, the heart was exposed and the pericardium was removed. A 1ml syringe was used to confluence the veins at the bottom of the heart. Blood was taken and the time taken for recording the blood was recorded as T 1. After the blood was taken, the blood was immediately transferred to a glass test tube pre-medicated, incubated at 37 ° C, and the timing was started immediately. When the time was 3 minutes, the glasses were tilted.
  • Test tube (45 °), observe whether the blood is coagulated, and then tilt the test tube every 30s until the blood does not flow when the test tube is poured. The time when the blood was not observed for the first time (ie, the clotting time) was recorded as T 2 .
  • Metrnl recombinant protein and t-PA recombinant protein have different characteristics against mouse whole blood coagulation: Metrnl recombinant protein can extend the whole blood coagulation time, but once the blood coagulates, it will no longer lyse ( The longest observation time is 60min); while t-PA recombinant protein has no significant effect on the whole blood coagulation time, but it can be dissolved after the blood coagulation and maintained in a flowing state (the longest observation time is 90min). The experiment was repeated three times.
  • Metrnl recombinant protein also inhibited the clot contraction response of platelets in Metrnl gene knockout mice.
  • Metrnl protein can inhibit platelet activation, which is manifested by a decrease in the level of platelet activation marker CD62p.
  • the anticoagulant characteristics of Metrnl can reduce bleeding Adverse reactions.

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Abstract

Disclosed in the present invention is an application of Metrnl in diagnosis of atherosclerosis. A Metrnl-/-ApoE-/-double knockout atherosclerotic rat model and an EC-Metrnl-/-ApoE-/-atherosclerotic rat model are constructed. The experiment shows that the severity of atherosclerosis, the area of atherosclerotic plaque, and the degree of atherosclerosis and inflammatory infiltration in an aortic root of two Metrnl knockout rats are higher than a rat in a control group. The experiment shows that a vascular endothelial cell can secrete Metrnl, and the vascular endothelium and blood Metrin levels decrease during atherosclerosis, and the degree of decrease in Metrnl levels increases as the disease is exacerbated. The antithrombotic effect of a Metrnl recombinant protein does not have a species difference, and represents a dose-effect relationship on the inhibition effect of a clot contraction reaction. By comparing the function and characteristic of the Metrnl protein with a classical thrombolytic drug, it is found that the antithrombotic effect of Metrnl can reduce an adverse reaction of bleeding.

Description

Metrnl蛋白或基因在血管堵塞性疾病中的应用Application of Metrnl protein or gene in vascular obstructive diseases 技术领域Technical field
本发明涉及医药技术领域，具体地说，涉及Metrnl蛋白或基因在血管堵塞性疾病中的应用。The invention relates to the technical field of medicine, in particular to the application of Metrnl protein or gene in vascular obstructive diseases.
背景技术Background technique
血管堵塞是指由于年龄、饮食等因素，使血流缓慢、血液成分改变或血液粘度增加形成血栓，导致血管闭塞。血管堵塞性疾病包括：动脉在硬化基础上的血栓形成、心脏血栓脱落后的动脉栓塞、静脉血栓形成、静脉曲张后的血液回流受阻等，较常见的包括脑血管堵塞、心脏冠状血管堵塞等，动脉粥样硬化、糖尿病、高脂血症和高血压等可加速它们的发展。血管堵塞性疾病的治疗目标是血管再通，目前常用的药物包括链激酶(SK)、酰基纤溶酶原链激酶活化剂复合物(APSAC)、尿激酶(UK)、组织型纤溶酶原活化剂(t-PA)等，然而现有的溶栓药物会引起纤维蛋白原及其他凝血因子降低所致的不良反应，临床表现为局部渗血、皮下片状出血，甚至颅内出血。Vascular occlusion refers to the occlusion of blood vessels due to slow blood flow, changes in blood composition, or increase in blood viscosity due to age, diet, and other factors. Vascular occlusive diseases include: thrombosis of arteries on the basis of sclerosis, arterial embolism after cardiac thrombolysis, venous thrombosis, impeded blood return after varicose veins, etc. The more common include cerebrovascular occlusion, coronary coronary occlusion, etc. Atherosclerosis, diabetes, hyperlipidemia, and hypertension can accelerate their development. The goal of treatment of vascular obstructive diseases is recanalization of blood vessels. Currently commonly used drugs include streptokinase (SK), acyl plasminogen streptokinase activator complex (APSAC), urokinase (UK), and tissue-type plasminogen. Activator (t-PA), etc. However, the existing thrombolytic drugs can cause adverse reactions caused by the reduction of fibrinogen and other coagulation factors, and the clinical manifestations are local bleeding, subcutaneous sheet bleeding, and even intracranial bleeding.
动脉粥样硬化(atherosclerosis，AS)，作为心脑血管疾病的共同病理基础，长期以来始终是心血管病研究领域的重点和热点之一，AS也是心血管疾病致残致死的主要原因。因此，如何防治AS对于心血管疾病患者的治疗及远期预后的改善至关重要。As the common pathological basis of cardiovascular and cerebrovascular diseases, atherosclerosis (AS) has long been one of the focuses and hotspots in the field of cardiovascular disease. AS is also the main cause of disability and death of cardiovascular disease. Therefore, how to prevent and treat AS is very important for the treatment of cardiovascular disease patients and the improvement of long-term prognosis.
Metrnl又称Subfatin、Cometin、Interleukin39，是本实验室(缪朝玉课题组)近年来发现的一个新型脂肪因子，其在皮下脂肪组织、肠道上皮组织等部位高表达，与Meteorin是同源蛋白，但表达分布与功能却大不相同。目前，关于Metrnl蛋白功能的研究较少，有限的研究表明，Metrnl在胰岛素敏感性、神经轴突生长、白色脂肪棕色化、软骨细胞分化等方面具有功能活性。国际专利公布号WO2010009732A1和中国专利公布号CN102164611A公开了Metrnl在减轻内耳听力损害方面的应用。中国专利公布号CN103536903A、CN103536904A和国际专利公布号WO2015062167A8公开了Metrnl在降血脂、降血糖中的应用。中国专利公布号CN104808004A公开了Metrnl在诊断结肠癌中的应用。但是，关于Metrnl蛋白或基因在血管堵塞性疾病中的用途目前还未见报道。Metrnl, also known as Subfatin, Cometin, Interleukin39, is a new type of adipokine discovered by our laboratory (Miao Chaoyu's research group) in recent years. It is highly expressed in subcutaneous adipose tissue, intestinal epithelial tissue and other parts. It is a homologous protein with Meteorin, but Expression distribution and function are quite different. At present, there are few studies on the function of Metrnl protein. Limited studies have shown that Metrnl is functionally active in aspects such as insulin sensitivity, axon growth, white fat browning, and chondrocyte differentiation. International Patent Publication No. WO2010009732A1 and Chinese Patent Publication No. CN102164611A disclose the application of Metrnl in reducing hearing loss in the inner ear. Chinese Patent Publication Nos. CN103536903A, CN103536904A and International Patent Publication No. WO2015062167A8 disclose the application of Metrnl in lowering blood lipids and blood glucose. Chinese Patent Publication No. CN104808004A discloses the application of Metrnl in the diagnosis of colon cancer. However, the use of Metrnl protein or gene in vascular obstructive diseases has not been reported yet.
发明内容Summary of the invention
本发明的第一个目的是针对现有技术中的不足，提供Metrnl蛋白或基因的新的治疗用途。The first object of the present invention is to provide a new therapeutic use of Metrnl protein or gene in view of the shortcomings in the prior art.
第一方面，提供了Metrnl蛋白或基因或它们的增效剂在制备抗动脉粥样硬化的药物中的应用。In a first aspect, the use of Metrnl proteins or genes or their synergists in the preparation of anti-atherosclerotic drugs is provided.
第二方面，提供了Metrnl蛋白或基因在治疗动脉粥样硬化的药物中的应用。In a second aspect, the application of Metrnl protein or gene in a drug for treating atherosclerosis is provided.
第三方面，提供了Metrnl蛋白或基因或它们的增效剂在制备药物中的应用，所述药物用于：In a third aspect, there is provided the use of a Metrnl protein or gene or their synergist in the preparation of a medicament, the medicament being used for:
a)抑制高脂饮食条件下主动脉内粥样斑块的形成和斑块面积；或a) Inhibiting the formation of atheromatous plaque and plaque area in the aorta under a high-fat diet; or
b)抑制高脂饮食条件下主动脉血管粥样硬化增厚；或b) Inhibiting thickening of aortic atherosclerosis under a high-fat diet; or
c)降低高脂饮食条件下主动脉内炎症相关因子VCAM-1、ICAM-1、TNFα、IL-6及巨噬细胞标记物分子F4/80、CD68的表达。c) Reduce the expression of inflammation-related factors VCAM-1, ICAM-1, TNFα, IL-6 and macrophage marker molecules F4 / 80, CD68 in the aorta under high-fat diet.
所述药物可采用医学领域常规的方法，将Metrnl蛋白作为活性成分，与药学上可接受的辅料制成各种剂型。当用于口服时，可将其制备成常规的固体制剂如片剂、粉剂或胶囊剂等；用于注射时，可将其制备成注射液。在各种制剂中，活性成分的重量含量为0.1～99.9％，优选的重量含量为0.5～90％。The medicament can be made into various dosage forms by using conventional methods in the medical field, using Metrnl protein as an active ingredient, and pharmaceutically acceptable excipients. When used for oral administration, it can be prepared into conventional solid preparations such as tablets, powders, or capsules; for injection, it can be prepared into injection solutions. In various preparations, the active ingredient has a weight content of 0.1 to 99.9%, and a preferred weight content is 0.5 to 90%.
所述药物可以按不同剂型通过腹腔注射、皮下注射、静脉注射、肌肉注射、***内注射、粘膜用药等途径应用于需要治疗的个体。个体可以是人或动物。剂量一般为0.01～1000mg/公斤体重/天，具体可根据个体的年龄、病情等进行变化。The medicament can be applied to individuals in need of treatment through different routes such as intraperitoneal injection, subcutaneous injection, intravenous injection, intramuscular injection, intra-lymph node injection, and mucosal medication. The individual can be a human or an animal. The dosage is generally 0.01 to 1000 mg / kg of body weight / day, which can be specifically changed according to the age and condition of the individual.
本发明的第二个目的在于，提供一种治疗动脉粥样硬化的方法。A second object of the present invention is to provide a method for treating atherosclerosis.
为实现上述目的，本发明采取的技术方案是：一种治疗动脉粥样硬化的方法，它包括将有效量的Metrnl蛋白或其增效剂给予需要受治疗的个体，所述的增效剂选自激动剂、上调剂或稳定剂。In order to achieve the above object, the technical solution adopted by the present invention is: a method for treating atherosclerosis, which comprises administering an effective amount of Metrnl protein or a synergist to an individual in need of treatment, Self-agonist, up-regulator or stabilizer.
本发明的第三个目的在于，提供Metrnl蛋白新的诊断用途。A third object of the present invention is to provide a new diagnostic application of Metrnl protein.
第一方面，提供了Metrnl蛋白或其特异性抗体的用途，用于制备检测动脉粥样硬化的诊断试剂或试剂盒。In a first aspect, the use of Metrnl protein or a specific antibody thereof is provided for preparing a diagnostic reagent or a kit for detecting atherosclerosis.
第二方面，提供了Metrnl蛋白或其特异性抗体的用途，用于制备检测动脉粥样硬化疾病程度的诊断试剂或试剂盒。In a second aspect, the use of Metrnl protein or a specific antibody thereof is provided for preparing a diagnostic reagent or a kit for detecting the degree of atherosclerotic disease.
第三方面，提供了Metrnl蛋白的用途，它被用作检测动脉粥样硬化的诊断标志物。In a third aspect, the use of Metrnl protein is provided as a diagnostic marker for detecting atherosclerosis.
本发明的第四个目的在于，提供一种检测动脉粥样硬化的方法。A fourth object of the present invention is to provide a method for detecting atherosclerosis.
为实现上述目的，本发明采取的技术方案是：一种检测动脉粥样硬化的方法，所述的方法是检测血清样品中Metrnl蛋白的含量，以此判断所述的血清样品来源的对象是否 患动脉粥样硬化。To achieve the above object, the technical solution adopted by the present invention is: a method for detecting atherosclerosis, the method is to detect the content of Metrnl protein in a serum sample, so as to determine whether the subject derived from the serum sample is suffering Atherosclerosis.
在另一优选例中，所述的检测是血清检测。In another preferred example, the test is a serum test.
在另一优选例中，所述的血清检测是ELISA法。In another preferred example, the serum detection is an ELISA method.
在另一优选例中，所述检测是以常规方法制备抗Metrnl蛋白的抗体，建立检测Metrnl蛋白的定性或定量方法，通过检测血清样品中Metrnl蛋白的含量，判断血清样品来源的对象是否患动脉粥样硬化。具体如下：首先检测待测血清样品中的Metrnl蛋白的含量，其次与正常人血清样品中Metrnl蛋白的含量进行比较，判断待测血清样品中的Metrnl蛋白的含量是否降低，根据比较结果判断待测血清样品来源的对象是否患动脉粥样硬化。In another preferred example, the detection is to prepare an antibody against Metrnl protein by a conventional method, and establish a qualitative or quantitative method for detecting Metrnl protein. By detecting the content of Metrnl protein in the serum sample, it is determined whether the subject from which the serum sample originates has an artery. Atherosclerosis. The specifics are as follows: first, measure the content of Metrnl protein in the serum sample to be tested, and then compare it with the content of Metrnl protein in the normal human serum sample to determine whether the content of Metrnl protein in the serum sample to be tested is reduced, and judge the test based on the comparison result Whether the subject of the serum sample has atherosclerosis.
本发明的第五个目的在于，提供一种检测动脉粥样硬化患者疾病程度的方法。A fifth object of the present invention is to provide a method for detecting the degree of disease in patients with atherosclerosis.
实现上述目的，本发明采取的技术方案是：一种检测动脉粥样硬化患者疾病程度的方法，所述的方法是检测患者血清样品中Metrnl蛋白的含量，以此判断所述的血清样品来源的对象动脉粥样硬化的疾病程度。To achieve the above object, the technical solution adopted by the present invention is: a method for detecting the degree of disease in atherosclerosis patients, the method is to detect the content of Metrnl protein in a patient's serum sample, thereby determining the origin of the serum sample The degree of atherosclerotic disease in the subject.
在另一优选例中，所述检测是以常规方法制备抗Metrnl蛋白的抗体，建立检测Metrnl蛋白的定性或定量方法，通过连续检测不同时间的动脉粥样硬化患者血清样品中Metrnl蛋白的含量，以此来判断血清样品来源的对象动脉粥样硬化的疾病程度。具体如下：连续多天检测动脉粥样硬化患者血清样品中的Metrnl蛋白的含量，进行比较，如果患者血清样品中的Metrnl蛋白的含量(浓度)下降，表示疾病程度加重；如果患者血清样品中的Metrnl蛋白的含量(浓度)升高，表示疾病程度减轻。In another preferred example, the detection is to prepare an antibody against Metrnl protein by a conventional method, establish a qualitative or quantitative method for detecting Metrnl protein, and continuously measure the content of Metrnl protein in serum samples of atherosclerotic patients at different times, Based on this, the degree of atherosclerotic disease in the subject from which the serum sample was derived was determined. The details are as follows: The Metrnl protein content in serum samples of patients with atherosclerosis is measured for multiple consecutive days and compared. If the Metrnl protein content (concentration) in the patient's serum sample decreases, it indicates that the disease is aggravated. An increase in the content (concentration) of Metrnl protein indicates a reduction in disease severity.
本发明优点在于：The advantages of the present invention are:
1、本发明首次通过实验发现，Metrnl可用于诊治动脉粥样硬化。1. The present invention found for the first time that Metrnl can be used to diagnose and treat atherosclerosis.
2、本发明构建Metrnl -/-ApoE -/-双敲小鼠动脉粥样硬化模型和EC-Metrnl -/-ApoE -/-小鼠动脉粥样硬化模型，实验显示：Metrnl -/-ApoE -/-双敲小鼠的主动脉较对照小鼠具有更多的白色粥样斑块，EC-Metrnl -/-ApoE -/-小鼠的主动脉较对照小鼠具有更多的白色粥样斑块；Metrnl -/-ApoE -/-双敲小鼠主动脉粥样斑块面积高于对照小鼠，EC-Metrnl -/-ApoE -/-小鼠主动脉粥样斑块面积高于对照小鼠；动脉粥样硬化小鼠主动脉根部血管发生明显病理性增厚，管腔变窄，Metrnl -/-ApoE -/-双敲小鼠的管壁增厚程度比对照组小鼠严重，EC-Metrnl -/-ApoE -/-小鼠的管壁增厚程度比对照组小鼠严重；动脉粥样硬化小鼠主动脉根部有明显的脂质斑块，Metrnl -/-ApoE -/-双敲小鼠的主动脉根部发生动粥样硬化的严重程度高于对照组小鼠，EC-Metrnl -/-ApoE -/-小鼠主动脉根部的动粥样硬化严重程度也高于对照组小鼠； EC-Metrnl -/-ApoE -/-小鼠主动脉根部的F4/80分子表达明显多于对照组小鼠，EC-Metrnl -/-ApoE -/-小鼠主动脉根部的α-SMA分子表达明显多于对照组小鼠；EC-Metrnl -/-ApoE -/-小鼠主动脉组织的促炎因子VCAM-1、ICAM-1、TNFα、IL-6及巨噬细胞标记物分子F4/80、CD68都上调，而抗炎因子IL-10、Fizz和Ym1表达水平无明显改变。 2, the present invention is constructed Metrnl - / - ApoE - / - double knockout mouse model of atherosclerosis and arterial EC-Metrnl - / - ApoE - / - mouse model of atherosclerotic arteries, experiments show: Metrnl - / - ApoE - / - double knockout mice aorta compared with the control mice had more white plaque, EC-Metrnl - / - ApoE - / - mice than in control mice aorta having more white atheroma Block; Metrnl -/- ApoE -/- double-knock mice have larger aortic plaque area than control mice, EC-Metrnl -/- ApoE -/- mice have aortic plaque area smaller than control Mice; atherosclerotic mice, the aortic root vessels have significant pathological thickening and narrowing of the lumen, and the thickness of the tube wall of Metrnl -/- ApoE -/- double knock mice is more severe than that of control mice, EC -Metrnl -/- ApoE -/- mice had a thicker wall than the control group; atherosclerotic mice had obvious lipid plaques at the root of the aorta, and Metrnl -/- ApoE -/- double knockout mice aortic root atherosclerosis occurs movable higher severity mice, EC-Metrnl - / - ApoE - / - movable severity of atherosclerosis in the aortic root of mice is also higher Murine; EC-Metrnl - / - ApoE - / - F4 / 80 molecule aortic root of mice was significantly greater than control mice, EC-Metrnl - / - ApoE - / - mice in the aortic root α-SMA The molecular expression was significantly more than that of control mice; EC-Metrnl -/- ApoE -/- mouse pro-inflammatory factors VCAM-1, ICAM-1, TNFα, IL-6 and macrophage marker molecule F4 / 80 and CD68 were up-regulated, while the expression levels of anti-inflammatory factors IL-10, Fizz and Ym1 were not significantly changed.
3、发现血管内皮细胞可分泌Metrnl，是通过经典分泌途径实现的；动脉粥样硬化时血管内皮和血液Metrnl水平下降，且随着疾病加重Metrnl水平下降程度增加。提示Metrnl的重要性，不但具有抗动脉粥样硬化的用途，而且可作为疾病诊断指标。3. It was found that Metrnl can be secreted by vascular endothelial cells, which is achieved through the classic secretion pathway; the level of vascular endothelial and blood Metrnl decreased during atherosclerosis, and the level of Metrnl decreased with the increase of disease. It is suggested that Metrnl is not only useful for anti-atherosclerosis, but also can be used as a diagnostic indicator of disease.
4、前期发现小鼠Metrnl抗血栓作用，本发明发现Metrnl重组蛋白抗血栓作用没有种属差异，小鼠Metrnl重组蛋白能够抑制大鼠和人血小板凝块收缩反应(clot retraction)，人Metrnl重组蛋白也可抑制小鼠和大鼠血小板凝块收缩反应。Metrnl重组蛋白对凝块收缩反应的抑制作用呈现量效关系，且不论人还是小鼠的Metrnl蛋白皆在低浓度(0.1μg/ml)即出现抑制作用，且随浓度增加抑制作用增强，其中Metrnl蛋白30μg/ml时凝块收缩启动时间较正常延迟1倍以上。Metrnl重组蛋白对Metrnl基因敲除小鼠血小板的凝块收缩反应也具有抑制作用。Metrnl蛋白能够抑制血小板活化，表现为血小板活化标记物CD62p水平的下降。此外，通过比较Metrnl蛋白与经典溶栓药物t-PA之间抗凝作用的特点，发现两者在血小板凝块收缩实验和全血凝固实验中的不同点，Metrnl的抗凝作用特点可减少出血不良反应。4. The anti-thrombotic effect of Metrnl in mice was found in the early stage. The present invention has found that there is no species difference in the anti-thrombotic effect of Metrnl recombinant protein. The mouse Metrnl recombinant protein can inhibit rat and human platelet clot retraction, and human Metrnl recombinant protein. Can also inhibit mouse and rat platelet clot contraction response. The inhibitory effect of Metrnl recombinant protein on the clot contraction reaction showed a dose-effect relationship, and both human and mouse Metrnl protein showed inhibitory effect at low concentration (0.1 μg / ml), and the inhibitory effect increased with increasing concentration, of which Metrnl When the protein was 30 μg / ml, the initiation time of clot contraction was more than doubled than normal. Metrnl recombinant protein also inhibited the clot contraction response of platelets in Metrnl gene knockout mice. Metrnl protein can inhibit platelet activation, which is manifested by a decrease in the level of platelet activation marker CD62p. In addition, by comparing the characteristics of anticoagulation between Metrnl protein and the classic thrombolytic drug t-PA, it is found that the differences between the two in the platelet clot contraction test and the whole blood coagulation test, the anticoagulant characteristics of Metrnl can reduce bleeding Adverse reactions.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
附图1：Metrnl -/-小鼠的培育流程和基因型鉴定。 Figure 1: Metrnl -/- mouse breeding process and genotype identification.
附图2：Metrnl -/-ApoE -/-双敲小鼠的培育流程和基因型鉴定。 Figure 2: Metrnl -/- ApoE -/- double-knock breeding process and genotype identification.
附图3：EC-Metrnl -/-小鼠的培育流程和基因型鉴定。 Figure 3: EC-Metrnl -/- mouse breeding process and genotype identification.
附图4：EC-Metrnl -/-ApoE -/-小鼠的培育流程和基因型鉴定。 Figure 4: The cultivation process and genotype identification of EC-Metrnl -/- ApoE -/- mice.
附图5：Metrnl -/-ApoE -/-双敲小鼠及其对照小鼠HFD第2.5个月时主动脉的在体形态。图中黑色箭头所示为粥样斑块的位置。 Figure 5: In vivo morphology of the aorta at 2.5 months of HFD in Metrnl -/- ApoE -/- double knock mice and their control mice. The black arrow in the figure shows the location of the atheromatous plaque.
附图6：EC-Metrnl -/-ApoE -/-小鼠及其对照小鼠HFD第5.5个月时主动脉的离体形态。图中白色箭头所示为粥样斑块的位置。 Figure 6: The aortic morphology of EC-Metrnl -/- ApoE -/- and its control mice at 5.5 months of HFD. The white arrow in the picture shows the location of the atheromatous plaque.
附图7：Metrnl -/-ApoE -/-双敲小鼠及其对照小鼠HFD第2.5个月时主动脉纵向剖面油红O染色结果。**P<0.01vs对照组小鼠。 Figure 7: Results of oil red O staining in longitudinal section of aorta at 2.5 months of HFD in Metrnl -/- ApoE -/- double knock mice and their control mice. ** P <0.01 vs control mice.
附图8：EC-Metrnl -/-ApoE -/-小鼠及其对照小鼠HFD第5.5个月时主动脉纵向剖面油红O染色结果。图A为雄性小鼠的结果，图B为雌性小鼠的结果。*P<0.05vs对照组小鼠。 Figure 8: Results of oil red O staining in longitudinal sections of the aorta at 5.5 months of HFD in EC-Metrnl -/- ApoE -/- and its control mice. Panel A shows the results of male mice, and Panel B shows the results of female mice. * P <0.05 vs control mice.
附图9：C57野生型小鼠和Metrnl -/-ApoE -/-双敲小鼠及其对照小鼠HFD第2.5个月时主动脉根部HE染色代表图。 Figure 9: Representative images of HE staining of aortic roots at 2.5 months of HFD in C57 wild-type mice and Metrnl -/- ApoE -/- double knock mice and their control mice.
附图10：EC-Metrnl -/-ApoE -/-小鼠及其对照小鼠HFD第5.5个月时主动脉根部HE染色代表图。 Figure 10: Representative diagram of aortic root HE staining in EC-Metrnl -/- ApoE -/- and its control mice at 5.5 months of HFD.
附图11：C57野生型小鼠和Metrnl -/-ApoE -/-双敲小鼠及其对照小鼠HFD第2.5个月时主动脉根部冰冻切片油红O染色结果。*P<0.05vs对照组小鼠。 Figure 11: Results of oil red O staining on frozen sections of aortic roots at 2.5 months of HFD in C57 wild-type mice and Metrnl -/- ApoE -/- double knock mice and their control mice. * P <0.05 vs control mice.
附图12：EC-Metrnl -/-ApoE -/-小鼠及其对照小鼠HFD第5.5个月时主动脉根部冰冻切片油红O染色结果。*P<0.05vs对照组小鼠。 Figure 12: Results of oil red O staining of frozen sections of aortic roots at 5.5 months of HFD in EC-Metrnl -/- ApoE -/- and its control mice. * P <0.05 vs control mice.
附图13：EC-Metrnl -/-ApoE -/-小鼠及其对照小鼠HFD第5.5个月时主动脉冰冻切片油红O染色代表图。 Figure 13: Representative diagram of oil red O staining of frozen sections of aorta at 5.5 months of HFD in EC-Metrnl -/- ApoE -/- and its control mice.
附图14：EC-Metrnl -/-ApoE -/-小鼠及其对照小鼠HFD第5.5个月时主动脉根部冰冻切片F4/80免疫组化代表图。 Figure 14: Representative diagram of immunohistochemistry of frozen section F4 / 80 of aortic root in EC-Metrnl -/- ApoE -/- and its control mice at 5.5 months of HFD.
附图15：EC-Metrnl -/-ApoE -/-小鼠及其对照小鼠HFD第5.5个月时主动脉根部冰冻切片α-SMA免疫组化代表图。 Figure 15: Representative map of α-SMA immunohistochemistry of frozen sections of aortic roots in EC-Metrnl -/- ApoE -/- mice and their control mice at 5.5 months of HFD.
附图16：EC-Metrnl -/-ApoE -/-小鼠及其对照小鼠HFD第5.5个月时主动脉炎症因子mRNA定量检测结果。*P<0.05，**P<0.01vs对照组小鼠。 Figure 16: Quantitative detection results of aortic inflammatory factor mRNA in EC-Metrnl -/- ApoE -/- and its control mice at 5.5 months of HFD. * P <0.05, ** P <0.01 vs control mice.
附图17：小鼠原代内皮细胞孵育液及其浓缩液中Metrnl的浓度。Figure 17: Metrnl concentration in mouse primary endothelial cell incubation solution and its concentrate.
附图18：人脐静脉内皮细胞经brefeldin A或eeyarestatin1处理后其孵育液中Metrnl的浓度。**P<0.01vs Vehicle对照组。Figure 18: Concentration of Metrnl in human umbilical vein endothelial cells treated with brefeldin A or eeyarestatin1. ** P <0.01 vs Vehicle Group.
附图19：EC-Metrnl -/-小鼠的血液Metrnl水平。每组n＝16-17。**P<0.01vs对照组小鼠。 Figure 19: Blood Metrnl levels of EC-Metrnl -/- mice. N = 16-17 in each group. ** P <0.01 vs control mice.
附图20：不同AS疾病程度的小鼠其主动脉和颈动脉血管形态。Figure 20: Aortic and carotid vascular morphology of mice with different degrees of AS disease.
附图21：不同AS疾病程度的小鼠其血液Metrnl水平。每组n＝4-5，*P<0.05vs C57正常小鼠, #P<0.05vs ApoE -/-小鼠。 Figure 21: Blood Metrnl levels in mice with different degrees of AS disease. N = 4-5 in each group, * P <0.05 vs C57 normal mice, # P <0.05 vs ApoE -/- mice.
附图22：Metrnl和CD31在小鼠主动脉AS斑块区与非AS斑块区的免疫荧光染色。Figure 22: Metrnl and CD31 immunofluorescence staining in AS plaque area and non-AS plaque area of mouse aorta.
附图23：动脉粥样硬化并发心梗(AS+MI)患者的血液Metrnl水平显著低于对照组。 对照组11例(男8例，女3例)，AS+MI患者组21例(男17例，女4例)。***P<0.001vs对照组患者。Figure 23: Blood Metrnl levels in patients with atherosclerosis complicated with myocardial infarction (AS + MI) are significantly lower than those in the control group. There were 11 patients in the control group (8 males and 3 females), and 21 patients in the AS + MI group (17 males and 4 females). *** P <0.001 vs control patients.
附图24：Metrnl抑制血小板凝块收缩的作用无种属差异。A.小鼠Metrnl重组蛋白对大鼠血小板凝块收缩反应的作用；B.小鼠Metrnl重组蛋白对人血小板凝块收缩反应的作用；C.人Metrnl重组蛋白对小鼠血小板凝块收缩反应的作用；D.人Metrnl重组蛋白对大鼠血小板凝块收缩反应的作用。Figure 24: There is no species difference in the effect of Metrnl in inhibiting platelet clot contraction. A. Effect of mouse Metrnl recombinant protein on rat platelet clot shrinkage; B. effect of mouse Metrnl recombinant protein on human platelet clot shrinkage; C. human Metrnl recombinant protein on mouse platelet clot shrinkage Effect; D. Effect of human Metrnl recombinant protein on contractile response of rat platelet clots.
附图25：Metrnl重组蛋白抑制血小板凝块收缩反应的量效关系。Figure 25: Dose-effect relationship of Metrnl recombinant protein inhibiting platelet clot shrinkage response.
附图26：Metrnl重组蛋白对Metrnl基因敲除小鼠血小板凝块收缩反应的抑制作用。Figure 26: Inhibition effect of Metrnl recombinant protein on platelet clot shrinkage response in Metrnl knockout mice.
附图27：Metrnl重组蛋白可降低血小板活化指标CD62p含量。每组n＝3，*P<0.05vs对照组。Figure 27: Metrnl recombinant protein can reduce platelet activation index CD62p content. Each group was n = 3, * P <0.05 vs control group.
附图28：人Metrnl重组蛋白与t-PA重组蛋白抗凝作用特点的比较。A.Metrnl和t-PA抗血小板凝块收缩实验；B.Metrnl和t-PA抗全血凝固实验；C.不同浓度t-PA抗全血凝固实验；D.Metrnl对全血凝固时间的影响，对照组n＝6，各实验组n＝3，*P<0.05vs对照组；E.t-PA对全血凝固时间无明显影响，对照组n＝7，各实验组n＝3。Figure 28: Comparison of anticoagulant characteristics of human Metrnl recombinant protein and t-PA recombinant protein. A. Metrnl and t-PA anti-platelet clot contraction experiment; B. Metrnl and t-PA anti-whole blood coagulation experiment; C. Different concentration t-PA anti-whole blood coagulation experiment; D. Effect of Metrnl on whole blood coagulation time , Control group n = 6, each experimental group n = 3, * P <0.05 vs control group; Et-PA had no significant effect on whole blood coagulation time, control group n = 7, and each experimental group n = 3.
具体实施方式detailed description
下面结合附图对本发明提供的具体实施方式做详细说明。The specific embodiments provided by the present invention will be described in detail below with reference to the drawings.
Metrnl蛋白及基因Metrnl protein and gene
本文中，所用的Metrnl蛋白可以是天然存在的，比如其可被分离纯化自哺乳动物。Herein, the Metrnl protein used may be naturally occurring, for example, it may be isolated and purified from mammals.
此外，所述Metrnl蛋白也可以是人工制备的，比如根据常规基因工程技术来制备得到。任何合适的Metrnl蛋白均可适用于本发明。所述Metrnl蛋白包括全长的Metrnl蛋白或其生物活性片段。作为一种具体实施方式，所述Metrnl蛋白可以是一种人Metrnl蛋白，大小为30kDa左右，氨基酸序列见NP_001004431.1。也可以是大鼠、小鼠等Metrnl蛋白。In addition, the Metrnl protein may also be artificially prepared, for example, according to conventional genetic engineering techniques. Any suitable Metrnl protein may be suitable for use in the present invention. The Metrnl protein includes a full-length Metrnl protein or a biologically active fragment thereof. As a specific embodiment, the Metrnl protein may be a human Metrnl protein with a size of about 30 kDa, and the amino acid sequence is shown in NP_001004431.1. Metrnl proteins such as rats and mice may also be used.
经过一个或多个氨基酸残基的取代、缺失或添加而形成的Metrnl蛋白的氨基酸序列也包括在本发明中。Metrnl蛋白或其生物活性片段包括一部分保守氨基酸的替代序列，所述氨基酸替代的序列并不影响其活性或保留了其部分活性。适当替换氨基酸是本领域内的公知技术，所述技术可以很容易地被实施并且确保不改变已知分子的生物活性。这些技术使本领域人员认识到，一般来说，在一种多肽的非必需氨基酸区域改变单个氨基酸并不会改变生物活性。The amino acid sequence of Metrnl protein formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention. The Metrnl protein or a biologically active fragment thereof includes a part of a conservative amino acid substitution sequence which does not affect its activity or retains a part of its activity. Proper substitution of amino acids is a well-known technique in the art that can be easily implemented and ensures that the biological activity of known molecules is not altered. These techniques have taught those skilled in the art that, in general, changing a single amino acid in a non-essential amino acid region of a polypeptide does not alter biological activity.
任何一种Metrnl蛋白的生物活性片段均可以应用到本发明中。在这里，Metrnl蛋白的生物活性片段的含义是指一种多肽，其仍然能保持全长的Metrnl蛋白的全部或部分功能。通常情况下，所述的生物活性片段至少保持50％、60％至99％或者100％的全长Metrnl蛋白的活性。Any biologically active fragment of Metrnl protein can be used in the present invention. Here, the meaning of the biologically active fragment of the Metrnl protein refers to a polypeptide that can still retain all or part of the function of the full-length Metrnl protein. Generally, the biologically active fragment retains at least 50%, 60% to 99%, or 100% of the activity of the full-length Metrnl protein.
本发明也可以采用经修饰或改良的全部或部分氨基酸的Metrnl蛋白，比如，可以为了促进半衰期、有效性、代谢和/或蛋白的效力而加以修饰或改良的Metrnl蛋白。所述经过修饰或改良的Metrnl蛋白可以是一种Metrnl蛋白的共轭物，或其可被取代的或人工的氨基酸。所述经过修饰或改良的Metrnl蛋白或基因可以与天然Metrnl蛋白或基因有一定的不同点，但也具有本发明所述的功能，且不会带来其它不良反应或毒性。也就是说，任何不影响Metrnl蛋白的生物活性或者说是基因的生物学功能的变化形式都可用于本发明。The present invention can also use modified or improved all or part of the amino acid Metrnl protein, for example, modified or improved Metrnl protein to promote half-life, effectiveness, metabolism and / or protein efficacy. The modified or modified Metrnl protein may be a conjugate of a Metrnl protein, or a substituted or artificial amino acid thereof. The modified or improved Metrnl protein or gene may have certain differences from the natural Metrnl protein or gene, but it also has the functions described in the present invention and does not cause other adverse reactions or toxicity. That is, any change that does not affect the biological activity of the Metrnl protein or the biological function of the gene can be used in the present invention.
Metrnl增效剂及其用途Metrnl synergist and use thereof
所述的“Metrnl增效剂”包括了激动剂、上调剂、稳定剂等，是指任何可提高Metrnl的活性、提高Metrnl的稳定性、上调Metrnl的表达、增加Metrnl有效作用时间的物质，这些物质均可用于本发明。它们可以是化合物、化学小分子、生物分子等。所述的生物分子可以是核酸水平(包括DNA、RNA)的，蛋白水平的，也可以是上调Metrnl表达的病毒产品等。The "Metrnl synergist" includes agonists, up-regulators, stabilizers, etc., and refers to any substance that can increase the activity of Metrnl, increase the stability of Metrnl, increase the expression of Metrnl, and increase the effective action time of Metrnl. Substances can be used in the present invention. They can be compounds, small chemical molecules, biomolecules, and the like. The biomolecule may be at a nucleic acid level (including DNA, RNA), at a protein level, or may be a virus product that up-regulates the expression of Metrnl.
实施例1：Metrnl基因工程小鼠动脉粥样硬化模型的构建Example 1: Construction of a atherosclerotic model of Metrnl genetically engineered mice
1.1Metrnl -/-ApoE -/-双敲小鼠动脉粥样硬化模型的构建 1.1 Construction of the Metrnl -/- ApoE -/- double knock mouse atherosclerosis model
(1)Metrnl全身性敲除小鼠(Metrnl -/-)的培育和基因型鉴定 (1) Breeding and genotyping of Metrnl systemic knockout mice (Metrnl -/- )
Metrnl全身性敲除小鼠(简称为Metrnl -/-)采用Cre-LoxP基因敲除***(Diabetes2015；64:4011-4022)，按图1A培育流程交配繁殖获得。其中Metrnl loxP/loxP来自本单位，Ella-Cre小鼠购自上海南方模式生物科技发展有限公司，C57BL/6J小鼠购自上海西普尔-必凯实验动物有限公司。基因型鉴定方法：需鉴定Ella-Cre基因和Metrnl基因。采用相应基因上下游引物分别进行鼠尾基因型鉴定，Ella-Cre基因阳性出现100bp条带；Metrnl敲除序列扩增产物为289bp，Metrnl野生序列扩增产物为150bp。如图1B所示，Metrnl +/-Ella-Cre小鼠出现100bp、150bp和289bp三条带(泳道1)；Metrnl +/-出现150bp和289bp两条带(泳道2)；Metrnl -/-全敲小鼠出现289bp一条带(泳道4)；野生对照小鼠出现150bp一条带(泳道3)。 Metrnl systemic knockout mice (Metrnl -/- for short) were obtained using the Cre-LoxP gene knockout system (Diabetes2015; 64: 4011-4022) according to the breeding process of FIG. 1A. Among them, Metrnl loxP / loxP came from this unit, Ella-Cre mice were purchased from Shanghai Nanfang Model Biotechnology Development Co., Ltd., and C57BL / 6J mice were purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd. Genotype identification method: Ella-Cre gene and Metrnl gene need to be identified. The mouse tail genotype was identified using upstream and downstream primers of the corresponding genes. Ella-Cre gene was positive with a 100bp band; the amplified product of the Metrnl knockout sequence was 289bp, and the amplified product of the Metrnl wild sequence was 150bp. As shown in Figure 1B, Metrnl +/- Ella-Cre mice showed three bands of 100bp, 150bp, and 289bp (lane 1); Metrnl +/- appeared two bands of 150bp and 289bp (lane 2); Metrnl -/- full knock The mouse showed a band of 289 bp (lane 4); the wild control mouse showed a band of 150 bp (lane 3).
(2)Metrnl -/-ApoE -/-双敲小鼠的培育和基因型鉴定 (2) Breeding and genotyping of Metrnl -/- ApoE -/- double knock mice
Metrnl -/-ApoE -/-双敲小鼠利用Metrnl -/-小鼠和ApoE -/-小鼠，按图2A培育流程交配繁殖获得，其中ApoE -/-小鼠购自上海南方模式生物科技发展有限公司。基因型鉴定方法：需鉴定Metrnl基因和ApoE基因。采用相应基因上下游引物分别进行鼠尾基因型鉴定，Metrnl敲除序列扩增产物为289bp，Metrnl野生序列扩增产物为150bp，ApoE敲除序列扩增产物为245bp，ApoE野生序列扩增产物为155bp。如图2B所示，Metrnl +/-ApoE +/-小鼠出现289bp、150bp、245bp和155bp三条带(泳道3)；Metrnl -/-ApoE -/-双敲小鼠出现289bp和245bp两条带(泳道6)；双敲小鼠的野生对照小鼠出现150bp和245bp两条带(泳道2)。 Metrnl -/- ApoE -/- double knock mice Metrnl -/- mice and ApoE -/- mice were obtained by mating and breeding according to the incubation process of Fig. 2A, of which ApoE -/- mice were purchased from Shanghai Nanfang Biotechnology Development Co., Ltd. Genotype identification method: Metrnl gene and ApoE gene need to be identified. The mouse tail genotype was identified using upstream and downstream primers of the corresponding genes. The amplified product of Metrnl knockout sequence was 289bp, the amplified product of Metrnl wild sequence was 150bp, the amplified product of ApoE knockout sequence was 245bp, and the amplified product of ApoE wild sequence was 155bp. As shown in FIG. 2B, three bands of 289bp, 150bp, 245bp, and 155bp appeared in Metrnl +/- ApoE +/- mice (lane 3); two bands of 289bp and 245bp appeared in Metrnl -/- ApoE -/- double-click mice (Lane 6); Wild control mice with double knock mice showed two bands of 150 bp and 245 bp (lane 2).
(3)利用西方高脂饮食饲料制备Metrnl -/-ApoE -/-双敲小鼠动脉粥样硬化模型 (3) Preparation of Metrnl -/- ApoE -/- double knock mouse atherosclerosis model using western high-fat diet
西方高脂饮食饲料购自上海普路腾生物科技有限公司，饲料配方为每100g饲料含：普通繁殖饲料60g，猪油16.4g，蔗糖10g，酪蛋白8.5g，胆固醇1.3g，胆盐0.3g，预混料1.6g，麦芽糊精1.9g。自小鼠2-3月龄起，开始将其正常普通饲料换以高脂饮食喂养(High Fat Diet,HFD)，在HFD第2.5个月时考察动脉粥样硬化各项指标。Western high-fat diet feed was purchased from Shanghai Pluton Biotechnology Co., Ltd. The feed formula was 100g of feed: 60g of common breeding feed, 16.4g of lard, 10g of sucrose, 8.5g of casein, 1.3g of cholesterol, and 0.3g of bile salt. , Premix 1.6g, Maltodextrin 1.9g. From 2-3 months of age, mice began to replace their normal diet with high-fat diet (HFD), and examined atherosclerotic indicators at 2.5 months of HFD.
1.2EC-Metrnl -/-ApoE -/-小鼠动脉粥样硬化模型的构建 1.2 Construction of EC-Metrnl -/- ApoE -/- mouse atherosclerosis model
(1)Metrnl内皮细胞特异性敲除小鼠(EC-Metrnl -/-)的培育和基因型鉴定 (1) Breeding and genotyping of Metrnl endothelial cell-specific knockout mice (EC-Metrnl -/- )
Metrnl内皮细胞特异性敲除小鼠(即Metrnl loxP/loxPTek-Cre小鼠，简称为EC-Metrnl -/-)采用Cre-LoxP基因敲除***按图3A培育流程交配繁殖获得，其中Tek-Cre小鼠购自上海南方模式生物科技发展有限公司。基因型鉴定方法：需鉴定Tek-Cre基因和Metrnl-flox基因。采用相应基因上下游引物分别进行鼠尾基因型鉴定，Tek-Cre基因阳性出现100bp条带；Metrnl-flox序列扩增产物为243bp，对应野生序列扩增产物为131bp。如图3B所示，Metrnl loxP/wtTek-Cre小鼠出现100bp、243bp和131bp(泳道1)；EC-Metrnl -/-小鼠出现100bp和243bp(泳道2)；特敲小鼠的对照小鼠出现243bp(泳道3)。 Metrnl endothelial cell-specific knockout mice (ie, Metrnl loxP / loxP Tek-Cre mice, referred to as EC-Metrnl -/- ) were obtained by mating and breeding using the Cre-LoxP gene knockout system according to FIG. 3A incubation process, where Tek- Cre mice were purchased from Shanghai Nanfang Model Biotechnology Development Co., Ltd. Genotype identification method: Tek-Cre gene and Metrnl-flox gene need to be identified. Using the upstream and downstream primers of the corresponding genes to identify rat tail genotypes, a 100bp band was positive for the Tek-Cre gene; the amplified product of the Metrnl-flox sequence was 243bp, and the corresponding amplified product of the wild sequence was 131bp. As shown in FIG. 3B, 100 bp, 243 bp, and 131 bp appeared in Metrnl loxP / wt Tek-Cre mice (lane 1); 100 bp and 243 bp appeared in EC-Metrnl -/- mice (lane 2); the control of special knockout mice was small The mouse appeared 243 bp (lane 3).
(2)EC-Metrnl -/-ApoE -/-小鼠的培育和基因型鉴定 (2) Breeding and genotyping of EC-Metrnl -/- ApoE -/- mice
EC-Metrnl -/-ApoE -/-双敲小鼠利用EC-Metrnl -/-小鼠和ApoE -/-小鼠，按图4A培育流程交配繁殖获得，基因型鉴定方法：需鉴定Tek-Cre基因、Metrnl-flox基因和ApoE基因。采用相应基因上下游引物分别进行鼠尾基因型鉴定，Tek-Cre基因阳性出现100bp条带；Metrnl-flox序列扩增产物为243bp，对应野生序列扩增产物为131bp；ApoE敲除序列扩增产物为245bp，ApoE野生序列扩增产物为155bp。如图4B所示，EC-Metrnl -/-ApoE -/- 双敲小鼠出现100bp、243bp和245bp(泳道2)，其对照小鼠出现243bp和245bp两条带(泳道3)。 EC-Metrnl -/- ApoE -/- double knock mice were obtained by mating and breeding according to the incubation flow of Figure 4A using EC-Metrnl -/- mice and ApoE -/- mice. Genotype identification method: Tek-Cre needs to be identified Gene, Metrnl-flox gene and ApoE gene. Using the upstream and downstream primers of the corresponding genes to identify rat tail genotypes, a 100bp band was positive for the Tek-Cre gene; the Metrnl-flox sequence amplification product was 243bp, and the corresponding wild sequence amplification product was 131bp; the ApoE knockout sequence amplification product It was 245bp, and the amplified ApoE wild sequence product was 155bp. As shown in FIG. 4B, EC-Metrnl -/- ApoE -/- double knock mice showed 100 bp, 243 bp, and 245 bp (lane 2), and control mice showed two bands of 243 bp and 245 bp (lane 3).
(3)利用西方高脂饮食饲料制备EC-Metrnl -/-ApoE -/-双敲小鼠动脉粥样硬化模型 (3) Preparation of EC-Metrnl -/- ApoE -/- double knock mouse atherosclerosis model using western high-fat diet feed
西方高脂饮食饲料同上。自小鼠2-3月龄起，开始将其正常普通饲料换以西方高脂饲料喂养，在HFD第5.5个月时考察动脉粥样硬化各项指标。Western high-fat diet feeds are the same as above. From 2-3 months of age, mice began to replace their normal ordinary feed with Western high-fat feed, and examined various indicators of atherosclerosis at 5.5 months of HFD.
实施例2：Metrnl抗动脉粥样硬化的实验方法和结果Example 2: Experimental methods and results of metrnl against atherosclerosis
2.1实验方法2.1 Experimental methods
(1)小鼠心脏和主动脉分离(1) Mouse heart and aorta separation
将小鼠腹腔注射1％戊巴比妥钠(100mg/kg)进行麻醉，酒精棉球擦湿其胸腹部，自胸骨柄下方依次剪开腹腔、胸腔，充分暴露心脏及其底部静脉，用1ml注射器从腔静脉取血，然后在心脏右心房剪一个小口，用10ml注射器装满预冷的1×PBS缓冲液，将针尖***心脏左室，缓慢推入PBS缓冲液进行灌流。之后剪去肺、肝、胃、脾、胰腺、肠、脂肪和生殖脏器，留心脏、主动脉和双肾，在显微镜下分离主动脉。分离时注意勿伤主动脉，先将主动脉各部脂肪去除干净，再逐一剪断主动脉与在体的连接，分离出主动脉和与其相连的心脏(保留左右心耳)。最后将心脏浸在4％多聚甲醛中固定；将主动脉浸在4％多聚甲醛中固定或者立即用液氮速冻用于提取mRNA。Mice were intraperitoneally injected with 1% sodium pentobarbital (100mg / kg) for anesthesia. The alcohol and cotton balls were used to moisten their thorax and abdomen. The abdominal cavity and thorax were cut in sequence from below the sternal handle to fully expose the heart and its bottom veins. Blood was taken from the vena cava with a syringe, then a small cut was made in the right atrium of the heart. A 10 ml syringe was filled with pre-chilled 1 × PBS buffer. Then the lungs, liver, stomach, spleen, pancreas, intestine, fat and genital organs were cut off, leaving the heart, aorta and kidneys, and the aorta was separated under a microscope. Take care not to injure the aorta when separating, first remove the fat from each part of the aorta, and then cut the aorta and the body one by one to separate the aorta and the heart connected to it (retain the left and right atrial appendages). Finally, the heart was immersed in 4% paraformaldehyde and fixed; the aorta was fixed in 4% paraformaldehyde or immediately frozen with liquid nitrogen to extract mRNA.
(2)小鼠主动脉纵向剖面粥样斑块油红O染色(2) Aortic plaque oil red O staining
油红O染液配法：先用异丙醇将油红O粉末(Sigma公司)配成0.5％油红O原液，用滤纸过滤，使用前将油红O原液和ddH 2O按照3:2体积比混匀。染色方法：当主动脉浸在4％多聚甲醛中固定24h后，将其转移至装有1×PBS缓冲液的硅胶皿中，纵向剖开主动脉，并用针将其平整地固定于硅胶平面，然后倒掉PBS缓冲液，加入油红O染液，静置15min，再倒掉油红O染液并加入70％乙醇，静置10-15min后再倒掉70％乙醇，加ddH 2O，拍照。 Oil red O dye solution preparation method: First use oil isopropyl alcohol to mix oil red O powder (Sigma) into 0.5% oil red O stock solution, filter with filter paper, and use oil red O stock solution and ddH 2 O according to 3: 2 Mix by volume. Staining method: When the aorta is immersed and fixed in 4% paraformaldehyde for 24 hours, it is transferred to a silicone dish filled with 1 × PBS buffer solution, the aorta is dissected longitudinally, and it is fixed flatly on a silicone surface with a needle. Then discard the PBS buffer, add oil red O staining solution, and let stand for 15 minutes, then drain oil red O staining solution and add 70% ethanol, leave it for 10-15 minutes, then drain 70% ethanol, add ddH 2 O, Take a picture.
(3)小鼠主动脉根部冰冻切片(3) Frozen section of mouse aortic root
当心脏浸在4％多聚甲醛中固定48h后，取出，沿左右心耳下缘将心脏的心尖部分切除，将心脏主动脉根部组织以切面平放于预冷的冰冻切片机样品头上，用冰冻包埋剂包埋组织，于-20℃冷冻30min，然后开始冰冻切片，切至主动脉根部时每张样品的切片厚度为10μm，室温放置30min后转存于-20℃。After the heart is immersed in 4% paraformaldehyde for 48 hours and fixed, remove it, remove the apical part of the heart along the left and right atrial appendages, and place the root tissue of the heart aorta on the sample surface of the pre-chilled frozen microtome. Tissues were embedded with frozen embedding agent, frozen at -20 ° C for 30 min, and then frozen sections were started. The thickness of each sample was 10 μm when cut to the root of the aorta, and stored at -20 ° C for 30 minutes at room temperature.
(4)小鼠主动脉根部冰冻切片HE染色(4) HE staining of frozen sections of mouse aortic roots
将冰冻切片从-20℃取出，于37℃烘1h，将片子依次放入：无水乙醇×1min→95％乙醇×1min→85％乙醇×1min→75％乙醇×1min→流水冲洗×5min→甩掉水滴→苏木素×5min→流水冲洗×5min→甩掉水滴→0.5％HCl-75％乙醇×3s→流水冲洗×5min→甩掉水滴→75％乙醇×1min→伊红×5min→无水乙醇×1min×4次→电吹风吹干→中性树胶封片。Remove the frozen section from -20 ° C and bake at 37 ° C for 1h. Place the pieces in order: absolute ethanol × 1min → 95% ethanol × 1min → 85% ethanol × 1min → 75% ethanol × 1min → rinsing under running water × 5min → Drop water drop → hematoxylin × 5min → running water wash × 5min → drop water drop → 0.5% HCl-75% ethanol × 3s → running water wash × 5min → drop water drop → 75% ethanol × 1min → eosin × 5min → absolute ethanol × 1min × 4 times → drying with electric hair dryer → neutral gum sealing sheet.
(5)小鼠主动脉根部冰冻切片油红O染色(5) Oil red O staining of frozen sections of mouse aortic roots
将冰冻切片从-20℃取出，于37℃烘1h，将片子依次放入：油红O染液×15min→60％乙醇×1min→ddH 2O×1min→甩掉水滴→稍晾干→甘油封片。 Remove the frozen section from -20 ° C and bake at 37 ° C for 1h. Put the pieces in order: Oil Red O dye solution × 15min → 60% ethanol × 1min → ddH 2 O × 1min → Drop off the water droplets → Slightly dry → Glycerin Cover film.
(6)小鼠主动脉根部冰冻切片免疫组化(6) Immunohistochemistry of frozen sections of mouse aortic roots
F4/80抗体购自abcam公司，α-SMA抗体和SABC试剂盒购自武汉博士德生物工程有限公司。免疫组化方法：将冰冻切片从-20℃取出，于37℃烘1h，用免疫组化笔在每张片子的组织周围画圈。向圈内加入3％H 2O 2，避光孵育×8min→ddH 2O洗×5min×3次→向圈内加入0.2％Triton X-100，5min→甩掉水滴→1×PBS缓冲液洗×5min×2次→向圈内加入5％BSA，30min→向圈内滴加适当稀释的抗体，置湿盒中4℃过夜→1×PBS缓冲液洗×5min×3次→向圈内滴加二抗，20min→1×PBS缓冲液洗×5min×3次→向圈内滴加SABC液体，20min→1×PBS缓冲液洗×5min×4次→取1ml ddH 2O至1.5mL EP管中，向里滴加ABC试剂各一滴，混匀后往圈内滴数滴，显微镜下控制反应时间(约1min)→流水冲洗×10min→苏木素×45s→流水冲洗×5min→甩掉水滴→0.5％HCl-75％乙醇×5s→流水冲洗×5min→无水乙醇×5s→二甲苯×5s→稍晾干→中性树胶封片。 The F4 / 80 antibody was purchased from abcam company, and the α-SMA antibody and SABC kit were purchased from Wuhan Baoshide Biological Engineering Co., Ltd. Immunohistochemical method: Take frozen sections from -20 ° C, bake at 37 ° C for 1h, and draw circles around the tissue of each piece with an immunohistochemical pen. Add 3% H 2 O 2 to the circle, and incubate in the dark × 8min → wash with ddH 2 O × 5min × 3 times → add 0.2% Triton X-100 to the circle, 5min → drop off the water → 1 × PBS buffer wash × 5min × 2 times → Add 5% BSA to the circle, 30min → Add appropriately diluted antibody to the circle, put in a wet box at 4 ° C overnight → 1 × PBS buffer solution wash × 5min × 3 times → drop into the circle Add secondary antibody, 20min → 1 × PBS buffer solution × 5min × 3 times → SABC liquid is added dropwise into the circle, 20min → 1 × PBS buffer solution × 5min × 4 times → 1ml ddH 2 O to 1.5mL EP tube In the middle, add one drop of ABC reagent to the inside, mix a few drops into the circle after mixing, control the reaction time under the microscope (about 1min) → running water washing × 10min → hematoxylin × 45s → running water washing × 5min → shake off the water droplet → 0.5 % HCl-75% ethanol × 5s → running water rinse × 5min → anhydrous ethanol × 5s → xylene × 5s → slightly dry → neutral gum seal.
(7)小鼠主动脉炎症因子表达检测(7) Detection of mouse aortic inflammatory factor expression
提取小鼠主动脉mRNA的试剂盒“RNeasy Micro Kit”购自QIAGEN公司。小鼠主动脉获取及冻存方法同实验方法“(1)小鼠心脏和主动脉分离”。按照试剂盒的说明操作，提取主动脉mRNA，并按照500ng/10ul的浓度将mRNA逆转成cDNA，进行QPCR扩增，采用ΔΔCт法进行定量分析。所用各引物序列如下：The kit "RNeasy Micro Kit" for extracting mouse aortic mRNA was purchased from QIAGEN. The method for obtaining and cryopreserving the mouse aorta was the same as the experimental method "(1) Mouse heart and aorta separation". Follow the instructions of the kit to extract aortic mRNA, reverse the mRNA into cDNA at a concentration of 500ng / 10ul, perform QPCR amplification, and use ΔΔCт method for quantitative analysis. The primer sequences used are as follows:
GeneGene Forward primer(5’→3’)Forward primer (5 ’→ 3’) Reverse primer(5’→3’)Reverse Primer (5 ’→ 3’)
VCAM-1VCAM-1 TGCCGGCATATACGAGTGTGATGCCGGCATATACGAGTGTGA CCCGATGGCAGGTATTACCAAGCCCGATGGCAGGTATTACCAAG
ICAM-1ICAM-1 CAATTCACACTGAATGCCAGCTCCAATTCACACTGAATGCCAGCTC CAAGCAGTCCGTCTCGTCCACAAGCAGTCCGTCTCGTCCA
TNFαTNFα AAGCCTGTAGCCCACGTCGTAAAGCCTGTAGCCCACGTCGTA GGCACCACTAGTTGGTTGTCTTTGGGCACCACTAGTTGGTTGTCTTTG
IL-6IL-6 CCACTTCACAAGTCGGAGGCTTACCACTTCACAAGTCGGAGGCTTA GCAAGTGCATCATCGTTGTTCATACGCAAGTGCATCATCGTTGTTCATAC
IL-1βIL-1β TCCAGGATGAGGACATGAGCACTCCAGGATGAGGACATGAGCAC GAACGTCACACACCAGCAGGTTAGAACGTCACACACCAGCAGGTTA
F4/80F4 / 80 GAGATTGTGGAAGCATCCGAGACGAGATTGTGGAAGCATCCGAGAC GATGACTGTACCCACATGGCTGAGATGACTGTACCCACATGGCTGA
CD68CD68 CATCAGAGCCCGAGTACAGTCTACCCATCAGAGCCCGAGTACAGTCTACC AATTCTGCGCCATGAATGTCCAATTCTGCGCCATGAATGTCC
IL-10IL-10 CTTACTGACTGGCATGAGGATCACTTACTGACTGGCATGAGGATCA GCAGCTCTAGGAGCATGTGGGCAGCTCTAGGAGCATGTGG
Fizz1Fizz1 TTGCAACTGCCTGTGCTTACTTGCAACTGCCTGTGCTTAC CAAGAAGCAGGGTAAATGGGCAAGAAGCAGGGTAAATGGG
Ym1Ym1 TTTGATGGCCTCAACCTGGATTTGATGGCCTCAACCTGGA AGTGAGTAGCAGCCTTGGAATGTCAGTGAGTAGCAGCCTTGGAATGTC
GAPDHGAPDH GTATGACTCCACTCACGGCAAAGTATGACTCCACTCACGGCAAA GGTCTCGCTCCTGGAAGATGGGTCTCGCTCCTGGAAGATG
2.2实验结果2.2 Experimental results
(1)小鼠主动脉粥样硬化造模后主动脉形态(1) Aortic morphology after mouse aortic atherosclerosis
小鼠心脏经1×PBS缓冲液充分灌流后，血管内血液被冲净，可在低倍显微镜下肉眼观察主动脉粥样斑块的分布情况。图5所示为Metrnl -/-ApoE -/-双敲小鼠及其对照小鼠在HFD第2.5个月时主动脉在体形态，可见心脏、主动脉和肌肉组织，结果显示，Metrnl -/-ApoE -/-双敲小鼠的主动脉具有更多的白色粥样斑块(图中黑色箭头所示)。图6所示为EC-Metrnl -/-ApoE -/-小鼠及其对照小鼠在HFD第5.5个月时主动脉离体形态，可见心脏和主动脉，结果显示，EC-Metrnl -/-ApoE -/-小鼠的主动脉具有更多的白色粥样斑块(图中黄色箭头所示)。 After the mouse heart was sufficiently perfused with 1 × PBS buffer, the blood in the blood vessels was washed away, and the distribution of aortic plaques could be observed with the naked eye under a low magnification microscope. Figure 5 shows the morphology of aorta in the body of Metrnl -/- ApoE -/- double knock mice and their control mice at 2.5 months of HFD. The heart, aorta and muscle tissue can be seen. The results show that Metrnl- / -The aorta of ApoE -/- double knock mice has more white atheromatous plaques (shown by the black arrows in the figure). Figure 6 shows the aortic morphology of EC-Metrnl -/- ApoE -/- and its control mice at 5.5 months of HFD. The heart and aorta can be seen. The results show that EC-Metrnl -/- The aorta of ApoE -/- mice has more white atheromatous plaques (shown by yellow arrows in the figure).
(2)小鼠主动脉纵向剖面粥样斑块油红O染色结果(2) Oil red O staining of atherosclerotic plaques in longitudinal sections of mouse aorta
油红O是一种脂溶性的呈明亮红色的染料，组织经油红O染色后，脂质沉积部位被染成红色，非脂质沉积部位不着色，因此可以反映主动脉组织粥样斑块的严重程度。如图7所示，Metrnl -/-ApoE -/-双敲小鼠在HFD第2.5个月时，其主动脉红色粥样斑块面积占整个主动脉纵向剖面面积的比例明显高于对照组。如图8所示，在HFD第5.5个月时的EC-Metrnl -/-ApoE -/-小鼠，其主动脉产生粥样斑块的面积比也高于对照组小鼠，雄鼠小鼠(图8A)和雌鼠小鼠(图8B)都得到证明。 Oil red O is a fat-soluble bright red dye. After oil red O staining, the lipid deposition site is stained red, and the non-lipid deposition site is not colored, so it can reflect the aortic atheromatous plaque. Severity. As shown in Figure 7, in the 2.5-month HFD of Metrnl -/- ApoE -/- double-click mice, the proportion of the area of the red aortic plaque in the longitudinal section of the entire aorta was significantly higher than that in the control group. As shown in Figure 8, the ratio of the area of atheromatous plaques in the aorta of EC-Metrnl -/- ApoE -/- mice at 5.5 months of HFD was also higher than that of control mice and male mice. (Figure 8A) and female mice (Figure 8B) were demonstrated.
(3)小鼠主动脉根部冰冻切片HE染色结果(3) HE staining results of frozen sections of mouse aortic roots
HE染色用于观察组织的形态。图9所示为HFD第2.5个月时C57、Metrnl -/-ApoE -/-双敲小鼠及其对照小鼠主动脉根部的HE染色结果。可发现，未发生动脉粥样硬化的C57野生型小鼠其主动脉根部的血管壁很薄，而发生了动脉粥样硬化的Metrnl -/-ApoE -/-双敲小 鼠及其对照小鼠，其主动脉根部血管发生明显病理性增厚，管腔变窄，且Metrnl -/-ApoE -/-双敲小鼠的管壁增厚程度比对照组小鼠严重。图10所示为EC-Metrnl -/-ApoE -/-小鼠及其对照小鼠在HFD第5.5个月时主动脉根部HE染色结果，同样，EC-Metrnl -/-ApoE -/-小鼠的管壁增厚程度比对照组小鼠严重。 HE staining is used to observe the morphology of the tissue. Figure 9 shows the results of HE staining of the aortic roots of C57, Metrnl -/- ApoE -/- double knock mice and their control mice at 2.5 months of HFD. It was found that the vascular wall of the aortic root of C57 wild-type mice without atherosclerosis was thin, while Metrnl -/- ApoE -/- double-knocking mice and their control mice with atherosclerosis The aortic root vessels had a significant pathological thickening and narrowed lumen, and the thickness of the tube walls of Metrnl -/- ApoE -/- double-knocked mice was more severe than that of the control group. Figure 10 shows the results of HE staining of aortic roots in EC-Metrnl -/- ApoE -/- and its control mice at 5.5 months of HFD. Similarly, EC-Metrnl -/- ApoE -/- mice The tube wall thickened more severely than the control mice.
(4)小鼠主动脉及主动脉根部冰冻切片油红O染色结果(4) Oil red O staining results on frozen sections of mouse aorta and aortic root
小鼠主动脉根部冰冻切片油红O染色反映主动脉根部粥样斑块的形成情况。如图11所示，未发生动脉粥样硬化的C57野生型小鼠其主动脉根部不存在脂质沉积，而发生了动脉粥样硬化的Metrnl -/-ApoE -/-双敲小鼠及其对照小鼠，其主动脉根部有明显的脂质斑块，导致原来的旧管腔变窄形成新管腔。以管腔内红色粥样斑块的面积与旧管腔的面积比表示主动脉根部受累情况，统计发现，Metrnl -/-ApoE -/-双敲小鼠的主动脉根部发生动脉粥样硬化的严重程度高于对照组小鼠。同样，如图12所示，EC-Metrnl -/-ApoE -/-小鼠主动脉根部的动粥样硬化严重程度也高于对照组小鼠。图13所示为EC-Metrnl -/-ApoE -/-小鼠及其对照组小鼠主动脉油红O染色的结果，与主动脉根部染色结果一致。 Oil red O staining of frozen sections of mouse aortic roots reflects the formation of atherosclerotic plaques in the aortic roots. As shown in Figure 11, C57 wild-type mice without atherosclerosis did not have lipid deposits in their aortic roots, but Metrnl -/- ApoE -/- double knock mice with atherosclerosis and their In control mice, aortic roots had obvious lipid plaques, which caused the old old lumen to narrow to form new ones. The ratio of the area of the red atheroma in the lumen to the area of the old lumen was used to indicate the involvement of the aortic root. Statistics showed that atherosclerosis in the aortic root of Metrnl -/- ApoE -/- double knock mice Severity was higher than that of control mice. Similarly, as shown in Fig. 12, the severity of arteriosclerosis in the aortic root of EC-Metrnl -/- ApoE -/- mice was also higher than that in the control group. Figure 13 shows the results of aortic oil red O staining in EC-Metrnl -/- ApoE -/- mice and their control mice, which is consistent with the aortic root staining results.
(5)小鼠主动脉根部冰冻切片免疫组化结果(5) Immunohistochemical results of frozen sections of mouse aortic roots
动脉粥样硬化为一种慢性炎症反应疾病，F4/80分子作为巨噬细胞的标记物，可以用于观察动脉粥样硬化的炎性反应程度。如图14所示，EC-Metrnl -/-ApoE -/-小鼠在HFD第5.5个月时其主动脉根部的F4/80分子表达明显多于对照组小鼠。 Atherosclerosis is a chronic inflammatory disease. The F4 / 80 molecule is used as a marker of macrophages and can be used to observe the inflammatory response of atherosclerosis. As shown in Figure 14, the expression of F4 / 80 molecules in the aortic roots of EC-Metrnl -/- ApoE -/- mice at 5.5 months of HFD was significantly higher than that of control mice.
在动脉粥样硬化发生发展过程中，平滑肌细胞由血管中膜向内膜迁移，并在内膜增生，α-SMA作为平滑肌细胞的标记物，可以反映动脉粥样硬化的严重程度。如图15所示，EC-Metrnl -/-ApoE -/-小鼠在HFD第5.5个月时其主动脉根部的α-SMA分子表达明显多于对照组小鼠。 During the development of atherosclerosis, smooth muscle cells migrate from the tunica media to the intima and proliferate. Α-SMA, as a marker of smooth muscle cells, can reflect the severity of atherosclerosis. As shown in FIG. 15, the expression of α-SMA molecules in the aortic root of EC-Metrnl -/- ApoE -/- mice at 5.5 months of HFD was significantly higher than that of control mice.
(6)小鼠主动脉炎症因子表达检测(6) Detection of mouse aortic inflammatory factor expression
动脉粥样硬化作为一种慢性炎症反应疾病，其发生发展过程中会出现很多炎性因子表达水平的改变。如图16所示，EC-Metrnl -/-ApoE -/-小鼠在HFD第5.5个月时，其主动脉组织的促炎因子VCAM-1、ICAM-1、TNFα、IL-6及巨噬细胞标记物分子F4/80、CD68都上调，IL-1β有上调趋势但无统计学意义，而抗炎因子IL-10、Fizz和Ym1表达水平无明显改变。 Atherosclerosis, as a chronic inflammatory disease, changes in the expression levels of many inflammatory factors during its development. As shown in Figure 16, EC-Metrnl -/- ApoE -/- mice had pro-inflammatory factors VCAM-1, ICAM-1, TNFα, IL-6 and macrophages in aortic tissues at 5.5 months of HFD Cell marker molecules F4 / 80 and CD68 were all up-regulated, IL-1β was up-regulated but not statistically significant, and the expression levels of anti-inflammatory factors IL-10, Fizz and Ym1 were not significantly changed.
实施例3：内皮细胞可分泌Metrnl，是通过经典分泌途径实现的Example 3: Endothelial cells can secrete Metrnl, which is achieved through the classical secretion pathway
3.1实验方法3.1 Experimental methods
(1)小鼠原代内皮细胞孵育液及其浓缩液搜集(1) Collection of mouse primary endothelial cell incubation solution and its concentrated solution
待细胞生长至约90％融合时，用1×PBS缓冲液将细胞轻柔漂洗3遍，加入不含血清的内皮培养基，置于细胞培养箱中培养12h。将细胞孵育液吸至15ml离心管中，离心：室温×1200rpm×5min，所得上清液即为细胞孵育液原液。取2ml孵育液原液加入至超滤离心管中，离心：室温×4000rpm×20min，浓缩管中液体(约0.7ml)即为孵育液的浓缩液。以无血清培养基为对照处理组。分装保存于-80℃。When the cells grow to about 90% confluence, the cells are gently rinsed 3 times with 1 × PBS buffer, serum-free endothelial medium is added, and the cells are cultured in a cell incubator for 12 hours. The cell incubation solution was aspirated into a 15 ml centrifuge tube, and centrifuged: room temperature × 1200 rpm × 5 min, and the obtained supernatant was the cell incubation solution. Take 2ml of the original incubation solution into an ultrafiltration centrifuge tube, centrifuge: room temperature × 4000rpm × 20min, the liquid in the concentration tube (about 0.7ml) is the concentrated solution of the incubation solution. Serum-free medium was used as a control treatment group. Store separately at -80 ° C.
(2)人脐静脉内皮细胞加药处理及孵育液搜集(2) Dosing and collection of human umbilical vein endothelial cells
待细胞生长至约90％融合时，用1×PBS缓冲液将细胞轻柔漂洗2遍。加入含有药物(brefeldin A或eeyarestatin1，终浓度各为5μg/ml)的无血清培养基，置于细胞培养箱中孵育24h。将孵育液吸至15mL离心管中，离心：室温×1200rpm×5min，吸取上清液。以无血清培养基为对照处理组。分装保存于-80℃。When the cells grew to about 90% confluence, the cells were gently rinsed twice with 1 × PBS buffer. Serum-free medium containing drugs (brefeldin A or eeyarestatin1, each with a final concentration of 5 μg / ml) was added and placed in a cell culture incubator for 24 h. Pipet the incubation solution into a 15 mL centrifuge tube, centrifuge: room temperature × 1200 rpm × 5 min, and suck the supernatant. Serum-free medium was used as a control treatment group. Store separately at -80 ° C.
(3)ELISA测定Metrnl浓度(3) Determination of Metrnl concentration by ELISA
使用ELISA试剂盒(#DY6679,R&D system公司)来测定小鼠原代内皮细胞孵育液中的Metrnl水平；使用ELISA试剂盒(#DY7867-05,R&D system公司)来测定人脐静脉内皮细胞孵育液中Metrnl水平。实验皆为双复孔加样检测，操作步骤均根据试剂盒提供的说明书严格执行，测定的标准曲线均具有高度的拟合度(R 2>0.99)。 ELISA kit (# DY6679, R & D system) was used to measure Metrnl levels in mouse primary endothelial cell incubation solution; ELISA kit (# DY7867-05, R & D system) was used to determine human umbilical vein endothelial cell incubation solution Metrnl level. The experiments were performed with double duplicate wells, and the operation steps were strictly performed according to the instructions provided by the kit. The standard curves were highly fit (R 2 > 0.99).
3.2实验结果3.2 Experimental results
(1)内皮细胞Metrnl的分泌性(1) Secretion of endothelial cells Metrnl
如图17所示，Metrnl在小鼠原代内皮细胞孵育液原液及其浓缩液中都被成功检测到，且原液中Metrnl浓度约为浓缩液中浓度的1/3，与浓缩步骤相符(2ml孵育液原液浓缩至约0.7ml浓缩液)。此外，Metrnl在空白无血清培养基中的浓度低于检测限。本结果证明了内皮细胞确实可以分泌Metrnl。As shown in Figure 17, Metrnl was successfully detected in the mouse primary endothelial cell incubation stock solution and its concentrated solution, and the Metrnl concentration in the stock solution was about 1/3 of the concentration in the concentrated solution, which was consistent with the concentration step (2ml The stock solution was concentrated to about 0.7 ml of concentrate). In addition, the concentration of Metrnl in the blank serum-free medium was below the detection limit. This result demonstrates that endothelial cells can indeed secrete Metrnl.
(2)内皮细胞Metrnl的分泌途径(2) Secretion pathway of endothelial cells Metrnl
brefeldin A是高尔基体转运功能抑制剂，eeyarestatin 1是内质网运输通道抑制剂。如图18所示，使用brefeldin A或eeyarestatin 1处理内皮细胞后，几乎检测不到培养基中Metrnl，说明内皮细胞Metrnl的分泌过程能被brefeldin A和eeyarestatin1成功抑制，反映内皮细胞Metrnl的分泌途径属于经典的内质网-高尔基体(ER-Golgi)蛋白分泌途径。Brefeldin A is an inhibitor of Golgi transport function, and eeyarestatin 1 is an inhibitor of endoplasmic reticulum transport channels. As shown in Figure 18, after treating endothelial cells with brefeldin A or eeyarestatin 1, Metrnl was hardly detected in the culture medium, which indicates that the secretion process of endothelial cells Metrnl can be successfully inhibited by brefeldin A and eeyarestatin1, reflecting that the secretion pathway of endothelial cells Metrnl belongs Classical ER-Golgi protein secretion pathway.
(3)内皮细胞特异性敲除Metrnl可明显降低血液Metrnl水平，平均下降74％(3) Endothelial cell-specific knockout of Metrnl can significantly reduce blood Metrnl levels, with an average decrease of 74%
如图19所示，Metrnl内皮细胞特异性敲除小鼠(EC-Metrnl -/-)的血液Metrnl水平较 其正常对照组小鼠显著降低，平均下降74％，说明内皮细胞是血液Metrnl的主要分泌来源。 As shown in Figure 19, the blood Metrnl level of Metrnl endothelial cell-specific knockout mice (EC-Metrnl -/- ) was significantly lower than that of normal control mice, with an average decrease of 74%, indicating that endothelial cells are the main blood Metrnl Source of secretion.
实施例4：动脉粥样硬化时血管内皮和血液Metrnl水平下降，而且随着疾病加重Metrnl水平下降程度增加Example 4: Endothelial and blood Metrnl levels decrease during atherosclerosis, and Metrnl levels decrease with increasing disease
4.1实验方法4.1 Experimental methods
(1)动脉粥样硬化小鼠模型的制备(1) Preparation of a mouse model of atherosclerosis
动脉粥样硬化(atherosclerosis,AS)易感小鼠ApoE -/-小鼠购自上海南方模式生物科技发展有限公司。不同AS程度小鼠模型的制备：在同样饲养环境下，将ApoE -/-小鼠以普通饲料喂养至7月龄，随机分为两组，一组继续喂以普通饲料，另一组将普通饲料换成西方高脂饮食饲料。22周后考察小鼠(12月龄)动脉粥样硬化情况。 ApoE -/- mice susceptible to atherosclerosis (AS) were purchased from Shanghai Nanfang Model Biotechnology Development Co., Ltd. Preparation of mouse models with different AS levels: Under the same feeding environment, ApoE -/- mice were fed with ordinary feed to 7 months of age and randomly divided into two groups. One group continued to be fed with ordinary feed, and the other group was fed with ordinary feed. Feed was changed to Western high-fat diet feed. After 22 weeks, the atherosclerosis of mice (12 months old) was examined.
(2)小鼠主动脉和颈动脉的分离(2) Isolation of mouse aorta and carotid artery
小鼠主动脉的分离方法同前。小鼠颈动脉的分离在主动脉分离完成后进行，将小鼠仰卧位，沿颈部前正中切开，分离直至暴露颈总、颈内和颈外动脉，取血管分叉处附近的颈动脉。Isolation of mouse aorta was the same as before. Isolation of the mouse carotid artery was performed after the aortic separation was completed. The mice were supine, cut along the front of the neck, and separated until the common, internal and external carotid arteries were exposed. The carotid arteries near the bifurcation of the blood vessels were taken. .
(3)小鼠主动脉免疫荧光染色(3) Mouse aortic immunofluorescence staining
分离小鼠胸主动脉，并将其剪成2-3mm一段，浸于4％多聚甲醛溶液中固定至少48h，制成冰冻切片冻存于-20℃。免疫组化实验前先将冰冻切片置37℃烘2h，之后用1×PBS缓冲液将片子漂洗2次；使用柠檬酸钠抗原修复液和微波炉加热的方式对片子进行抗原修复；用免疫组化笔在组织周围画圈；将片子漂洗3次；向圈内滴加封闭血清，室温下湿盒封闭2h；甩去封闭血清，向圈内滴加稀释好的Metrnl一抗(Abcam公司，#ab121775，稀释比例1:200)或CD31一抗(Abcam公司，#ab121775，稀释比例1:200)，于4℃湿盒孵育过夜。次日将片子漂洗3次；向圈内滴加二抗，室温下湿盒避光孵育1h；将片子漂洗3次；向圈内滴加DAPI染液，室温下湿盒避光孵育3-5min；将片子漂洗3次；向圈内滴加抗荧光淬灭剂，封片，激光共聚焦显微镜下观察拍照。The mouse thoracic aorta was isolated and cut into 2-3mm sections, immersed in 4% paraformaldehyde solution and fixed for at least 48 hours, and frozen sections were made and stored at -20 ° C. Before the immunohistochemistry experiment, the frozen sections were dried at 37 ° C for 2 hours, and then the tablets were rinsed twice with 1 × PBS buffer; the tablets were antigen-recovered using sodium citrate antigen repair solution and microwave heating; immunohistochemistry was used. The pen draws a circle around the tissue; rinses the film 3 times; drops the blocking serum into the circle, and blocks the wet box at room temperature for 2h; shakes off the blocking serum, and drops the diluted Metrnl primary antibody (Abcam, # ab121775) into the circle. , Dilution ratio 1: 200) or CD31 primary antibody (Abcam, # ab121775, dilution ratio 1: 200), and incubated overnight at 4 ° C in a wet box. The next day, the film was rinsed 3 times; the secondary antibody was added dropwise to the circle, and the wet box was incubated at room temperature for 1 hour in the dark; the film was rinsed 3 times; the DAPI dye solution was added to the circle, and the wet box was incubated at room temperature for 3-5 minutes, protected from light. ; Rinse the film 3 times; add anti-fluorescent quencher to the circle, seal the slide, observe and take a picture under a laser confocal microscope.
4.2实验结果4.2 Experimental results
(1)不同AS疾病程度的小鼠其主动脉和颈动脉血管形态(1) Aortic and carotid vascular morphology in mice with different degrees of AS disease
ApoE -/-小鼠广泛用于AS疾病的临床前研究，其在正常饮食条件下即可自发AS，而HFD可加速AS的发生发展。如图20所示，未发生AS疾病的C57正常小鼠，其血管内干净无异物；正常饮食条件下自发AS的ApoE -/-小鼠(12月龄)其主动脉和颈动脉内都 出现了类白色的AS斑块；相比之下，经过22周HFD的ApoE -/-小鼠(12月龄)，其AS情况更严重，可见主动脉和颈动脉内出现大量AS斑块。 ApoE -/- mice are widely used in preclinical studies of AS diseases. They can spontaneously AS under normal dietary conditions, and HFD can accelerate the occurrence and development of AS. As shown in Fig. 20, C57 normal mice without AS disease had clean blood vessels without foreign bodies; ApoE -/- mice (12 months old) with spontaneous AS under normal diet had both aorta and carotid arteries. White AS plaques were compared; in contrast, after 22 weeks of HFD in ApoE -/- mice (12 months of age), the AS condition was more severe, showing a large number of AS plaques in the aorta and carotid arteries.
(2)不同AS疾病程度的小鼠其血液Metrnl浓度(2) Metrnl concentration in blood of mice with different levels of AS disease
取不同AS疾病程度的小鼠(如图20所示)进行血清Metrnl浓度检测，如图21所示，血清Metrnl水平随着AS疾病程度加重而下降。Mice with different levels of AS disease (as shown in FIG. 20) were tested for serum Metrnl concentration. As shown in FIG. 21, serum Metrnl levels decreased with the increase of AS disease severity.
(3)Metrnl和CD31在小鼠主动脉AS斑块区与非斑块区免疫荧光染色(3) Metrnl and CD31 immunofluorescence staining in mouse plaque and non-plaque areas
CD31分子是非常丰富的内皮细胞间连接蛋白，常作为内皮细胞分子标记物。取C57小鼠和正常饮食条件下自发AS的ApoE -/-小鼠(如图20所示)，对其胸主动脉冰冻切片进行免疫荧光染色(如图22所示)，可见CD31分子(绿色荧光)在正常C57小鼠的血管壁呈非常明显的连续表达，与Metrnl染色(红色荧光)重叠；ApoE -/-小鼠非斑块区的CD31分子在血管壁也有线性表达，提示非斑块区存在较完整的内皮细胞，同时也可检测到Metrnl在血管壁的表达减弱；相比之下，斑块区的CD31和Metrnl表达都非常少，几乎缺乏，提示斑块区内皮细胞被破坏。Metrnl是一种分泌性蛋白，该结果不仅与AS发展过程中血管内皮受损加重的特点相符，也与上述血清Metrnl水平随着AS疾病程度加重而下降的结果相一致。 CD31 molecule is a very abundant endothelial cell-to-cell junction protein, often used as a molecular marker of endothelial cells. Take C57 mice and ApoE -/- mice with spontaneous AS under normal diet (as shown in Figure 20), and perform immunofluorescence staining on frozen sections of their thoracic aorta (as shown in Figure 22). CD31 molecules (green (Fluorescence) showed very obvious continuous expression in the vascular wall of normal C57 mice, overlapping with Metrnl staining (red fluorescence); CD31 molecules in non-plaque areas of ApoE -/- mice also linearly expressed in the vascular wall, suggesting non-plaque There are relatively complete endothelial cells in the region, and at the same time, the weakened expression of Metrnl in the vascular wall can also be detected. In contrast, the expression of CD31 and Metrnl in the plaque area are very low, almost lacking, indicating that the endothelial cells in the plaque area are damaged. Metrnl is a secreted protein, and this result is not only consistent with the characteristics of exacerbation of vascular endothelium during the development of AS, but also consistent with the above-mentioned decrease in serum Metrnl levels with the increase in AS disease severity.
(4)AS疾病并发心肌梗死患者的血液Metrnl水平也明显下降(4) The blood Metrnl level of patients with AS disease complicated by myocardial infarction also decreased significantly.
根据病历、心电图、生化指标、血管影像学等诊断AS合并心梗患者，采血、抗凝、离心，取血浆测定，如图23所示，AS并发心梗患者血液Metrnl水平显著低于对照组患者。Diagnose AS patients with myocardial infarction according to medical records, ECG, biochemical indicators, and vascular imaging. Blood collection, anticoagulation, centrifugation, and plasma measurement are performed. As shown in Figure 23, the blood Metrnl level in AS patients with myocardial infarction is significantly lower than that in the control group. .
上述实验证实血管内皮细胞可分泌Metrnl，是通过经典分泌途径实现的；动脉粥样硬化时血管内皮和血液Metrnl水平下降，且随着疾病加重Metrnl水平下降程度增加。提示Metrnl的重要性，不但具有抗动脉粥样硬化的用途，而且可作为疾病诊断指标。The above experiments confirmed that Metrnl can be secreted by vascular endothelial cells, which is achieved through the classic secretion pathway; the level of vascular endothelial and blood Metrnl decreases during atherosclerosis, and the level of Metrnl decreases with the increase of disease. It is suggested that Metrnl is not only useful for anti-atherosclerosis, but also can be used as a diagnostic indicator of disease.
实施例5：Metrnl重组蛋白抑制血小板凝块收缩反应无种属差异Example 5: Metrnl recombinant protein inhibits platelet clot contraction response without species differences
5.1实验方法5.1 Experimental methods
(1)血小板悬液制备(1) Preparation of platelet suspension
小鼠血小板悬液制备：腹腔注射10％水合氯醛(300mg/kg)对小鼠进行麻醉，取1ml注射器用2％枸橼酸钠抗凝剂湿润注射器内壁，同时预留100μl抗凝剂于注射器内。小鼠麻倒后固定其四肢，沿腹正中线将其腹部打开(避免损伤到血管)，小心拨开内脏，暴露出腹主动脉，用带针头的1ml注射器斜45°刺进腹主动脉取血(约取到1ml)，取血完 毕后立即轻柔颠倒注射器，将动脉血和抗凝剂混匀，并转移至15ml离心管。离心(1750rpm×2min，室温)，吸取血小板层至新15ml离心管，继续往装有剩余血细胞的15ml离心管中加约300μl生理盐水，轻柔混匀，再次离心(1750rpm×30s，室温)，吸取血小板层。两次血小板层溶液混在一起，离心(2750rpm×3min，室温)，获得血小板沉淀，先将上层透明PPP(Poor Platelet Plasma)溶液转移至新的1.5ml离心管中于37℃孵育用于后续实验，之后继续向15ml离心管中加入适量37℃TB(Tyrode’s Buffer)溶液(含0.04U/ml Apyrase)，小心吹打重悬血小板，获得血小板悬液用于后续实验。Preparation of mouse platelet suspension: intraperitoneally inject 10% chloral hydrate (300mg / kg) to anesthetize the mice, take a 1ml syringe and wet the inner wall of the syringe with 2% sodium citrate anticoagulant, and reserve 100 μl of anticoagulant in Inside the syringe. After the mouse was numbed, its limbs were fixed, and its abdomen was opened along the midline of the abdomen (to avoid damage to the blood vessels). Carefully poke out the internal organs to expose the abdominal aorta. Use a 1ml syringe with a needle to pierce the abdominal aorta at an angle of 45 ° Blood (about 1ml), immediately after the blood collection, gently invert the syringe, mix the arterial blood and the anticoagulant, and transfer to a 15ml centrifuge tube. Centrifuge (1750rpm × 2min, room temperature), draw the platelet layer into a new 15ml centrifuge tube, continue to add about 300μl physiological saline to the 15ml centrifuge tube containing the remaining blood cells, gently mix, and centrifuge again (1750rpm × 30s, room temperature). Platelet layer. The two platelet layer solutions were mixed together and centrifuged (2750 rpm × 3min, room temperature) to obtain platelet precipitation. The upper transparent PPP (Poor Platelet Plasma) solution was first transferred to a new 1.5 ml centrifuge tube and incubated at 37 ° C for subsequent experiments. Then continue to add an appropriate amount of 37 ° C TB (Tyrode's Buffer) solution (containing 0.04U / ml Apyrase) to a 15ml centrifuge tube, and carefully resuspend the platelets by pipetting to obtain a platelet suspension for subsequent experiments.
大鼠血小板悬液制备：腹腔注射10％水合氯醛(350mg/kg)对大鼠进行麻醉，取10ml注射器用3.8％枸橼酸钠抗凝剂湿润注射器内壁，同时预留1ml抗凝剂于注射器内。大鼠麻倒后将其仰卧位，沿腹正中线将其腹部打开(避免损伤血管)，小心拨开内脏，暴露出腹主动脉，用带针头的10ml注射器斜45°刺进腹主动脉取血(约取到10ml)，取血完毕后立即轻柔颠倒注射器，使血液与抗凝剂混匀，并转移至15ml离心管中。离心(1750rpm×10min，室温)，吸取血小板层至新的15ml离心管，离心(2750rpm×5min，室温)，获得血小板沉淀，先将上层透明PPP溶液转移至新的1.5ml离心管中于37℃孵育用于后续实验，之后继续往15ml离心管中加入适量37℃TB溶液(含0.04U/ml Apyrase)，小心吹打重悬血小板，获得血小板悬液用于后续实验。Preparation of rat platelet suspension: intraperitoneally inject 10% chloral hydrate (350mg / kg) to anaesthetize the rats, take a 10ml syringe and wet the inner wall of the syringe with 3.8% sodium citrate anticoagulant, and reserve 1ml of anticoagulant in Inside the syringe. After the rat is anesthetized, place it in the supine position, open its abdomen along the midline of the abdomen (to avoid damaging blood vessels), carefully open the internal organs, and expose the abdominal aorta. Use a 10ml syringe with a needle to pierce the abdominal aorta at an angle of 45 ° Blood (about 10ml), immediately after taking the blood, gently invert the syringe, mix the blood with the anticoagulant, and transfer to a 15ml centrifuge tube. Centrifuge (1750rpm × 10min, room temperature), draw the platelet layer to a new 15ml centrifuge tube, centrifuge (2750rpm × 5min, room temperature) to obtain platelet precipitation, first transfer the upper transparent PPP solution to a new 1.5ml centrifuge tube at 37 ° C Incubate for subsequent experiments, and then continue to add an appropriate amount of 37 ° C TB solution (containing 0.04U / ml Apyrase) to a 15ml centrifuge tube, and carefully resuspend the platelets to obtain platelet suspensions for subsequent experiments.
人血小板悬液制备：由医院护士采集志愿者肘部静脉血液，收集至加有ACD抗凝剂的真空采血管中，立即将血液与抗凝剂混匀，转移至15ml离心管。离心(1750rpm×4min，室温)，吸取血小板层至新的15ml离心管，离心(2550rpm×2.5min，室温)，获得血小板沉淀，先将上层透明PPP溶液转移至新的1.5ml离心管中于37℃孵育用于后续实验，之后继续往15ml离心管中加入适量37℃TB溶液(含0.04U/ml Apyrase)，小心吹打重悬血小板，获得血小板悬液用于后续实验。Human platelet suspension preparation: Volunteer elbow vein blood was collected by a hospital nurse, collected into a vacuum blood collection tube with ACD anticoagulant, and the blood was immediately mixed with the anticoagulant and transferred to a 15 ml centrifuge tube. Centrifuge (1750 rpm × 4min, room temperature), aspirate the platelet layer to a new 15ml centrifuge tube, centrifuge (2550rpm × 2.5min, room temperature) to obtain platelet precipitation, first transfer the upper transparent PPP solution to a new 1.5ml centrifuge tube at 37 Incubate at ℃ for subsequent experiments, and then continue to add an appropriate amount of 37 ° C TB solution (containing 0.04U / ml Apyrase) to a 15ml centrifuge tube, and carefully resuspend the platelets by pipetting to obtain platelet suspensions for subsequent experiments.
(2)药物孵育(2) Drug incubation
向血小板悬液(250μl)中加入Metrnl重组蛋白溶液或等体积对照溶剂，混匀后于37℃孵育15min用于实验。人和小鼠Metrnl重组蛋白均购自R&D Systems公司，加至血小板悬液后终浓度均为1.923μg/ml。Metrnl recombinant protein solution or an equal volume of a control solvent was added to the platelet suspension (250 μl), and the mixture was incubated at 37 ° C. for 15 minutes for experiments. Both human and mouse Metrnl recombinant proteins were purchased from R & D Systems, and the final concentration after adding to platelet suspension was 1.923 μg / ml.
(3)凝块收缩实验(3) clot shrinkage test
向血小板悬液中依次加入10μl自体PPP溶液、2.5μl 100mM CaCl 2溶液、1μl 10U/ml thrombin，轻柔混匀，转移至透明玻璃管中，静置观察悬液收缩情况，实时拍照。 10 μl of autologous PPP solution, 2.5 μl of 100 mM CaCl 2 solution, and 1 μl of 10 U / ml thrombin were added to the platelet suspension in order, mixed gently, transferred to a transparent glass tube, and the suspension was allowed to stand to observe the shrinkage of the suspension. Real-time photographing was performed.
5.2实验结果5.2 Experimental results
如图24所示，小鼠Metrnl重组蛋白能够明显抑制大鼠(图24A)和人(图24B)的血小板凝块收缩速度；同样，人Metrnl重组蛋白亦能够明显抑制小鼠(图24C)和大鼠(图24D)的血小板凝块收缩速度。说明Metrnl重组蛋白对血小板凝块收缩反应的抑制作用没有种属差异。实验重复3次。As shown in Figure 24, mouse Metrnl recombinant protein can significantly inhibit platelet clot contraction speed in rats (Figure 24A) and human (Figure 24B); similarly, human Metrnl recombinant protein can also significantly inhibit mice (Figure 24C) and Platelet clot contraction rate in rats (Figure 24D). This shows that there is no species difference in the inhibitory effect of Metrnl recombinant protein on platelet clot shrinkage response. The experiment was repeated three times.
实施例6：Metrnl重组蛋白抑制血小板凝块收缩反应呈现量效关系Example 6: Metrnl Recombinant Protein Inhibits Platelet Clot Shrinkage Response in a Dose-Effect Relationship
6.1实验方法6.1 Experimental method
(1)血小板悬液制备(1) Preparation of platelet suspension
小鼠血小板悬液制备同前。Mouse platelet suspensions were prepared as before.
(2)药物孵育(2) Drug incubation
向血小板悬液(250μl)中加入等体积不同浓度的Metrnl重组蛋白或对照溶剂，混匀后于37℃孵育15min用于实验。人和小鼠Metrnl重组蛋白均购自R&D Systems公司，加至血小板悬液后终浓度分别为0μg/ml、0.1μg/ml、0.3μg/ml、1μg/ml、3μg/ml、10μg/ml、30μg/ml。To the platelet suspension (250 μl), equal volumes of different concentrations of Metrnl recombinant protein or a control solvent were added, and the mixture was incubated at 37 ° C. for 15 minutes for experiments. Human and mouse Metrnl recombinant proteins were purchased from R & D Systems, and the final concentrations after being added to the platelet suspension were 0 μg / ml, 0.1 μg / ml, 0.3 μg / ml, 1 μg / ml, 3 μg / ml, 10 μg / ml, 30 μg / ml.
(3)凝块收缩实验(3) clot shrinkage experiment
同前。Cit.
6.2实验结果6.2 Experimental results
如图25所示，低剂量(0.1μg/ml)小鼠Metrnl重组蛋白即可表现出抑制血小板凝块收缩反应的作用，且这种抑制作用随着Metrnl浓度的增加(0.1～10μg/ml)而增强。继续提高Metrnl浓度，这种抑制作用继续加强，其中30μg/ml时可以观察到血小板在25min时仍未见明显凝块收缩反应，该组反应较10μg/ml组的启动时间更晚，较Vehicle组的启动时间延迟1倍以上，并且收缩反应速度降低。另外，人Metrnl重组蛋白也表现出相似的量效关系。实验各重复2次。As shown in Figure 25, low-dose (0.1 μg / ml) mice Metrnl recombinant protein can inhibit the platelet clot shrinkage response, and this inhibitory effect increases with the concentration of Metrnl (0.1-10 μg / ml) And enhanced. Continue to increase the concentration of Metrnl, and this inhibitory effect continues to increase. Among them, platelets can be observed at 30μg / ml without obvious clot contraction response at 25min. The response time of this group is later than that of the 10μg / ml group, and is shorter than that of the Vehicle group. The start-up time is delayed more than 1 time, and the contraction response speed is reduced. In addition, human Metrnl recombinant protein also showed a similar dose-effect relationship. The experiment was repeated twice each.
实施例7：Metrnl重组蛋白对Metrnl基因敲除小鼠血小板凝块收缩反应具有抑制作用Example 7: Metrnl recombinant protein has an inhibitory effect on platelet clot shrinkage in Metrnl knockout mice
7.1实验方法7.1 Experimental method
(1)血小板悬液制备(1) Preparation of platelet suspension
小鼠血小板悬液制备同前。Mouse platelet suspensions were prepared as before.
(2)药物孵育(2) Drug incubation
向血小板悬液(250μl)中加入Metrnl重组蛋白溶液或等体积对照溶剂，混匀后于37℃孵育15min用于实验。人和小鼠Metrnl重组蛋白均购自R&D Systems公司，加至血小板悬液后终浓度均为1.923μg/ml。Metrnl recombinant protein solution or an equal volume of a control solvent was added to the platelet suspension (250 μl), and the mixture was incubated at 37 ° C. for 15 minutes for experiments. Both human and mouse Metrnl recombinant proteins were purchased from R & D Systems, and the final concentration after adding to platelet suspension was 1.923 μg / ml.
(3)凝块收缩实验(3) clot shrinkage experiment
同前。Cit.
7.2实验结果7.2 Experimental results
如图26A所示，Metrnl全敲小鼠(Metrnl -/-,KO)的血小板凝块收缩反应速度明显比野生对照小鼠(WT)快，与前期观察结果一致。然而加入Metrnl重组蛋白后，KO小鼠的血小板收缩速度明显减慢，与野生对照组速度相当。类似地，如图26B所示，Metrnl内皮细胞特异性敲除小鼠(EC-Metrnl -/-)的血小板凝块收缩反应速度也比对照小鼠(WT)快，在加入Metrnl重组蛋白后，EC-Metrnl -/-小鼠的血小板收缩速度明显减慢，甚至慢于野生对照组。说明Metrnl重组蛋白对Metrnl基因敲除小鼠血小板凝块收缩反应具有抑制作用。实验各重复3次。 As shown in FIG. 26A, the platelet clot contraction response speed of Metrnl fully knocked mice (Metrnl -/- , KO) was significantly faster than that of wild control mice (WT), which was consistent with the previous observation results. However, after the addition of Metrnl recombinant protein, the platelet contraction speed of KO mice was significantly reduced, which was comparable to that of the wild control group. Similarly, as shown in FIG. 26B, the platelet clot contraction response speed of Metrnl endothelial cell-specific knockout mice (EC-Metrnl -/- ) is also faster than that of control mice (WT). After adding Metrnl recombinant protein, The platelet contraction speed of EC-Metrnl -/- mice was significantly slower, even slower than that of the wild control group. This indicates that Metrnl recombinant protein can inhibit the platelet clot contraction response of Metrnl gene knockout mice. The experiment was repeated 3 times each.
实施例8：Metrnl重组蛋白能够抑制血小板活化Example 8: Metrnl Recombinant Protein Can Inhibit Platelet Activation
8.1实验方法8.1 Experimental methods
(1)富含血小板血浆(Platelet Rich Plasma，PRP)制备(1) Preparation of Platelet Rich Plasma (PRP)
腹腔注射10％水合氯醛(300mg/kg)对小鼠进行麻醉，取1ml注射器用2％枸橼酸钠抗凝剂湿润注射器内壁，同时预留100μl抗凝剂在注射器内。小鼠麻倒后将其仰卧位，充分打开胸腔，暴露心脏并去除心包膜，用1ml注射器自心脏底部静脉汇合处缓慢抽取血液(约取到1ml)，取血完毕后立即轻柔颠倒注射器，使血液和抗凝剂混匀，并转移至15ml离心管中。离心(2000rpm×8min，室温)，吸取上层PRP溶液至新的1.5ml离心管中，用于后续实验。The mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg / kg). A 1 ml syringe was used to wet the inner wall of the syringe with 2% sodium citrate anticoagulant, while leaving 100 μl of the anticoagulant in the syringe. After the mouse was anesthetized, place it in the supine position, fully open the chest cavity, expose the heart and remove the pericardium. Use a 1ml syringe to slowly draw blood (about 1ml) from the confluence of veins at the bottom of the heart. Immediately after taking the blood, reverse the syringe gently. The blood and anticoagulant were mixed and transferred to a 15 ml centrifuge tube. Centrifuge (2000 rpm x 8 min, room temperature), pipette the upper PRP solution into a new 1.5 ml centrifuge tube for subsequent experiments.
(2)药物孵育(2) Drug incubation
PRP等体积分为4份，分别向其中加入等体积不同浓度的人Metrnl重组蛋白或对照溶剂，混匀后于37℃孵育15～20min，之后分别加入1.5μl 1U/ml thrombin，混匀，于37℃继续孵育5min，用于后续实验。加至PRP后的Metrnl终浓度分别为10μg/ml、30μg/ml、100μg/ml。PRP is divided into 4 equal volumes. Add equal volumes of human Metrnl recombinant protein or control solvent at different concentrations, mix and incubate at 37 ° C for 15-20 minutes, and then add 1.5 μl 1U / ml thrombin, mix, and mix. Continue to incubate at 37 ° C for 5 min for subsequent experiments. The final Metrnl concentrations after addition to PRP were 10 μg / ml, 30 μg / ml, and 100 μg / ml, respectively.
(3)ELISA检测CD62p含量(3) ELISA to detect CD62p content
使用ELISA试剂盒(上海哈灵生物科技有限公司，#Hl1006)来检测小鼠PRP中CD62p 蛋白的含量，实验均为双复孔加样检测，操作步骤根据试剂盒提供的说明书严格执行，测定的标准曲线均具有高度的拟合度(R 2>0.98)。 The ELISA kit (Shanghai Haring Biotechnology Co., Ltd., # Hl1006) was used to detect the content of CD62p protein in mouse PRP. The experiments were performed in duplicate wells. The operation steps were strictly performed according to the instructions provided in the kit. The standard curves all have a high degree of fit (R 2 > 0.98).
8.2实验结果8.2 Experimental results
如图27所示，人Metrnl重组蛋白可以抑制血小板活化，表现为在thrombin的刺激条件下，Metrnl重组蛋白能够降低PRP中血小板活化指标CD62p蛋白的浓度。本实验设置3个剂量组，每个剂量组3例，实验重复2次。As shown in FIG. 27, human Metrnl recombinant protein can inhibit platelet activation, and it is shown that under the stimulation condition of thrombin, Metrnl recombinant protein can reduce the concentration of CD62p protein, a platelet activation index in PRP. This experiment consists of 3 dose groups, with 3 cases in each dose group, and the experiment was repeated twice.
实施例9：Metrnl重组蛋白与t-PA抗凝作用特点的比较Example 9: Comparison of anticoagulant characteristics between Metrnl recombinant protein and t-PA
9.1血小板凝块收缩实验9.1 Platelet clot contraction test
9.1.1实验方法9.1.1 Experimental method
(1)血小板悬液制备(1) Preparation of platelet suspension
小鼠血小板悬液制备同前。Mouse platelet suspensions were prepared as before.
(2)药物孵育(2) Drug incubation
向血小板悬液中加入人Metrnl重组蛋白、t-PA重组蛋白或等体积对照溶剂，混匀后于37℃孵育15min用于实验。人Metrnl重组蛋白购自R&D Systems公司，t-PA重组蛋白购自novoprotein公司，加至血小板悬液后的蛋白终浓度均为1.923μg/ml。Add human Metrnl recombinant protein, t-PA recombinant protein or an equal volume of control solvent to the platelet suspension, mix and incubate at 37 ° C for 15 min for experiment. Human Metrnl recombinant protein was purchased from R & D Systems, and t-PA recombinant protein was purchased from novoprotein. The final protein concentration after added to the platelet suspension was 1.923 μg / ml.
(3)血凝块收缩实验(3) blood clot contraction test
同前。Cit.
9.1.2实验结果9.1.2 Experimental results
如图28A所示，人Metrnl重组蛋白和t-PA重组蛋白对小鼠血小板凝块收缩反应的抑制作用表现不同。其中，Metrnl重组蛋白可抑制血小板凝块收缩反应的速度；而t-PA作用下的血小板悬液不收缩成凝块而呈絮状液，即无明显凝块收缩反应。实验重复3次。As shown in FIG. 28A, the inhibitory effects of the human Metrnl recombinant protein and the t-PA recombinant protein on the platelet clot shrinkage response in mice were different. Among them, Metrnl recombinant protein can inhibit the speed of platelet clot shrinkage reaction; while the platelet suspension under t-PA does not shrink into a clot but flocculent, that is, no obvious clot shrinkage reaction. The experiment was repeated three times.
9.2全血凝固时间测定(试管法)9.2 Determination of whole blood coagulation time (test tube method)
9.2.1实验方法9.2.1 Experimental method
(1)药物准备(1) Drug preparation
向干净的玻璃试管(内径为10mm)中加入等体积10μg/ml、20μg/ml人Metrnl重组蛋白，10μg/ml t-PA重组蛋白或对照溶剂。To a clean glass test tube (with an inner diameter of 10 mm), an equal volume of 10 μg / ml, 20 μg / ml human Metrnl recombinant protein, 10 μg / ml t-PA recombinant protein, or a control solvent was added.
(2)凝血时间测定(2) Determination of clotting time
腹腔注射10％水合氯醛(300mg/kg)对小鼠进行麻醉，待小鼠麻倒后将其仰卧位，充分打开胸腔，暴露心脏并去除心包膜，用1ml注射器自心脏底部静脉汇合处抽取血 液，记录抽血所用的时间为T 1，待取血完毕后立即将血液转移至预先加有药物的玻璃试管中，37℃孵育，并立即开始计时，至计时第3min时，倾斜各玻璃试管(45°)，观察血液是否凝固，以后每隔30s将试管倾斜一次，直至试管倾倒时血液不流动为止。记录首次观察血液不流动的时间(即凝血时间)为T 2。 The mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (300mg / kg). After the mice were anesthetized, they were placed in the supine position, the chest cavity was fully opened, the heart was exposed and the pericardium was removed. A 1ml syringe was used to confluence the veins at the bottom of the heart. Blood was taken and the time taken for recording the blood was recorded as T 1. After the blood was taken, the blood was immediately transferred to a glass test tube pre-medicated, incubated at 37 ° C, and the timing was started immediately. When the time was 3 minutes, the glasses were tilted. Test tube (45 °), observe whether the blood is coagulated, and then tilt the test tube every 30s until the blood does not flow when the test tube is poured. The time when the blood was not observed for the first time (ie, the clotting time) was recorded as T 2 .
9.2.2实验结果9.2.2 Experimental results
如图28B-E所示，人Metrnl重组蛋白与t-PA重组蛋白抗小鼠全血凝固的作用特点不同：Metrnl重组蛋白可使全血凝固时间延长，但血液一旦凝固就不再发生溶解(最长观察时间为60min)；而t-PA重组蛋白对全血凝固时间无明显影响，但能在血液凝固后使其发生溶解，并保持在流动状态(最长观察时间为90min)。实验重复3次。As shown in Figure 28B-E, human Metrnl recombinant protein and t-PA recombinant protein have different characteristics against mouse whole blood coagulation: Metrnl recombinant protein can extend the whole blood coagulation time, but once the blood coagulates, it will no longer lyse ( The longest observation time is 60min); while t-PA recombinant protein has no significant effect on the whole blood coagulation time, but it can be dissolved after the blood coagulation and maintained in a flowing state (the longest observation time is 90min). The experiment was repeated three times.
上述实验证实Metrnl重组蛋白抗血栓作用没有种属差异，小鼠Metrnl重组蛋白能够抑制大鼠和人血小板凝块收缩反应(clot retraction)，人Metrnl重组蛋白也可抑制小鼠和大鼠血小板凝块收缩反应。Metrnl重组蛋白对凝块收缩反应的抑制作用呈现量效关系，且不论人还是小鼠的Metrnl蛋白在低浓度(0.1μg/ml)即出现抑制作用，随浓度增加抑制作用增强，其中Metrnl蛋白30μg/ml时凝块收缩启动时间较正常延迟1倍以上。Metrnl重组蛋白对Metrnl基因敲除小鼠血小板的凝块收缩反应也具有抑制作用。Metrnl蛋白能够抑制血小板活化，表现为血小板活化标记物CD62p水平的下降。此外，通过比较Metrnl蛋白与经典溶栓药物t-PA之间抗凝作用的特点，发现两者在血小板凝块收缩实验和全血凝固实验中的不同点，Metrnl的抗凝作用特点可减少出血不良反应。The above experiments confirmed that there is no species difference in the antithrombotic effect of Metrnl recombinant protein. Mouse Metrnl recombinant protein can inhibit clot retraction in rats and human platelets. Human Metrnl recombinant protein can also inhibit platelet clots in mice and rats. Shrink response. The inhibitory effect of Metrnl recombinant protein on clot contraction showed a dose-effect relationship, and the inhibitory effect of Metrnl protein at low concentration (0.1 μg / ml) appeared in both human and mouse, and the inhibitory effect increased with increasing concentration, of which Metrnl protein 30 μg At ml / ml, the initiation time of clot contraction was more than doubled than normal. Metrnl recombinant protein also inhibited the clot contraction response of platelets in Metrnl gene knockout mice. Metrnl protein can inhibit platelet activation, which is manifested by a decrease in the level of platelet activation marker CD62p. In addition, by comparing the characteristics of anticoagulation between Metrnl protein and the classic thrombolytic drug t-PA, it is found that the differences between the two in the platelet clot contraction test and the whole blood coagulation test, the anticoagulant characteristics of Metrnl can reduce bleeding Adverse reactions.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention. It should be noted that, for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and additions can be made, and these improvements and additions should also be considered as It is the protection scope of the present invention.

Claims (9)

  1. Metrnl蛋白或基因或它们的增效剂在制备抗动脉粥样硬化的药物中的应用，所述的增效剂选自激动剂、上调剂或稳定剂。Application of Metrnl proteins or genes or their synergists in the preparation of anti-atherosclerotic drugs, said synergists are selected from agonists, up-regulators or stabilizers.
  2. Metrnl蛋白或基因在治疗动脉粥样硬化的药物中的应用。Application of Metrnl protein or gene in medicine for treating atherosclerosis.
  3. Metrnl蛋白或基因或它们的增效剂在制备药物中的应用，其特质在于，所述的增效剂选自激动剂、上调剂或稳定剂，所述药物用于：The application of Metrnl proteins or genes or their synergists in the preparation of medicaments is characterized in that the synergists are selected from agonists, up-regulators or stabilizers, and the medicaments are used for:
    a)抑制高脂饮食条件下主动脉内粥样斑块的形成和斑块面积；或a) Inhibiting the formation of atheromatous plaque and plaque area in the aorta under a high-fat diet; or
    b)抑制高脂饮食条件下主动脉血管粥样硬化增厚；或b) Inhibiting thickening of aortic atherosclerosis under a high-fat diet; or
    c)降低高脂饮食条件下主动脉内炎症相关因子VCAM-1、ICAM-1、TNFα、IL-6及巨噬细胞标记物分子F4/80、CD68的表达。c) Reduce the expression of inflammation-related factors VCAM-1, ICAM-1, TNFα, IL-6 and macrophage marker molecules F4 / 80, CD68 in the aorta under high-fat diet.
  4. 一种治疗动脉粥样硬化的方法，其特征在于，它包括将有效量的Metrnl蛋白或其增效剂给予需要受治疗的个体，所述的增效剂选自激动剂、上调剂或稳定剂。A method for treating atherosclerosis, characterized in that it comprises administering an effective amount of Metrnl protein or a synergist to an individual in need of treatment, the synergist is selected from an agonist, an upregulator or a stabilizer .
  5. Metrnl蛋白或其特异性抗体的用途，其特征在于，用于制备检测动脉粥样硬化的诊断试剂或试剂盒。The use of Metrnl protein or a specific antibody thereof is characterized in that it is used for preparing a diagnostic reagent or a kit for detecting atherosclerosis.
  6. Metrnl蛋白或其特异性抗体的用途，其特征在于，用于制备检测动脉粥样硬化疾病程度的诊断试剂或试剂盒。The use of Metrnl protein or a specific antibody thereof is characterized in that it is used for preparing a diagnostic reagent or a kit for detecting the degree of atherosclerotic disease.
  7. Metrnl蛋白的用途，其特征在于，它被用作检测动脉粥样硬化的诊断标志物。The use of Metrnl protein is characterized in that it is used as a diagnostic marker for detecting atherosclerosis.
  8. 一种检测动脉粥样硬化的方法，其特征在于，所述的方法是检测血清样品中Metrnl蛋白的含量，以此判断所述的血清样品来源的对象是否患动脉粥样硬化。A method for detecting atherosclerosis, characterized in that the method is to detect the content of Metrnl protein in a serum sample, so as to determine whether the subject from which the serum sample is derived has atherosclerosis.
  9. 一种检测动脉粥样硬化患者疾病程度的方法，其特征在于，所述的方法是检测患者血清样品中Metrnl蛋白的含量，以此判断所述的血清样品来源的对象动脉粥样硬化的疾病程度。A method for detecting the degree of disease in atherosclerotic patients, characterized in that the method is to detect the content of Metrnl protein in a patient's serum sample, thereby determining the degree of atherosclerotic disease in the subject from which the serum sample is derived .
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