CN108210915A - Improve drug of heart disease and application thereof - Google Patents

Improve drug of heart disease and application thereof Download PDF

Info

Publication number
CN108210915A
CN108210915A CN201710467076.9A CN201710467076A CN108210915A CN 108210915 A CN108210915 A CN 108210915A CN 201710467076 A CN201710467076 A CN 201710467076A CN 108210915 A CN108210915 A CN 108210915A
Authority
CN
China
Prior art keywords
plasminogen
pro
gly
glu
thr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201710467076.9A
Other languages
Chinese (zh)
Inventor
李季男
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Life Science Research Institute Co Ltd
Original Assignee
Shenzhen Life Science Research Institute Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Life Science Research Institute Co Ltd filed Critical Shenzhen Life Science Research Institute Co Ltd
Publication of CN108210915A publication Critical patent/CN108210915A/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/484—Plasmin (3.4.21.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin

Abstract

The present invention relates to for preventing or treating the method for subject's myocardial damage, including a effective amount of plasminogen of subject is administered, wherein the subject has myocardial damage risk, suspection to have myocardial damage or suffer from myocardial damage.The invention further relates to for being used to prevent or treat the drug comprising plasminogen, pharmaceutical composition, product, the kit of subject's myocardial damage.

Description

Improve drug of heart disease and application thereof
The present invention relates to myocardial damage, heart dysfunctions caused by the treatment of heart disease, especially a variety of causes.
Background technology
Heart disease is a kind of common disease, including coronary atherosclerosis, coronary heart diseases and angina pectoris, myocardial infarction, Arrhythmia cordis, heart failure, pericarditis etc..It causes the factor of heart disease more and influences each other, such as diabetes can be because of fat Fat metabolism disorder leads to hyperlipidemia, atherosclerosis, and hyperlipidemia and atherosclerosis aggravate glycosuria in turn Disease.In this interactional relationship each other, atherosclerosis is the common pathologic basis of numerous cardiovascular and cerebrovascular diseases, It is most common disease in disease of cardiovascular system, seriously endangers human health.The occurrence and development of atherosclerosis include fat Matter invasion, platelet activation, thrombosis, inner film injury, inflammatory reaction, oxidative stress, vascular smooth muscle cells (VSMC) swash Living, Basic selective material metabolism and reconstructing blood vessel etc..Only for atherosclerosis, the imperceptible any symptom of people.Only After the artery being connected with some internal vitals is blocked, this disease can be just found.Due to the artery in the organ is obstructed Caused symptom is more apparent.For example, if heart blood supply arterial portions are obstructed, people may feel angina pectoris；But such as Fruit is blocked completely, it is possible to lead to heart disease (dead by the heart tissue for artery blood supply of being obstructed).
Diabetes are also to cause one of latency of heart disease.In diabetic, glycosylation end products (advanced glycation end products, AGEs) and generation and the progress that atherosclerotic lesion can be promoted. Glucose and protein and the non-enzymatic glycation product of lipoprotein, pass through after being combined with corresponding receptor in AGEs arterial walls Following mechanism accelerates atherosclerosis：Long term hyperglycemia can make AGEs generate increase, AGEs can modify protein, nucleic acid and Lipid makes reactive oxygen species generate increase, enhances oxidative stress, AGEs is in the generation by increasing polymorphonuclear Neutrophil NADPH oxidase activity can be increased simultaneously, promote blood vessel oxidative stress, so as to increase diabetic's painstaking effort The incidence of pipe disease.Long-term hyperglycemia state can lead to serious diabetic complication caused by diabetes, wherein just wrapping Include diabetic cardiomyopathy etc..
And on the other hand, since disorders of lipid metabolism usually merges generation with diabetes, diabetes are also known as " sugar Fat disease ".The pathogenesis of diabetes is related to B cell function damage and insulin resistance, shows as chronic hyperglycemia, and sugared generation The disorder and the often disorder of merging lipid-metabolism thanked.Lipids metabolic disturbance in diabetes mellitus have become angiocardiopathy independence it is dangerous because Element is mainly shown as that Hypertriglyceridemia, low-level HDL and LDL concentration increase.Research shows that diabetic The morbidity and mortality of angiocardiopathy are apparently higher than non-diabetic patients, and diabetes have become the independence of angiocardiopathy Risk factor.
Research shows that the morbidity and mortality of diabetic's angiocardiopathy are apparently higher than non-diabetic patients, sugar Urine disease has become the independent hazard factor of angiocardiopathy[3], in the angiocardiopathy, the incidence of atherosclerosis Occupy it is high, usually with it is diabetes complicated.
The generation of diabetic's atherosclerosis is related to many factors, but the exception of plasma lipid level is most main The factor wanted.In recent years, nephrosis and the relationship of disorders of lipid metabolism are more and more noticeable, in chronic progressive injury of kidney companion often With Anomalous lipid metablism, and hyperlipidemia can promote and aggravate the damage of kidney, other than mediating glomerular injury, between tubule It also works in matter damage.
Clinical research confirmation also has certain correlation between disorders of lipid metabolism and diabetic nephropathy.Diabetic Disorders of lipid metabolism, raised lipid deposition is in glomerular basement membrane, stimulation basilar memebrane cell Proliferation and extracellular matrix generation.It is early In 1936, Kimmelstiel and Wilson just had found in the renal arterioles of Diabetic Nephropathy patients, glomerulus and renal tubule There is a large amount of lipidosis[7].Anomalous lipid metablism makes glomerulus and renal interstitial fibrosis be that renal function progressive is caused to damage One of the major reasons[8].And disorders of lipid metabolism increases the probability that heart disease occurs for human body, such as the danger of fatty liver in itself One of evil is exactly to induce or aggravate hypertension, coronary heart disease, is easy to cause myocardial infarction and dies suddenly.
Present invention research finds that plasminogen can targetedly treat heart disease, is opened up for cardiopathic treatment Completely new thinking.
Summary of the invention
The present invention relates to following items：
On the one hand, the present invention relates to：1. it is a kind of prevent or the methods for the treatment of subject's myocardial damage, including administration by A effective amount of plasminogen of examination person, wherein the subject has myocardial damage risk, suspection to have myocardial damage or suffer from myocardium damage Wound.
The method of 2. 1, wherein the myocardial damage includes ischemic, inflammation, allergy, autoimmunity, thrombus, micro- Dyshaemia, wound, radiation injury, carbohydrate metabolism disturbance, fat metabolic disturbance myocardial injury.
The method that item is 3. 1 or 2, wherein the myocardial damage is selected from myocardial damage caused by following disease：Myocarditis, Pericarditis, hypertension, atherosclerosis, coronary heart diseases and angina pectoris, myocardial infarction, arrhythmia cordis, heart failure, shock, diffusivity blood Intravascular coagulation, microcirculation disorder, diabetes, hyperlipidemia, Artery, Vein blood bolt, fat embolism, ischemia-reperfusion, systematicness Sclerosis, systemic loupus erythematosus, coronary artery stenosis, rheumatic heart disease, mitral stenosis/incompetence, aorta petal Narrow/incompetence.
The method that item is 4. 1 or 2, wherein the myocardial damage is myocardial damage caused by ischemic heart disease.
The method that item is 5. 4, wherein the ischemic heart disease is atherosclerosis, coronary heart diseases and angina pectoris, cardiac muscle Infarct, arrhythmia cordis, heart failure, shock, disseminated intravascular coagulation, microcirculation disorder, ischemia-reperfusion, coronary artery stenosis, Mitral stenosis/incompetence, aortic stenosis/incompetence.
The method that item is 6. 1 or 2, wherein the myocardial damage is cardiac muscle caused by Artery, Vein blood bolt or fat embolism Damage.
The method that item is 7. 6, wherein the thrombus or embolism are caused by atherosclerosis.
On the other hand, the present invention relates to：A kind of methods prevented or treat subject's myocardial damage of item 8., including administration A effective amount of plasminogen protection cardiac muscular tissue of subject.
The method that item is 9. 8, wherein the plasminogen mitigates the cardiac muscle cell apoptosis caused by myocardial cell injury.
The method that item is 10. 8 or 9, wherein the plasminogen promotes the reparation of injury of myocardium.
The method of any one of 11. 8-10 of item, wherein the plasminogen mitigates the fibrosis of injury of myocardium.
The method of any one of 12. 8-11 of item, wherein the plasminogen promotes myocardial function to restore.
The method of any one of 13. 8-12 of item, wherein expansion and heart after plasminogen mitigation myocardial damage Compensatory hypertrophy.
On the other hand, the present invention relates to：A kind of methods prevented or treat the lipid myocardial damage of subject of item 14., Including a effective amount of plasminogen protection cardiac muscle of administration subject.
The method that item is 15. 14, wherein the plasminogen mitigates lipid heart tissue deposition.
The method that item is 16. 14 or 15, wherein the plasminogen promotes the reparation of injury of myocardium.
The method of any one of 17. 14-16 of item, wherein the plasminogen mitigates the fibrosis of injury of myocardium tissue.
The method of any one of 18. 14-17 of item, wherein the plasminogen mitigates the apoptosis of damaged cardiomyocytes.
The method of any one of 19. 14-18 of item, wherein the plasminogen promotes myocardial function to restore.
The method of any one of 20. 14-19 of item, wherein expansion and heart after plasminogen mitigation myocardial damage Compensatory hypertrophy.
The method of any one of 21. 14-20 of item, wherein the plasminogen passes through following one or more mitigation blood fat： It reduces serum triglyceride, low-density lipoprotein, very low density lipoprotein, serum cholesterol, improve serum high-density LP.
On the other hand, the present invention relates to：A kind of methods prevented or treat the inflammatory myocardial damage of subject of item 22., Including a effective amount of plasminogen protection cardiac muscle of administration subject.
The method that item is 23. 22, wherein the inflammation is inflammation caused by subject's autoimmunity.
The method that item is 24. 23, wherein the inflammation is systemic loupus erythematosus, systemic sclerosis, myocarditis, pericardium It is scorching.
The method of any one of 25. 22-24 of item, wherein the plasminogen promotes the reparation of injury of myocardium.
The method of any one of 26. 22-25 of item, wherein the plasminogen mitigates the fibrosis of injury of myocardium tissue.
The method of any one of 27. 22-26 of item, wherein the plasminogen mitigates the apoptosis of damaged cardiomyocytes.
The method of any one of 28. 22-27 of item, wherein the plasminogen promotes myocardial function to restore.
The method of any one of 29. 22-28 of item, wherein expansion and heart after plasminogen mitigation myocardial damage Compensatory hypertrophy.
On the other hand, the present invention relates to：A kind of 30. prevention of item or treatment subject's coronary arteriosclerotic myocardial damage Method, including administration subject a effective amount of plasminogen protection cardiac muscle.
The method that item is 31. 30, wherein the myocardial damage is caused by subject's coronary heart disease.
The method that item is 32. 31, wherein the plasminogen promotes the reparation of injury of myocardium.
The method of any one of 33. 30-32 of item, wherein the plasminogen mitigates the fibrosis of injury of myocardium tissue.
The method of any one of 34. 30-33 of item, wherein the plasminogen mitigates the apoptosis of damaged cardiomyocytes.
The method of any one of 35. 30-34 of item, wherein the plasminogen promotes myocardial function to restore.
The method of any one of 36. 30-35 of item, wherein expansion and heart after plasminogen mitigation myocardial damage Compensatory hypertrophy.
On the other hand, the present invention relates to：A kind of 37. prevention of item or treatment sub-ject initiation or concurrent cardiac muscle Damage method, including a effective amount of plasminogen protection cardiac muscle of administration subject.
The method that item is 38. 37, wherein the plasminogen promotes the reparation of injury of myocardium.
The method that item is 39. 37 or 38, wherein the plasminogen mitigates the fibrosis of injury of myocardium tissue.
The method of any one of 40. 37-39 of item, wherein the plasminogen mitigates the apoptosis of damaged cardiomyocytes.
The method of any one of 41. 37-40 of item, wherein the plasminogen promotes myocardial function to restore.
The method of any one of 42. 37-41 of item, wherein expansion and heart after plasminogen mitigation myocardial damage Compensatory hypertrophy.
On the other hand, the present invention relates to：Myocardial damage caused by a kind of 43. prevention of item or treatment subject's lipidosis Method, including be administered a effective amount of plasminogen of subject.
The method that item is 44. 43, wherein the lipidosis is due to subject's fat metabolism exception or abnormal carbohydrate metabolism Caused by caused hyperlipidemia.
On the other hand, the present invention relates to：A kind of 45. prevention of item or the initiation for the treatment of subject hyperlipidemia or adjoint kidney The method of tissue damage, including a effective amount of plasminogen of subject is administered.
On the other hand, the present invention relates to：A kind of 46. prevention of item or treatment subject's ischaemia-reperfusion cardiac muscular tissue damage The method of wound, including a effective amount of plasminogen of subject is administered.
47. according to the method for any one of claim 1-46, wherein the plasminogen can with it is one or more other Drug or treatment means are administered in combination.
The method of 48. claims 47 of item, wherein one or more other medicines include hypertension therapeutic drug, sugar The sick treatment drug of urine, treatment of atherosclerosis drug, chronic glomerulonephritis medicine, chronic pyelonephritis treatment Drug, Therapy of Nephrotic Syndrome drug, renal insufficiency treatment drug, uremia therapy drug, kidney transplantation treatment are used Drug, fatty liver treatment drug, cirrhosis treatment drug, bariatrician drug.
49. according to the method for claim 48, wherein the other medicines include：Blood lipid-lowering medicine, antiplatelet drug Object, blood-pressure drug, vasodilator drug, hypoglycemic drug, anticoagulation medicine, thrombolytic agent, hepatic, the anti-rhythm of the heart lose Normal drug, cardiac drug, diuretic, anti-infectives, antiviral drugs, immunoregulation medicament, inflammation adjust class drug, resist Tumour medicine, hormone medicine, thyroxine.
The method of 50. claims 49 of item, wherein the drug includes blood lipid-lowering medicine：Statins；Fibrates；Niacin； Cholestyramine；Clofibrate；Unrighted acid such as yishouning, lipid level and heart arteries and veins pleasure；Sodium alginate；Antiplatelet drug：Ah Take charge of a woods；Persantine；Clopidogrel；Xi Luota；Vasodilator drug：Hydralazine；Nitroglycerin and isosorbide dinitrate；Sodium nitroprussiate；α Nitre receptor blocking pharmacon such as prazosin；Alpha receptor blocking agent such as phentolamine；β draws receptor agonist such as albuterol；Captopril, according to That Puli；Nifepine, thiazolone；Willow Gamma Amino Butyric Acid, minoxidil, prostaglandin, atrial natriuretic peptide；Thrombolytic agent：Urokinase and chain Kinases；Tissue-type plasminogen activator；Single-chain Urokinase-type Plasminogen Activator；TNK- Plasminogens Agent；Anticoagulation medicine：Heparin；Enoxaparin；Nagqu heparin；Bivalirudin.
The method of any one of 51. claim 1-50 of item, wherein the plasminogen has with sequence 2,6,8,10 or 12 The sequence identity of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, and still there is fibre Molten zymogen activity.
The method of any one of 52. claim 1-51 of item, the plasminogen is on the basis of sequence 2,6,8,10 or 12 On, addition deletes and/or replaces 1-100,1-90,1-80,1-70,1-60,1-50,1-45,1-40,1-35,1-30,1- 25th, 1-20,1-15,1-10,1-5,1-4,1-3,1-2,1 amino acid, and still there is the albumen of activities of endothelial tissue plasminogen Matter.
The method of any one of 53. claim 1-52, the plasminogen be comprising activities of endothelial tissue plasminogen segment and Still there is the protein of activities of endothelial tissue plasminogen.
The method of any one of 54. claim 1-53 of item, the plasminogen are selected from Glu- plasminogens, Lys- fibrinolytics Proenzyme, Miniplasminogen, Microplasminogen, delta- plasminogens or their variant for retaining activities of endothelial tissue plasminogen.
The method of any one of 55. claim 1-54, the plasminogen for natural or synthetic human plasminogen or It still retains the variant of activities of endothelial tissue plasminogen or segment.
The method of any one of 56. claim 1-54 of item, the plasminogen is moves from primate or rodent The human plasminogen of object directly to homologue or its still retain the variant of activities of endothelial tissue plasminogen or segment.
The method of any one of 57. claim 1-56 of item, the amino acid such as sequence 2,6,8,10 or 12 of the plasminogen It is shown.
The method of any one of 58. claim 1-57 of item, wherein the plasminogen is naive plasminogen.
The method of any one of 59. claim 1-58 of item, wherein the subject is people.
The method of any one of 60. claim 1-59 of item, wherein the subject lacks or missing plasminogen.
The method of 61. claims 60 of item, wherein the shortage or missing are inborn, secondary and/or local.
On the other hand, the present invention relates to：A kind of fibrinolysins of the methods for any one of claim 1-61 of item 62. It is former.
On the other hand, the present invention relates to：A kind of 63. pharmaceutical compositions, it includes pharmaceutically acceptable supporting agent and For the plasminogen of any one of claim 1-61 the methods.
On the other hand, the present invention relates to：A kind of preventative or therapeutic agent boxes of item 64., it includes：(i) for weighing Profit requires the plasminogen and (ii) of any one of 1-61 the methods for delivering the plasminogen to the subject's Component (means).
65. kits according to claim 64 of item, wherein the component is syringe or bottle.
The kit of 66. claims 64 or 65 of item, also includes label or operation instructions, the label or operation instruction The plasminogen is administered the subject to implement any one of claim 1-46 the methods by book instruction.
On the other hand, the present invention relates to：A kind of 67. products of item, it includes：
Container containing label；With
Include the drug of (i) for the plasminogen of any one of claim 1-61 the methods or comprising plasminogen Composition, wherein the plasminogen or composition are administered the subject to implement claim 1-61 by label instruction Any one of the method.
The kit of any one of 68. claim 64-66 of item or the product of claim 67, also comprising other one Or multiple components or container, contain other drugs in the component or container.
The kit or product of 69. claims 68 of item, wherein the other drugs are selected from the group：Blood lipid-lowering medicine resists Antiplatelet drug, blood-pressure drug, vasodilator drug, hypoglycemic drug, anticoagulation medicine, thrombolytic agent, hepatic, Antiarrhythmic drug, cardiac drug, diuretic, anti-infectives, antiviral drugs, immunoregulation medicament, inflammation are adjusted Class drug, antitumor drug, hormone medicine, thyroxine.
The invention further relates to plasminogen for the purposes of the method for any one of practical matter 1-61.
The invention further relates to plasminogen in preparation for the drug of the method for any one of item 1-61, pharmaceutical composition, system Purposes in product, kit.
In some embodiments, the kit or product, should also comprising other one or more components or container Contain other drugs in component or container.In some embodiments, the other drugs are selected from the group：Blood lipid-lowering medicine resists Antiplatelet drug, blood-pressure drug, vasodilator drug, hypoglycemic drug, anticoagulation medicine, thrombolytic agent, hepatic, Antiarrhythmic drug, cardiac drug, diuretic, anti-infectives, antiviral drugs, immunoregulation medicament, inflammation are adjusted Class drug, antitumor drug, hormone medicine, thyroxine.
In some embodiments of preceding method, the plasminogen by administered either systemically or locally, preferably by with Lower approach application：Intravenously, intramuscular, subcutaneous administration fibrinolysin was treated originally.In some embodiments of preceding method, The plasminogen is applied with appropriate peptide carrier or combination of stabilizers.It is described in some embodiments of preceding method Plasminogen is with daily 0.0001- 2000mg/kg, 0.001-800mg/kg, 0.01-600mg/kg, 0.1-400mg/kg, 1- 200mg/kg, 1-100mg/kg, 10-100mg/kg (being calculated with per kilogram of body weight) or 0.0001-2000mg/cm2,0.001- 800mg/cm2、0.01-600mg/cm2、0.1-400mg/cm2、1-200mg/cm2、1- 100mg/cm2、10-100mg/cm2 The dosage of (being calculated with body surface area every square centimeter) is applied, and is preferably at least repeated once, preferably at least application daily.
The present invention clearly covers all combinations for belonging to the technical characteristic between embodiment of the present invention, and these groups Technical solution after conjunction clearly discloses in this application, just as above-mentioned technical proposal is individually and clearly disclosing. In addition, the present invention also clearly covers the combination between each embodiment and its element, the technical solution after the combination is at this It is clearly disclosed in text.
Detailed description of the invention
Fibrinolysin is the key component of plasminogen activating system system (PA systems).It is a kind of protease of wide spectrum, can Several components of hydrolyzed cellular epimatrix (ECM), gather including fibrin, gelatin, fibronectin, laminin and albumen Sugar.In addition, fibrinolysin, which can activate some metalloprotein enzyme precursors (pro-MMPs), forms active metalloproteinases (MMPs).Therefore fibrinolysin is considered as an important upstream regulation object of extracellular proteolysis effect.Fibrinolysin is by fibre The PAs that lyase original passes through two kinds of physiologicals：Tissue-type plasminogen activator (tPA) or urokinase type plasminogen activator (uPA) proteolysis is formed.Due to plasminogen, relative level is higher in blood plasma and other body fluid, conventionally PA systems The adjusting of system is mainly realized by the synthesis of PAs and activity level.The synthesis of PA system components is strictly adjusted by different factors, Such as hormone, growth factor and cell factor.In addition, also there are fibrinolysins and the specific physiological inhibitor of PAs.Fibrinolysin it is main Inhibitor is α 2- antiplasmins (α 2-antiplasmin).The activity of PAs is simultaneously by the plasminogen activator of uPA and tPA Inhibitor -1 (PAI-1) inhibits and the main plasminogen activator agent inhibitor -2 (PAI-2) for inhibiting uPA is adjusted.Certain cells Surface has the uPA specific cell surface receptors (uPAR) of direct hydrolysis activity.
Plasminogen is a single chain glycoprotein, is made of 791 amino acid, and molecular weight is about 92 kDa.Plasminogen It mainly synthesizes in liver, is largely present in extracellular fluid.Content of plasminogen is about 2 μM in blood plasma.Therefore plasminogen is group Knit a huge potential source with proteolytic activity in body fluid.There are two kinds of molecular forms for plasminogen：Glutamic acid- Plasminogen (Glu- plasminogen) and lysine-plasminogen (Lys-plasminogen).Natural secretion and uncracked The plasminogen tool of form is referred to as glutamic acid-plasminogen there are one amino terminal (N- ends) glutamic acid.However, In the presence of fibrinolysin, glutamic acid-plasminogen is hydrolyzed at Lys76-Lys77 as lysine-plasminogen.With paddy ammonia Acid-plasminogen is compared, and lysine-plasminogen has higher affinity with fibrin, and can be with higher rate quilt PAs is activated.The Arg560-Val561 peptide bonds of the plasminogen of both forms can be cut by uPA or tPA, and disulfide bond is caused to connect The formation of dichain proteins enzyme fibrinolysin connect.The amino terminus portion of plasminogen includes five homologous tricyclics, i.e., so-called Kringles, carboxy-terminal sections include protease domain.Some kringles contain mediation plasminogen and fibrin And its lysine-binding site of inhibitor α 2-AP specificity interaction.One plasminogen of latest find is the piece of 38kDa Section, is effective inhibitor of angiogenesis including kringles1-4.This segment is named as angiostatin, can pass through Several protease hydrolytic plasminogens generate.
The main substrate of fibrinolysin is fibrin, and fibrinous dissolving is the key that prevention pathologic thrombus formation. Fibrinolysin also has the substrate specificity of several components to ECM, including laminin, fibronectin, proteoglycans and gelatin, Show that fibrinolysin also plays an important role in ECM reconstructions.Indirectly, fibrinolysin can also be by converting certain protease precursors It is active protease come the other components for the ECM that degrades, including MMP-1, MMP-2, MMP-3 and MMP-9.It is thus proposed that Fibrinolysin may be an important upstream regulator of extracellular proteolysis.In addition, fibrinolysin is certain potential with activating The ability of the growth factor of form.In vitro, fibrinolysin can also hydrolyze the component of complement system and discharge chemotactic complement fragment.
" fibrinolysin " is a kind of very important enzyme being present in blood, and fibrin clot can be hydrolyzed to fiber egg White catabolite and d-dimer.
" plasminogen " is the zymogen forms of fibrinolysin, according to the sequence in swiss prot, by the day containing signal peptide Right people source plasminogen amino acid sequence (sequence 4) calculating is made of 810 amino acid, and molecular weight is about 90kD, mainly in liver The dirty middle glycoprotein that synthesizes and can recycle in blood, encodes the cDNA sequence of the amino acid sequence as shown in sequence 3.Overall length Plasminogen include seven structural domains：The Pan Apple (PAp) of serine protease domain, N-terminal positioned at C-terminal Structural domain and 5 Kringle structural domains (Kringle1-5).With reference to the sequence in swiss prot, signal peptide includes residual Base Met1-Gly19, PAp include residue Glu20-Val98, and Kringle1 includes residue Cys103-Cys181, Kringle2 packet It includes residue Glu184-Cys262, Kringle3 and includes residue Cys275-Cys352, Kringle4 includes residue Cys377- Cys454, Kringle5 include residue Cys481-Cys560.According to NCBI data, serine protease domain includes residue Val581- Arg804。
Glu- plasminogens are the plasminogens of Native full-length, are made of 791 amino acid and (do not contain 19 amino acid Signal peptide), encode the cDNA sequence of the sequence as shown in sequence 1, amino acid sequence is as shown in sequence 2.In vivo, it also deposits It is a kind of be at the 76-77 amino acids of Glu- plasminogens hydrolysis so as to formed Lys- plasminogens, such as 6 institute of sequence Show, encode the cDNA sequence of the amino acid sequence as shown in sequence 5.Delta- plasminogens (δ-plasminogen) are overall lengths Plasminogen has lacked the segment of Kringle2-Kringle5 structures, only containing Kringle1 and serine protease domain, there is text The amino acid sequence (sequence 8) for reporting delta- plasminogens is offered, encodes the cDNA sequence such as sequence 7 of the amino acid sequence. Miniplasminogen (Mini-plasminogen) is made of Kringle5 and serine protease domain, and having document report, it includes Residue Val443-Asn791 (using do not contain signal peptide Glu- plasminogen sequences Glu residues as initial amino acid), Amino acid sequence encodes the cDNA sequence of the amino acid sequence as shown in sequence 9 as shown in sequence 10.And Microplasminogen (Micro-plasminogen) only contain serine protease domain, there is its amino acid sequence of document report to include residue Ala543-Asn791 (using do not contain signal peptide Glu- plasminogen sequences Glu residues as initial amino acid), also have patent Document CN102154253A reports that its sequence includes residue Lys531-Asn791 (not contain the Glu- plasminogens of signal peptide The Glu residues of sequence are initial amino acid), this patent sequence reference patent document CN102154253A, amino acid sequence is such as Shown in sequence 12, the cDNA sequence of the amino acid sequence is encoded as shown in sequence 11.
" fibrinolysin " and " fibrinolysin ", " fibrinoclase " of the present invention is used interchangeably, and meaning is identical； " plasminogen " is used interchangeably with " plasminogen ", " plasminogen ", and meaning is identical.
In this application, content or active ratio of the meaning of the plasminogen " shortage " for plasminogen in subject's body Normal person is low, down to the normal physiological function for being enough to influence the subject；The meaning of the plasminogen " missing " is tested The content of plasminogen or activity are substantially less than normal person or even activity in person's body or expression is atomic, are only provided by external source It could maintain normal physiological function.
It will be understood by those skilled in the art that all technical solutions of plasminogen of the present invention are suitable for fibrinolysin, therefore, The technical solution that the present invention describes covers plasminogen and fibrinolysin.
In cyclic process, plasminogen uses the nonactive conformation of closing, but when being bound to thrombus or cell surface, Under the mediation of plasminogen activator (plasminogen activator, PA), it is changed into the activity in open conformation Fibrinolysin.Fibrin clot further can be hydrolyzed to fibrin degradation product (FDP) and D- dimerization by active fibrinolysin Body, and then thrombus.Wherein the PAp structural domains of plasminogen include the weight that plasminogen is maintained to be in nonactive closing conformation Determinant is wanted, and KR structural domains can then be combined with the lysine residue being present on receptor and substrate.It is known a variety of to make For the enzyme of plasminogen activator, including：Tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), Kallikrein and Hageman factor (Hageman factor (HF)) etc..
" activities of endothelial tissue plasminogen segment " refers in plasminogen protein, can be combined and played with the target sequence in substrate The active fragment of proteolysis function.Technical solution the present invention relates to plasminogen was covered with activities of endothelial tissue plasminogen segment generation For the technical solution of plasminogen.Activities of endothelial tissue plasminogen segment of the present invention is the serine protease comprising plasminogen The protein in domain, it is preferable that activities of endothelial tissue plasminogen segment of the present invention include sequence 14, with sequence 14 have at least 80%, 90%th, the protein of the amino acid sequence of 95%, 96%, 97%, 98%, 99% homology.Therefore, fibrinolytic of the present invention Proenzyme includes containing the activities of endothelial tissue plasminogen segment and still maintains the albumen of the activities of endothelial tissue plasminogen.
At present, plasminogen in blood and its activity determination method are included：To tissue plasminogen activator's activity Detection (t-PAA), Plasma Tissue-Type Plasminogen Activitor antigen detection (t-PAAg), to plasma tissue activities of endothelial tissue plasminogen Detection (plgA), the detection (plgAg) of plasma tissue plasminogen antigen, Plasma Tissue-Type Plasminogen Activitor mortifier The detection of activity, the detection of Plasma Tissue-Type Plasminogen Activitor mortifier antigen, plasma fibrin lyase-antifibrin The compound analyte detection of lyase (PAP).The detection method of most common of which is Chromogenic assay：To by inspection blood plasma in plus streptokinase (SK) and chromophoric substrate, the PLG in by inspection blood plasma are transformed into PLM, the latter acts on chromophoric substrate, then under the action of SK With spectrophotometric determination, absorbance increases directly proportional to activities of endothelial tissue plasminogen.In addition immuno-chemical method, gel electricity can also be used Swimming, immunoturbidimetry, radioimmunodiffusion etc. are measured the activities of endothelial tissue plasminogen in blood.
" ortholog thing or lineal homologue (ortholog) " refers to the homologue between different plant species, both same including albumen Source object also includes DNA homology object, also referred to as ortholog, Paralog object.It is referred specifically in different plant species by same ancestors Gene evolution and come albumen or gene.The plasminogen of the present invention includes the natural plasminogen of people, further includes from not Plasminogen ortholog thing infraspecific, that there is activities of endothelial tissue plasminogen or lineal homologue.
" conservative substitution variant " refer to one of them given amino acid residue change but do not change protein or enzyme it is whole Body conformation and function, this include but not limited to similar characteristic (as acid, alkalinity, hydrophobicity, etc.) amino acid substitution parent Amino acid in Amino Acids in Proteins sequence.Amino acid with similarity is well-known.For example, arginine, group Propylhomoserin and lysine are hydrophilic basic amino acids and can be interchanged.Equally, isoleucine is hydrophobic amino acid, then can be bright Propylhomoserin, methionine or valine are replaced.Therefore, two albumen of identity function or the similitude of amino acid sequence may not Together.For example, 70% to 99% similarity (homogeneity) based on MEGALIGN algorithms." conservative substitution variant ", which further includes, to be passed through BLAST or fasta algorithm determine polypeptide or enzyme with more than 60% amino acid identities, if can be more preferable up to more than 75%, It is preferably best up to more than 85% or even up to more than 90%, and with identical compared with natural or parent protein or enzyme Or substantially similar property or function.
" separation " plasminogen refers to the plasminogen protein for detaching and/or recycling from its natural surroundings.In some realities It applies in scheme, the plasminogen can purify (1) and extremely be more than the 90%, purity (by weight) more than 95% or more than 98%, As determined by by Lowry methods, such as more than 99% (by weight), (2) are to being enough by using rotating cup sequence analysis To homogeney, which is by making for the degree of at least 15 residues of instrument acquisition N-terminal or internal amino acid sequence or (3) With the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Coomassie blue or silver staining under reproducibility or non-reducing conditions (SDS-PAGE) it determines.The plasminogen of separation also include by biotechnology from recombinant cell prepare, and pass through to The plasminogen of few purification step separation.
Term " polypeptide ", " peptide " and " protein " is used interchangeably herein, and refers to the polymerization of the amino acid of any length Form can include the amino acid of genetic coding and non-genetic coding, chemical or biochemical modification or derivatization Amino acid and the polypeptide with modified peptide backbone.The term includes fusion protein, including but not limited to heterologous amino The fusion protein of acid sequence has heterologous and homologous leader sequences (with and without N-terminal methionine residues) fusions；Deng Deng.
It is defined as introducing notch if necessary about " amino acid sequence identity percentage (%) " with reference to peptide sequence It is candidate after realizing largest percentage sequence identity, and when any conservative replacement not being considered as a part for sequence identity The percentage of the amino acid residue identical with the amino acid residue in reference peptide sequence in sequence.To measure percent amino acid The comparison of sequence identity purpose can be realized, such as using publicly available with the various ways in the range of art technology Computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine Surely the suitable parameter of aligned sequences is used for, any algorithm needed including realizing maximum contrast to compared sequence.However, For the purposes of the present invention, amino acid sequence identity percent value is to compare computer program ALIGN-2 using sequence to generate 's.
In the case of using ALIGN-2 come comparing amino acid sequence, amino acid sequence A is given relative to given amino acid Sequence B % amino acid sequence identities (or can be expressed as having or comprising relative to, with or for given amino acid sequence Arrange the given amino acid sequence A of a certain % amino acid sequence identities of B) it is calculated as below：
Score X/Y multiplies 100
Wherein X is that scoring is the amino of identical match in the A and B of the program are compared by alignment programs ALIGN-2 The number of sour residue, and wherein Y is the sum of the amino acid residue in B.It will be appreciated that in the length and ammonia of amino acid sequence A In the case of the length of base acid sequence B is unequal, A can be not equal to B relative to A relative to the % amino acid sequence identities of B % amino acid sequence identities.Unless expressly stated otherwise, all % amino acid sequence identities values used herein are all It is according to described in the preceding paragraph, is obtained using ALIGN-2 computer programs.
As used in this article, term " treatment " and " processing ", which refer to, obtains desired pharmacology and/or physiologic effect.The effect Fruit can be complete or partial prevention disease or its symptom and/or partially or completely cure disease and/or its symptom, and wrap It includes：(a) prevention disease occurs in subject's body, and the subject can have the procatarxis of disease, but not yet be diagnosed as having There is disease；(b) inhibit disease, that is, block its formation；(c) mitigate disease and/or its symptom, that is, cause disease and/or its disease Shape subsides.
Term " individual ", " subject " and " patient " is used interchangeably herein, and refers to mammal, including but not limited to Mouse (rat, mouse), non-human primates, people, dog, cat, ungulate (such as horse, ox, sheep, pig, goat) etc..
" therapeutically effective amount " or " effective quantity " refers to be enough when applying mammal or other subjects to treat disease Realize the amount to the prevention of disease and/or the plasminogen for the treatment of." therapeutically effective amount " can be according to used fibrinolysin The disease of subject former, to be treated and/or the severity of its symptom and age, weight etc. and change.
The preparation of plasminogen of the present invention
Plasminogen can be detached from nature and be purified for further treatment purposes, can also pass through the change of standard Peptide symthesis technology is learned to synthesize.When passing through chemically synthesized polypeptide, can be synthesized through liquid phase or solid phase.Solid phase peptide synthssis (SPPS) (the C-terminal amino acid of sequence is wherein attached into insoluble support, then remaining amino in sequential addition sequence Acid) it is the method for being suitble to plasminogen chemical synthesis.Various forms of SPPS, such as Fmoc and Boc can be used for synthesizing fibrinolysin It is former.Barany and Solid-Phase Peptide Synthesis are described in for the technology of synthesis in solid state；The 3-284 pages in The Peptides:Analysis, Synthesis, Biology. volume 2：Special Methods in Peptide Synthesis, Part A., Merrifield, wait J.Am.Chem.Soc., and 85:2149-2156 (1963)；Stewart etc., Solid Phase Peptide Synthesis,2nd ed.Pierce Chem.Co., Rockford,Ill.(1984)；With Ganesan A.2006Mini Rev.Med Chem.6:The 2005Protein such as 3-10 and Camarero JA Pept Lett.12:In 723-8.In short, handle small insoluble porous bead with the functional element for being built with peptide chain thereon.In idol After connection/de-protected repetitive cycling, the solid phase of attachment is dissociated N-terminal amine and single Amino Acid Unit coupling protect by N.So Afterwards, this element is deprotected, exposes the new N-terminal amine that can be attached with other amino acid.Peptide is remained fixed in solid phase, it It is cut away afterwards.
The plasminogen of the present invention can be produced using Standard recombinant methods.For example, the nucleic acid by encoding plasminogen It is inserted into expression vector, it is made to be operatively connected with the regulating and controlling sequence in expression vector.Expression regulation sequence includes but not limited to Promoter (such as naturally associated or heterologous promoter), signal sequence, enhancer element and transcription terminator.Expression Regulation and control can be eukaryotic promoter system in carrier, the carrier can convert or transfect eukaryotic host cell (such as COS or Chinese hamster ovary celI).Once carrier is mixed in suitable host, in the high level expression and plasminogen for being suitable for nucleotide sequence Collection and purifying under conditions of maintain host.
Suitable integration of the expression vector usually in host organisms as episome or as host chromosome DNA It replicates part.In general, expression vector contains selection marker, (such as amicillin resistance, hygromycin resistance, tetracycline resist Property, kalamycin resistance or neomycin resistance) to help to examine those cells that external source is converted with desired DNA sequence dna It surveys.
Escherichia coli (Escherichia coli) can be used for the protokaryon place of clone's theme antibody coding polynucleotides The example of chief cell.The other microbial hosts being adapted for use with include bacillus, such as bacillus subtilis (Bacillus Subtilis) and other enterobacteriaceaes (enterobacteriaceae), such as Salmonella (Salmonella), sand Lei Shi Pseudomonas (Serratia) and various pseudomonas (Pseudomonas) species.In these prokaryotic hosts, it can also generate Expression vector would generally contain the expression control sequence (such as replication orgin) compatible with host cell.Permitted in addition, can exist More well known promoters, such as lactose promoter system, tryptophan (trp) promoter systems, beta- iactamase promoters system System or the promoter systems from phageλ.Promoter would generally control expression, optionally in the case of operator sequence, And with ribosome bind site sequence etc., to start and complete transcription and translation.
Other microorganisms, such as yeast can also be used for expressing.Yeast (such as saccharomyces cerevisiae (S. cerevisiae)) and finish Red yeast (Pichia) is the example of suitable yeast host cell, wherein suitable carrier has expression control sequence as needed Arrange (such as promoter), replication orgin, termination sequence etc..Typical promoter includes glycerol 3-phosphate acid kinase and other sugars Solve enzyme.Induction type yeast starts in particularly including from alcohol dehydrogenase, different cell pigment C and responsible maltose and galactolipin profit The promoter of enzyme.
Outside microorganism, mammalian cell (such as the mammalian cell cultivated in cell culture in vitro) also may be used For expressing and generating the anti-Tau antibody (such as polynucleotides of the anti-Tau antibody of encoding schemes) of the present invention.Referring to Winnacker,From Genes to Clones,VCH Publishers, N.Y.,N.Y.(1987).Suitable mammal Host cell include Chinese hamster ovary celI system, various Cos cell lines, HeLa cells, myeloma cell line and inverted B cell or Hybridoma.Expression control sequence, such as replication orgin, promoter and enhancer can be included for the expression vector of these cells (Queen etc., Immunol.Rev.89:49 (1986)) and required machining information site, such as ribosome bind site, RNA splice sites, polyadenylation site and transcription terminator sequences.The example of suitable expression control sequence is white exempts from Promoter derived from epidemic disease globulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus etc..Referring to Co etc., J.Immunol.148:1149 (1992)。
Once synthesis (chemistry or recombination form), can be affine including ammonium sulfate precipitation according to the standard schedule of this field Column, column chromatography, high performance liquid chroma- tography (HPLC), gel electrophoresis etc. purify plasminogen of the present invention.The plasminogen It is substantially pure, for example, at least about 80% to 85% is pure, and at least about 85% to 90% is pure, and at least about 90% to 95% is pure Or it is 98% to 99% pure or purer, such as without pollutant, the pollutant such as cell fragment, except theme antibody with Outer macromolecular, etc..
Pharmaceutical formulation
Can be by by plasminogen with the desired purity and optional pharmaceutical carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences, 16 editions, Osol, A.ed. (1980)) be mixed to form lyophilized preparation or Aqueous solution prepares treatment preparaton.Acceptable carrier, excipient, stabilizer are nontoxic to receptor under dosage used and concentration Property, and including buffer such as phosphate, citrate and other organic acids；Antioxidant includes ascorbic acid and methionine； Preservative (such as stearyl dimethyl benzyl ammonium chloride；Hexamethonium chloride；Benzalkonium chloride (benzalkonium Chloride), benzethonium chloride；Phenol, butanol or benzyl alcohol；Alkyl parabens such as methyl or propyl p-hydroxybenzoate Ester；Catechol；Resorcinol；Cyclohexanol；3- amylalcohols；Metacresol)；Low molecular weight polypeptide (less than about 10 residues)；Albumen Matter such as seralbumin, gelatin or immunoglobulin；Hydrophilic polymer such as polyvinylpyrrolidone；Amino acid such as glycine, paddy Glutamine, asparagine, histidine, arginine or lysine；Monosaccharide, disaccharides and other carbohydrate include glucose, sweet Dew sugar or dextrin；Chelating agent such as EDTA；Carbohydrate such as sucrose, mannitol, fucose or sorbierite；Into salt counter ion such as sodium；Metal Compound (such as zinc-albumen composition)；And/or nonionic surfactant, such as TWEENTM, PLURONICSTM or poly- second Glycol (PEG).It is preferred that the anti-VEGF antibody formulations being lyophilized are described in WO 97/04801, it includes herein as With reference to.
The preparaton of the present invention also contains more than one the reactive compound needed for the specific illness that need to be treated, preferably Complementary activities and those being free from side effects between each other.For example, antihypertensive drug, antiarrhythmic drug, are controlled Treat drug of diabetes etc..
The plasminogen of the present invention can be wrapped in the microcapsules prepared by such as condensation technique or interfacial polymerization, example Such as, colloidal drug delivery systems (for example, liposome, albumin microsphere, microemulsion, nano particle and Nano capsule) can be embedded in In or merging macro emulsion in hydroxymethyl cellulose or gelatin-microcapsule and poly- (methyl methacrylate) microcapsules in. These technologies are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)。
Plasminogen for the present invention of vivo medicine-feeding is necessarily sterile.This can be by freeze-drying and again It is realized easily by degerming membrane filtration before or after preparation.
The plasminogen of the present invention can prepare sustained release preparation.The appropriate example of sustained release preparation includes with definite shape and contains There are the half penetrating matrix of solid hydrophobic polymers of glycoprotein, such as film or microcapsules.Sustained-release matrix example includes polyester, hydrogel (such as poly- (2- hydroxyethyl-methacrylates) (Langer, J. Biomed.Mater.Res., 15:167-277(1981)； Langer,Chem.Tech.,12:98- 105 (1982)) or it is poly- (vinyl alcohol), polyactide (United States Patent (USP) 3773919, EP 58, 481), the copolymer (Sidman, etc. Biopolymers 22 of Pidolidone and γ ethyl-L-glutamates:547 (1983)), no Degradable ethylene vinyl acetate (ethylene-vinyl acetate) (Langer, etc. ibid) or degradable Poly lactic coglycolic acid such as Lupron DepotTM (by poly lactic coglycolic acid and leucyl proline (leuprolide) microsphere of the injectable of acetic acid esters composition) and poly- D- (-) -3- hydroxybutyric acids.Polymer such as ethylene-second Vinyl acetate and lactic-co-glycolic acid energy sustained release molecule 100 days or more, and the time of some hydrogels release proteins It is shorter.Can protein stabilized rational strategy be made to design according to Related Mechanism.For example, if it find that the mechanism of cohesion is to pass through Thio Disulfide interchange and form intermolecular S -- S, then can by modify sulfhydryl residue, be lyophilized from acid solution, control it is wet Degree realizes stabilization using suitable additive and the specific polymer matrix composition of exploitation.
Administration and dosage
Can be by different modes, such as by intravenous, in peritonaeum, subcutaneously, encephalic is intrathecal, intra-arterial (such as via Arteria carotis), it is intramuscular to realize the application of pharmaceutical composition of the present invention.
Include sterile aqueous or non-aqueous solution, suspension and emulsion for the prepared product of parenteral administration.It is non-aqueous molten The example of agent is propylene glycol, polyethylene glycol, vegetable oil such as olive oil and injectable organic ester, such as ethyl oleate.Aqueous carrier packet Water, alcohol/aqueous solution, emulsion or suspension are included, including brine and buffer medium.It is molten that parenteral medium includes sodium chloride Liquid, woods grignard dextrose, dextrose and sodium chloride or fixing oil.Intravenous vehicles include liquid and nutritional supplement, electrolysis Matter supplement, etc..There may also be preservatives and other additives, such as, antimicrobial, antioxidant, chelating Agent and inert gas, etc..
Medical worker can determine dosage based on various clinical factors.As well known in medical domain, any patient's Dosage depends on many factors, build, body surface area, age including patient, the particular compound to be applied, gender, application Number and path, the general health and other medicines being administered simultaneously.The dosage of pharmaceutical composition of the present invention comprising plasminogen Range can be, for example, for example daily about 0.0001 to 2000mg/kg or about 0.001 to 500mg/kg (such as 0.02mg/kg, 0.25 mg/kg, 0.5mg/kg, 0.75mg/kg, 10mg/kg, 50mg/kg etc.) subject's weight.For example, dosage can be 1mg/kg weight or 50mg/kg weight or the range or at least 1mg/kg in 1-50mg/kg.Higher or lower than this illustrative model Including the dosage enclosed is also covered by, it is especially considering that above-mentioned factor.Intermediate dosage in above range is also contained in the present invention In the range of.Subject can apply this daily, every other day, weekly or according to any other schedule determined by empirical analysis Class dosage.Illustrative dosage schedule includes continuous several days 1-10mg/kg.Reality is needed during the medicament administration of the present invention When assessment therapeutic effect and safety.
Product or medicine box
One embodiment of the invention is related to a kind of product or medicine box, and it includes available for treating as caused by diabetes The plasminogen or fibrinolysin of the present invention of cardiovascular disease and its associated disease.The product preferably includes a container, label or Package insert.Appropriate container has bottle, bottle, syringe etc..Container can be made of a variety of materials such as glass or plastics.It is described Container contains composition, and the composition can effectively treat the disease of the present invention or illness and (such as described with sterile access port Container can be intravenous solution packet or bottle, and containing can be by the plug that hypodermic needle penetrates).In the composition at least A kind of activating agent is plasminogen/fibrinolysin.On the container or appended label illustrates the composition for treating this hair The bright cardiovascular disease as caused by diabetes and its associated disease.The product can further include containing pharmaceutically acceptable buffer solution Second container, the brine of such as phosphate-buffered, Ringer's solution and glucose solution.It can further include from business With other materials required from the point of view of user's angle, including other buffer solutions, diluent, filtrate, needle and syringe.In addition, The product includes the package insert with operation instruction, including for example indicating the user of the composition by plasminogen group It closes object and treats the other medicines administered patient of adjoint disease.
Brief description
Fig. 1 24-25 week old diabetic mices give plasminogen serum cTnT content detection result after 31 days. The results show that plasminogen group cardiac muscle troponin I concentration significantly lower than giving solvent PBS control group, and statistical difference is heteropolar Significantly (* * represent P<0.01).Illustrate that plasminogen can remarkably promote the reparation of diabetes later stage mouse cardiac muscle damage.
Fig. 2 ApoE Atherosclerosis Model mouse give plasminogen heart organ coefficient statistical result after 30 days.Knot Fruit shows, is significantly lower than to plasminogen group mouse heart organ coefficient and gives solvent PBS control group.Illustrate that plasminogen can change Cardiac compensation caused by kind ApoE Atherosclerosis Models mouse heart damage is loose.
Fig. 3 ApoE Atherosclerosis Model mouse give 30 days posterior sinus of Valsalva oil red O stains of plasminogen and represent Property picture.A is gives solvent PBS control group, and B is gives plasminogen group.The results show that give plasminogen group mouse aorta sinus fat Fat deposition (arrow logo) is considerably less than and gives solvent PBS control groups.Illustrate that plasminogen can improve fat in the heavy of aortic sinus Product.
Fig. 4 ApoE Atherosclerosis Model mouse give plasminogen 30 days aorta posterior valve HE dyeing representative diagrams Piece.A, C is gives solvent PBS control group, and B, D is give plasminogen group.The results show that give plasminogen group mouse aorta valve spot Block deposition (arrow logo) is considerably less than and gives solvent PBS control groups, and aortic valve fusion degree the former be less than the latter.It says Bright plasminogen can improve the damage of Atherosclerosis Model mouse aorta valve.
Fig. 5 ApoE Atherosclerosis Model mouse give plasminogen heart IgM immunostaining representative diagrams after 30 days Piece.A is gives solvent PBS control group, and B is gives plasminogen group.The results show that the positive to plasminogen group mouse heart IgM Expression (arrow logo) is considerably less than and gives solvent PBS control group, illustrates that plasminogen can promote heart caused by atherosclerosis The reparation of damage.
Fig. 6 ApoE Atherosclerosis Model mouse give plasminogen 30 days posterior sinus of Valsalva sirius red stains generations Table picture.A, C is gives solvent PBS control group, and B, D is give plasminogen group.As a result it is shown to plasminogen group aortic sinus The area of blood vessel collagen deposition (arrow logo), which is significantly less than, gives solvent PBS control group, illustrates that plasminogen can Cut down artery congee hardening model mouse aorta sinus Fibrosis levels.
Fig. 7 shows that 3% cholesterol hyperlipemia model mouse gives plasminogen 20 days heart risk index result of calculation. Solvent PBS control group is given, and statistical discrepancy is extremely notable the results show that being significantly less than to plasminogen group CRI.Illustrate fibrinolysin Proper energy effectively reduces the risk that heart disease occurs for hyperlipemia model mouse.
16 weeks hyperlipemia model mouse of Fig. 8 give plasminogen 30 days posterior sinus of Valsalva oil red O stain observation results. A, C is gives solvent PBS control group, and to give plasminogen group, E is quantitative analysis results by B, D.The results show that give plasminogen group Mouse aorta sinus fat deposition, which is considerably less than, gives solvent PBS control group, and significantly (* represents P to statistical discrepancy<0.05).Explanation Plasminogen can improve deposition of the fat in hyperlipemia model mouse aorta sinus.
16 weeks hyperlipemia model mouse of Fig. 9 give plasminogen 30 days representative pictures of posterior sinus of Valsalva HE dyeing.A、 C is gives solvent PBS control group, and B, D is give plasminogen group.The results show that the visible bubble of solvent PBS control group active blood vessel wall Foam cell deposition (arrow is signified), plaque deposition is serious；Give plasminogen group active blood vessel wall only visible slight foam cells Deposition, and apparent atheromatous plaque deposition is had no under inner membrance, it is lighter to plasminogen group Aortic injury.Illustrate plasminogen energy Improve vessel wall damage of the hyperlipemia model rat aorta sinus tube wall caused by lipidosis
16 weeks hyperlipemia model mouse of Figure 10 give plasminogen cardiac fibers protein immunization histochemical staining figure after 30 days Piece.A is gives solvent PBS control group, and for B to give plasminogen group, C is quantitative analysis results.It is the results show that small to plasminogen group The fibrinous positive expression of rat heart, which is considerably less than, gives solvent PBS control group, and significantly (* represents P to statistical discrepancy<0.05). Illustrate that plasminogen can reduce the heart injury caused by hyperlipidemia.
16 weeks hyperlipemia model mouse of Figure 11 give plasminogen heart IgM immunostaining representativeness pictures after 30 days. A is gives solvent PBS control group, and B is gives plasminogen group.The results show that the positive expression to plasminogen group mouse heart IgM Solvent PBS control group is considerably less than given, illustrates that plasminogen can mitigate heart injury caused by hyperlipidemia.
16 weeks hyperlipemia model mouse of Figure 12 give plasminogen heart sirius red stains representative diagram after 30 days Piece.A is gives solvent PBS control group, and B is gives plasminogen group.The results show that it is considerably less than to the deposition of plasminogen group collagen Solvent PBS control group is given, illustrates that plasminogen can mitigate hyperlipemia model mouse heart fibrosis.
16 weeks hyperlipemia model mouse of Figure 13 give plasminogen serum cardiac troponin I testing result after 30 days.As a result It has been shown that, is apparently higher than to solvent PBS control group serum cardiac troponin concentration and gives plasminogen group, and statistical discrepancy significantly (* Represent P<0.05).Illustrate that plasminogen can repair the damage of hyperlipidemia heart.
26 week old diabetic mices of Figure 14 give plasminogen ventricle oil red O stain representativeness picture after 35 days.A be to Solvent PBS control group, B is gives plasminogen group.The results show that plasminogen group mouse ventricle lipidosis (arrow logo) Considerably less than give solvent PBS control group.Illustrate that plasminogen can reduce diabetic mice ventricle lipidosis, promote ventricle damage Reparation.
16 week old hyperlipemia model mouse of Figure 15 give 30 days posterior sinus of Valsalva sirius red stains of plasminogen and represent Property picture.A, C is gives solvent PBS control group, and B, D is give plasminogen group.The results show that give plasminogen group aortic sinus blood The area of inside pipe wall collagen deposition (arrow logo), which is significantly less than, gives solvent PBS control group, illustrates that plasminogen can be cut down Hyperlipemia model mouse aorta sinus Fibrosis levels.
The systemic sclerosis model mice of Figure 16 bleomycin induceds give plasminogen after 21 days heart Picro-Sirius red contaminate Color representativeness picture.A is gives solvent PBS control group, and B is gives plasminogen group.The study found that in the system of bleomycin induced Property hardening disease mouse model in, to solvent PBS control group myocardial collagen deposit (arrow logo) degree ratio to plasminogen Group is high.Illustrate that plasminogen can effectively reduce the cardiac fibrosis of bleomycin induced.
Figure 17 24-25 week old diabetic mices give plasminogen after 31 days heart masson dyeing observation result.A is Solvent PBS control group is given, B is gives plasminogen group.The results show that visible blue between solvent PBS control group cardiac muscle fibre The collagenous fibres (arrow logo) of the hyperplasia of color, in slight myocardial fibrosis；To visible few between plasminogen group cardiac muscle fibre The collagenous fibres of the hyperplasia azury of amount, compared to control group, myocardial fibrosis is substantially reduced.Illustrate that plasminogen can change The fibrosis of kind diabetic mice heart.
Figure 18 17-18 week old diabetic mices give plasminogen heart sirius red stains representativeness picture after 35 days. A is gives solvent PBS control group, and B is gives plasminogen group.The results show that deposition (the arrow to plasminogen group mouse collagen fiber Leader is known) it is considerably less than and gives solvent PBS control group.Illustrate that plasminogen can reduce the fibrosis of diabetic mice heart.
Figure 19 26-27 week old diabetic mices give plasminogen heart sirius red stains representativeness picture after 35 days. A is gives solvent PBS control group, and B is gives plasminogen group.The results show that deposition (the arrow to plasminogen group mouse collagen Leader is known) it is considerably less than and gives solvent PBS control group.Illustrate that plasminogen can weaken the fibrosis of diabetic mice heart.
Figure 20 gives plasminogen 30 days ApoE Atherosclerosis Model mouse ventricles oil red O stain observation results.A is Solvent PBS control group is given, for B to give plasminogen group, C is quantitative analysis results.The results show that give plasminogen group mouse ventricle Lipidosis (arrow logo), which is considerably less than, gives solvent PBS control group, and significantly (* represents P to statistical discrepancy<0.05).Illustrate fibre Lyase proper energy enough reduces Atherosclerosis Model mouse ventricle lipidosis, and what ventricle caused by promoting lipidosis damaged repaiies It is multiple.
Figure 21 ApoE Atherosclerosis Model mouse give plasminogen after 30 days heart sirius red stains it is representative Picture.A is gives solvent PBS control group, and B is gives plasminogen group.The results show that deposition (the arrow to plasminogen group collagen Mark) it is considerably less than and gives solvent PBS control group, illustrate that plasminogen can mitigate ApoE Atherosclerosis Models mouse heart fibre Dimensionization.
Embodiment：
Protective effect of 1 plasminogen of embodiment to diabetic mice myocardial damage
Diabetes generally entail cardiovascular atherosclerotic[1,2].Cardiovascular atherosclerotic can lead to cardiac muscle cell Ischemic injuries.Cardiac muscle troponin I (cardiac troponin I, CTNI) is the important symbol object of myocardial damage, blood Clear concentration is capable of the degree of reflecting myocardium damage[3].This experiment observes plasminogen to cardiac muscle by detecting cardiac muscle troponin I The repair of damage.
24-25 week old db/db male mices 28, are randomly divided into two groups, to solvent PBS control group 12, to fibrinolysin Original group 16.Experiment is denoted as the 0th day and grouping of weighing on the day of starting, and second day after grouping starts to plasminogen or PBS and remember It is the 1st day, successive administration 31 days.To plasminogen group mouse by 2mg/0.2ml/ pcs/day of tail vein injection plasminogen, to molten Matchmaker's PBS control group tail vein injection gives the PBS of same volume.It extracts eyeball within 32nd day and takes blood, 15- is centrifuged with 3500r/min 20 minutes, and take the concentration mensuration of supernatant detection progress cardiac muscle troponin I.The results show that give plasminogen group myocardium myo calcium The concentration of protein I, which is significantly lower than, gives solvent PBS control group, and statistical discrepancy is extremely significantly (Fig. 1).Illustrate that plasminogen can be significantly Promote the reparation of diabetic mice myocardial damage.
2 plasminogen of embodiment improves ApoE atherosclerosis mouse heart compensatory hypertrophies
6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model[4,5].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1mL/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, continues to feed high cholesterol diet high in fat during administration.In to Mouse is put to death in the 31st day of medicine after weighing, dirty weigh, and calculate heart coefficient of coring.Heart coefficient (%)=cardiac weight/body Weight × 100.
The results show that it is significantly lower than to plasminogen group mouse heart coefficient to solvent PBS control group (Fig. 2).Illustrate fibre It is loose that lyase proper energy mitigates Cardiac compensation of the ApoE Atherosclerosis Model mouse caused by heart injury.
3 plasminogen of embodiment improves ApoE atherosclerosis mouse aorta sinus lipidosis
6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model[4,5].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1mL/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, and mouse was put to death in the 31st day, it cores dirty in 4% poly first Aldehyde fixes 24-48 hours, is sunk to the bottom overnight respectively at 4 DEG C in 15%, 30% sucrose, OCT embeddings, 8 μm of frozen section thickness, oil red O dyes 15min, and 75% alcohol breaks up 5 seconds, bush uniformly dyeing core 30 seconds, glycerin gelatine mounting.Slice is in 200 times of light microscopes Lower observation.
It is given the results show that being considerably less than to plasminogen group (Fig. 3 B) mouse aorta sinus fat deposition (arrow logo) Solvent PBS control group (Fig. 3 A).Illustrate that plasminogen can improve aortic sinus fat deposition in atherosclerosis.
4 plasminogen of embodiment improves the damage of ApoE atherosclerosis mouse aortas sinus
6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model[4,5].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1ml/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, continues to feed high cholesterol diet high in fat during administration.In It puts to death mouse within 31 days, cores dirty in 4% paraformaldehyde fixation 24-48 hours.Tissue samples after fixation are through alcohol serial dehydration With the transparent rear progress paraffin embedding of dimethylbenzene.Aortic sinus histotomy thickness be 3 μm, slice dewaxing rehydration and with haematoxylin with Eosin stains (HE dyeing), ammonium hydroxide returns blue and alcohol serial dehydration mounting after the differentiation of 1% hydrochloride alcohol, is sliced respectively 40 (Fig. 4 A and 4B) and 200 times of (Fig. 4 C and 4D) optical microphotograph Microscopic observations again.
Coloration result is shown, (arrow logo) is deposited to plasminogen group (Fig. 4 B, 4D) mouse aorta sinus Lipid Plaque Considerably less than give solvent PBS control group (Fig. 4 A, 4C), and aortic valve fusion degree the former be less than the latter.Illustrate fibrinolysin Proper energy improves aorta petal membrane damage in atherosclerosis.
5 plasminogen of embodiment improves the damage of ApoE atherosclerosis mouse heart
6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model[4,5].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1ml/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, continues to feed high cholesterol diet high in fat during administration.In It puts to death mouse within 31 days, cores dirty in 4% paraformaldehyde fixation 24-48 hours.Tissue after fixation is through alcohol serial dehydration and two Paraffin embedding is carried out after toluene is transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, With 3% dioxygen water incubation 15 minutes, 0.01MPBS was washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector Laboratories, Inc., USA) it closes 30 minutes；After time arrives, it is anti-that mountain sheep anti mouse IgM (HRP) is added dropwise in reject sheep blood serum liquid Body (Abcam) is incubated at room temperature 1 hour, and 0.01MPBS is washed 2 times, every time 5 minutes.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing, and flowing water rinses 5 minutes.Gradient alcohol dehydration, two Toluene is transparent and neutral gum mounting, is sliced in 200 times of optical microphotograph Microscopic observations.IgM antibody is thin in removing apoptosis and necrosis Born of the same parents play an important role in the process, and the level of injuries of tissues and organs local I gM antibody is proportionate with degree of injury[6,7].Cause This, the level of detection histoorgan local I gM antibody can reflect the degree of impairment of the histoorgan.Experiment is found, to fibrinolytic The positive expression of proenzyme group mouse (Fig. 5 B) heart IgM is considerably less than and gives solvent PBS control group (Fig. 5 A).Illustrate plasminogen energy Enough it is obviously improved the damage of ApoE mouse cardiac muscles.
6 plasminogen of embodiment reduces ApoE atherosclerosis mouse heart Fibrosis levels
6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model[4,5].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1ml/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, continues to feed high cholesterol diet high in fat during administration.In Mouse is put to death within 31 days, materials heart fixes 24-48 hours in 4% paraformaldehyde.Tissue after fixation through alcohol serial dehydration and Paraffin embedding is carried out after dimethylbenzene is transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration, with 0.1% day wolf After the red saturation picric acid of star dyes 30 minutes, flowing water rinses 2min, haematoxylin dyeing 1 minute, and flowing water rinses, 1% hydrochloride alcohol Differentiation, ammonium hydroxide return indigo plant, and flowing water rinses, neutral gum mounting after drying, in 200 times of optical microphotograph Microscopic observations.
Sirius red stains can be such that collagen persistently dyes, and be the specific stain method to collagen tissue in pathological section, with Special display collagen tissue.
Coloration result is shown, to the deposition (arrow of plasminogen group (Fig. 6 B) heart arter sinus atherosis part collagen Mark) it is considerably less than and gives solvent PBS control group (Fig. 6 A), illustrate that plasminogen can reduce ApoE Atherosclerosis Model mouse The deposition of collagen in heart tissue reduces cardiac fibrosis.
7 plasminogen of embodiment reduces by 3% cholesterol hyperlipemia model mouse heart onset risk
9 week old male C57 mouse 16 feed 3% cholesterol high lipid food (Nantong Te Luofei) 4 weeks, induce hyperlipemia Disease[8,9], this model is set to 3% cholesterol hyperlipemia model.Mouse after Cheng Mo continues to feed 3% cholesterol high lipid food. Three days every mouse take 50 μ l of blood, detection T-CHOL (T-CHO) before administration, and are randomly divided into two according to total cholesterol concentration Group, every group each 8.Start administration to be denoted as the 1st day, give plasminogen group mouse tail vein injection people source plasminogen 1mg/ 0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume.Mouse is fasted after administration in the 20th day, Fasting 16 hours pricks orbital venous plexus on the 21st day and takes 50 μ L of blood, and centrifugation obtains supernatant, and total cholesterol level is examined using T-CHOL Test agent box (Bioengineering Research Institute, article No. A111-1 are built up in Nanjing) is detected；High-density lipoprotein cholesterol (HDL-C) Content is carried out using high-density lipoprotein cholesterol detection kit (Bioengineering Research Institute, article No. A112-1 are built up in Nanjing) Detection.
Cardiac risk index (cardiac risk index, CRI) to assess blood fat disorder induce heart disease wind Danger[10]。
Cardiac risk index=T-CHO/HDL-C.
Solvent PBS control group is given the results show that being significantly less than to plasminogen group CRI, and statistical discrepancy extremely significantly (figure 7).Illustrate that plasminogen can effectively reduce the risk that heart disease occurs for hyperlipemia model mouse.
8 plasminogen of embodiment reduces deposition of the lipid in 16 weeks hyperlipemia model mouse aorta sinus
6 week old male C57 mouse 11 feed high cholesterol diet (Nantong Te Luofei, article No. TP2031) high in fat 16 weeks With induced hyperlipidemia model[8,9]], this model is set to 16 weeks hyperlipemia models.Mouse after Cheng Mo, which continues to feed high courage, consolidates Alcohol feed.Before administration three days every take 50 μ l of blood to detect T-CHOL (T-CHO) content, and random according to T-CHO contents It is divided into two groups, to solvent PBS control groups 6, to plasminogen group 5.Start administration to be denoted as the 1st day, give plasminogen group tail 1mg/0.1ml/ pcs/day of intravenous injection human source plasminogen, to the PBS of solvent PBS control group tail vein injection same volume.Administration 30 days, and mouse was put to death in the 31st day, dirty 4% paraformaldehyde that is organized in of coring fixes 24-48 hours, respectively at 15%, 30% It is sunk to the bottom overnight for 4 DEG C in sucrose, OCT embeddings, 8 μm of aortic sinus frozen section thickness, oil red O stain 15min, the differentiation of 75% alcohol 5 seconds, bush uniformly dyeing core 30s, glycerin gelatine mounting.Slice is in 40 (Fig. 8 A, 8B), 200 times of (Fig. 8 C, 8D) times light microscopes Lower observation.
The results show that it is considerably less than to plasminogen group (Fig. 8 B, 8D) mouse aorta sinus fat deposition to PBS pairs of solvent According to group (Fig. 8 A, 8C), and statistical discrepancy is significantly (Fig. 8 E).Illustrate that plasminogen can reduce lipid in hyperlipemia model mouse master Deposition in artery sinus.
9 plasminogen of embodiment improves hyperlipemia model mouse aorta sinus damage in 16 weeks
6 week old male C57 mouse 11 feed high cholesterol diet (Nantong Te Luofei, article No. TP2031) high in fat 16 weeks With induced hyperlipidemia model[8,9], this model is set to 16 weeks hyperlipemia models.Mouse after Cheng Mo continues to feed high cholesterol Feed.Before administration three days every take 50 μ l of blood to detect T-CHOL (T-CHO) content, and divide at random according to T-CHO contents It it is two groups, to solvent PBS control groups 6, to plasminogen group 5.Start administration to be denoted as the 1st day, it is quiet to plasminogen group tail 1mg/0.1ml/ pcs/day of arteries and veins injection people source plasminogen, to the PBS of solvent PBS control group tail vein injection same volume.Administration 30 My god, and mouse was put to death in the 31st day, dirty 4% paraformaldehyde that is organized in of coring fixes 24-48 hours.Tissue after fixation is through alcohol Paraffin embedding is carried out after serial dehydration and dimethylbenzene are transparent.Tissue samples after fixation are transparent through alcohol serial dehydration and dimethylbenzene After carry out paraffin embedding.Aortic sinus histotomy thickness is 3 μm, slice dewaxing rehydration and with haematoxylin and eosin stains (HE Dyeing), ammonium hydroxide returns blue and alcohol serial dehydration mounting after the differentiation of 1% hydrochloride alcohol, is sliced in 40 (Fig. 9 A, B), 200 times of (figures 9C, D) optical microphotograph Microscopic observation.
The results show that give solvent PBS control group (Fig. 9 A, C) aortic sinus inner wall foam cells deposition (arrow is signified), spot Block heavy deposition；It is deposited, and under inner membrance not to the only visible slight foam cells of plasminogen group (Fig. 9 B, D) aortic sinus inner wall See apparent atheromatous plaque deposition, damaged to plasminogen group aorta inner wall lighter.Illustrate that plasminogen can improve hyperlipemia Disease model mice artery sinus inner wall damages.
10 plasminogen of embodiment reduces the fibrinous expression of hyperlipemia model mouse heart in 16 weeks
6 week old male C57 mouse 11 feed high cholesterol diet (Nantong Te Luofei, article No. TP2031) high in fat 16 weeks With induced hyperlipidemia model[8,9], this model is set to 16 weeks hyperlipemia models.Mouse after Cheng Mo continues to feed high cholesterol Feed.Before administration three days every take 50 μ l of blood to detect T-CHOL (T-CHO) content, and divide at random according to T-CHO contents It it is two groups, to solvent PBS control groups 6, to plasminogen group 5.Start administration to be denoted as the 1st day, it is quiet to plasminogen group tail 1mg/0.1ml/ pcs/day of arteries and veins injection people source plasminogen, to the PBS of solvent PBS control group tail vein injection same volume.Administration 30 My god, and mouse was put to death in the 31st day, dirty 4% paraformaldehyde that is organized in of coring fixes 24-48 hours.Tissue after fixation is through alcohol Paraffin embedding is carried out after serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.With 3% dioxygen water incubation 15 minutes is washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) it closes 30 minutes；After time arrives, reject sheep blood serum liquid irises out tissue with PAP.Divided with 3% dioxygen water incubation 15 Clock is washed 2 times, every time 5 minutes.4 DEG C of rabbit anti-mouse fibrin antibody (Abcam) overnight incubation, 0.01MPBS wash 2 times, often Secondary 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and PBS is washed 2 times, every time 5 minutes.By DAB Kit (Vector laboratories, Inc., USA) develops the color, and haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 points Clock.Gradient alcohol dehydration, dimethylbenzene is transparent and neutral gum mounting, is sliced in 200 times of optical microphotograph Microscopic observations.
Fibrinogen is fibrinous precursor, is deposited in the case of a fracture in tissue, as body to the one of damage Kind stress reaction, fibrinogen are hydrolyzed into fibrin deposition in damage location[11,12].Therefore, local fiber will can be damaged A mark of the protein level as degree of injury.
Immunohistochemical staining is the results show that bright to the positive expression of plasminogen group mouse (Figure 10 B) cardiac fibers albumen Aobvious be less than gives solvent PBS control group (Figure 10 A), and statistical discrepancy is significantly (Figure 10 C), illustrates that plasminogen can reduce hyperlipidemia Caused myocardial damage.
The hyperlipemia model mouse cardiac muscle damage in 16 weeks of 11 plasminogen effective protection of embodiment
6 week old male C57 mouse 11 feed high cholesterol diet (Nantong Te Luofei, article No. TP2031) high in fat 16 weeks With induced hyperlipidemia model[8,9], this model is set to 16 weeks hyperlipemia models.Mouse after Cheng Mo continues to feed high cholesterol Feed.Before administration three days every take 50 μ l of blood to detect T-CHOL (T-CHO) content, and divide at random according to T-CHO contents It it is two groups, to solvent PBS control groups 6, to plasminogen group 5.Start administration to be denoted as the 1st day, it is quiet to plasminogen group tail 1mg/0.1ml/ pcs/day of arteries and veins injection people source plasminogen, to the PBS of solvent PBS control group tail vein injection same volume.Administration 30 My god, and mouse was put to death in the 31st day, dirty 4% paraformaldehyde that is organized in of coring fixes 24-48 hours.Tissue after fixation is through alcohol Paraffin embedding is carried out after serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.With 3% dioxygen water incubation 15 minutes is washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) it closes 30 minutes；After time arrives, reject sheep blood serum liquid irises out tissue with PAP.Divided with 3% dioxygen water incubation 15 Clock is washed 2 times, every time 5 minutes.Mountain sheep anti mouse IgM (HRP) antibody (Abcam) is incubated at room temperature 1 hour, and PBS washes 2 times, and every time 5 Minute.It develops the color by DAB kits (Vector laboratories, Inc., USA), bush uniformly dyeing core 30 seconds after washing 3 times, Flowing water rinses 5 minutes.Gradient alcohol dehydration, dimethylbenzene is transparent, and neutral gum mounting is sliced and is seen under 200 times of light microscopes It examines.
IgM antibody plays an important role during apoptosis and non-viable non-apoptotic cell is removed, the histoorgan local I gM of damage The level of antibody is proportionate with degree of injury[6,7].Therefore, the level of detection histoorgan local I gM antibody can reflect this The degree of injury of histoorgan.
Immunostaining results are shown, are considerably less than to the positive expression of plasminogen group mouse (Figure 11 B) heart IgM to molten Matchmaker's PBS control group (Figure 11 A), illustrates that plasminogen can reduce the damage of hyperlipemia model animal hearts.
12 plasminogen of embodiment mitigates 16 weeks hyperlipemia model mouse heart fibrosis
6 week old male C57 mouse 11 feed high cholesterol diet (Nantong Te Luofei, article No. TP2031) high in fat 16 weeks With induced hyperlipidemia model[8,9], this model is set to 16 weeks hyperlipemia models.Mouse after Cheng Mo continues to feed high cholesterol Feed.Before administration three days every take 50 μ l of blood to detect T-CHOL (T-CHO) content, and divide at random according to T-CHO contents It it is two groups, to solvent PBS control groups 6, to plasminogen group 5.Start administration to be denoted as the 1st day, it is quiet to plasminogen group tail 1mg/0.1ml/ pcs/day of arteries and veins injection people source plasminogen, to the PBS of solvent PBS control group tail vein injection same volume.Administration 30 My god, and mouse was put to death in the 31st day, dirty 4% paraformaldehyde that is organized in of coring fixes 24-48 hours.Tissue after fixation is through alcohol Paraffin embedding is carried out after serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration, with After 0.1% Picro-Sirius red saturation picric acid dyes 30 minutes, flowing water rinses 2min, haematoxylin dyeing 1 minute, and flowing water rinses, 1% hydrochloride alcohol breaks up, and ammonium hydroxide returns indigo plant, and flowing water rinses, neutral gum mounting after drying, in 200 times of optical microphotograph Microscopic observations.
Sirius red stains can be such that collagen persistently dyes, and as pathological section specific stain method, sirius red stains can Specifically to show collagen tissue.
Coloration result is shown, is considerably less than to the deposition of plasminogen group (Figure 12 B) collagen and is given solvent PBS control group (figure 12A), illustrate that plasminogen can mitigate the deposition of hyperlipemia model mouse heart tissue collagen, mitigate myocardial fibrosis.
13 plasminogen of embodiment repairs hyperlipemia model mouse cardiac muscle damage in 16 weeks
6 week old male C57 mouse 11 feed high cholesterol diet (Nantong Te Luofei, article No. TP2031) high in fat 16 weeks With induced hyperlipidemia model[8,9], this model is set to 16 weeks hyperlipemia models.Mouse after Cheng Mo continues to feed high cholesterol Feed.Before administration three days every take 50 μ l of blood to detect T-CHOL (T-CHO) content, and divide at random according to T-CHO contents It it is two groups, to solvent PBS control groups 6, to plasminogen group 5.Start administration to be denoted as the 1st day, it is quiet to plasminogen group tail 1mg/0.1ml/ pcs/day of arteries and veins injection people source plasminogen, to the PBS of solvent PBS control group tail vein injection same volume.Administration 30 My god, mouse is fasted after administration in the 30th day, fasting 16 hours, plucks within the 31st day eyeball and takes blood, centrifugation obtains supernatant, using the heart Flesh troponin (Cardiac troponin I, CTNI) detection kit (Nanjing is built up) detects the dense of serum cTnT Degree.
Cardiac muscle troponin I is the important symbol object of myocardial damage, and serum-concentration is capable of the journey of reflecting myocardium damage Degree[3]。
Testing result is shown, is apparently higher than to solvent PBS control group serum cardiac troponin concentration and is given plasminogen group, And statistical discrepancy is significantly (Figure 13).Illustrate that plasminogen can significantly repair the damage of hyperlipemia model mouse heart.
14 plasminogen of embodiment reduces diabetic mice ventricle lipidosis
26 week old male db/db mouse 9 are grouped at random, to plasminogen group 4, to solvent PBS control group 5.It gives 2mg/0.2ml/ pcs/day of plasminogen group tail vein injection people source plasminogen gives solvent PBS control group tail vein injection consubstantiality Long-pending PBS is administered 35 days.In the 36th day put to death mouse, core it is dirty in 4% paraformaldehyde fix 24-48 hours, respectively at 15%th, in 30% sucrose 4 DEG C sink to the bottom overnight, OCT embeddings, 8 μm of frozen section thickness, oil red O stain 15min, 75% alcohol point Change 5 seconds, bush uniformly dyeing core 30s, glycerin gelatine mounting.Slice is in 200 times of optical microphotograph Microscopic observations.
The results show that it is considerably less than to plasminogen group mouse (Figure 14 B) ventricle lipidosis (arrow logo) to solvent PBS control group (Figure 14 A).Illustrate that plasminogen can reduce fat and be deposited in diabetic mice ventricle, promote repairing for ventricle damage It is multiple.
15 plasminogen of embodiment reduces by 16 weeks hyperlipemia model mouse aorta sinus fibrosis
6 week old male C57 mouse 11 feed high cholesterol diet (Nantong Te Luofei, article No. TP2031) high in fat 16 weeks With induced hyperlipidemia model[8,9], this model is set to 16 weeks hyperlipemia models.Mouse after Cheng Mo continues to feed high cholesterol Feed.Before administration three days every take 50 μ l of blood to detect T-CHOL (T-CHO) content, and divide at random according to T-CHO contents It it is two groups, to solvent PBS control groups 6, to plasminogen group 5.Start administration to be denoted as the 1st day, it is quiet to plasminogen group tail 1mg/0.1ml/ pcs/day of arteries and veins injection people source plasminogen, to the PBS of solvent PBS control group tail vein injection same volume.Administration 30 My god, mouse was put to death in the 31st day, is cored dirty in 4% paraformaldehyde fixation 24-48 hours.Tissue after fixation is through alcohol gradient Paraffin embedding is carried out after dehydration and dimethylbenzene are transparent.Aortic sinus slice thickness is 3 μm, is washed 1 time after slice dewaxing rehydration, with After 0.1% Picro-Sirius red saturation picric acid dyes 30 minutes, flowing water rinses 2min, haematoxylin dyeing 1 minute, and flowing water rinses, 1% hydrochloride alcohol breaks up, and ammonium hydroxide returns indigo plant, and flowing water rinses, neutral gum mounting after drying, 40 (15A, 15B), 200 times (15C, 165) optical microphotograph Microscopic observation.
The results show that give plasminogen group (Figure 15 B, 15D) aortic sinus blood vessel collagen deposition (arrow mark Know) area be significantly less than and give solvent PBS control group (Figure 15 A, 15C), illustrate that plasminogen can cut down hyperlipemia model Mouse aorta sinus Fibrosis levels.
16 plasminogen of embodiment reduces systemic sclerosis mouse heart fibrosis
12 week old C57 heros mouse 10 are taken, are randomly divided into two groups, to solvent PBS control group and each 5 to plasminogen group. Experiment is denoted as the 0th day and grouping of weighing on the day of starting, start within the 1st day modeling administration, by 0.1mg/0.1ml/ pcs/day of hypodermic injection Bleomycin induced systemic sclerosis[13], and start to plasminogen or PBS, successive administration 21 days.It is small to plasminogen group Mouse presses 1mg/0.1ml/ pcs/day of tail vein injection plasminogen, to solvent PBS control group tail vein injection same volume PBS put to death mouse at the 22nd day and cores and dirty fixes 24 hours in 4% paraformaldehyde fixer.Heart after fixation is through wine Paraffin embedding is carried out after smart serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, and 1 is washed after slice dewaxing to water Secondary, after being dyed 30 minutes with 0.1% Picro-Sirius red saturation picric acid, flowing water rinses 2min, haematoxylin dyeing 1 minute, flowing water punching It washes, 1% hydrochloride alcohol differentiation, ammonium hydroxide returns indigo plant, and flowing water rinses, and neutral gum mounting after drying is seen under 200 times of light microscopes It examines.
The study found that in the systemic sclerosis disease mouse model of bleomycin induced, Microscopic observation is to PBS pairs of solvent Plasminogen group (Figure 16 B) height is given according to group (Figure 16 A) myocardial collagen deposition rate.It is rich to illustrate that plasminogen can be reduced effectively The cardiac fibrosis of Lay mycin induction.
17 plasminogen of embodiment improves 24-25 week old diabetic mice cardiac fibrosis
24-25 week old db/db heros mouse 10, is randomly divided into two groups, to solvent PBS control group and gives plasminogen group, often Each 5 of group.Experiment is denoted as the 0th day and grouping of weighing on the day of starting, and the 1st day starts to plasminogen or PBS, successive administration 31 My god.To plasminogen group mouse by 2mg/0.2ml/ pcs/day of tail vein injection plasminogen, phase is given to solvent PBS control group The PBS of same volume.To putting to death mouse after plasminogen 31 days and that heart tissue is taken to fix 24 in 4% paraformaldehyde fixer is small When.Heart tissue after fixation carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 4 μ M is put into potassium bichromate solution and stays overnight after slice dewaxing rehydration.Garapa uniformly dyeing 3 to 5 minutes, flowing water is slightly washed.1% hydrochloride alcohol Differentiation, AMMONIA TREATMENT 1 second, washing.Ponceaux acid fuchsin liquid contaminates 8 minutes, short rinse in water.At 1% phosphomolybdic acid aqueous solution Reason about 2 minutes, aniline blue liquid is redyed 6 minutes.1% glacial acetic acid rinses general 1 minute or so.Absolute ethyl alcohol dehydration dimethylbenzene is transparent Mounting afterwards is sliced in 200 times of optical microphotograph Microscopic observations.
The most common complication of diabetes is connective tissue excessive accumulation (pathologic fibrosis), and Myocardial Interstitial Fibrosis can It can be the characteristic pathology change of Leonurus heterophyllus sweet[14,15]。
Masson dyes the fibrosis that can show tissue.It is the results show that fine to solvent PBS control group (Figure 17 A) cardiac muscle The collagenous fibres (arrow logo) of the hyperplasia of visible blue between dimension, in slight myocardial fibrosis；Give plasminogen group (figure 17B) between cardiac muscle fibre visible a small amount of hyperplasia azury collagenous fibres, compared to control group, myocardial fibrosis is apparent Mitigate.Illustrate that plasminogen can improve the fibrosis of diabetic mice heart.
18 plasminogen of embodiment reduces 17-18 week old diabetic mices myocardial collagen deposition
17-18 week old db/db heros mouse 8, is randomly divided into two groups, to solvent PBS control group and gives plasminogen group, every group Each 4.Experiment is denoted as the 0th day and grouping of weighing on the day of starting, and starts within the 1st day to plasminogen or PBS, successive administration 35 days. To plasminogen group mouse by 2mg/0.2ml/ pcs/day of tail vein injection plasminogen, given to solvent PBS control group identical The PBS of volume.To putting to death mouse after plasminogen 35 days and that heart tissue is taken to fix 24 in 4% paraformaldehyde fixer is small When.Heart after fixation carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, is cut Piece is washed 1 time after dewaxing to water, and after being dyed 30 minutes with 0.1% Picro-Sirius red saturation picric acid, flowing water rinses 2min, bush Uniformly dyeing color 1 minute, flowing water rinse, and 1% hydrochloride alcohol differentiation, ammonium hydroxide returns indigo plant, and flowing water rinses, neutral gum mounting after drying, 200 times of optical microphotograph Microscopic observations.
The results show that the deposition (arrow logo) to plasminogen group (Figure 18 B) mouse collagen fiber is considerably less than to molten Matchmaker's PBS control group (Figure 18 A).Illustrate that plasminogen can reduce the deposition of heart tissue collagen, plasminogen is prompted to be expected to By reducing the deposition of heart tissue collagen, mitigate versus young (17-18 week old) diabetic mice heart tissue fiber Change.
19 plasminogen of embodiment reduces 26-27 week old diabetic mices myocardial collagen deposition
26-27 week old db/db heros mouse 9, is randomly divided into two groups, to solvent PBS control group 5 and to plasminogen group 4 Only.Experiment is denoted as the 0th day and grouping of weighing on the day of starting, and starts within the 1st day to plasminogen or PBS, successive administration 35 days.To fibre Lyase original group mouse presses 2mg/0.2ml/ pcs/day of tail vein injection plasminogen, and same volume is given to solvent PBS control group PBS.To putting to death mouse after plasminogen 35 days and heart tissue taken to fix 24 hours in 4% paraformaldehyde fixer.It is fixed Heart afterwards carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, slice dewaxing It is washed after to water 1 time, with 0.1% sirius red stains after sixty minutes, flowing water rinses, haematoxylin dyeing 1 minute, and flowing water rinses, Indigo plant is returned in 1% hydrochloride alcohol and ammonium hydroxide differentiation, and flowing water rinses, mounting after drying, in 200 times of optical microphotograph Microscopic observations.
The results show that the deposition (arrow logo) to plasminogen group (Figure 19 B) mouse collagen fiber is considerably less than to molten Matchmaker's PBS control group (Figure 19 A).Illustrate that plasminogen can reduce the deposition of heart tissue collagen, plasminogen is prompted to be expected to By reducing the deposition of heart tissue collagen, mitigate relatively old (26-27 week old) diabetic mice heart tissue fiber Change.
20 plasminogen of embodiment improves ApoE atherosclerosis mouse ventricle lipidosis
6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model[4,5].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1mL/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, and mouse was put to death in the 31st day, it cores dirty in 4% poly first Aldehyde fixes 24-48 hours, is sunk to the bottom overnight respectively at 4 DEG C in 15%, 30% sucrose, OCT embeddings, 8 μm of ventricle frozen section thickness, Oil red O stain 15min, 75% alcohol break up 5 seconds, bush uniformly dyeing core 30 seconds, glycerin gelatine mounting.Slice is shown in 200 times of optics Micro- Microscopic observation.
The results show that it is considerably less than to plasminogen group mouse (Figure 20 B) ventricle lipidosis (arrow logo) to solvent PBS control group (Figure 20 A), and statistical discrepancy is significantly (Figure 20 C).It is small to illustrate that plasminogen can reduce Atherosclerosis Model Mouse ventricle lipidosis, the reparation that ventricle caused by promoting lipidosis damages.
21 plasminogen of embodiment reduces ApoE atherosclerosis mouse heart Fibrosis levels
6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model[4,5].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1ml/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, continues to feed high cholesterol diet high in fat during administration.In Mouse is put to death within 31 days, materials heart fixes 24-48 hours in 4% paraformaldehyde.Tissue after fixation through alcohol serial dehydration and Paraffin embedding is carried out after dimethylbenzene is transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration, with 0.1% day wolf After the red saturation picric acid of star dyes 30 minutes, flowing water rinses 2min, haematoxylin dyeing 1 minute, and flowing water rinses, 1% hydrochloride alcohol Differentiation, ammonium hydroxide return indigo plant, and flowing water rinses, neutral gum mounting after drying, in 200 times of optical microphotograph Microscopic observations.
Sirius red stains can be such that collagen persistently dyes, and be the specific stain method to collagen tissue in pathological section, with Special display collagen tissue.
Coloration result is shown, is considerably less than to the deposition (arrow logo) of plasminogen group (Figure 21 B) collagen and is given solvent PBS Control group (Figure 21 A) illustrates that plasminogen can reduce the deposition of collagen in ApoE Atherosclerosis Model mouse heart tissues, Reduce cardiac fibrosis.
Bibliography
[1]Sun Mi Hwang,Jin Sook Kim,Yun Jung Lee et al.Anti-Diabetic Atherosclerosis Effect of Prunella vulgaris in db/db Mice with Type 2Diabetes.The American Journal of Chinese Medicine,Vol.40,No.5,937–951.
[2]Hardy,D.S.,D.M.Hoelscher,C.Aragaki et al.Association of glycemic index and glycemic load with risk of incident coronary heart disease among Whites and African Americans with and without type 2diabetes:the atherosclerosis risk in communities study.Ann.Epidemiol.20: 610–616,2010.
[3]R.Langhorn and J.L.Willesen.Cardiac Troponins in Dogs and Cats.J Vet Intern Med 2016；30:36–50.
[4]Yutaka Nakashima,Andrew S.Plump,Elaine W.Raines et al.Arterioscler Thromb.1994 Jan；14(1):133-40.
[5]Yvonne Nitschke,Gabriele Weissen-Plenz,Robert Terkeltaub et al.Npp1promotes atherosclerosis in ApoE knockout mice.J.Cell.Mol.Med.Vol 15, No 11,2011pp.2273-2283. [6]Zhang M,Takahashi K,Alicot EM,Vorup-Jensen T, Kessler B,et al.(2006)Activation of the lectin pathway by natural IgM in a model of ischemia/reperfusion injury.J Immunol 177: 4727–4734.
[7]Kim SJ,Gershov D,Ma X,Brot N,Elkon KB(2002)I-PLA2Activation during Apoptosis Promotes the Exposure of Membrane Lysophosphatidylcholine Leading to Binding by Natural Immunoglobulin M Antibodies and Complement.
[8]Dominika Nackiewicz,Paromita Dey,Barbara Szczerba et al.Inhibitor of differentiation 3, a transcription factor regulates hyperlipidemia associated kidney disease.Nephron Exp Nephrol.2014；126(3):141–147.
[9]Ming Gu1,Yu Zhang.,Shengjie Fan et al.Extracts of Rhizoma Polygonati Odorati Prevent High-Fat Diet-Induced Metabolic Disorders in C57BL/6 Mice.PLoS ONE 8(11):e81724.
[10]Hao W,Friedman A(2014)The LDL-HDL Profile Determines the Risk of Atherosclerosis:A Mathematical Model.PLoS ONE 9(3):e90497
[11]Siobhan M.Craige,PhD,Shashi Kant et al.Endothelial NADPH oxidase 4 protects ApoE-/-mice from atherosclerotic lesions.Free Radic Biol Med.2015 December；89:1–7.
[12]Dimitrios Davalos,Katerina Akassoglou.Fibrinogen as a key regulator of inflammation in disease.Seminars in Immunopathology,2012.34(1): 43-62.
[13]Yosuke Kanno,En Shu,Hiroyuki Kanoh et al.The Antifibrotic Effect of a2AP Neutralization in Systemic Sclerosis Dermal Fibroblasts and Mouse Models of Systemic Sclerosis.J Invest Dermatol.2016 Apr；136(4):762-9.
[14]Ashish Aneja,W.H.Wilson Tang,Sameer Bansilal et al.Diabetic Cardiomyopathy: Insights into Pathogenesis,Diagnostic Challenges,and Therapeutic Options.Am J Med.2008 Sep；121(9):748-57.
[15]SamuelCS1,HewitsonTD,ZhangYetal.Relaxin ameliorates fibrosis in experimental diabet c cardiomyopathy.Endocrinology.2008 Jul；149(7):3286-93.
Sequence table
<110>Shenzhen Co., Ltd of Rui Jian life sciences institute
<120>A kind of method for improving heart disease
<130> PDK03595
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 2376
<212> DNA
<213>Natural plasminogen (Glu-PLG, Glu- plasminogen) nucleic acid sequence of signal peptide is not contained
<400> 1
gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60
cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120
acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180
aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240
ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300
aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360
gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420
caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480
gagtgtgaag aggaatgtat gcattgcagt ggagaaaact atgacggcaa aatttccaag 540
accatgtctg gactggaatg ccaggcctgg gactctcaga gcccacacgc tcatggatac 600
attccttcca aatttccaaa caagaacctg aagaagaatt actgtcgtaa ccccgatagg 660
gagctgcggc cttggtgttt caccaccgac cccaacaagc gctgggaact ttgtgacatc 720
ccccgctgca caacacctcc accatcttct ggtcccacct accagtgtct gaagggaaca 780
ggtgaaaact atcgcgggaa tgtggctgtt accgtgtccg ggcacacctg tcagcactgg 840
agtgcacaga cccctcacac acataacagg acaccagaaa acttcccctg caaaaatttg 900
gatgaaaact actgccgcaa tcctgacgga aaaagggccc catggtgcca tacaaccaac 960
agccaagtgc ggtgggagta ctgtaagata ccgtcctgtg actcctcccc agtatccacg 1020
gaacaattgg ctcccacagc accacctgag ctaacccctg tggtccagga ctgctaccat 1080
ggtgatggac agagctaccg aggcacatcc tccaccacca ccacaggaaa gaagtgtcag 1140
tcttggtcat ctatgacacc acaccggcac cagaagaccc cagaaaacta cccaaatgct 1200
ggcctgacaa tgaactactg caggaatcca gatgccgata aaggcccctg gtgttttacc 1260
acagacccca gcgtcaggtg ggagtactgc aacctgaaaa aatgctcagg aacagaagcg 1320
agtgttgtag cacctccgcc tgttgtcctg cttccagatg tagagactcc ttccgaagaa 1380
gactgtatgt ttgggaatgg gaaaggatac cgaggcaaga gggcgaccac tgttactggg 1440
acgccatgcc aggactgggc tgcccaggag ccccatagac acagcatttt cactccagag 1500
acaaatccac gggcgggtct ggaaaaaaat tactgccgta accctgatgg tgatgtaggt 1560
ggtccctggt gctacacgac aaatccaaga aaactttacg actactgtga tgtccctcag 1620
tgtgcggccc cttcatttga ttgtgggaag cctcaagtgg agccgaagaa atgtcctgga 1680
agggttgtag gggggtgtgt ggcccaccca cattcctggc cctggcaagt cagtcttaga 1740
acaaggtttg gaatgcactt ctgtggaggc accttgatat ccccagagtg ggtgttgact 1800
gctgcccact gcttggagaa gtccccaagg ccttcatcct acaaggtcat cctgggtgca 1860
caccaagaag tgaatctcga accgcatgtt caggaaatag aagtgtctag gctgttcttg 1920
gagcccacac gaaaagatat tgccttgcta aagctaagca gtcctgccgt catcactgac 1980
aaagtaatcc cagcttgtct gccatcccca aattatgtgg tcgctgaccg gaccgaatgt 2040
ttcatcactg gctggggaga aacccaaggt acttttggag ctggccttct caaggaagcc 2100
cagctccctg tgattgagaa taaagtgtgc aatcgctatg agtttctgaa tggaagagtc 2160
caatccaccg aactctgtgc tgggcatttg gccggaggca ctgacagttg ccagggtgac 2220
agtggaggtc ctctggtttg cttcgagaag gacaaataca ttttacaagg agtcacttct 2280
tggggtcttg gctgtgcacg ccccaataag cctggtgtct atgttcgtgt ttcaaggttt 2340
gttacttgga ttgagggagt gatgagaaat aattaa 2376
<210> 2
<211> 791
<212> PRT
<213>Natural plasminogen (Glu-PLG, Glu- plasminogen) amino acid sequence of signal peptide is not contained
<400> 2
Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser
1 5 10 15
Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala
20 25 30
Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His
35 40 45
Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser
50 55 60
Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr
65 70 75 80
Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met
85 90 95
Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser
100 105 110
Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu
115 120 125
Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp
130 135 140
Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu
145 150 155 160
Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly
165 170 175
Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser
180 185 190
Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys
195 200 205
Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro
210 215 220
Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile
225 230 235 240
Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys
245 250 255
Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val
260 265 270
Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His
275 280 285
Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr
290 295 300
Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn
305 310 315 320
Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser
325 330 335
Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr
340 345 350
Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly
355 360 365
Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser
370 375 380
Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala
385 390 395 400
Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro
405 410 415
Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu
420 425 430
Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val
435 440 445
Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe
450 455 460
Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly
465 470 475 480
Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile
485 490 495
Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys
500 505 510
Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn
515 520 525
Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro
530 535 540
Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly
545 550 555 560
Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln
565 570 575
Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu
580 585 590
Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser
595 600 605
Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val
610 615 620
Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu
625 630 635 640
Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala
645 650 655
Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr
660 665 670
Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr
675 680 685
Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val
690 695 700
Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val
705 710 715 720
Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser
725 730 735
Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys
740 745 750
Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro
755 760 765
Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile
770 775 780
Glu Gly Val Met Arg Asn Asn
785 790
<210> 3
<211> 2433
<212> DNA
<213>Natural plasminogen containing signal peptide（From swiss prot）Nucleic acid sequence
<400> 3
atggaacata aggaagtggt tcttctactt cttttatttc tgaaatcagg tcaaggagag 60
cctctggatg actatgtgaa tacccagggg gcttcactgt tcagtgtcac taagaagcag 120
ctgggagcag gaagtataga agaatgtgca gcaaaatgtg aggaggacga agaattcacc 180
tgcagggcat tccaatatca cagtaaagag caacaatgtg tgataatggc tgaaaacagg 240
aagtcctcca taatcattag gatgagagat gtagttttat ttgaaaagaa agtgtatctc 300
tcagagtgca agactgggaa tggaaagaac tacagaggga cgatgtccaa aacaaaaaat 360
ggcatcacct gtcaaaaatg gagttccact tctccccaca gacctagatt ctcacctgct 420
acacacccct cagagggact ggaggagaac tactgcagga atccagacaa cgatccgcag 480
gggccctggt gctatactac tgatccagaa aagagatatg actactgcga cattcttgag 540
tgtgaagagg aatgtatgca ttgcagtgga gaaaactatg acggcaaaat ttccaagacc 600
atgtctggac tggaatgcca ggcctgggac tctcagagcc cacacgctca tggatacatt 660
ccttccaaat ttccaaacaa gaacctgaag aagaattact gtcgtaaccc cgatagggag 720
ctgcggcctt ggtgtttcac caccgacccc aacaagcgct gggaactttg tgacatcccc 780
cgctgcacaa cacctccacc atcttctggt cccacctacc agtgtctgaa gggaacaggt 840
gaaaactatc gcgggaatgt ggctgttacc gtgtccgggc acacctgtca gcactggagt 900
gcacagaccc ctcacacaca taacaggaca ccagaaaact tcccctgcaa aaatttggat 960
gaaaactact gccgcaatcc tgacggaaaa agggccccat ggtgccatac aaccaacagc 1020
caagtgcggt gggagtactg taagataccg tcctgtgact cctccccagt atccacggaa 1080
caattggctc ccacagcacc acctgagcta acccctgtgg tccaggactg ctaccatggt 1140
gatggacaga gctaccgagg cacatcctcc accaccacca caggaaagaa gtgtcagtct 1200
tggtcatcta tgacaccaca ccggcaccag aagaccccag aaaactaccc aaatgctggc 1260
ctgacaatga actactgcag gaatccagat gccgataaag gcccctggtg ttttaccaca 1320
gaccccagcg tcaggtggga gtactgcaac ctgaaaaaat gctcaggaac agaagcgagt 1380
gttgtagcac ctccgcctgt tgtcctgctt ccagatgtag agactccttc cgaagaagac 1440
tgtatgtttg ggaatgggaa aggataccga ggcaagaggg cgaccactgt tactgggacg 1500
ccatgccagg actgggctgc ccaggagccc catagacaca gcattttcac tccagagaca 1560
aatccacggg cgggtctgga aaaaaattac tgccgtaacc ctgatggtga tgtaggtggt 1620
ccctggtgct acacgacaaa tccaagaaaa ctttacgact actgtgatgt ccctcagtgt 1680
gcggcccctt catttgattg tgggaagcct caagtggagc cgaagaaatg tcctggaagg 1740
gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 1800
aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 1860
gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 1920
caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 1980
cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 2040
gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 2100
atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 2160
ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 2220
tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 2280
ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 2340
ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 2400
acttggattg agggagtgat gagaaataat taa 2433
<210> 4
<211> 810
<212> PRT
<213>Natural plasminogen containing signal peptide（From swiss prot）Amino acid sequence
<400> 4
Met Glu His Lys Glu Val Val Leu Leu Leu Leu Leu Phe Leu Lys Ser
1 5 10 15
Gly Gln Gly Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser
20 25 30
Leu Phe Ser Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu
35 40 45
Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe
50 55 60
Gln Tyr His Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg
65 70 75 80
Lys Ser Ser Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys
85 90 95
Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg
100 105 110
Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser
115 120 125
Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser
130 135 140
Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln
145 150 155 160
Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys
165 170 175
Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn
180 185 190
Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala
195 200 205
Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe
210 215 220
Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu
225 230 235 240
Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu
245 250 255
Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr
260 265 270
Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala
275 280 285
Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro
290 295 300
His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp
305 310 315 320
Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His
325 330 335
Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys
340 345 350
Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro
355 360 365
Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser
370 375 380
Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser
385 390 395 400
Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr
405 410 415
Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp
420 425 430
Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr
435 440 445
Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro
450 455 460
Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp
465 470 475 480
Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr
485 490 495
Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg
500 505 510
His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys
515 520 525
Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr
530 535 540
Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys
545 550 555 560
Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys
565 570 575
Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp
580 585 590
Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly
595 600 605
Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu
610 615 620
Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His
625 630 635 640
Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg
645 650 655
Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser
660 665 670
Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser
675 680 685
Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp
690 695 700
Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln
705 710 715 720
Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn
725 730 735
Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly
740 745 750
Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu
755 760 765
Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys
770 775 780
Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val
785 790 795 800
Thr Trp Ile Glu Gly Val Met Arg Asn Asn
805 810
<210> 5
<211> 2145
<212> DNA
<213> LYS77-PLG（Lys- plasminogens）Nucleic acid sequence
<400> 5
aaagtgtatc tctcagagtg caagactggg aatggaaaga actacagagg gacgatgtcc 60
aaaacaaaaa atggcatcac ctgtcaaaaa tggagttcca cttctcccca cagacctaga 120
ttctcacctg ctacacaccc ctcagaggga ctggaggaga actactgcag gaatccagac 180
aacgatccgc aggggccctg gtgctatact actgatccag aaaagagata tgactactgc 240
gacattcttg agtgtgaaga ggaatgtatg cattgcagtg gagaaaacta tgacggcaaa 300
atttccaaga ccatgtctgg actggaatgc caggcctggg actctcagag cccacacgct 360
catggataca ttccttccaa atttccaaac aagaacctga agaagaatta ctgtcgtaac 420
cccgataggg agctgcggcc ttggtgtttc accaccgacc ccaacaagcg ctgggaactt 480
tgtgacatcc cccgctgcac aacacctcca ccatcttctg gtcccaccta ccagtgtctg 540
aagggaacag gtgaaaacta tcgcgggaat gtggctgtta ccgtgtccgg gcacacctgt 600
cagcactgga gtgcacagac ccctcacaca cataacagga caccagaaaa cttcccctgc 660
aaaaatttgg atgaaaacta ctgccgcaat cctgacggaa aaagggcccc atggtgccat 720
acaaccaaca gccaagtgcg gtgggagtac tgtaagatac cgtcctgtga ctcctcccca 780
gtatccacgg aacaattggc tcccacagca ccacctgagc taacccctgt ggtccaggac 840
tgctaccatg gtgatggaca gagctaccga ggcacatcct ccaccaccac cacaggaaag 900
aagtgtcagt cttggtcatc tatgacacca caccggcacc agaagacccc agaaaactac 960
ccaaatgctg gcctgacaat gaactactgc aggaatccag atgccgataa aggcccctgg 1020
tgttttacca cagaccccag cgtcaggtgg gagtactgca acctgaaaaa atgctcagga 1080
acagaagcga gtgttgtagc acctccgcct gttgtcctgc ttccagatgt agagactcct 1140
tccgaagaag actgtatgtt tgggaatggg aaaggatacc gaggcaagag ggcgaccact 1200
gttactggga cgccatgcca ggactgggct gcccaggagc cccatagaca cagcattttc 1260
actccagaga caaatccacg ggcgggtctg gaaaaaaatt actgccgtaa ccctgatggt 1320
gatgtaggtg gtccctggtg ctacacgaca aatccaagaa aactttacga ctactgtgat 1380
gtccctcagt gtgcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 1440
tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 1500
agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 1560
gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 1620
ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 1680
ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 1740
atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 1800
accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 1860
aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1920
ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1980
cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 2040
gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 2100
tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 2145
<210> 6
<211> 714
<212> PRT
<213> LYS77-PLG（Lys- plasminogens）Amino acid sequence
<400> 6
Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg
1 5 10 15
Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser
20 25 30
Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser
35 40 45
Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln
50 55 60
Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys
65 70 75 80
Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn
85 90 95
Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala
100 105 110
Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe
115 120 125
Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu
130 135 140
Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu
145 150 155 160
Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr
165 170 175
Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala
180 185 190
Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro
195 200 205
His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp
210 215 220
Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His
225 230 235 240
Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys
245 250 255
Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro
260 265 270
Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser
275 280 285
Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser
290 295 300
Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr
305 310 315 320
Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp
325 330 335
Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr
340 345 350
Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro
355 360 365
Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp
370 375 380
Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr
385 390 395 400
Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg
405 410 415
His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys
420 425 430
Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr
435 440 445
Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys
450 455 460
Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys
465 470 475 480
Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp
485 490 495
Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly
500 505 510
Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu
515 520 525
Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His
530 535 540
Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg
545 550 555 560
Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser
565 570 575
Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser
580 585 590
Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp
595 600 605
Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln
610 615 620
Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn
625 630 635 640
Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly
645 650 655
Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu
660 665 670
Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys
675 680 685
Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val
690 695 700
Thr Trp Ile Glu Gly Val Met Arg Asn Asn
705 710
<210> 7
<211> 1245
<212> DNA
<213> delta-plg（Delta- plasminogens）Nucleic acid sequence
<400> 7
gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60
cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120
acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180
aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240
ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300
aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360
gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420
caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480
gagtgtgaag aggcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 540
tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 600
agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 660
gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 720
ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 780
ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 840
atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 900
accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 960
aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1020
ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1080
cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 1140
gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 1200
tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 1245
<210> 8
<211> 414
<212> PRT
<213> delta-plg（Delta- plasminogens）Amino acid sequence
<400> 8
Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser
1 5 10 15
Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala
20 25 30
Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His
35 40 45
Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser
50 55 60
Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr
65 70 75 80
Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met
85 90 95
Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser
100 105 110
Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu
115 120 125
Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp
130 135 140
Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu
145 150 155 160
Glu Cys Glu Glu Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val
165 170 175
Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His
180 185 190
Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met
195 200 205
His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala
210 215 220
Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile
225 230 235 240
Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile
245 250 255
Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu
260 265 270
Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala
275 280 285
Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe
290 295 300
Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu
305 310 315 320
Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr
325 330 335
Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His
340 345 350
Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu
355 360 365
Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp
370 375 380
Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val
385 390 395 400
Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn
405 410
<210> 9
<211> 1104
<212> DNA
<213> Mini-plg（Small plasminogen）Nucleic acid sequence
<400> 9
gtcaggtggg agtactgcaa cctgaaaaaa tgctcaggaa cagaagcgag tgttgtagca 60
cctccgcctg ttgtcctgct tccagatgta gagactcctt ccgaagaaga ctgtatgttt 120
gggaatggga aaggataccg aggcaagagg gcgaccactg ttactgggac gccatgccag 180
gactgggctg cccaggagcc ccatagacac agcattttca ctccagagac aaatccacgg 240
gcgggtctgg aaaaaaatta ctgccgtaac cctgatggtg atgtaggtgg tccctggtgc 300
tacacgacaa atccaagaaa actttacgac tactgtgatg tccctcagtg tgcggcccct 360
tcatttgatt gtgggaagcc tcaagtggag ccgaagaaat gtcctggaag ggttgtaggg 420
gggtgtgtgg cccacccaca ttcctggccc tggcaagtca gtcttagaac aaggtttgga 480
atgcacttct gtggaggcac cttgatatcc ccagagtggg tgttgactgc tgcccactgc 540
ttggagaagt ccccaaggcc ttcatcctac aaggtcatcc tgggtgcaca ccaagaagtg 600
aatctcgaac cgcatgttca ggaaatagaa gtgtctaggc tgttcttgga gcccacacga 660
aaagatattg ccttgctaaa gctaagcagt cctgccgtca tcactgacaa agtaatccca 720
gcttgtctgc catccccaaa ttatgtggtc gctgaccgga ccgaatgttt catcactggc 780
tggggagaaa cccaaggtac ttttggagct ggccttctca aggaagccca gctccctgtg 840
attgagaata aagtgtgcaa tcgctatgag tttctgaatg gaagagtcca atccaccgaa 900
ctctgtgctg ggcatttggc cggaggcact gacagttgcc agggtgacag tggaggtcct 960
ctggtttgct tcgagaagga caaatacatt ttacaaggag tcacttcttg gggtcttggc 1020
tgtgcacgcc ccaataagcc tggtgtctat gttcgtgttt caaggtttgt tacttggatt 1080
gagggagtga tgagaaataa ttaa 1104
<210> 10
<211> 367
<212> PRT
<213> Mini-plg（Small plasminogen）Amino acid sequence
<400> 10
Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala
1 5 10 15
Ser Val Val Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr
20 25 30
Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly
35 40 45
Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala
50 55 60
Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg
65 70 75 80
Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly
85 90 95
Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys
100 105 110
Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln
115 120 125
Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala
130 135 140
His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly
145 150 155 160
Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr
165 170 175
Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val
180 185 190
Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu
195 200 205
Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala
210 215 220
Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro
225 230 235 240
Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys
245 250 255
Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu
260 265 270
Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg
275 280 285
Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly
290 295 300
His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro
305 310 315 320
Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser
325 330 335
Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg
340 345 350
Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn
355 360 365
<210> 11
<211> 750
<212> DNA
<213> Micro-plg（Fibrillin lyase is former）Nucleic acid sequence
<400> 11
gccccttcat ttgattgtgg gaagcctcaa gtggagccga agaaatgtcc tggaagggtt 60
gtaggggggt gtgtggccca cccacattcc tggccctggc aagtcagtct tagaacaagg 120
tttggaatgc acttctgtgg aggcaccttg atatccccag agtgggtgtt gactgctgcc 180
cactgcttgg agaagtcccc aaggccttca tcctacaagg tcatcctggg tgcacaccaa 240
gaagtgaatc tcgaaccgca tgttcaggaa atagaagtgt ctaggctgtt cttggagccc 300
acacgaaaag atattgcctt gctaaagcta agcagtcctg ccgtcatcac tgacaaagta 360
atcccagctt gtctgccatc cccaaattat gtggtcgctg accggaccga atgtttcatc 420
actggctggg gagaaaccca aggtactttt ggagctggcc ttctcaagga agcccagctc 480
cctgtgattg agaataaagt gtgcaatcgc tatgagtttc tgaatggaag agtccaatcc 540
accgaactct gtgctgggca tttggccgga ggcactgaca gttgccaggg tgacagtgga 600
ggtcctctgg tttgcttcga gaaggacaaa tacattttac aaggagtcac ttcttggggt 660
cttggctgtg cacgccccaa taagcctggt gtctatgttc gtgtttcaag gtttgttact 720
tggattgagg gagtgatgag aaataattaa 750
<210> 12
<211> 249
<212> PRT
<213> Micro-plg（Fibrillin lyase is former）Amino acid sequence
<400> 12
Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys
1 5 10 15
Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro
20 25 30
Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly
35 40 45
Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu
50 55 60
Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln
65 70 75 80
Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu
85 90 95
Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser
100 105 110
Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro
115 120 125
Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly
130 135 140
Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu
145 150 155 160
Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly
165 170 175
Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr
180 185 190
Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys
195 200 205
Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala
210 215 220
Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr
225 230 235 240
Trp Ile Glu Gly Val Met Arg Asn Asn
245
<210> 13
<211> 684
<212> DNA
<213>Serine protease（Structure）The nucleic acid sequence in domain
<400> 13
gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 60
aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 120
gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 180
caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 240
cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 300
gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 360
atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 420
ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 480
tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 540
ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 600
ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 660
acttggattg agggagtgat gaga 684
<210> 14
<211> 228
<212> PRT
<213>Serine protease（Structure）The amino acid sequence in domain
<400> 14
Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val
1 5 10 15
Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile
20 25 30
Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro
35 40 45
Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn
50 55 60
Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu
65 70 75 80
Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val
85 90 95
Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val
100 105 110
Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln
115 120 125
Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile
130 135 140
Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln
145 150 155 160
Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys
165 170 175
Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr
180 185 190
Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn
195 200 205
Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu
210 215 220
Gly Val Met Arg
225

Claims (10)

1. a kind of method prevented or treat subject's myocardial damage, including a effective amount of plasminogen of subject is administered, wherein The subject has myocardial damage risk, suspection to have myocardial damage or suffer from myocardial damage.
2. the method for claim 1 wherein the myocardial damage includes ischemic, inflammation, allergy, autoimmunity, thrombus, micro- Dyshaemia, wound, radiation injury, carbohydrate metabolism disturbance, fat metabolic disturbance myocardial injury.
3. the method for claims 1 or 2, wherein the myocardial damage is selected from myocardial damage caused by following disease：Myocarditis, Pericarditis, hypertension, atherosclerosis, coronary heart diseases and angina pectoris, myocardial infarction, arrhythmia cordis, heart failure, shock, diffusivity blood Intravascular coagulation, microcirculation disorder, diabetes, hyperlipidemia, Artery, Vein blood bolt, fat embolism, ischemia-reperfusion, systematicness Sclerosis, systemic loupus erythematosus, coronary artery stenosis, rheumatic heart disease, mitral stenosis/incompetence, aorta petal Narrow/incompetence, necrosis, infection.
4. the method for claims 1 or 2, wherein the myocardial damage is myocardial damage caused by ischemic heart disease.
5. the method for claim 4, wherein the ischemic heart disease is atherosclerosis, coronary heart diseases and angina pectoris, cardiac muscle Infarct, arrhythmia cordis, heart failure, shock, disseminated intravascular coagulation, microcirculation disorder, ischemia-reperfusion, coronary artery stenosis, Mitral stenosis/incompetence, aortic stenosis/incompetence.
6. the method for claims 1 or 2, wherein the myocardial damage is cardiac muscle caused by Artery, Vein blood bolt or fat embolism Damage.
7. the method for claim 6, wherein the thrombus or embolism are caused by atherosclerosis.
8. a kind of method prevented or treat subject's myocardial damage, including a effective amount of plasminogen protection heart of administration subject Muscular tissue.
9. the method for claim 8, wherein the plasminogen mitigates the cardiac muscle cell apoptosis caused by myocardial cell injury.
10. the method for claim 8 or 9, wherein the plasminogen promotes the reparation of injury of myocardium.
CN201710467076.9A 2016-12-15 2017-06-19 Improve drug of heart disease and application thereof Pending CN108210915A (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN2016111622708 2016-12-15
CN201611162237 2016-12-15
CN201611162270 2016-12-15
CN2016111622375 2016-12-15
CN201611161347X 2016-12-15
CN201611161347 2016-12-15

Publications (1)

Publication Number Publication Date
CN108210915A true CN108210915A (en) 2018-06-29

Family

ID=62657340

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710467076.9A Pending CN108210915A (en) 2016-12-15 2017-06-19 Improve drug of heart disease and application thereof

Country Status (2)

Country Link
CN (1) CN108210915A (en)
HK (1) HK1257598A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021170099A1 (en) * 2020-02-26 2021-09-02 泰伦基国际有限公司 Method and drug for preventing and treating abnormal blood pressure condition

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0192667B1 (en) * 1984-08-08 1994-10-26 Survival Technology, Inc. Device for initiating reperfusion treatment when heart attack symptoms are present
CN1768138A (en) * 2002-02-06 2006-05-03 N-酶生物技术有限公司 Method for producing recombinant proteins in micro-organisms
CN101132788A (en) * 2005-03-01 2008-02-27 瑟维尔实验室 Diosmetin derivative for treating and preventing disease of thrombosis
CN102154253A (en) * 2011-01-06 2011-08-17 郑州大学 Micro plasminogen mutant with function of inhibiting platelet aggregation and preparation method and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0192667B1 (en) * 1984-08-08 1994-10-26 Survival Technology, Inc. Device for initiating reperfusion treatment when heart attack symptoms are present
CN1768138A (en) * 2002-02-06 2006-05-03 N-酶生物技术有限公司 Method for producing recombinant proteins in micro-organisms
CN101132788A (en) * 2005-03-01 2008-02-27 瑟维尔实验室 Diosmetin derivative for treating and preventing disease of thrombosis
CN102154253A (en) * 2011-01-06 2011-08-17 郑州大学 Micro plasminogen mutant with function of inhibiting platelet aggregation and preparation method and application thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
QING XIAO ET AL.: "Plasminogen deficiency accelerates vessel wall disease in mice predisposed to atherosclerosis", 《PROC. NATL. ACAD. SCI. USA》 *
中国医学百科全书编辑委员会编，《基础医学》编辑委员会编: "《中国医学百科全书 基础医学》", 31 December 1998, 上海：上海科学技术出版社 *
王恩华主编: "《病理学》", 30 September 2003, 北京：高等教育出版社 *
赵雅静,张雯: "代谢综合征患者凝血纤溶系统功能的变化", 《疑难病杂志》 *
黄翠瑶等: "重组组织型纤溶酶原激活剂对实验性缺血心肌的保护", 《实用医学杂志》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021170099A1 (en) * 2020-02-26 2021-09-02 泰伦基国际有限公司 Method and drug for preventing and treating abnormal blood pressure condition

Also Published As

Publication number Publication date
HK1257598A1 (en) 2019-10-25

Similar Documents

Publication Publication Date Title
TWI684459B (en) A method for treating atherosclerosis and its complications
CN110114081A (en) A method of improving heart disease
CN108210902A (en) Prevent and treat drug of fatty liver and application thereof
CN108463240A (en) A method of preventing or treat diabetic retinopathy
CN110139668A (en) A method of preventing and treating liver fibrosis
CN109125715A (en) A kind of method and drug of regulation GLP-1/GLP-1R
KR20140026399A (en) Factor xii inhibitors for the treatment of silent brain ischemia and ischemia of other organs
CN109925507A (en) A kind of method and drug prevented or treat osteoarthritis
CN110114079A (en) A kind of method and drug preventing and treating obesity
CN108210899A (en) Prevent and treat drug of histoorgan fibrosis and application thereof
CN106890324A (en) A kind of method for preventing and treating diabetic nephropathy
CN106890320A (en) It is a kind of for preventing or treating acute and Chronic Thrombotic method
CN108210909A (en) Prevent and treat drug of lipid injury of kidney and application thereof
CN108210915A (en) Improve drug of heart disease and application thereof
CN108210893A (en) Prevent and treat drug of kidney fibrosis and application thereof
CN108210917A (en) A kind of method and drug for preventing and treating obesity
CN106890318A (en) A kind of new method for preventing and treating diabetic cardiomyopathy
CN106890325A (en) A kind of method prevented or treat diabetic nerve damage and its associated conditions
CN108210916A (en) A kind of drug for preventing and treating osteoporosis and application thereof
CN106890319A (en) A kind of method prevented or treat BDR
CN106890323A (en) A kind of method for preventing and treating liver tissue injury and its associated conditions
CN106890322A (en) A kind of prevention or the method for therapeutic radiation and chemical lesion

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 1257598

Country of ref document: HK

RJ01 Rejection of invention patent application after publication

Application publication date: 20180629

RJ01 Rejection of invention patent application after publication