CN108210908A - Prevent and treat drug of Drug injury of kidney and application thereof - Google Patents

Abstract

The present invention relates to the method for subject's Renal tissues damage and its associated disease caused by prevention and/or medicine, including a effective amount of plasminogen of subject is administered.The invention further relates to subject's Renal tissues damage caused by for prevention and/or medicine and its drug comprising plasminogen, pharmaceutical composition, product, the kits of associated disease.

Description

Prevent and treat drug of Drug injury of kidney and application thereof

Technical field

It is led the present invention relates to effect of the plasminogen in terms of renal lesions are prevented and treated, and then to treat different reasons The renal lesions and its associated disease of cause provide completely new therapeutic strategy.

Background of invention

Renal lesions are Renal Structure variation and dysfunction caused by a variety of causes.Renal lesions lead to nephridial tissue structure Damage, then influence its function.Renal lesions can be primary, such as the kidney of the initiations such as infection, inflammation, allergy is small Between ball ephritis, chronic pyelonephritis, nephrotic syndrome, renal insufficiency, glomerulosclerosis, glomerular mesangial matrixes, renal tubule Matter lesion, renal tubule atrophy etc.；Can also be secondary to Other diseases, such as can be due to ischemic, metabolic disorder, such as sugared generation It thanks, the other diseases such as fat metabolic disturbance, tumour lead to renal lesions.

Such as the diseases such as hypertension, diabetes, atherosclerosis are often accompanied by renal lesions.Hypertension is common chronic One of disease, which is mainly shown as that systemic arterial pressure increases, if the controlling of blood pressure to hypertensive patient is bad, easily The complication such as cerebral apoplexy, coronary heart disease, retinopathy and chronic renal disease occur.And prolonged hypertension can make More and more histoorgans are affected.So to strengthen and the complication of preventing hypertension and hypertension, so as to reduce height The harm that sphygmomanometer cuff comes^[1]。

Diabetic nephropathy (diabetic nephropathy, DN), is the common important complication of diabetes, is diabetes Lethal, the main reason for disabling.Such as diagnosis and treatment not in time, when DN develops to end-stage of renal disease, dialysis even kidney can only be used to move It plants.The major pathologic features of DN early stages are glomerulus hypertrophys, glomerulus and renal tubular basement membrane thickens and the extracellular base of mesangial region The progressive accumulation of matter；Later stage is glomerulus, renal interstitial fibrosis.Glomerular filtration rate is clinically can behave as in early days to subtract It is few, then there is microalbuminuria, arterial pressure raising, albuminuria and fluid retention, eventually lead to kidney failure^[2]。

Diabetic nephropathy category Diabetic microvascular complication, the factors such as generation and hyperglycemia, oxidative stress are related, wherein Hyperglycemia is an important factor for generating microalbuminuria^[3].Microdose urine protein can indicate the progress of diabetic nephropathy.And Diabetic artherosclerosis belongs to great vessels complication in diabetic patients, with hyperglycemia, vascular endothelial dysfunction, insulin resistance Etc. factors it is closely related.In recent years studies have found that, albuminuria is closely related with atherosclerosis^[4]。

In developed country, the main reason for diabetic nephropathy, hypertensive renal arteriolar sclerosis have become chronic kidney disease. China after both diseases still occupy primary glomerulonephritis in the various causes of disease, but also has and significantly increases trend in recent years.

Systemic allergic disease, such as systemic sclerosis, systemic loupus erythematosus, systemic vasculitis, allergy Property purpura, polymyositis, thrombotic microvascular disease etc. usually involve kidney.

Most of drug and its metabolite are excreted through kidney, thus drug-induced renal damage incidence is very high. Researches show that drug leads to acute tubular necrosis (acute tubular necrosis, ATN) or acute interstitial nephritis The incidence of (acute interstitial nephritis, AIN) is up to 18.3%, the wherein generation of nephropathy due to antibiotics Rate is up to 36%^[5]。

Anti-infection drug, such as aminoglycoside aminoglycoside antibiotics are widely used in gram-negative bacterial infection Treatment, however renal toxicity limits its clinical practice^[6]。

Anti-virus formulation acyclovir (aciclovir, ACV) is the cyclic analogs of the deoxyguanosine of anti-herpesvirus. Parenteral high-dose of acyclovir of giving can cause 10%~48% patient acute renal failure (acute renal occur Failure, ARF), this may be deposited due to acyclovir in renal tubule, toxic immune reaction or hypersensitivity etc. occurs draws Rise kidney intrinsic obstruction caused by^[7].Adefovirdipivoxil (adedovir, ADV) and Qi Duofuwei (cidofovir, CDV) are similar as nucleosides Object is clinically then commonly used to treatment hepatitis B and AIDS.The generation of its renal toxicity shows as renal tubular necrosis often and interstitial is fine Dimensionization^[8]。

Immunosuppressor such as cyclosporine cyclosporine (cyclosporine A, CsA) is widely used in as immunosuppressor Organ transplant and the treatment of autoimmune disease.It is reported that the patient for receiving CsA treatments there are about 30% arrives severe in will appear Renal dysfunction.Its injury of kidney mechanism is since Renal vascular is shunk and endothelial cell damage causes ischemic and CsA to renal tubule The direct toxic effect of epithelial cell^[9]。

Antitumor drug such as cis-platinum (cisplatin, Cis) is a kind of inhibition of cell proliferation, be widely used in carcinoma of testis, In the treatment of the solid tumors such as oophoroma, prostate cancer, lung cancer, osteocarcinoma and head and neck cancer.Cis's is antitumor efficient, but has simultaneously There is dose-dependent renal toxicity etc.^[10], azotemia, polyuria and renal failure are mainly shown as, with glomerulus and kidney Tubule is impaired to be characterized.

Non-steroid anti-inflammatory drug such as acetylsalicylic acid acetylsalicylic acid (acetylsalicylic acid, ASA) is generation Most widely used antipyretic-antalgic anti-inflammatory agent in boundary.It is used for the dosage of antipyretic-antalgic and seldom causes adverse reaction, but long-term big It is then easier to side effect occur when measuring medication, shows as potassium ion being made to escape from renal tubular cell due to oxidative phosphorylation solution couples, Cause potassium deficiency, urine uric acid discharge excessively high；During larger damage, interstitial nephritis, necrosis of renal papillae and renal function can occur and subtract It moves back^[11]。

Natural product active ingredient such as aristolochic acid aristolochic acid (aristolochic acid, AA) is as birthwort The nephrotoxin of section's plant origin, it is famous Aristolochic acid nephropathy (aristolochic acid nephropathy, AAN) The cause of disease^[12].Kidney injury position caused by AA mainly in renal tubule, causes Apoptosis or death, and inhibit renal interstitial into Fibroblast proliferation leads to renal tubule atrophy and few cellularity interstitial fibrosis.

Diuretics is a kind of by inhibiting reabsorption of the renal tubule to water, electrolyte, the drug for increasing urine volume discharge.Respectively Kind diuretics has potential renal toxicity, there is the possibility for causing renal damage after.Renal toxicity caused by diuretics and such The adverse reactions such as the cytotoxicity of drug, immune response, allergic reaction and metabolic disorder are related, should avoid as possible and renal toxicity Drug combination prevents from aggravating renal damage.In addition, also injury of kidney can be caused there are many drug, to be formed as sulfa drugs often causes Sulfanilamide crystal obstruction ureter causes obstructive nephropathy^[13].Lipid-lowering medicine Statins can cause rhabdomyolysis, then cause kidney small Pipe necrosis^[14]。

Kidney trouble, which develops to the later stage, can all cause some or all of forfeiture of renal function, that is, lead to the pathology shape of kidney failure State.Kidney failure can be divided into acute renal failure and chronic renal failure, and the disease progression of acute renal failure is quick, typically because of kidney blood Stream is insufficient, kidney function caused by certain factor is blocked is damaged or is injured by poisonous substance, causes acute renal failure It generates.And chronic renal failure main cause is long-term renal lesions, with the progress of time and disease, the function of kidney is gradual Decline, cause the generation of kidney failure.

Slow progressive kidney function damage caused by chronic renal failure refers to various kidney troubles, finally results in uremic Disease and renal function completely lose, and cause a series of clinical syndrome of the metabolic disorders composition such as clinical symptoms and biochemical endocrine, Since protopathy onset to renal failure, interval time can be the several years to more than ten years.

Uremia is the whole latter stage of chronic renal failure.Chronic renal failure be the various causes of disease cause kidney damage and The result that progressive deteriorates.Common underlying diseases have primary glomerulonephritis, tubulointerstitial nephritis, diabetic nephropathy Deng.Its clinical manifestation is mainly renal hypofunction, metabolic waste retention, water, electrolyte and acid-base imbalance, so that cannot tie up The stabilization of organismic internal environment is held, according to the different degrees of of kidney function damage, chronic renal failure can be divided into four-stage： Compensatory stage of renal insufficiency, renal insufficiency Decompensated stage, renal failure stage, Uremic.

The early stage of chronic renal insufficiency, the clinically only symptom of primary disease, only visible creatinine is clear in inspection Except rate declines.The patient of uremia compensatory phase often in the case that stress, renal function deteriorates suddenly, and water, electrolyte, soda acid occurs Metabolic disorder, protein, carbohydrate, fat and vitamin metabolism are disorderly, loss of appetite or indigestion, and when aggravation may occur in which The uremic early symptom such as the gastrointestinal symptoms such as apocleisis, Nausea and vomiting or diarrhea, clinically referred to as Reversible Uremic, one Denier stress factor removes, and renal function can often be restored to the compensatory phase.If progression of the disease cannot adapt to body most to " strong to deposit " nephron Low even if without stress factor, uremic symptom also can be showed gradually when requiring, and show as that water-electrolyte metabolism is disorderly, body Constitutional symptoms and the cardiovascular systems such as the accumulation of intracellular metabolite product, respiratory system, hematological system moderate symptoms.

The drug therapy of chronic renal insufficiency, which is concentrated mainly on, to be alleviated symptom, delays disease progression and prevention complication Three aspects specifically for example correct acid poisoning and water and rock-soil coupling, treatment hypertension, prevention infection, treatment hyperlipidemia, And the treatment for cardiovascular system, respiratory system and hematological system complication, while uremic substitute can be carried out and dialysed Treatment, such as haemodialysis or peritoneal dialysis or progress kidney transplant.

" acute kidney injury (acute kidney injury, AKI) " is the new term proposed in recent years, for taking In generation, has been widely recognized as using acute renal failure (acute renal failure, ARF) for many years.It is a kind of common Clinical syndrome, be mainly shown as the accumulation of the rapid decrease and metabolic waste of renal function.AKI incidence is high, and in year by year Ascendant trend.The incidence of foreign countries report AKI in the past 10 years rises to 5 ‰ from 0.65 ‰, needs the AKI to dialyse the incidence to be 0.295‰^[15-16].Inpatient AKI incidences are 1.9%, and intensive care unit then may be up to 60%^[17].It does not control still at present The specific medicament of AKI is treated, patient with severe symptoms needs renal replacement therapies^[18].The prognosis of AKI also allows of no optimist, and ATN and RENAL are ground Study carefully^[19-20]It is respectively 53.0% and 44.7% that case fatality rate after AKI, which occurs, for report patient with severe symptoms, and the patient survived also easily into It opens up to chronic kidney disease or even end-stage renal disease^[21].Therefore, AKI increasingly causes the attention of clinician.

For Renal tissues damage caused by a variety of causes, people thirst for searching out more effective medicine always.This hair The bright renal tissue reparation for passing through the damage the study found that plasminogen can promote damage, the recovery of renal function, while fibrinolytic Proenzyme can also inhibit the renal tissue Apoptosis of damage, reduce its fibrosis.Therefore, plasminogen is expected to become a kind of novel Kidney diseases drug.

Summary of the invention

The present invention relates to following items：

On the one hand, the present invention relates to：Subject's Renal tissues damage caused by a kind of 1. preventions and/or medicine and The method of its associated disease, including a effective amount of plasminogen of subject is administered.

The method that item is 2. 1, wherein the drug is nephrotoxic drugs.

The method that item is 3. 1 or 2, wherein the drug is the drug of kidney excretion.

The method of any one of 4. 1-3, wherein the drug include chemotherapeutics, blood-pressure drug, blood lipid-lowering medicine, Hypoglycemic drug, nonsteroidal antiinflammatory drug, antibiotic medicine, antiviral drugs.

The method of any one of 5. 1-4 of item, wherein the plasminogen promotes the reparation of the nephridial tissue of damage.

The method of any one of 6. 1-5 of item, wherein the plasminogen mitigates the fibroid of the nephridial tissue of the damage.

The method of any one of 7. 1-6 of item, wherein the plasminogen mitigates the apoptosis of the nephridial tissue of the damage.

The method of any one of 8. 1-7 of item, wherein drug induced subject's Renal tissues damage is acute nephridial tissue Damage.

The method of any one of 9. 1-7 of item, wherein drug induced subject's Renal tissues damage is chronic nephridial tissue Damage.

The method of any one of item 10, item 1-9, wherein the plasminogen promotes renal function to restore.

On the other hand, the present invention relates to：Item 11. is a kind of to protect subject's kidney that it is made to mitigate or from drug induced injury Method is included in before giving the drug while, and/or gives a effective amount of plasminogen of the subject later.

On the other hand, the present invention relates to：Kidney group caused by a kind of 12. prevention of item and/or treatment subject's renal lesions The method for knitting damage and its associated disease, including a effective amount of plasminogen of subject is administered.

The method that item is 13. 13, wherein the drug is cis-platinum.

The method that item is 14. 12 or 13, wherein the kidney tissue damage includes infection, inflammation, allergy, itself exempts from Kidney group caused by renal lesions caused by epidemic disease, ischemic, thrombus, wound, radiation injury, carbohydrate metabolism disturbance, fat metabolic disturbance, tumour Knit damage.

The method of any one of 15. 12-14 of item, wherein the renal lesions are caused by systemic disease chosen from the followings Renal lesions：Hypertension, diabetes, atherosclerosis, systemic sclerosis, systemic loupus erythematosus, hyperlipidemia, it is non-what Outstanding gold lymphomas, Huppert's disease, systemic vasculitis, anaphylactoid purpura, polymyositis, thrombotic microvascular disease.

The method of any one of 16. 12-15 of item, wherein the renal lesions are chronic renal disease.

The method of any one of 17. 12-16 of item, wherein the chronic renal disease is chronic glomerulonephritis, chronic renal Nephropyelitis, nephrotic syndrome, renal insufficiency or uremia.

The method that item is 18. 17, wherein the chronic renal disease is glomerulosclerosis, glomerular mesangial matrixes；Kidney is small Pipe interstitial lesion, kidney region fibrosis, renal tubule atrophy.

The method of any one of 19. 12-18 of item, wherein the chronic renal disease is drug induced Chronic Renal Impairment.

The method of 20. 19, wherein the chronic renal disease is chemotherapeutics, blood-pressure drug, blood lipid-lowering medicine, Chronic Renal Impairment caused by hypoglycemic drug, nonsteroidal antiinflammatory drug, antibiotic medicine, antiviral drugs.

The method that item is 21. 20, wherein the chemotherapeutics is cis-platinum.

The method of any one of 22. 12-15 of item, wherein the renal lesions are acute kidney diseases.

The method of any one of 23. 12-15 of item, wherein the acute kidney diseases are acute glomerulonephritis, acute kidney Nephropyelitis, acute kidney injury, acute renal failure, acute renal insufficiency, acute tubular necrosis.

The method that item is 24. 22 or 23, wherein the acute kidney injury is acute kidney injury caused by chemotherapeutics.

On the other hand, the present invention relates to：A kind of methods prevented and/or treat Chronic Renal Impairment of item 25., including administration Subject prevents and/or the plasminogen of therapeutically effective amount.

The method that item is 26. 25, wherein the Chronic Renal Impairment is Renal tissues damage caused by chronic renal disease.

The method that item is 27. 26, the chronic renal disease is chronic glomerulonephritis, chronic pyelonephritis, nephrosis Syndrome, renal insufficiency or uremia.

The method that item is 28. 26, wherein the chronic renal disease is glomerulosclerosis, glomerular mesangial matrixes；Kidney is small Pipe interstitial lesion, kidney region fibrosis, kidney failure, renal tubule atrophy.

The method of any one of 29. 25-28, wherein the Chronic Renal Impairment for systemic disease with nephridial tissue damage Wound.

The method that item is 30. 29, wherein the systemic disease is selected from：Hypertension diabetes, atherosclerosis, is System property sclerosis, systemic loupus erythematosus, hyperlipidemia, non_hodgkin lymphoma, Huppert's disease, systemic vascular Inflammation, anaphylactoid purpura, polymyositis, thrombotic microvascular disease.

The method that item is 31. 30, wherein the systemic disease is systemic sclerosis.

The method of any one of 32. 1-31 of item, wherein the plasminogen can promote the renal tissue reparation of damage.

The method of any one of 33. 1-31 of item, wherein the plasminogen can reduce the renal tissue fibrosis of damage.

The method of any one of 34. 1-31 of item, wherein the plasminogen can promote the table of apoptosis inhibitory protein Bcl-2 It reaches, inhibits the apoptosis of renal tissue cell.

The method of any one of 35. 1-31 of item, wherein the plasminogen can promote the recovery of renal function.

The method that item is 36. 35, wherein the plasminogen can promote removing of the kidney to urea nitrogen and/or creatinine.

On the other hand, the present invention relates to：A kind of methods prevented and/or treat acute kidney injury of item 37., including administration The subject's prevention needed and/or the plasminogen of therapeutically effective amount.

The method of 38. 37, wherein the acute kidney injury is acute glomerulonephritis, acute pyelonephritis, acute Renal failure, acute renal insufficiency, acute tubular necrosis.

The method that item is 39 37 or 38, wherein the acute kidney injury is acute kidney injury caused by chemotherapeutics.

The method that item is 40. 39, wherein the chemotherapeutics is cis-platinum.

The method of any one of 41. 37-40 of item, wherein the plasminogen can reduce the renal tissue fiber of damage Change.

The method of any one of 42. 37-40 of item, wherein the plasminogen can promote apoptosis inhibitory protein Bcl-2's Expression inhibits the apoptosis of renal tissue cell.

The method of any one of 43. 37-40 of item, wherein the plasminogen can promote the recovery of renal function.

The method that item is 44. 43, wherein the plasminogen can promote removing of the kidney to urea nitrogen and/or creatinine.

On the other hand, the present invention relates to：A kind of methods prevented and/or treat Renal tissues damage associated disease of item 45., Plasminogen including subject's prevention in need and/or therapeutically effective amount is administered.

The method that item is 46. 45, wherein the Renal tissues damage associated disease is selected from：Blood urine, albuminuria, cylindruria, kidney Glomerular filtration rate reduction, oliguresis, anuria, metabolin retention, water, electrolyte and acid-base imbalance, kidney fibrosis, kidney failure, Uremia.

The method of any one of 47. 1-46 of item, wherein the plasminogen has at least with sequence 2,6,8,10 or 12 75%th, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is fibrinolysin Former activity.

The method of any one of 48. 1-47 of item, the plasminogen is on the basis of sequence 2,6,8,10 or 12, is added Add, delete and/or replace 1-100,1-90,1-80,1-70,1-60,1-50,1-45,1-40,1-35,1-30,1-25,1- 20th, 1-15,1-10,1-5,1-4,1-3,1-2,1 amino acid, and still there is the protein of activities of endothelial tissue plasminogen.

The method of any one of 49. 1-48 of item, the plasminogen are comprising activities of endothelial tissue plasminogen segment and still have There is the protein of activities of endothelial tissue plasminogen.

The method of any one of 50. 1-49, the plasminogen are selected from Glu- plasminogens, Lys- plasminogens, small Plasminogen, Microplasminogen, delta- plasminogens or their variant for retaining activities of endothelial tissue plasminogen.

51. according to the method for any one of item 1-50, wherein the plasminogen can with it is one or more chosen from the followings Medication combined application：Blood-pressure drug, antibiotic medicine, anticoagulant, thrombolytic agent, diuretics, resists and swells blood lipid-lowering medicine Knurl medicine, hypoglycemic agent, non-steroid anti-inflammatory drug, immunoregulation medicament, anti-infectives, antiviral drugs, hormone, natural products Active constituent.

The method of any one of 52. 1-51, the plasminogen for natural or synthetic human plasminogen or its still Retain the variant or segment of activities of endothelial tissue plasminogen.

The method of any one of 53. 1-51 of item, the plasminogen are the people from primate or rodent Plasminogen directly to homologue or its still retain the variant of activities of endothelial tissue plasminogen or segment.

The method of any one of 54. 1-53 of item, the amino acid of the plasminogen is as shown in sequence 2,6,8,10 or 12.

The method of any one of 55. 1-54 of item, wherein the plasminogen is naive plasminogen.

The method of any one of 56. 1-55 of item, wherein the subject is people.

The method of any one of 57. 1-26 of item, wherein the subject lacks or missing plasminogen.

The method that item is 58. 57, wherein the shortage or missing are inborn, secondary and/or local.

On the other hand, the present invention relates to：A kind of plasminogens of the methods for any one of item 1-58 of item 59..

On the other hand, the present invention relates to：A kind of 60. pharmaceutical compositions, it includes pharmaceutically acceptable supporting agent and For the plasminogen of any one of item 1-58 the methods.

On the other hand, the present invention relates to：A kind of preventative or therapeutic agent boxes of item 61., it includes：(i) for item The plasminogen of any one of 1-58 the methods and (ii) are for delivering the plasminogen to the component of the subject (means)。

62. kit according to item 61 of item, wherein the component is syringe or bottle.

The kit that item is 63. 61 or 62 is also indicated comprising label or operation instructions, the label or operation instructions The plasminogen is administered into the subject with any one of practical matter 1-58 the methods.

On the other hand, the present invention relates to：A kind of 64. products of item, it includes：

Container containing label；With

Include the pharmaceutical composition of (i) for the plasminogen of any one of item 1-58 the methods or comprising plasminogen Object, wherein the plasminogen or composition are administered the subject with any one of practical matter 1-58 institutes by label instruction State method.

The kit of any one of 65. 61-63 or the product of item 64, also comprising other one or more components or Container contains other drugs in the component or container.

The kit or product that item is 66. 65, wherein the other drugs are selected from the group：Blood lipid-lowering medicine, antiplatelet Drug, blood-pressure drug, vasodilator drug, hypoglycemic drug, anticoagulation medicine, thrombolytic agent, hepatic, the anti-rhythm of the heart Arrhythmic agents, cardiac drug, diuretic, anti-infectives, antiviral drugs, immunoregulation medicament, inflammation adjust 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 1-58.

The invention further relates to plasminogen in preparation for the drug of the method for any one of item 1-58, pharmaceutical composition, system Purposes in product, kit.

The invention further relates to following items：

On the one hand, the present invention relates to：A kind of methods prevented or treat subject's Renal tissues damage of item 1., including administration A effective amount of plasminogen of subject, wherein the subject has Renal tissues damage risk, suspection to have Renal tissues damage or suffer from Renal tissues damage.

The method of 2. 1, wherein the Renal tissues damage include infection, inflammation, allergy, autoimmunity, ischemic, Renal tissues damage caused by thrombus, wound, radiation injury, carbohydrate metabolism disturbance, fat metabolic disturbance, cancer.

The method that item is 3. 1 or 2, wherein the Renal tissues damage is nephridial tissue caused by systemic disease chosen from the followings Damage：Hypertension, diabetes, atherosclerosis, systemic sclerosis, systemic loupus erythematosus, hyperlipidemia, non-hodgkin's Lymphomas, Huppert's disease, systemic vasculitis, anaphylactoid purpura, polymyositis, thrombotic microvascular disease.

The method that item is 4. 1 or 2, wherein the Renal tissues damage is Renal tissues damage caused by chronic renal disease.

The method that item is 5. 4, wherein the chronic renal disease is chronic glomerulonephritis, chronic pyelonephritis, nephrosis Syndrome, renal insufficiency, renal failure or uremia.

The method that item is 6. 1 or 2, wherein the chronic renal disease is drug induced Chronic Renal Impairment.

The method that item is 7. 6, wherein the drug includes chemotherapeutics, blood-pressure drug, blood lipid-lowering medicine, hypoglycemic agent Object, nonsteroidal antiinflammatory drug, antibiotic medicine, antiviral drugs.

The method that item is 8. 7, wherein the drug is chemotherapeutics, specially cis-platinum.

On the other hand, the present invention relates to：A kind of methods prevented or treat the acute Renal tissues damage of subject of item 9., packet Include a effective amount of plasminogen protection nephridial tissue of administration subject.

The method that item is 10. 9, wherein the nephridial tissue cell caused by the plasminogen mitigates acute Renal tissues damage withers It dies.

The method that item is 11. 9 or 10, wherein the plasminogen promotes the reparation of damage nephridial tissue.

The method of any one of 12. 9-11 of item, wherein the plasminogen mitigates the fibrosis of damage nephridial tissue.

The method of any one of 13. 9-12 of item, wherein the plasminogen promotes graft function.

The method of any one of 14. 1-13, wherein the injury of kidney is acute glomerulonephritis, acute pyelonephritis, Acute kidney injury, acute renal failure, acute renal insufficiency, acute tubular necrosis.

The method of 15. 1-13 of item, wherein the acute kidney injury is acute kidney injury caused by chemotherapeutics.

On the other hand, the present invention relates to：A kind of methods prevented or treat the chronic Renal tissues damage of subject of item 16., Including a effective amount of plasminogen protection nephridial tissue of administration subject.

The method that item is 17. 16, wherein the plasminogen mitigates nephridial tissue Apoptosis.

The method that item is 18. 16 or 17, wherein the plasminogen promotes the reparation of damage nephridial tissue.

The method of any one of 19. 16-18 of item, wherein the plasminogen mitigates the fibrosis of damage nephridial tissue.

The method of any one of 20. 16-19 of item, wherein the plasminogen promotes graft function.

The method of any one of 21. 16-20 of item, wherein the Chronic Renal Impairment is caused by the chronic disease of renal tissue Renal tissues damage.

The method of any one of 22. 16-20, wherein the Chronic Renal Impairment cause for Other diseases or with kidney group Knit damage.

The method that item is 23. 22, wherein the Other diseases include hypertension, diabetes, atherosclerosis, hyperlipemia Disease, hepatitis, hepatic sclerosis, coronary heart diseases and angina pectoris, myocardial infarction.

The method that item is 24. 23, wherein the Other diseases are hypertension, diabetes, atherosclerosis, hyperlipemia Disease.

On the other hand, the present invention relates to：Nephridial tissue caused by a kind of 25. prevention of item or treatment subject's lipidosis is damaged The method of wound, including a effective amount of plasminogen of subject is administered.

The method that item is 26. 25, 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 27. prevention of item or treatment sub-ject initiation or adjoint kidney group The method for knitting damage, including a effective amount of plasminogen of subject is administered.

On the other hand, the present invention relates to：A kind of 28. 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 29. prevention of item or treatment subject artery atherosis initiation or adjoint Renal tissues damage method, including be administered a effective amount of plasminogen of subject.

On the other hand, the present invention relates to：A kind of sides prevented or treat subject's ischemic Renal tissues damage of item 30. Method, including a effective amount of plasminogen of subject is administered.

The method that item is 31. 30, wherein the plasminogen mitigates nephridial tissue Apoptosis.

The method that item is 32. 30 or 31, wherein the plasminogen promotes the reparation of damage nephridial tissue.

The method of any one of 33. 30-32 of item, wherein the plasminogen mitigates the fibrosis of damage nephridial tissue.

The method of any one of 34. 30-33 of item, wherein the plasminogen promotes graft function.

The method of any one of 35. 30-34 of item, wherein the ischemic is caused by vessel lumen is narrow.

The method of any one of 36. 30-34 of item, wherein the ischemic is caused by thrombus blocks blood vessel.

The method that item is 37. 35 or 36, wherein the ischemic is by hypertension, diabetes, atherosclerosis, heart disease institute It causes.

On the other hand, the present invention relates to：A kind of 38. prevention of item or treatment subject's ischaemia-reperfusion Renal tissues damage Method, including be administered a effective amount of plasminogen of subject.

On the other hand, the present invention relates to：A kind of 39. prevention of item or treatment subject's autoimmune response nephridial tissue damage The method of wound, including a effective amount of plasminogen of subject is administered.

The method that item is 40. 39, wherein the autoimmune response Renal tissues damage is caused by systemic sclerosis.

On the other hand, the present invention relates to：A kind of sides prevented or treat the inflammatory Renal tissues damage of subject of item 41. Method, including a effective amount of plasminogen of subject is administered

The method that item is 42. 41, wherein the inflammation is acute and chronic kidney parenchyma inflammation or renal interstitial inflammation.

The method that item is 43. 42, wherein the plasminogen mitigates nephridial tissue Apoptosis.

The method that item is 44. 42 or 43, wherein the plasminogen promotes the reparation of damage nephridial tissue.

The method of any one of 45. 41-44 of item, wherein the plasminogen mitigates the fibrosis of damage nephridial tissue.

The method of any one of 46. 41-45 of item, wherein the plasminogen promotes graft function.

The method of any one of 47. 1-46 of item, wherein the plasminogen has at least with sequence 2,6,8,10 or 12 75%th, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is fibrinolysin Former activity.

The method of any one of 48. 1-47 of item, the plasminogen is on the basis of sequence 2,6,8,10 or 12, is added Add, delete and/or replace 1-100,1-90,1-80,1-70,1-60,1-50,1-45,1-40,1-35,1-30,1-25,1- 20th, 1-15,1-10,1-5,1-4,1-3,1-2,1 amino acid, and still there is the protein of activities of endothelial tissue plasminogen.

The method of any one of 49. 1-48 of item, the plasminogen are comprising activities of endothelial tissue plasminogen segment and still have There is the protein of activities of endothelial tissue plasminogen.

The method of any one of 50. 1-49, the plasminogen are selected from Glu- plasminogens, Lys- plasminogens, small Plasminogen, Microplasminogen, delta- plasminogens or their variant for retaining activities of endothelial tissue plasminogen.

51. according to the method for any one of item 1-50, wherein the plasminogen can with it is one or more chosen from the followings Medication combined application：Blood-pressure drug, antibiotic medicine, anticoagulant, thrombolytic agent, diuretics, resists and swells blood lipid-lowering medicine Knurl medicine, hypoglycemic agent, non-steroid anti-inflammatory drug, immunoregulation medicament, anti-infectives, antiviral drugs, hormone, natural products Active constituent.

The method of any one of 52. 1-51, the plasminogen for natural or synthetic human plasminogen or its still Retain the variant or segment of activities of endothelial tissue plasminogen.

The method of any one of 53. 1-51 of item, the plasminogen are the people from primate or rodent Plasminogen directly to homologue or its still retain the variant of activities of endothelial tissue plasminogen or segment.

The method of any one of 54. 1-53 of item, the amino acid of the plasminogen is as shown in sequence 2,6,8,10 or 12.

The method of any one of 55. 1-54 of item, wherein the plasminogen is naive plasminogen.

The method of any one of 56. 1-55 of item, wherein the subject is people.

The method of any one of 57. 1-26 of item, wherein the subject lacks or missing plasminogen.

The method that item is 58. 57, wherein the shortage or missing are inborn, secondary and/or local.

On the other hand, the present invention relates to：A kind of plasminogens of the methods for any one of item 1-58 of item 59..

On the other hand, the present invention relates to：A kind of 60. pharmaceutical compositions, it includes pharmaceutically acceptable supporting agent and For the plasminogen of any one of item 1-58 the methods.

On the other hand, the present invention relates to：A kind of preventative or therapeutic agent boxes of item 61., it includes：(i) for item The plasminogen of any one of 1-58 the methods and (ii) are for delivering the plasminogen to the component of the subject (means)。

62. kit according to item 61 of item, wherein the component is syringe or bottle.

The kit that item is 63. 61 or 62 is also indicated comprising label or operation instructions, the label or operation instructions The plasminogen is administered into the subject with any one of practical matter 1-58 the methods.

On the other hand, the present invention relates to：A kind of 64. products of item, it includes：

Container containing label；With

Include the pharmaceutical composition of (i) for the plasminogen of any one of item 1-58 the methods or comprising plasminogen Object, wherein the plasminogen or composition are administered the subject with any one of practical matter 1-58 institutes by label instruction State method.

The kit of any one of 65. 61-63 or the product of item 64, also comprising other one or more components or Container contains other drugs in the component or container.

The kit or product that item is 66. 65, wherein the other drugs are selected from the group：Blood lipid-lowering medicine, antiplatelet Drug, blood-pressure drug, vasodilator drug, hypoglycemic drug, anticoagulation medicine, thrombolytic agent, hepatic, the anti-rhythm of the heart Arrhythmic agents, cardiac drug, diuretic, anti-infectives, antiviral drugs, immunoregulation medicament, inflammation adjust 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 1-58.

The invention further relates to plasminogen in preparation for the drug of the method for any one of item 1-58, pharmaceutical composition, system Purposes in product, kit.

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

" renal lesions " are kidney structure and function obstacles caused by a variety of causes.

" 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 92kD, 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), the cDNA sequence of the sequence is encoded as shown in sequence 1, and amino acid sequence is as shown in sequence 2.In vivo, also exist A kind of is that hydrolysis is so as to the Lys- plasminogens formed at the 76-77 amino acids of Glu- plasminogens, such as 6 institute of sequence Show, encode the cDNA sequence of the amino acid sequence as shown in sequence 5.δ-plasminogen (δ-plasminogen) is overall length fibrinolytic Proenzyme has lacked the segment of Kringle2-Kringle5 structures, only containing Kringle1 and serine protease domain^[22,23], have The document report amino acid sequence (sequence 8) of δ-plasminogen^[23], encode the cDNA sequence such as sequence 7 of the amino acid sequence. Miniplasminogen (Mini-plasminogen) is made of Kringle5 and serine protease domain, have document report it include it is residual Base Val443-Asn791 (using do not contain signal peptide Glu- plasminogen sequences Glu residues as initial amino acid)^[24], 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)^[25], also have Patent document CN102154253A reports that its sequence includes residue Lys531-Asn791 (not contain the Glu- fibrinolytics of signal peptide The Glu residues of proenzyme sequence are initial amino acid), this patent sequence reference patent document CN102154253A, amino acid sequence Row encode the cDNA sequence of the amino acid sequence as shown in sequence 11 as shown in sequence 12.

" 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 live The detection of property, 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 plasminogen in by inspection blood plasma are transformed into fibrinolysin under the action of SK, the latter acts on chromophoric substrate, Then with spectrophotometric determination, absorbance increases directly proportional to activities of endothelial tissue plasminogen.In addition immuno-chemical method can also be used, coagulate Gel electrophoresis, 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； Etc..

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 The given amino acid sequence A of a certain % amino acid sequence identities of sequence 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.

2. 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 portion of the expression vector usually in host organisms as episome or as host chromosome DNA Divide and replicate.In general, expression vector contain selection marker (such as amicillin resistance, hygromycin resistance, tetracyclin resistance, Kalamycin resistance or neomycin resistance) to help to be detected those cells that external source is converted with desired DNA sequence dna.

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), Sha Lei Bordetella (Serratia) and various pseudomonas (Pseudomonas) species.In these prokaryotic hosts, it can also give birth to Into expression vector, the expression control sequence (such as replication orgin) compatible with host cell would generally be contained.In addition, can exist Many well known promoters, such as lactose promoter system, tryptophan (trp) promoter systems, beta-lactam enzyme 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 plasminogen (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 according to the standard schedule of this field, including ammonium sulfate precipitation, affine 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..

3. pharmaceutical formulation

It 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)) are 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 antibodies preparaton being lyophilized, described in WO 97/04801, it includes herein as ginseng It examines.

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, controlling 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 poly- (vinyl alcohol), polyactide (United States Patent (USP) 3773919, EP 58, 481), the copolymer of Pidolidone and ethyl-L-glutamate (Sidman, etc. Biopolymers 22:547 (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.

4. administration and dosage

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

In some cases, the plasminogen pharmaceutical composition of the present invention can be modified or prepare in the following manner, so as to carry The ability of blood-brain barrier is passed through for it.It can be by various enteral and parenteral administration routes, including oral, intravenous etc. to suffering from There is the composition that the individual of thrombus and/or thrombus relevant disease applies this type plasminogen.

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..

In some embodiments, plasminogen of the invention is formulated together with promoting the medicament across blood-brain barrier. In some cases, plasminogen of the invention is directly or through connector with promoting carrier molecule, peptide or egg across blood-brain barrier White matter merges.In some embodiments, plasminogen of the invention melts with combining the polypeptide of endogenous blood-brain barrier (BBB) receptor It closes.Plasminogen is connected with combining the polypeptide of endogenous BBB receptors, is promoted across BBB.With reference to the suitable more of endogenous BBB receptors Peptide includes antibody, such as monoclonal antibody or its antigen-binding fragment, specifically binds endogenous BBB receptors.It is suitable endogenous BBB receptors include but not limited to insulin by some cases, and antibody is encapsulated in liposome.It is special see, for example, the U.S. Sharp disclosure No.2009/0156498.

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 May range from for example daily about 0.0001 to 2000mg/kg or about 0.001 to 500mg/kg (such as 0.02mg/kg, 0.25mg/kg, 0.5mg/kg, 0.75mg/kg, 10mg/kg, 50mg/kg etc.) subject's 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 the safety of periodical evaluation thrombus and thrombus relevant disease.

5. treat effect

One embodiment of the invention is related to after treating subject using plasminogen, to treatment effect and treatment safety The judgement of property.Wherein clinically the judgment method for treating effect is included but not limited to detect following index, so as to assess kidney function Energy：Serum creatinine level, creatinine clearance, 24- hour urine protein excretion rate (UAER), glomerular filtration rate, the white egg of urine In vain/creatinine ratio, albumin secretion rate and biopsy etc..For example, glomerular filtration rate can illustrate that glomerulus is excessively filtered It crosses and the situation of hyperperfusion, instruction diabetic nephropathy early symptom alleviates degree.Glomerular filtration rate is kidney production per minute The volume of raw filtrate can be determined with various methods, and it is clear such as to measure the filtering marker such as urine of glycan, iothalamate or Iohexol Except rate.More common method is, can be by determining kreatinin (a kind of is generated by muscle and be discharged into the protein in blood) clearly Except rate carrys out estimated glomerular filtration rate.The intoxicated clearance rate of creatine (being typically expressed as ml/min) can be by comparing given time Creatinine levels in the creatinine levels and blood collected in (such as 12 or 24 hours) interior urine determine.Adult male Typical creatinine clearance is about 97-137 ml/mins, and adult female is about 88-128 ml/mins.The intoxicated clearance rate of creatine It is directly proportional to the excretion of urinary creatine acid anhydride, it is inversely proportional with serum creatinine concentration.

Usually using creatinine clearance/glomerular filtration rate or urinary albumin discharge rate as main efficacy evaluation index. Can increase other secondary indexs simultaneously to assess effect of the drug of the present invention to related complication, for example, increase triglycerides, Blood Lipid is assessed in the detections such as T-CHOL, low-density lipoprotein, increases pretherapy and post-treatment systolic pressure, diastolic pressure level detection comes Assess hypertension alleviation degree etc..

6. product or medicine box

One embodiment of the invention is related to a kind of product or medicine box, and it includes the sheets available for treating diabetic nephropathy Invention plasminogen.The product preferably includes a container, label or package insert.Appropriate container has bottle, bottle, note Emitter etc..Container can be made of a variety of materials such as glass or plastics.The container contains composition, and the composition can be controlled effectively Treat the present invention disease or illness and with sterile access port (such as the container can be intravenous solution packet or bottle, contain It can be by the plug that hypodermic needle penetrates).At least one of composition activating agent is plasminogen.On the container or Appended label illustrates the composition for treating diabetic nephropathy and diabetic nephropathy relevant disease of the present invention.It is described Product can further include the second container containing pharmaceutically acceptable buffer solution, the brine of such as phosphate-buffered, Ringer's solution with And glucose solution.It can further include required other materials from the point of view of business and user's angle, including other bufferings Liquid, diluent, filtrate, needle and syringe.In addition, the product includes the package insert with operation instruction, including for example Indicate the user of the composition by the other medicines administered patient of the adjoint disease of plasminogen composition and treatment.

Brief description

Fig. 1 cis-platinum damage model mouse give plasminogen after 7 days kidney IgM immunostainings observe result.A is to solvent PBS control group, for B to give plasminogen group, C is quantitative analysis results.It is the results show that bright to plasminogen group IgM positive expressions It shows to be less than and gives solvent PBS control groups, and significantly (* represents P to statistical discrepancy<0.05).Caused by illustrating plasminogen and promoting cis-platinum The reparation of kidney injury.

Fig. 2 cis-platinum damage model mouse give plasminogen kidney IV Collagen Type VIs immunostaining representativeness picture after 7 days.A To give solvent PBS control group, B is gives plasminogen group.It is the results show that significantly low to plasminogen group IV Collagen Type VI positive expressions In giving solvent PBS control group.Illustrate that plasminogen can improve the renal fibrosis caused by cis-platinum.

Fig. 3 purine induction Chronic Renal Impairment model mice give plasminogen after 10 days kidney HE dye representative diagram Piece.A is gives solvent PBS control group, and B is gives plasminogen group, C, D PLG^-/-Group.The results show that PLG^-/-Mouse Kidney popular name for Become most serious, can be seen that a large amount of purulence cast (shown in block arrow), a small amount of purine crystallization (triangle mark), the big face of renal tubule Product atrophy, epithelial cell flattening；With PLG^-/-Mouse is compared, lighter to the damage of solvent PBS control group mouse kidney, although kidney Bead atrophy and renal tubular necrosis are still serious, but do not find apparent purine crystallization, and purulence cast is relatively light；To plasminogen The area of group Mouse Kidney tubular atrophy is smaller than PBS control group, and tubular ectasia does not find purulence cast compared with light yet.Illustrate fibrinolytic Proenzyme can mitigate the damage of chronic renal failure model mice kidney.

The Chronic Renal Impairment model mice of Fig. 4 purine induction gives plasminogen kidney sirius red stains representative after 10 days Property picture.A is gives solvent PBS control group, and B is gives plasminogen group, C PLG^-/-Group, D are quantitative analysis results.As a result it shows Show, PLG is considerably less than to plasminogen group and to the deposition of solvent PBS control group collagen^-/-Group, and statistical discrepancy significantly (* tables Show P<0.05, * * represents P<0.01).In addition, the deposition to plasminogen group collagen considerably less than gives solvent PBS control group.It says Bright plasminogen plays a crucial role during the renal fibrosis for repairing chronic kidney hypofunction model.

Fig. 5 purine induction Chronic Renal Impairment model mice give plasminogen after 10 days kidney Bcl-2 immunohistochemistry contaminate Color representativeness picture.A is blank control group, and B is gives solvent PBS control groups, and C is gives plasminogen group.The results show that fibre The coloring of the lyase original group kidney positive is significantly deeper than and gives solvent PBS control group, and with the expression phase of blank control group mouse Closely.This shows that plasminogen can promote the expression of chronic renal failure model mice kidney cell Apoptosis inhibitor molecule Bcl-2, so as to Inhibit the apoptosis of mouse kidney tissue cell.

The Chronic Renal Impairment model mice of Fig. 6 purine induction gives plasminogen kidney IgM immunohistochemical stainings after 10 days Picture.A is blank control group, and B is gives solvent PBS control group, and C is gives plasminogen group.The results show that give plasminogen group The positive coloring of mouse kidney IgM, which is shallower than, gives solvent PBS control group, and range is small compared with control group, and coloring and normal mouse are more Adjunction is near.This shows that the damage to kidney after plasminogen has obtained apparent improvement, reflects that plasminogen damages chronic renal Hindering mouse kidney damage has notable repair.

The Chronic Renal Impairment model mice of Fig. 7 purine induction gives plasminogen kidney fibrous protein immunization group after 4 days The representative picture of dyeing.A is gives solvent PBS control group, and for B to give plasminogen group, C is PLG-/- group.The results show that solvent The positive coloring of PBS control group kidney fibrous albumen, which is deeper than, gives plasminogen group mouse, and PLG-/- group coloring is deeper than to solvent PBS control group.This shows that plasminogen can mitigate the damage of kidney.

Fig. 8 24-25 week old diabetic mices give plasminogen kidney IV Collagen Type VI immunostaining representative diagrams after 31 days Piece.A is gives solvent PBS control group, and B is gives plasminogen group.The results show that it is coloured to the plasminogen group IV collagens positive apparent It is shallower than and gives solvent PBS control group, illustrates that plasminogen can improve diabetic mice renal fibrosis.

26 week old diabetic mices of Fig. 9 give plasminogen representative picture of kidney masson dyeing after 35 days.A be to Solvent PBS control group, B is gives plasminogen group.The results show that solvent PBS control group renal interstitial mild fibrosis, hyperplasia Fibrosis is in blue.It is significantly reduced to plasminogen group kidney region fibrosis.Illustrate that plasminogen can reduce diabetic mice kidney The fibrosis of dirty interstitial.

The systemic sclerosis model mice of Figure 10 bleomycin induceds gives plasminogen kidney Picro-Sirius red after 21 days The representative picture of dyeing.A is gives solvent PBS control group, and B is gives plasminogen group.The results show that it is in bleomycin induced In system property hardening disease mouse model, it is significantly more than to solvent PBS control group renal collagen deposition and gives plasminogen group.Illustrate fibrinolytic Proenzyme effectively reduces the renal fibrosis of systemic sclerosis mouse.

Figure 11 purine inducing chronic injury of kidney mice serum urea nitrogen testing results.The results show that PLG^+/+In group serum Urea nitrogen concentration is significantly lower than PLG^-/-Group, and significantly (* represents P to statistical discrepancy<0.05).Illustrate that plasminogen can significantly improve The renal function of chronic renal failure model mice.

Figure 12 purine induction Chronic Renal Impairment model mice give plasminogen after 4 days serum creatinine concentration detection knot Fruit.The results show that it is significantly lower than PLG to solvent PBS control group and to plasminogen group creatinine in serum concentration^-/-Group mouse, and Significantly (* represents P to statistical discrepancy<0.05).Solvent PBS is given in addition, being significantly lower than to plasminogen group creatinine in serum concentration Control group.Illustrate that plasminogen can significantly improve the renal function of Chronic Renal Impairment model mice.

Figure 13 folic acid induces acute kidney injury mice serum urea nitrogen testing result.The results show that give plasminogen group blood Urea nitrogen concentration, which is significantly lower than, in clear gives solvent PBS control group, and statistical discrepancy is close to significantly (P=0.06).Illustrate fibrinolysin Proper energy significantly improves the renal function of acute kidney injury model mice.

Figure 14 give plasminogen after 30 days 3% cholesterol hyperlipemia model mouse kidney oil red O stain observe result. A is blank control group, and B is gives solvent PBS control group, and for C to give plasminogen group, D is quantitative analysis results.The results show that it gives Plasminogen group mouse kidney fat deposition (arrow logo) is considerably less than and gives solvent PBS control group, and quantitative analysis statistical difference It is different notable；It is in addition, similar to blank control group mouse to plasminogen group lipidosis level.Illustrate that plasminogen can cut down fat Deposition of the fat in hyperlipemia model mouse kidney, so as to reduce the kidney injury caused by fat deposition.

Figure 15 gives plasminogen representative picture of folic acid acute kidney injury model mice kidney HE dyeing after 7 days.A is sky White control group, B is gives solvent PBS control group, and C is gives plasminogen group.The results show that blank control group kidney core it is rounded or Ellipse, the red dye of endochylema, glomerulus and the equal form of renal tubule are normal；Observe that a large amount of kidney is small to solvent PBS control group kidney Pipe epithelial cell flattening (block arrow mark), brush border comes off, part karyopyknosis, and only light dye is presented in part renal tubule Endochylema, part renal tubule is also shown purulence cast (thin arrow logo), glomerulus and the slight cell infiltration of renal interstitial；To fibre Compared with to solvent PBS control group, tubular ectasia and epithelial cell flattening are clearly better lyase original group, it is seen that most Red dye is presented in renal tubule endochylema, has no purulence cast.Illustrate that fibrinolysin can improve the acute kidney injury of folic acid induction.

Figure 16 gives plasminogen folic acid acute kidney injury model mice kidney Bcl-2 immunohistochemical observation knots after 7 days Fruit.A is gives solvent PBS control group, and for B to give plasminogen group, C is quantitative analysis results.The results show that give plasminogen group The coloring of the kidney Bcl-2 positives, which is significantly more than, gives solvent PBS control group, and significantly (* represents P to statistical discrepancy<0.05).This shows fibre Lyase proper energy promotes the expression of iap protein Bcl-2 in acute kidney injury model mice kidney, acute so as to protect Injury of kidney mouse kidney tissue cell is from apoptosis.

Figure 17 gives plasminogen folic acid acute kidney injury model mice kidney IgM immunohistochemical observation results after 7 days.A To give solvent PBS control group, B is gives plasminogen group.The results show that the positive to plasminogen group mouse kidney IgM colours It is more shallow than to solvent PBS control group, and range is small compared with control group.This show inject plasminogen after kidney IgM expression it is apparent It reduces, reflects that plasminogen can effectively reduce the damage of folic acid acute kidney injury mouse kidney.

Figure 18 give plasminogen after 30 days 3% cholesterol hyperlipemia model mouse kidney sirius red stains observation knot Fruit.A is blank control group, and B is gives solvent PBS control group, and for C to give plasminogen group, D is quantitative analysis results.As a result it shows Show, be considerably less than to plasminogen group renal collagen proteinosis (arrow logo) and give solvent PBS control group, and statistical discrepancy is shown It writes；Normal level is substantially returned to plasminogen group fibrosis.It is high in fat to illustrate that plasminogen can effectively reduce by 3% cholesterol Blood stasis model mouse kidney fibrosis.

Figure 19 give plasminogen after 7 days ischemia-reperfusion acute kidney injury model mice kidney HE dye representative diagram Piece.A is sham-operation group, and B is gives solvent PBS control group, and C is gives plasminogen group.The results show that sham-operation group glomerulus capillary Vascular patency, the red dye of renal tubule endochylema, in normal renal tubule form；To the inflammation that the visible glomerulus of solvent PBS control group is slight Cellular infiltration (triangle mark), a large amount of cell infiltration of renal interstitial, purulence cast (thin arrow logo) is presented in part renal tubule, few Measure renal tubular cell karyopycnosis, the epithelial cell flattening (block arrow mark) of large area, tubular ectasia；Compared to solvent PBS control group only has a small amount of renal tubular epithelial flattening, the recovered normal kidney of most renal tubule to plasminogen group Tubule form, the red dye of endochylema do not find apparent renal tubule atrophy, and only renal interstitial has slight cell infiltration, approached Sham-operation group form.Illustrate that fibrinolysin can improve ischemia-reperfusion acute kidney injury model mice kidney injury.

Embodiment

The reparation of kidney injury caused by 1 plasminogen of embodiment promotion cis-platinum

The male C57 mouse 10 of 8-9 week old health, are randomly divided into two groups, to solvent PBS control groups and to fibrinolysin Former group, every group each 5.After the completion of grouping, by 10mg/Kg weight single intraperitoneal injection cis-platinums^[33], establish chemotherapy damage model. After establishing model, plasminogen is given by 1mg/0.1ml/ pcs/day of tail vein to plasminogen group, to solvent PBS control group with Same way gives the PBS of same volume.Experiment was weighed in and was grouped for the 0th day on the day of starting, and the 1st day starts to be injected intraperitoneally Cis-platinum modeling, gives plasminogen or solvent PBS in 3 hours after modeling, administration phase is 7 days.It puts to death mouse within 8th day, takes kidney 24-48 hours are fixed in 10% neutral formalin fixer.Renal tissue after fixation is through alcohol serial dehydration and diformazan Paraffin embedding is carried out after benzene is transparent.Histotomy thickness is 4 μm, is washed 1 time after slice dewaxing rehydration.Citric acid repairs 30 points Clock, water softly rinses after ten minutes for room temperature cooling.PAP are irised out tissue, and 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) is closed 30 minutes；Time After arriving, reject sheep blood serum liquid is added dropwise mountain sheep anti mouse IgM (HRP) antibody (Abcam) and is incubated at room temperature 1 hour, and PBS washes 2 times, every time 5 minutes.It developing the color by DAB kits (Vector laboratories, Inc., USA), haematoxylin is redyed 30 seconds after washing 3 times, Flowing water rinses 5 minutes.Serial dehydration is transparent and mounting, is sliced in 200 times of optical microphotograph Microscopic observations.

IgM antibody plays an important role during apoptosis and non-viable non-apoptotic cell is removed, and injuries of tissues and organs local I gM resists Level and the degree of injury of body are proportionate [29^,30].Therefore, the level of detection histoorgan local I gM antibody can reflect this The degree of impairment of histoorgan.

The results show that be significantly lower than to plasminogen group (Figure 1B) IgM positive expressions to solvent PBS control group (Figure 1A), And statistical discrepancy is significantly (Fig. 1 C).Illustrate that plasminogen can promote the reparation of kidney injury.

2 plasminogen of embodiment mitigates the fibrosis of cisplatin chemotherapy damage model mouse kidney

The male C57 mouse 10 of 8-9 week old health, are randomly divided into two groups, to solvent PBS control groups and to fibrinolysin Former group, every group each 5.After the completion of grouping, chemotherapy damage model is established, by 10mg/Kg weight single intraperitoneal injection cis-platinums^[33]。 After establishing model, give plasminogen through tail vein injection for 1mg/0.1ml/ pcs/day to plasminogen group, give solvent PBS control Group tail vein injection gives the PBS of same volume.Experiment starts the same day as the 0th day and grouping of weighing, and the 1st day starts to be injected intraperitoneally Cis-platinum modeling, gives plasminogen or solvent PBS in 3 hours after modeling, administration phase is 7 days.It puts to death mouse within 8th day, takes kidney 24 hours are fixed in 4% paraformaldehyde fixer.Liver organization after fixation is after alcohol serial dehydration and dimethylbenzene are transparent Carry out paraffin embedding.Histotomy thickness is 4 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, with 3% dioxygen Water incubation 15 minutes, 0.01MPBS are 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 is added dropwise 4 DEG C of rabbit anti-mouse IV collagen antibodies (Abcam) and incubates It educates overnight, TBS is washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and TBS is washed 2 times, every time 5 minutes.It develops the color by DAB kits (Vector laboratories, Inc., USA), haematoxylin after washing 3 times It redyes 30 seconds, flowing water returns indigo plant 5 minutes, and then TBS is washed 1 time.Serial dehydration is transparent and mounting, is sliced in 200 times of light microscopes Lower observation.

The results show that it is apparently higher than to solvent PBS control group (Fig. 2A) kidney IV Collagen Type VI positive expressions to plasminogen Group (Fig. 2 B).Illustrate that plasminogen can mitigate the fibrosis of cis-platinum damage model mouse kidney.

Protective effect of 3 plasminogen of embodiment to Chronic Renal Impairment model kidney

The PLG of 8-9 week old^+/+Mouse 20 and PLG^-/-Mouse 6, PLG^+/+Mouse is randomly divided into two groups, to fibrinolytic Proenzyme group and solvent PBS control group is given, every group each 10.To plasminogen group, to solvent PBS control group and PLG^-/-Mouse 0.25% purine feed (Nantong Te Luofei) of feeding daily, establishes Chronic Renal Impairment model^[26].It is denoted as the 1st day on the day of modeling, Start simultaneously at administration.Plasminogen is given by 1mg/0.1ml/ pcs/day of tail vein to plasminogen group, gives solvent PBS control group The PBS of same volume is given in the same manner, and continuous modeling is administered 10 days, PLG^-/-Mouse does not process.It puts to death within 11st day small Mouse takes kidney to fix 24 hours in 4% paraformaldehyde.Kidney after fixation is transparent through alcohol serial dehydration and dimethylbenzene After carry out paraffin embedding.Histotomy thickness is 3 μm, slice dewaxing rehydration and with haematoxylin and eosin stains (HE dyeing), 1% hydrochloride alcohol breaks up, and ammonium hydroxide returns indigo plant, and alcohol serial dehydration, and dimethylbenzene is transparent, neutral gum mounting, is sliced in 200 (figures 3A, B, C), 400 (3D) times of optical microphotograph Microscopic observation.

The results show that PLG^-/-Mouse kidney (Fig. 3 C, 3D) lesion most serious, can be observed a large amount of purulence cast (arrow It is shown), a small amount of purine crystallization (triangle mark), renal tubule large area atrophy, epithelial cell flattening；With PLG^-/-Mouse phase Than, it is damaged gently to solvent PBS control group mouse kidney (Fig. 3 A), although glomerulus atrophy and renal tubular necrosis are also very serious, But apparent purine crystallization is not found, purulence cast is relatively light；To the area of plasminogen group mouse (Fig. 3 B) renal tubule atrophy Smaller than PBS control group, tubular ectasia does not find purulence cast compared with light yet.Illustrate that plasminogen can repair Chronic Renal Impairment mould The damage of type mouse kidney.

4 plasminogen of embodiment repairs the fibrosis of Chronic Renal Impairment model kidney

8-9 week old males PLG^+/+Mouse 12 and PLG^-/-Mouse 6, PLG^+/+Mouse is randomly divided into two groups, to fibrinolytic Proenzyme group and each 6 to solvent PBS control group, Chronic Renal Impairment model modeling mode is the same as described in embodiment 1.It is denoted as on the day of modeling 1st day, start simultaneously at administration.Plasminogen is given by 1mg/0.1ml/ pcs/day of tail vein to plasminogen group, gives solvent PBS Control group gives the PBS of same volume in the same manner, and continuous modeling is administered 10 days, PLG^-/-Mouse does not process.11st day Putting to death mouse takes kidney to fix 24 hours in 4% paraformaldehyde.Kidney after fixation is saturating through alcohol serial dehydration and dimethylbenzene Paraffin embedding is carried out after bright.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing to water, with 0.1% sirius red stains After sixty minutes, flowing water rinses, haematoxylin dyeing 1 minute, and flowing water rinses, and indigo plant, flowing water punching are returned in 1% hydrochloride alcohol and ammonium hydroxide differentiation It washes, mounting after drying, is sliced 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.

The results show that it is considerably less than to plasminogen group (Fig. 4 B) and to the deposition of solvent PBS control group (Fig. 4 A) collagen PLG^-/-Group (Fig. 4 C), and statistical discrepancy is significantly (Fig. 4 D).In addition, the deposition to plasminogen group collagen is considerably less than to solvent PBS control group.Illustrate that plasminogen plays a crucial role during the renal fibrosis for repairing Chronic Renal Impairment model.

5 plasminogen of embodiment promotes the apoptosis inhibitory protein Bcl-2 expression of Chronic Renal Impairment mouse kidney

8-9 week old males PLG^+/+Mouse 18, is randomly divided into three groups, blank control group, to plasminogen group and to solvent PBS control group, every group each 6.Chronic Renal Impairment model modeling mode is the same as described in embodiment 1.It is denoted as the 1st day on the day of modeling, together When start to be administered.Blank control group feeding is normal to maintain feed.It is given to plasminogen group by 1mg/0.1ml/ pcs/day of tail vein Plasminogen gives the PBS of same volume to solvent PBS control group in the same manner, and continuous modeling is administered 10 days, blank control Group mouse group is without any processing.Start modeling administration to be set to the 1st day, put to death mouse within the 11st day and take kidney in 4% paraformaldehyde In fix 24 hours.Kidney after fixation carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy is thick It is 3 μm to spend, and is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, and 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) is closed 30 minutes；Time After arriving, reject sheep blood serum liquid is added dropwise rabbit anti-mouse Bcl-2 antibody 4 DEG C of (Abcam) overnight incubation, and 0.01M PBS wash 2 times, often Secondary 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and 0.01M PBS are washed 2 times, every time 5 points Clock.It develops the color by DAB kits (Vector laboratories, Inc., USA), haematoxylin is redyed 30 seconds after washing 3 times, is flowed 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.

Bcl-2 is iap protein, expresses and lowers under the effect of the apoptotic stimulus factor^[27,28].Bcl-2 immune groups Change the results show that being significantly deeper than to the coloring of plasminogen group (Fig. 5 C) the kidney Bcl-2 positives and give solvent PBS control group (Fig. 5 B), and It is and approximate with blank control group (Fig. 5 A) Bcl-2 positive coloring degrees.This shows that plasminogen can promote Chronic Renal Impairment model Apoptosis inhibits the expression of molecule Bcl-2 in mouse kidney, so as to help to protect Chronic Renal Impairment mouse kidney tissue thin Born of the same parents are from apoptosis.

6 plasminogen of embodiment improves the local damage of Chronic Renal Impairment mouse kidney

8-9 week old males PLG^+/+Mouse 18, is randomly divided into three groups, blank control group, to plasminogen group and to solvent PBS control group, every group each 6.Chronic Renal Impairment model modeling mode is the same as described in embodiment 1.It is denoted as the 1st day on the day of modeling, together When start to be administered, blank control group feeding is normal to maintain feed.It is given to plasminogen group by 1mg/0.1ml/ pcs/day of tail vein Plasminogen gives the PBS of same volume to solvent PBS control group in the same manner, and continuous modeling is administered 10 days, blank control Group is without any processing.Putting to death mouse within 11st day takes kidney to fix 24 hours in 4% paraformaldehyde.Kidney after fixation is through wine Paraffin embedding is carried out after smart serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration. PAP are irised out tissue, and 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) is closed 30 minutes；After time arrives, reject sheep blood serum liquid is added dropwise goat and resists Mouse IgM (HRP) antibody (Abcam) is incubated at room temperature 1 hour, and PBS is washed 2 times, every time 5 minutes.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Serial dehydration is saturating Bright and mounting is sliced in 200 times of optical microphotograph Microscopic observations.

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^[29,30].Therefore, the level of detection histoorgan local I gM antibody can reflect The degree of injury of the histoorgan.

The results show that the positive coloring to plasminogen group (Fig. 6 C) mouse kidney IgM is more shallow than to solvent PBS control group (Fig. 6 B), and range is small compared with control group, and coloring is very close with blank control mouse (Fig. 6 A).This shows to inject plasminogen The damage of glomerulus has obtained apparent improvement afterwards, reflects that plasminogen has the damage of Chronic Renal Impairment mouse kidney and significantly repaiies Multiple effect.

7 plasminogen of embodiment reduces the fibrinous expression of Chronic Renal Impairment mouse kidney

8-9 week old heros PLG^+/+Mouse 12 and PLG^-/-Mouse 6, PLG^+/+Mouse is randomly divided into two groups, to fibrinolysin Former group and solvent PBS control group is given, every group each 6.Chronic Renal Impairment model modeling mode is the same as described in embodiment 1.On the day of modeling It is denoted as the 1st day, starts simultaneously at administration, modeling dosage period 4 days, blank control group feeding is normal to maintain feed.To plasminogen Group gives plasminogen by 1mg/0.1ml/ pcs/day of tail vein, and same volume is given in the same manner to solvent PBS control group PBS, PLG^-/-It is without any processing.Putting to death mouse within 5th day takes kidney to fix 24 hours in 4% paraformaldehyde.Kidney after fixation It is dirty to carry out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, water after slice dewaxing rehydration It washes 1 time.PAP are irised out tissue, and with 3% dioxygen water incubation 15 minutes, 0.01MPBS was washed 2 times, every time 5 minutes.5% it is normal Sheep blood serum liquid (Vector laboratories, Inc., USA) is closed 30 minutes；After time arrives, rabbit is added dropwise in reject sheep blood serum liquid 4 DEG C of overnight incubations of anti-mouse fibrin antibody (Abcam), PBS are washed 2 times, every time 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 kits (Vector laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Serial dehydration is transparent and mounting, is sliced 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^[31,32].Therefore, local fiber will can be damaged A mark of the protein level as degree of injury.

The results show that the positive coloring of solvent PBS control group (Fig. 7 A) kidney fibrous albumen is given, which to be deeper than, gives plasminogen group Mouse (Fig. 7 B), and PLG^-/-Group (Fig. 7 C) coloring, which is deeper than, gives solvent PBS control group.This shows that plasminogen can be to a certain degree The upper damage for repairing renal tissue.

8 plasminogen of embodiment mitigates diabetic mice renal fibrosis

24-25 week old male db/db mouse 10, are randomly divided into two groups, to solvent PBS control group and to plasminogen Group, every group each 5.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, continuously to give Medicine 31 days.To plasminogen group mouse by 2mg/0.2ml/ pcs/day of tail vein injection plasminogen, solvent PBS control group tail is given Vein gives the PBS of same volume.To putting to death mouse after plasminogen 31 days and take renal tissue in 4% paraformaldehyde fixer In fix 24 hours.Renal tissue after fixation carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Tissue is cut Piece thickness is 4 μm, is washed 1 time after slice dewaxing rehydration.Tissue is irised out with PAP, with 3% dioxygen water incubation 15 minutes, 0.01MPBS is washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) closing 30 Minute；After time arrives, reject sheep blood serum liquid, 4 DEG C of the rabbit polyclonal antibody (Abcam) that IV collagens are added dropwise is incubated overnight, and TBS washes 2 It is secondary, 5 minutes every time.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and TBS is washed 2 times.By DAB reagents Box (Vector laboratories, Inc., USA) develops the color, and haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes, Serial dehydration is transparent and mounting, is sliced in 200 times of optical microphotograph Microscopic observations.

Diabetic nephropathy is chronic complicating diseases of diabetes, and glomerulosclerosis and kidney region fibrosis are its typical pathology Change^[34].Experimental result of the present invention is shown, is significantly shallower than to the coloring of plasminogen group (Fig. 8 B) the IV collagens positive and is given solvent PBS Control group (Fig. 8 A), illustrates that plasminogen can mitigate the fibrosis of diabetic mice kidney.

9 plasminogen of embodiment mitigates diabetic mice renal fibrosis

26 week old male db/db mouse 10, are randomly divided into two groups, to solvent PBS control group and give plasminogen group, often Each 5 of group.Experiment is denoted as grouping of weighing in the 0th day 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 tail vein The PBS of same volume.It puts to death within 36th day mouse and renal tissue is taken to fix 24 hours in 4% paraformaldehyde fixer.It is fixed Renal tissue afterwards carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 4 μm, and slice is de- Potassium bichromate solution is put into after wax rehydration to stay overnight.Garapa uniformly dyeing 3 to 5 minutes, flowing water is washed.1% hydrochloride alcohol breaks up, at ammonium hydroxide Reason 1 second, washing.Ponceaux acid fuchsin liquid contaminates 8 minutes, short rinse in water.1% phosphomolybdic acid aqueous solution is handled about 2 minutes, benzene Amine indigo plant liquid is redyed 6 minutes.1% glacial acetic acid rinses about 1 minute.Mounting after absolute ethyl alcohol dehydration dimethylbenzene is transparent, is sliced at 200 times Optical microphotograph Microscopic observation.

Masson dyes the fibrosis that can show tissue.The results show that solvent PBS control group (Fig. 9 A), renal interstitial Mild fibrosis, the fibrosis of hyperplasia is in blue.To plasminogen group (Fig. 9 B), renal interstitial is fine compared with to solvent PBS control group Dimensionization significantly reduces.Illustrate that plasminogen can reduce the fibrosis of diabetic mice kidney.

10 plasminogen of embodiment reduces systemic sclerosis mouse kidney fibrosis

12 week old C57 heros mouse 10 are taken, two groups are randomly divided into after weighing, to solvent PBS control groups and give plasminogen group Each 5.All mouse are by 0.1mg/0.1ml/ pcs/day of hypodermic injection bleomycin induced systemic sclerosis^【35】, the same day starts To plasminogen or PBS and it is denoted as the 1st day, successive administration 21 days.It is quiet by 1mg/0.1ml/ pcs/day of tail to plasminogen group mouse Arteries and veins injects plasminogen, and the PBS of same volume is given to solvent PBS control group tail vein injection.Mouse was put to death at the 22nd day simultaneously Kidney is taken to fix 24 hours in 4% paraformaldehyde fixer.Kidney after fixation is transparent through alcohol serial dehydration and dimethylbenzene After carry out paraffin embedding.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing to water, with 0.1% Picro-Sirius red saturation hardship After sour dyeing 30 minutes, flowing water rinses 2min, haematoxylin dyeing 1 minute, and flowing water rinses, and 1% hydrochloride alcohol differentiation, ammonium hydroxide returns Indigo plant, flowing water rinse, neutral gum mounting after drying, in 200 times of optical microphotograph Microscopic observations.

The results show that in the systemic sclerosis disease mouse model of bleomycin induced, solvent PBS control group (figure is given 10A) renal collagen deposition, which is significantly more than, gives plasminogen group (Figure 10 B).Illustrate that plasminogen effectively reduces systemic sclerosis The renal fibrosis of mouse.

11 plasminogen of embodiment promotes the reparation of Chronic Renal Impairment model mice renal function

The PLG of 8-9 week old^+/+Mouse 10 and PLG^-/-Mouse 6, the same embodiment of Chronic Renal Impairment model modeling mode Described in 1.It is denoted as the 1st day on the day of modeling, 10 days modeling periods.Eyeball was extractd at the 11st day and takes blood, centrifuging and taking supernatant detects serum Middle urea nitrogen concentration.Using Urea nitrogen detection reagent box, (Bioengineering Research Institute, article No. C013- are built up in Nanjing to urea nitrogen content 2) it, and by the Urea nitrogen detection reagent box the method is detected.

The results show that PLG^+/+Group urea in serum nitrogen concentration is significantly lower than PLG^-/-Group, and statistical discrepancy is significantly (Figure 11). Illustrate that plasminogen can significantly improve the renal function of Chronic Renal Impairment model mice.

12 plasminogen of embodiment promotes the reparation of Chronic Renal Impairment model mice renal function

The PLG of 8-9 week old^+/+Mouse 20 and PLG^-/-Mouse 6, PLG^+/+Mouse is randomly divided into two groups, to fibrinolysin Former group and each 10 to solvent PBS control group.Chronic Renal Impairment model modeling mode is the same as described in embodiment 1.It is denoted as on the day of modeling 1st day, start simultaneously at administration, dosage period 4 days.To plasminogen group fibrinolysin is given by 1mg/0.1ml/ pcs/day of tail vein Original gives the PBS, PLG of same volume to solvent PBS control group tail vein^-/-Mouse is without any processing.Extract eyeball within 5th day Blood is taken, centrifuging and taking supernatant detects creatinine in serum concentration.Using creatinine detection reagent box, (life is built up in Nanjing to serum creatinine concentration Object Graduate School of Engineering, article No. C011-2), and be detected by the kit the method.

The results show that it is significantly lower than PLG to solvent PBS control group and to plasminogen group creatinine in serum concentration^-/-Group is small Mouse, and statistical discrepancy is notable.Solvent PBS control group (figure is given in addition, being significantly lower than to plasminogen group creatinine in serum concentration 12).Illustrate that plasminogen can significantly improve the renal function of Chronic Renal Impairment model mice.

13 plasminogen of embodiment promotes the reparation of acute kidney injury model mice renal function

7 week old male C57 mouse 9, are randomly divided into two groups, to plasminogen group 5, to solvent PBS control group 4. All mouse induce acute kidney injury according to 250mg/kg weight single intraperitoneal injections folic acid (sigma A7876) solution^【36】。 Folic acid is dissolved in 0.3mol/L NaHCO₃.It is denoted as the 1st day, is started simultaneously to plasminogen or solvent PBS, to fibre on the day of modeling Lyase original group gives plasminogen by 1mg/0.1ml/ pcs/day of tail vein, and same volume is given to solvent PBS control group tail veins Long-pending PBS is administered 7 days by a definite date.Eyeball was plucked at the 8th day and takes blood, centrifuging and taking supernatant detects urea in serum nitrogen concentration.Urea nitrogen Content uses Urea nitrogen detection reagent box (Bioengineering Research Institute, article No. C013-2 are built up in Nanjing), and is detected by the urea nitrogen Kit the method is detected.

Solvent PBS control groups are given the results show that being significantly lower than to plasminogen group urea in serum nitrogen concentration, and are counted Difference is close to significantly (P=0.06) (Figure 13).Illustrate that plasminogen can significantly improve the renal function of acute kidney injury model mice.

14 plasminogen of embodiment reduces by 3% cholesterol hyperlipemia model mouse kidney fat deposition

9 week old male C57 mouse 16 feed 3% cholesterol high lipid food (Nantong Te Luofei) 4 weeks, induce hyperlipemia Disease^[37,38], this model is set to 3% cholesterol hyperlipemia model, and the mouse after Cheng Mo continues to feed the feeding high in fat of 3% cholesterol Material.The male C57 mouse 5 of identical week old is separately taken to be only used as blank control group, during the experiment feeding is common to maintain feed.It is being administered Every mouse of first three day takes 50 μ L of blood, detects T-CHOL, and model mice is randomly divided into two according to total cholesterol concentration and weight Group to plasminogen group and gives solvent PBS control group, every group each 8.Start administration to be denoted as the 1st day, it is small to plasminogen group 1mg/0.1ml/ pcs/day of tail vein injection people source plasminogen, to the PBS of solvent PBS control group tail vein injection same volume, Administration 30 days.It puts to death mouse within 31st day, kidney is taken to fix 24-48 hours in 4% paraformaldehyde, respectively at 15%, 30% sucrose In 4 DEG C sink to the bottom overnight, OCT embedding, 8 μm of 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 in 200 times of optical microphotograph Microscopic observations.

Oil red O stain can show lipidosis, reflect the degree of lipidosis^[37].The results show that give plasminogen group (Figure 14 C) mouse kidney fat deposition (arrow logo), which is considerably less than, gives solvent PBS control group (Figure 14 B), and quantitative analysis is united Count significant difference (Figure 14 D)；It is in addition, similar to blank control group mouse (Figure 14 A) to plasminogen group lipidosis level.It says Bright plasminogen can cut down deposition of the fat in hyperlipemia model mouse kidney, dirty so as to kidney caused by reducing fat deposition Damage.

15 plasminogen of embodiment improves folic acid acute kidney injury model mice kidney injury

7 week old male C57 mouse 15 are randomly divided into three groups, blank control group 3, to plasminogen group 7, to molten Matchmaker's PBS control group 5.To plasminogen group and to solvent PBS control group mouse according to 250mg/kg weight single intraperitoneal injections Folic acid (sigma A7876) solution induces acute kidney injury model^[36], blank control group single intraperitoneal injection respective volume NaHCO₃Solution.Folic acid is dissolved in 0.3mol/L NaHCO₃In solution.It is denoted as the 1st day, is started simultaneously to fibrinolysin on the day of modeling Former or solvent PBS, gives plasminogen by 1mg/0.1ml/ pcs/day of tail vein to plasminogen group, gives solvent PBS control group Tail vein gives the PBS of same volume, is administered 7 days by a definite date.Putting to death mouse within 8th day takes kidney to fix 24 in 4% paraformaldehyde Hour.Histotomy thickness be 3 μm, slice dewaxing rehydration and use haematoxylin and eosin stains (HE dyeing), 1% hydrochloride alcohol divide Change, ammonium hydroxide returns indigo plant, and alcohol serial dehydration, dimethylbenzene is transparent, neutral gum mounting, is sliced in 200 times of optical microphotograph Microscopic observations.

The results show that blank control group (Figure 15 A) kidney core is rounded or oval, the red dye of endochylema, glomerulus and kidney are small It is normal to manage equal form；It is in most renal cells flattening (block arrow to solvent PBS control group (Figure 15 B) kidney Mark), brush border comes off, part karyopyknosis, and only the endochylema of light dye is presented in part renal tubule, and part renal tubule is also shown Purulence cast (thin arrow logo), glomerulus and the slight cell infiltration of renal interstitial；To plasminogen group (Figure 15 C) with giving solvent PBS control group is compared, and tubular ectasia and epithelial cell flattening are clearly better, it is seen that most renal tubule endochylema presents red Dye, has no purulence cast.Illustrate, fibrinolysin can improve the acute kidney injury of folic acid induction.

16 plasminogen of embodiment promotes the expression of folic acid acute kidney injury model mice kidney Bcl-2

7 week old male C57 mouse 12, are randomly divided into two groups, to plasminogen group 7, to solvent PBS control group 5. All mouse induce acute kidney injury according to 250mg/kg weight single intraperitoneal injections folic acid (sigma A7876) solution^[36]。 Folic acid is dissolved in 0.3mol/L NaHCO₃.It is denoted as the 1st day, is started simultaneously to plasminogen or solvent PBS, to fibre on the day of modeling Lyase original group gives plasminogen by 1mg/0.1ml/ pcs/day of tail vein, and same volume is given to solvent PBS control group tail veins Long-pending PBS is administered 7 days by a definite date.Putting to death mouse within 8th day takes kidney to fix 24 hours in 4% paraformaldehyde.Kidney after fixation Paraffin embedding is carried out after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, is washed after slice dewaxing rehydration 1 time.PAP are irised out tissue, and with 3% dioxygen water incubation 15 minutes, 0.01MPBS was washed 2 times, every time 5 minutes.5% normal sheep Serum solution (Vector laboratories, Inc., USA) is closed 30 minutes；After time arrives, rabbit-anti is added dropwise in reject sheep blood serum liquid 4 DEG C of overnight incubations of mouse Bcl-2 antibody (Abcam), 0.01M PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and 0.01M PBS are washed 2 times, every time 5 minutes.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Graded ethanol takes off Water, dimethylbenzene is transparent and neutral gum mounting, is sliced in 200 times of optical microphotograph Microscopic observations.

Bcl-2 Showed by immune group result is significantly more than to the coloring of plasminogen group (Figure 16 B) the kidney Bcl-2 positives and gives Solvent PBS control group (Figure 16 A), and statistical discrepancy is significantly (Figure 16 C).This shows that plasminogen can promote acute kidney injury mould The expression of iap protein Bcl-2 in type mouse kidney, so as to help to protect acute kidney injury mouse kidney tissue Cell is from apoptosis.

17 plasminogen of embodiment reduces folic acid acute kidney injury model mice kidney injury

7 week old male C57 mouse 12, are randomly divided into two groups, to plasminogen group 7, to solvent PBS control group 5. All mouse induce acute kidney injury according to 250mg/kg weight single intraperitoneal injections folic acid (sigma A7876) solution^[36]。 Folic acid is dissolved in 0.3mol/L NaHCO₃In solution.It is denoted as the 1st day, is started simultaneously to plasminogen or solvent on the day of modeling PBS gives plasminogen to plasminogen group by 1mg/0.1ml/ pcs/day of tail vein, is given to solvent PBS control group tail vein The PBS of same volume is administered 7 days by a definite date.Putting to death mouse within 8th day takes kidney to fix 24 hours in 4% paraformaldehyde.After fixation Kidney carry out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, slice dewaxing rehydration It washes 1 time afterwards.PAP are irised out tissue, and 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) is closed 30 minutes；After time arrives, reject sheep blood serum liquid, drop Mountain sheep anti mouse IgM (HRP) antibody (Abcam) is added to be incubated at room temperature 1 hour, PBS is washed 2 times, every time 5 minutes.By DAB kits (Vector laboratories, Inc., USA) develops the color, and haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Gradient Transparent and mounting is dehydrated, is sliced in 200 times of optical microphotograph Microscopic observations.

The results show that the positive coloring ratio to plasminogen group (Figure 17 B) mouse kidney IgM gives solvent PBS control group (figure It is 17A) shallow, and range is small compared with control group.This shows that the expression of kidney IgM is substantially reduced after injecting plasminogen, reflects fiber Lyase proper energy effectively reduces the damage of folic acid acute kidney injury mouse kidney.

18 plasminogen of embodiment reduces by 3% cholesterol hyperlipemia model mouse kidney fibrosis

9 week old male C57 mouse 16 feed 3% cholesterol high lipid food (Nantong Te Luofei) 4 weeks, induce hyperlipemia Disease^[37,38], this model is set to 3% cholesterol hyperlipemia model, and the mouse after Cheng Mo continues to feed the feeding high in fat of 3% cholesterol Material.The male C57 mouse 5 of identical week old is separately taken to be only used as blank control group, during the experiment feeding is common to maintain feed.It is being administered Every mouse of first three day takes 50 μ L of blood, detects T-CHOL, and model mice is randomly divided into two according to total cholesterol concentration and weight Group to plasminogen group and gives solvent PBS control group, every group each 8.Start administration to be denoted as the 1st day, it is small to plasminogen group 1mg/0.1ml/ pcs/day of tail vein injection people source plasminogen, to the PBS of solvent PBS control group tail vein injection same volume. Mouse is administered 30 days after administration in the 30th day, and mouse was put to death in the 31st day, cores dirty in 4% paraformaldehyde fixation 24-48 hours. Tissue after fixation carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Aortic sinus slice thickness is 3 μm, is cut It is washed 1 time after piece dewaxing rehydration, 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 it is considerably less than to plasminogen group (Figure 18 C) renal collagen proteinosis (arrow logo) to solvent PBS control group (Figure 18 B), and statistical discrepancy is significantly (Figure 18 D)；Normal level is substantially returned to plasminogen group fibrosis (Figure 18 A).Illustrate that plasminogen can effectively reduce by 3% cholesterol hyperlipemia model mouse kidney fibrosis.

19 plasminogen of embodiment reduces ischemia-reperfusion acute kidney injury model mice kidney injury

7-9 week old males PLG^+/+Mouse 9, is randomly divided into three groups, and sham-operation group to plasminogen group and gives solvent PBS Control group, every group each 3.All mouse are with yellow Jackets according to 50mg/kg weight intraperitoneal injection of anesthesia.Give plasminogen group A notch is opened in abdomen with to solvent PBS control group mouse, exposure kidney detaches bilateral arteriovenous, bilateral is clamped with blood vessel clip Arteriovenous clips rear kidney and moves into abdominal cavity, is closed wound.Time exposes kidney again after arriving, and removes blood vessel clip, observes kidney feelings Condition after determining Reperfu- sion, is sewed up a wound.Sham-operation group group abdomen opens a notch, only exposes kidney, does not do ischemic preconditioning, time It sews up a wound after^[39].Every mouse peritoneal injection 37 DEG C of physiological saline of 1mL after the completion of operation.Keeping body temperature exists during operation 36.5-38℃.It is denoted as the 1st day, is started simultaneously to plasminogen or solvent PBS on the day of modeling, 1mg/ is pressed to plasminogen group 0.1ml/ pcs/day of tail vein gives plasminogen, and the PBS of same volume is given to solvent PBS control group tail vein, and sham-operation is small Mouse is not cooked injection treatment, is administered 7 days by a definite date.Putting to death mouse within 8th day takes kidney to fix 24 hours in 4% paraformaldehyde.Tissue Slice thickness is 3 μm, and slice dewaxing rehydration simultaneously uses haematoxylin and eosin stains (HE dyeing), and 1% hydrochloride alcohol breaks up, and ammonium hydroxide returns Indigo plant, alcohol serial dehydration, dimethylbenzene is transparent, neutral gum mounting, is sliced in 200 times of optical microphotograph Microscopic observations.

The results show that sham-operation group (Figure 19 A) glomerular capillary is unobstructed, the red dye of renal tubule endochylema, in normal kidney Tubule form；To the slight cell infiltration of solvent PBS control group (Figure 19 B) visible glomerulus (triangle mark), renal interstitial is big The cell infiltration of amount, part renal tubule presentation purulence cast (thin arrow logo), a small amount of renal tubular cell karyopycnosis, large area Epithelial cell flattening (block arrow mark), tubular ectasia；Compared to solvent PBS control group is given, plasminogen group (figure is given 19C) there was only a small amount of renal tubular epithelial flattening, the recovered normal renal tubule form of most renal tubule, the red dye of endochylema, Apparent renal tubule atrophy is not found, and only renal interstitial has slight cell infiltration, close to sham-operation group form.Illustrate fibre Lyase can improve ischemia-reperfusion acute kidney injury model mice kidney injury.

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Sequence table

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

<120>A kind of method for preventing and treating Drug injury of kidney

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<160> 14

<170> PatentIn version 3.5

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

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

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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 of subject's Renal tissues damage and its associated disease caused by prevention and/or medicine, including administration A effective amount of plasminogen of subject.

2. the method for claim 1 wherein the drug is nephrotoxic drugs.

3. the method for claims 1 or 2, wherein the drug is the drug of kidney excretion.

4. the method for any one of claim 1-3, wherein the drug include chemotherapeutics, blood-pressure drug, blood lipid-lowering medicine, Hypoglycemic drug, nonsteroidal antiinflammatory drug, antibiotic medicine, antiviral drugs.

5. the method for any one of claim 1-4, wherein the plasminogen promotes the reparation of the nephridial tissue of damage.

6. the method for any one of claim 1-5, wherein the plasminogen mitigates the fibroid of the nephridial tissue of the damage.

7. the method for any one of claim 1-6, wherein the plasminogen mitigates the apoptosis of the nephridial tissue of the damage.

8. the method for any one of claim 1-7, wherein drug induced subject's Renal tissues damage is acute nephridial tissue Damage.

9. the method for any one of claim 1-7, wherein drug induced subject's Renal tissues damage is chronic nephridial tissue Damage.

10. the method for any one of claim 1-9, wherein the plasminogen promotes renal function to restore.