CN100493604C - Application of hepatocyte growth promoting factors in medicine for treating diffusive interstitial pneumonia - Google Patents
Application of hepatocyte growth promoting factors in medicine for treating diffusive interstitial pneumonia Download PDFInfo
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Abstract
The invention relates to an application of liver cell accelerator auxin, that it is used to treat disperse acute nonsuppurative hepatitis drug, wherein the experiment has proves that the liver cell accelerator auxin can be used to treat kinds of liver damages and liver fiber, with high protective function. It can treat or auxiliary treat disperse acute nonsuppurative hepatitis.
Description
Affiliated technical field
The present invention relates to a kind of new purposes of hepatocyte growth-promoting factors, relate in particular to the application of hepatocyte growth-promoting factors in treatment diffuse interstitial pneumonia medicine, hepatocyte growth-promoting factors can be used as the treatment or the adjuvant therapy medicaments of treatment diffuse interstitial pneumonia.
Background technology
Hepatocyte growth-promoting factors is that molecular weight is 10, below the 000Da from fresh piglets liver or the multicomponent small-molecule peptide mixture that do not extract the suckling new born bovine liver.Its main pharmacological is to synthesize by the newborn hepatocellular DNA of obvious stimulation; improve the phagocytic function of liver Kupffer Cell; hepatocyte injury to tetrachloro-methane induction has the better protect effect, the be significantly improved effect of viability of the liver failure that D-Gal is led to.Clinically be used for various Severe Viral Hepatitis, as the auxiliary treatment in early stage or mid-term of acute, subacute, chronic hepatitis gravis etc.These product have been extensive use of for many years since listing in 1992, and clinical efficacy is definite.Hepatocyte growth-promoting factors system adopts modern biotechnology means such as homogenate, degeneration, centrifugal, classification ultrafiltration to extract purification and is prepared from.
Diffusivity interstitial lung disease be one group nonspecific, invade the disease of alveolar wall and alveolar surrounding tissue, main pathology is changed into interstitial pulmonary fibrosis.Kind causes in the interstitial pulmonary fibrosis disease surplus in the of 180, and it is unclear to account for 64% the cause of disease.Its typical clinical performance is gradual, labour's property tachypnea, dry cough, and dyspnea shows as respiratory failure whole latter stage.At present pulmonary fibrosis still there is not a kind of gratifying medicine.Clinical medicine commonly used has glucocorticoid, immunosuppressant/cell toxicity medicament and anti-fibrosis medicine.Glucocorticoid is topmost medicine, but untoward reaction is very many, can bring out or increase the weight of to infect, and is not suitable for long-term heavy dose of the use.
The medicine of exploitation treatment or auxiliary treatment pulmonary fibrosis reduces or alternative hormone, alleviates the injury that the hormone untoward reaction causes human body, current so that all have social meaning very widely from now on.
Summary of the invention
Technical problem to be solved by this invention provides a kind of new purposes of hepatocyte growth-promoting factors, the both application of hepatocyte growth-promoting factors in treatment diffuse interstitial pneumonia medicine, hepatocyte growth-promoting factors can be used for the treatment or the auxiliary treatment of diffuse interstitial pneumonia.
The therapeutic dose that is used for diffuse interstitial pneumonia is: intravenous drip, and this product 100mg adds the slow intravenous drip of 5% Glucose Liquid 250ml, and every day 1 time, decide on the state of an illness course of treatment, is generally 4-12 week.
The application of hepatocyte growth-promoting factors of the present invention in treatment diffuse interstitial pneumonia medicine, it leads to acute lung injury to have the better protect effect to oleic acid, and the injury of lung pulmonary fibrosis that bleomycin is led to has protective effect.
It is long-term in a large number about the experimental study checking of the treatment diffuse interstitial pneumonia of hepatocyte growth-promoting factors that the present invention is that the inventor passes through, and do not see record and report so far.Pharmacodynamics test lung index variation of the present invention, SOD (superoxide dismutase) value, MDA (malonaldehyde) value and HYP (hydroxyproline) value change and the matched group comparing difference all has significance.The proof hepatocyte growth-promoting factors to multiple factor cause injury of lung, pulmonary fibrosis has protective effect.The prompting hepatocyte growth-promoting factors has therapeutical effect to clinical diffuse interstitial pneumonia.
One, the pathogenesis of diffuse interstitial pneumonia
Alveolar epithelial cells, immunocyte, inflammatory cell, fibrocyte and collagen and cytokine etc. all play a role in diffuse interstitial pneumonia.
1. the impaired lung epithelial damage of alveolar epithelial cells is one of key factor that causes pulmonary fibrosis, and I type epithelial cell is impaired, II type epithelial hyperplasia.Comprise platelet derived growth factor (PDGF), transforming growth factor (TGF-α, TGF-β), endothelin-1 (ET-1), prostaglandin F
2a(PGF
1a) wait the injury of lung process that participates in.
2. (alveolar macrophage, AM) cytokine of Shi Fanging and inflammatory mediator play a major role in the injury of lung the activatory pulmonary alveolar macrophage of macrophage activation in early days.AM activation back TGF-β, PDGF and IGF-1 (insulin like growth factor) cause Fibrotic somatomedin, and secretion increasing in early days starts the process of pulmonary fibrosis simultaneously, forms the irreversibility injury of lung.TGF-β is the important fibrotic growth factor that causes, and can stimulate fibroblast proliferation and collagen synthetic, promotes collagen protein, fibronectin, hyaluronic acid, Dan Baijutang etc. to deposit in extracellular matrix, reduces degradation of extracellular matrix.PDGF and IGF-1 also are the important fibrotic growth factors that causes, and can stimulate smooth muscle cell, fibroblast proliferation and collagen synthetic.
3. inflammatory cell is assembled and activation
Lymphocyte often exists in patient's interstitial lung of interstitial pulmonary fibrosis, can discharge interleukin-4 (IL-4), fibroblast chemotactic factor, monocyte chemotactic factor and macrophage chemotactic factor etc.Interaction between these pulmonary parenchyma cells, the inflammatory cell, the inflammatory mediator of generation and somatomedin act on fibroblast at last, cause fibroblast hypertrophy and collagen to form.
4. fibroblast proliferation and collagen produce
PGE-2, interferon-(INF-γ), fibroblast migration inhibitory factor etc. all belong to negative growth factor.The unconventionality expression of I, III Collagen Type VI mainly betides intermediary and later stages of pulmonary fibrosis, and the variation of IV Collagen Type VI is early stage just very outstanding in pulmonary fibrosis.In recent years, the effect in the disorder of pulmonary fibrosis collagenic supersession out of proportion of metalloproteases (MMPs) and tectotype inhibitors of metalloproteinase (TIMP) draws attention.The TIMP-2 activity increases in idiopathic pulmonary fibrosis (IPF) the patient fibrosis focus.
5. apoptosis
There has been lot of experiment results to show that apoptosis plays an important role in pulmonary fibrosis.Fas, cascade enzyme, tumor necrosis factor-alpha (TNF-α), reactive oxygen free radical (ROS), NO, Angiotensin-Converting (ACE), transforming growth factor-beta (TGF-β), p53 and p21 (oncogene protein), interleukin-6 (IL-6) all can promote the FAS mediated Apoptosis.The apoptosis that Fas-FasL brings out promotes IL-1
βSecretion, neutrophil accumulation and epithelial disappearance.In the pulmonary disease model, inflammation and fibrosis can be controlled by the interaction or the downward modulation cascade reaction that disturb Fas-FasL.
6. effect and the mechanism of cytokine in pulmonary fibrosis morbidity
Between a large amount of cytokines and and inflammatory cell, lungs histiocyte between can interact, complex series of reactions takes place, increase the weight of lung tissue inflammation or immunologic injury, stimulate the fibroblast division growth, promote the generation and the deposition of extracellular matrix.Thereby pulmonary fibrosis is to cause a large amount of chemotactic factors release causing inflammatory cells to assemble, activate so that the cytokine network regulation and control are unbalance.
Somatomedin TGF-β mainly shows two broad aspect to the fibroblast effect: it can stimulate fibroblast synthetic cell epimatrix composition on the one hand; Suppress zymolysis on the other hand, also have inhibition alveolar epithelial cells (alveolar epithelial cell, AECs) Zeng Zhi effect new synthetic stromatin generation.IGF-1 in the lung is important one of the pulmonary fibrosis factor that causes, and its content increases with the order of severity of pulmonary fibrosis and is directly proportional.After IGF-1 and the rat fibroblast IGF-1R receptors bind, the interior DNA of inducing cell synthesizes and plays a role.
Tumor necrosis factor TNF-alpha is under pro-inflammatory cytokine stimulates, a kind of important cellular immunization defense factor that body tissue produces.In BLM induced mice pulmonary fibrosis generating process, mainly produce by macrophage and II type alveolar epithelial cells.Stimulate the hypertrophy of people's lung fibroblast and synthesizing of collagen.
The interleukin zoopery confirms, IL-1 receptor antagonist (IL one 1ra) can obviously alleviate the pulmonary fibrosis that bleomycin (BLM) or silicon dioxide bring out.IL-4 has the pulmonary surfactant of making SP-D and expresses increase, promotes fibroblast proliferation, collagen gene expression and collagen complex functionality.IL 1 is proinflammatory factors important in the pulmonary fibrosis, not only participates in keeping, developing of early stage injury of lung and inflammation, also may participate in Fibrotic formation.
It is synthetic that gamma interferon INF-γ suppresses collagen.
Chemotactic factor macrophage inflammatory protein-1 α (MIP-1 α), monocyte chemoattractant protein-1 (MCP-1) are chemotactic factor and the immunoregulatory factor relevant with pulmonary fibrosis, but the significant stimulation lung fibroblast synthesizes collagen, and the cytokine or the medium that can cause having into fiber properties produce.
Endothelin, Angiotensin II, transcription factor, matrix metalloproteinase-7 (MMP-7), iuntercellular adhesion molecule etc. are all brought into play certain effect to the generation of pulmonary fibrosis.
In the following test used hepatocyte growth-promoting factors can be commercially available also can be to be prepared from by the embodiment of the invention 1 method.
Two, the pharmacological testing of medicine of the present invention
------basement membrane of epithelium causes destruction, and---fibrosis in the alveolar space---final alveolar structure destroys---forms pulmonary fibrosis and vesicle honeycomb lung to the alveolar inflammation to the pathology process of diffuse interstitial pneumonia: various not clear virulence factors.Suppress acute stage the alveolar inflammation with chronic phase fibrosis make progress interstitial pneumonia all had positive effect.We adopt the protective effect of hepatocyte growth-promoting factors to acute and chronic injury of lung and pulmonary fibrosis that caused the scale-model investigation of induced lung damage fibrosis of oleic acid induced mice acute lung injury model and bleomycin.
(1) hepatocyte growth-promoting factors is to the protective effect of acute lung injury due to the oleic acid:
1, ultimate principle: acute lung injury often can cause pulmonary fibrosis.Adopt the oleic method of intravenous injection, causing the acute lung injury model is a common method studying acute lung injury (ALI) at present.We are with preventative hepatocyte growth-promoting factors three days of giving of mice, 30min after the last administration, vein causes ALI to oleic acid, judges with variation and the lung tissue disease variation of science of anti-peroxidation material superoxide dismutase (SOD), the variation of lung coefficient whether hepatocyte growth-promoting factors has the effect of antagonism ALI by measuring serum lipid overoxidation product malonaldehyde (MDA).
2, experimental technique: 60 mices are divided into 6 groups at random, and 10 every group, male and female half and half.Before the oil supply processed with acid is equipped with the ALI model, each treated animal preventive administration three times, every day 1 time.C group intraperitoneal injection dexamethasone 2mg/kg, D group intravenous injection hepatocyte growth-promoting factors 8mg/kg, E group hepatocyte growth-promoting factors 15mg/kg, F group hepatocyte growth-promoting factors 30mg/kg.The administration volume is 0.4～0.5ml, A group and B group tail vein injection equivalent normal saline, every day 1 time, continuous three times.Last administration 30min, except that A normal control group intravenous injection 0.15ml/kg normal saline, other respectively organizes equal tail vein injection 0.15ml/kg oleic acid, causes ALI.Respectively organize mice after 6 hours for oleic acid and pluck the eyeball blood sampling, centrifugalize serum ,-20 ℃ of refrigerators are preserved to be checked; Take off neck then and put to death mice, take out lungs, observe general form, blot, claim the lung weight in wet base with filter paper, calculate lung coefficient (lung weight in wet base ÷ body weight * 100), get its lobus dexter put in 4% formalin fixing, routine paraffin wax section after 72 hours, HE dyeing, light microscopy, active (pyrogallol autoxidation performance rate method), the MDA content detection [thiobarbituricacid (TBA) development process] that detect of SOD adopt Nanjing to build up the SOD of bio-engineering research institute, the detection of MDA kit method.
3, result of the test:
A lung index variation lung coefficient=lung weight/body weight * 100%.Each test group lung coefficient is all apparently higher than the normal control group, and difference all has significance (P＜0.01); The hepatocyte growth-promoting factors group is respectively organized the lung coefficient and is lower than the oleic acid model group, and wherein dosage group lung coefficient value is minimum among the E, significant difference (P＜0.01).
Table 1, lung index variation table (X ± SD) (g of unit)
Annotate:
▲Compare P＜0.05 with the oleic acid model group
▲ ▲Compare P＜0.01 with the oleic acid model group
* compare P＜0.01 with normal control group comparison P＜0.05 * * and normal control group
The variation of b serum MDA, SOD content
Compare with the normal control group, each test group SOD all is lower than the normal control group.Oleic acid model group SOD is minimum; The hepatocyte growth-promoting factors group is respectively organized the SOD value and all is higher than the oleic acid model group, and difference is all remarkable.Wherein the dosage group is the highest in the E hepatocyte growth-promoting factors.
Compare with the normal control group, each test group MDA is apparently higher than A normal control group, and difference all has significance; Hepatocyte growth-promoting factors group MDA is lower than the oleic acid model group, wherein dosage group and F high dose group significant difference (P＜0.05) among the E, and D low dose group MDA value also is lower than the B model group, but difference is not had a significance.
Table 2, serum malonaldehyde (MDA) and superoxide dismutase (SOD) result (X ± SD)
Annotate:
▲Compare P＜0.05 with the oleic acid model group
▲ ▲Compare P＜0.01 with the oleic acid model group
* compare P＜0.05 * * and end relatively P＜0.01 of normal matched group with the normal control group
C lung tissue disease is of science to change
Each test group lung volume of lung tissue finding of naked eye obviously increases, and the lung surface is congested, edema and petechia; The oleic acid model group is the most serious, and the visible pathological changes of dosage group naked eyes obviously is lighter than the oleic acid model group in the hepatocyte growth-promoting factors, and roughly similar to Dexamethasone group, it is less to show as lung volume, and the petechia is less, and lesion degree is lighter.
The all visible pulmonary capillary expansion of each test group of light microscopy checking, congested, hemorrhage, pulmonary interstitial edema, neutrophil infiltration, hyaline membrane forms, alveolar focal necrosis and pulmonary atelectasis.Hepatocyte growth-promoting factors high dose and middle dosage group lesion degree obviously are lighter than B oleic acid model group.
4, experiment conclusion:
This experiment adopts oleic acid to duplicate the ALI model, and the result shows that oleic acid model group serum MDA increases, and SOD reduces, and the lung coefficient raises, and pathological change is the acute inflammation performance, lung enlargement, hyperemia, inflammatory cell infiltration.Adopt dexamethasone as positive drug; the result of the test serum MDA reduces, SOD raises, the lung coefficient descends, and histopathology changes and alleviates, and dexamethasone is used in prompting can suppress lipid peroxidation; alleviate the degree of lung tissue damage, ALI has protective effect to oleic induced.Also confirmed simultaneously this animal model replication success.
Hepatocyte growth-promoting factors administration group serum MDA reduces than the oleic acid model group, significant difference; SOD in serum raises than the oleic acid model, and significant difference illustrates that hepatocyte growth-promoting factors has suppressed lipid peroxidation.Hepatocyte growth-promoting factors group lung pathology changes lighter, and the swelling degree is little, and the lung coefficient is low than model group.This result of the test shows, hepatocyte growth-promoting factors has protective effect to acute lung injury due to the oleic acid, and the result is similar to positive control medicine Dexamethasone group.
This test finds that simultaneously hepatocyte growth-promoting factors dosage is the most remarkable when 15mg/kg, and relatively poor in the 8mg/kg effect, with this inference people medication as a result, becomes body weight for humans 60-70kg meter, and clinical using dosage was advisable for 100mg/ day.
(2) hepatocyte growth-promoting factors is to being led to induced lung damage and fibrosis protective effect research by bleomycin
1, ultimate principle: bleomycin is a kind of antitumor drug, and one of its toxic and side effects is to cause pulmonary fibrosis, and it is approximate to have proved that its pathology and physiology change with people's pulmonary fibrosis.Adopting bleomycin to make the animal pulmonary fibrosis model is pharmacological testing method commonly used.We adopt, and disposable injection bleomycin duplicates pulmonary fibrosis model in the rat trachea; the hepatocyte growth-promoting factors of intravenous injection simultaneously carries out prophylactic treatment; by measuring in the serum hydroxyproline (HYP) content in malonaldehyde (MDA) content and lung tissue, measure the lung coefficient and carry out index judgement hepatocyte growth-promoting factors such as pneumonopathy inspections of science and whether injury of lung due to the bleomycin is had protective effect and reach whether have the pulmonary fibrosis resistant effect.
2, test method: 180 of Wistar rats, body weight 200～220g, male and female half and half are divided into 6 groups at random, and 30 every group, male and female half and half.Duplicate pulmonary fibrosis model with injecting bleomycin in the disposable trachea: concrete steps are with 1% pentobarbital sodium intraperitoneal injection of anesthesia, getting dorsal position is fixed on the flat board, the row aseptic operation, cut off skin of neck and isolate trachea, except that the normal control group, each is organized and injects bleomycin 0.2～0.3ml (5mg/kg) in the equal trachea.The normal control group is injected isopyknic normal saline.Upright and the rotation with rat immediately makes medicine uniform distribution in lung, skin suture after the administration.Behind operation on trachea, promptly begin Drug therapy: the about 0.4ml of Dexamethasone group intraperitoneal injection every day (3.33mg/kg); When using, dilutes Hepatocyte Growth-Promtting Factors For Injection with sodium chloride injection, basic, normal, high three dosage groups respectively by 5,10, the 20mg/kg administration, the administration volume is 1ml/200g, the tail intravenously administrable, normal control group and bleomycin group intravenous injection every day normal saline 1ml/200g, be administered once every day, to putting to death day.Respectively at the 3rd, 14,28 of administration, divide 3 batches of execution with animal, put to death 10 for every batch every group.After the administration about 1 hour, rat is used etherization, ventral aorta blood sampling (the 3rd, 14), centrifugalize serum ,-20 ℃ are freezing standby, malonaldehyde to be measured (MDA) content; Win lungs, blot, claim full lung heavy, calculate the lung coefficient, promptly full lung weight/body weight * 100% with filter paper; Get a part of right lung fixation of tissue in formaldehyde fixed liquid, do the pathology section; Other lung is made hydroxyproline content and is measured usefulness.
3, result of the test:
A lung index variation: lung coefficient=lung weight/body weight * 100%.
Test each treated animal body weight and all descend to some extent, pulmonary congestion swelling, weight increases, and causing the lung coefficient increases.Each group of hepatocyte growth-promoting factors compares with the bleomycin group, and the lung coefficient all decreases, and middle dosage group reduces the amplitude maximum, and there were significant differences (P＜0.01).
Table 3, different time lung index variation table (X ± SD) (% of unit)
Annotate:
▲Compare P＜0.05 with the bleomycin model group
▲ ▲Compare P＜0.01 with the bleomycin model group
* compare P＜0.01 with normal control group comparison P＜0.05 * * and normal control group
The variation of b serum malonaldehyde (MDA) content
Administration the 3rd day and the 14th day, each test group serum MDA all raise than normal matched group; Each group of hepatocyte growth-promoting factors compares with the bleomycin group, and MDA all decreases, and middle dosage group reduces the amplitude maximum.Significant difference (P＜0.01, the 3 day; P＜0.05, the 14 day).
Table 4, different time serum malonaldehyde (MDA) change list (X ± SD) (nmol/ml of unit)
Annotate:
▲Compare P＜0.05 with the bleomycin model group
▲ ▲Compare P＜0.01 with the bleomycin model group
* compare P＜0.01 with normal control group comparison P＜0.05 * * and normal control group
The variation of c hydroxyproline (HYP) content: on the 14th and 28, the normal matched group of each test group lung HYP content generally raise, and the 28th earning in a day is higher than the 14th earning in a day.With the bleomycin model group for the highest.Middle and high dosage group of hepatocyte growth-promoting factors group and bleomycin model group be tool significant difference (P＜0.01) relatively all.
Table 5, different time lung tissue hydroxyproline (HYP) content HYP change list (X ± SD) (mg/g of unit)
Annotate:
▲Compare P＜0.05 with the bleomycin model group
▲ ▲Compare P＜0.01 with the bleomycin model group
* compare P＜0.01 with normal control group comparison P＜0.05 * * and normal control group
The d histopathology changes: administration the 3rd day, the enlargement of test group lung outward appearance has the congested point of peony, and was rough, and on the 14th and 28, the two lungs of experimental group were pale, and congested point of peony and canescence lesser tubercle are arranged, and hardness increases, and volume dwindled than the 14th day.Each time period is all obvious with the bleomycin group, and dosage group situation is lighter in the hepatocyte growth-promoting factors.
HE dyeing light microscopic is observed pathological change down and is gradual fibrosis formation, early stage edema, and inflammatory cell infiltration mainly shows as blood capillary proliferation late period, fibroblast proliferation, collagen increases.Hepatocyte growth-promoting factors is respectively organized pathological change and also is lighter than the bleomycin group, and middle dosage group situation wherein is best.
4, conclusion (of pressure testing):
This experimental study finds that hepatocyte growth-promoting factors can suppress lung lipid peroxidation deposits yields significantly, and administration the 3rd day and the 14th day, model group MDA had downward trend.Three hepatocyte growth-promoting factors group MDA content all are lower than the bleomycin group, middle dosage group amplitude maximum wherein.The prompting hepatocyte growth-promoting factors may alleviate the damaging action of bleomycin to lungs by suppressing peroxidization; The hydroxyproline content of administration group meanwhile also significantly is lower than the bleomycin group; Pathological change also alleviates to some extent; This test finds that also hepatocyte growth-promoting factors treated animal lung coefficient obviously reduces than the bleomycin group, cooperates pathological change, can think that hepatocyte growth-promoting factors alleviates inflammatory activity such as lung swelling, hyperemia; The result shows that hepatocyte growth-promoting factors has inhibition and causes injury of lung and pulmonary fibrosis effect by bleomycin.
In this test, hepatocyte growth-promoting factors dosage is the most remarkable when 10mg/kg, and relatively poor at the 5mg/kg exercising result, and with this inference people medication as a result, proportionately body weight for humans 60-70kg counts, and clinical using dosage was advisable for 100mg/ day.
Above-mentioned test hepatocyte growth-promoting factors leads to the effect of acute lung injury better protect to reach the protective effect of the injury of lung pulmonary fibrosis that bleomycin is led to oleic acid, and the prompting hepatocyte growth-promoting factors has treatment or auxiliary treatment effect to clinical diffuse interstitial pneumonia.
The specific embodiment
Embodiment 1
The preparation method of hepatocyte growth-promoting factors comprises the steps:
1. choose health, non-infection district, through qualified piglets or the suckling new born bovine not of quarantining, get fresh liver (require the surface should be bright and clean, fine and smooth, color and luster is normal, encapsulation, tuberosity and parasite must not be arranged), be sub-packed in the sterilization container after cleaning with water for injection, directly use or-20 ℃ down freezing preservations be no more than use in 3 months;
2. organized processing: fresh or freezing liver are removed adventitia, clean, shred or shred with water for injection;
3. homogenate: by weight: volume is that 1:1 adds sterilized water for injection, homogenate 2 minutes repeatedly in the colloid mill under low temperature (4 ℃) condition, the test under microscope cell is broken fully, homogenate;
4. freeze thawing: with homogenate multigelation three times, take the slow method of melting of quick-freezing, cryogenic temperature-20 ℃～-35 ℃, melt temperature is lower than 40 ℃;
5. thermal denaturation and remove high molecular weight protein: fast melting liquid is warming up to 85 ℃ rapidly, and constant temperature 5 minutes reaching the high molecular weight protein thermal denaturation, and is removed high molecular weight protein;
6. centrifugal: after the thermal denaturation, 4 ℃, 4000 rev/mins, centrifugal 20 minutes, abandon precipitation, get supernatant;
7. classification ultrafiltration and inactivation of virus: centrifuged supernatant is carried out the classification ultrafiltration with ultrafilter.The one-level ultrafiltration---collect molecular weight 30, the component that 000Da is following; Two-stage ultrafiltering---collecting molecular weight is 10, the polypeptide fractions that 000Da is following, and 60 ℃ are incubated 10 hours, inactivation of viruses;
8. aseptic filtration, packing: with 0.45 μ m, 0.22 μ m filtering with microporous membrane, the filtrate that is up to the standards is sub-packed in the disposable use plastic infusion bag, low temperature (18 ℃ ± 2 ℃) is stored, and promptly gets promoting hepatocyte growth factor solution.
The promoting hepatocyte growth factor solution character of using said method to obtain is little yellow clear liquid, and its detection case is as follows:
[discriminating] adds biuret reaction solution and shows bluish violet to aubergine.
[inspection] pH value is 6.0～7.0.(two appendix VIH of Chinese Pharmacopoeia version in 2005)
The protein feminine gender.
High molecular weight material must not cross 6.0% less than the peak area sum of insulin spikes retention time.
Vitality test adopts mtt assay to measure the promoting hepatocyte growth factor solution activity, and stimulation index is not less than 4.0.
The undue toxicity gets this product, adds the chlorination sodium injection and makes the solution that contains polypeptide 5.0mg among every 1ml, checks in accordance with the law and (two appendix XI of Chinese Pharmacopoeia version in 2005 C) presses the intravenous injection administration, should be up to specification.
Pyrogen is got this product, adds the chlorination sodium injection and makes the solution that contains polypeptide 5.0mg among every 1ml, checks (two appendix XI of Chinese Pharmacopoeia version in 2005 D) in accordance with the law, and dosage should be up to specification by the every 1kg injection of rabbit body weight 1ml.
It is an amount of that sensitivity test is got this product, adds the chlorination sodium injection and make the solution that contains polypeptide 5.0mg among every 1ml, as test sample liquid.Get 6 of body weight 250～350g healthy guinea pigs, every other day lumbar injection test sample 0.5ml, continuous 3 times, for the third time the injection after 14 days, from intravenous injection this product 1.0ml, inject in back 15 minutes and observe animal, all must not occur continuous dry cough, continuously fore paw grab nose, obviously alarm that hair, extremity feel like jelly, couch, dyspnea, spasm, collapse and the phenomena of mortality.Observed 3 days continuously, animal should be good for and be deposited.
Aseptic in accordance with the law check (two appendix XI of Chinese Pharmacopoeia version in 2005 H) should be up to specification.
[assay] measured according to forint phenol algoscopy, contains polypeptide among every 1ml and must not be less than 10mg.
The application of above-mentioned hepatocyte growth-promoting factors in treatment diffuse interstitial pneumonia medicine, it leads to acute lung injury to have the better protect effect to oleic acid, and the injury of lung pulmonary fibrosis that bleomycin is led to has protective effect.
Claims (1)
1, the application of hepatic stimulator substance in preparation treatment diffuse interstitial pneumonia medicine.
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