CN100417412C - Liver cell growing promotor injection and its preparation method and application - Google Patents
Liver cell growing promotor injection and its preparation method and application Download PDFInfo
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- CN100417412C CN100417412C CNB031160522A CN03116052A CN100417412C CN 100417412 C CN100417412 C CN 100417412C CN B031160522 A CNB031160522 A CN B031160522A CN 03116052 A CN03116052 A CN 03116052A CN 100417412 C CN100417412 C CN 100417412C
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Abstract
The present invention relates to a hepatocyte growth promotion factor injection, and a preparation method and an application for the injection. In the hepatocyte growth promotion factor injection disclosed by the present invention, a selected hepatocyte growth promotion factor is active protein extracted and separated from the liver of a porket, the protein is composed of 15 amino acids, and the total amount of the amino acids is 135.21(+/-)10.23 n mol/ml; the molecular weight of the main active component protein is 2 1, 000 D and accounts for more than 60% of total content, and the protein has high activity of stimulating hepatocyte DNA synthesis to regenerate hepatocytes. The injection can be used for treating chronic hepatitis, serious hepatitis, viral hepatitis, toxic hepatitis and liver cirrhosis.
Description
Technical field
The present invention relates to biological technical field.Be specifically related to hepatocyte growth-promoting factor injection and preparation method and application.
Background technology
As far back as early seventies, it is found that: only need just can make liver return to the original volume size in 7-10 days behind the human liver of 2/3 or the excision major part damage of experimental excision rat liver and also stop voluntarily; Embryonic liver homogenate is simultaneously injected in the hepatic injury Mus body can obviously promote liver cell regeneration, therefore thinks to exist certain material to regulate the regeneration of cell in vivo.1973 LaBrecque morning a kind of liver regeneration of extraction from rat regeneration liver promotes material, 70% liver is cut the remaining adult rat hepatocyte in Xu back prepare homogenate and high speed centrifugation, increase but the normal rat hepatocyte DNA of the material obvious stimulation monolayer culture of extracting from supernatant is synthetic, and called after " hepatic stimulator substance (Hepatic Stimulator Substance, HSS).The Hepatopoietin and use thereof that the remaining hepatic tissue of Goldberg behind the rat partially hepatectomized purified can make the synthetic 3-4 of increasing of hepatocyte DNA doubly in vivo; Starzl also finds similar substance in the Liver Regeneration of Canis familiaris L..Hepatopoietin and use thereof in the proof platelet such as Russell etc. and the youth of salt pan nation also promotes hepatocyte DNA significantly synthetic; Waiting in liver with a man of virtue and ability synchronously divides the Hepatopoietin and use thereof that extracts in the solubility composition of the back 48 hours rat small intestine mucosa of excision different with the Hepatopoietin and use thereof in the hepatic tissue; People's tire Hepatopoietin and use thereof that Xie Ming etc. extract from human foetus liver cell has the stronger synthetic effect of promotion Hepar Mus cell DNA." hepatocyte growth factor (HGF) " that the old light of Guangzhou air hospital etc. extracts from the piglets liver is applied to the various hepatitis of clinical treatment curative effect preferably.Therefore, found that up till now a series of materials all can promote hepatocyte DNA synthetic, regulated liver cell regeneration, it originates different, and liver source property, platelet source property and intestinal source property are arranged; Physicochemical properties such as molecular weight are also inequality, even the material of the preparation of same source distinct methods also has difference, so name has difference, as " the liver cell regeneration factor ", " hepatocyte growth factor (HGF) ", " short liver auxin (Hepatopoietin; HPP) ", " hepatic stimulator substance (HepaticStimulator Substance; HSS) ", " the synthetic factor (DNA SynthesisPromoter) that promotes of DNA ", report about this class molecular weight of material differs bigger, and to separate back its molecular weight of survey through Sephadex G-200 from the acute severe hepatitis patient blood be 230000 for Bo Ren etc. in the level ground; Goldberg etc. separate its molecular weight of back and are about 38000 from rat plasma; LaBrecque is 1-2 ten thousand from the HSS molecular weight that the neonatal rat liver extracts; The piglets HCF molecular weight of Chen Guangming preparation is 10685, its molecular weight size no matter, and they have its biological activity, make that hepatocyte DNA is synthetic to be increased.
The research of the liver regeneration factor is at home and abroad all reported a lot, but be applied to clinical research seldom, at present, " the hepatocyte growth factor HGF " of domestic Guangzhou air hospital development Preliminary Applications in the various hepatopaths of clinical treatment, be low molecular weight polypeptide, certain therapeutic effect is arranged.
Viral hepatitis is commonly encountered diseases, frequently-occurring disease, its mortality rate height in China, does not have ideal medicine at present, has a strong impact on people's life and health.
Summary of the invention
Technical problem to be solved by this invention is to adopt the method for optimizing to extract active high, the ideal hepatocyte growth-promoting factors of purity from the piglets liver, and a kind of treatment liver disease drug of determined curative effect is provided.
The invention discloses a kind of hepatocyte growth-promoting factor injection that contains, described hepatocyte growth factor is a kind of reactive protein that extraction separation obtains from the piglets liver, and this protein is made up of 15 seed amino acids, and total amino acid content is 135.21 ± 10.23nmol/ml; Main active constituent protein molecular weight is 2.1 ten thousand dalton, accounts for more than 60% of total protein content, has the synthetic activity of higher cell cultured supernatant DNA.
15 seed amino acids of described constitutive protein matter are: Asp, Glu, Ser, Thr, Gly, His, Tyr, Arg, Ala, Met, Val, Ile, Leu, Lys, Phe.
Hepatocyte growth-promoting factor injection of the present invention is every milliliter of liquid drugs injection or the freeze-dried powder that contain protein 15-25 μ g, pH 6.0-8.0.
Another technical problem to be solved by this invention is the preparation method that discloses above-mentioned hepatocyte growth-promoting factor injection.
Hepatocyte growth-promoting factor injection disclosed by the invention is by obtaining piglets liver homogenate, stirring, heating, filtration, absorption, eluting, desalination, degerming, packing.This injection specifically makes by following technical proposals:
1. homogenate:
The piglets liver of learning from else's experience after washing tentatively blends on meat mincer earlier, and the reuse milling treatment of colloid obtains the homogenate of piglets liver.
2. stir:
Homogenate is placed in the extraction pot, add extracting solution in proportion, ratio is 1: 3W/V, and simultaneously to stir at a slow speed one hour.
3. heating:
Mode with water-bath heats to extraction pot, stops heating when feed temperature reaches 95 ℃, and keeps 15-20 minute on this temperature spot.
4. filter:
After heating is finished, earlier feed liquid is cooled to room temperature, carries out solid-liquid separation with filtering mode then, keep filtrate.
5. purify:
5.1. last sample:
The filtrate that obtains through solid-liquid separation with 10 liters/time flow velocity on sample to installed and counter-balanced DEAE Sepharose FF post in, applied sample amount is 6 times of column volume.
5.2. eluting:
After last sample finishes, carry out stepwise elution with A, B eluent respectively, and collect B liquid eluting peak.
5.3. desalination:
With ultrafilter membrane ultrafilter desalination;
6. semi-finished product calibrating:
Measure protein content with the Lowry method, adjust its content, make it g/ml into 15-25 μ with water for injection;
Measure pH value with pH meter, the adjusting pH value is 6.0-8.0;
The control chlorine ion concentration is less than 1.0mmol/L;
7. degerming
Carry out aseptic filtration with 0.2 μ micropore filter.
9. packing promptly gets aqueous injection or lyophilization gets freeze-dried powder, preserves below 4 ℃.
Extracting solution described in the present invention's preparation is a pH value 7.6, the phosphate buffer of 0.02M (PBS); Described A washing liquid is meant the PBS solution of sodium chloride-containing 0.28M; Described B washing liquid is meant the PBS solution of sodium chloride-containing 0.65M.
A technical problem more to be solved by this invention is to disclose the application of above-mentioned hepatocyte growth-promoting factor injection in preparation treatment liver disease drug.
Hepatocyte growth-promoting factor injection of the present invention can promote hepatocyte DNA synthetic, makes liver cell regeneration, can be used for chronic hepatitis, hepatitis gravis, viral hepatitis, the hardened treatment of toxic hepatitis regulating liver-QI.
Its therapeutic dose is hepatitis gravis and liver cirrhosis: intravenous injection, each 4 milliliters (60 μ g), be a course of treatment twice, 30 day every day.Chronic hepatitis: intravenous injection, each 2 milliliters (30 μ g), every day twice, 30-60 days is a course of treatment.
Hepatocyte growth-promoting factor injection of the present invention is to thermo-responsive, and activity below 4 ℃ can be preserved 1 year, and 14 days (25 ℃) active reductions of room temperature, lost activity in 30 minutes by 95 ℃.
Injection is stable in pH 6.0-8.0 scope, does not lose activity; To the trypsin sensitivity, insensitive to SDS.The determination of activity of injection can adopt body inside and outside
3H-TdR mixes method and detects, and compares with matched group, and obvious biological activity is all arranged; Its radiocounting (cpm) is more than 1.7 times of matched group.
Carry out relevant toxicity, medicine generation, drug effect and clinical trial with hepatocyte growth-promoting factor injection of the present invention:
One, toxicological test
1. the local test of hepatocyte growth-promoting factor injection:
Adopt experimental guinea pig, through intramuscular injection, breathe steady, palmic rate no abnormal change in normal range, do not have irritated reaction, the part is also not rubescent, swollen, thermal phenomenon, no heat source response.
2. the acute and long term toxicity test of hepatocyte growth-promoting factor injection:
Healthy mice is selected in acute toxicity test for use, and two kinds of administrations of muscle and vein are done an intramuscular injection and once quiet envenoemation test respectively, muscle and intravenous administration dosage reach maximum permissible dosage promptly during 750 μ g/kg body weight, all do not have animal dead, and animal behavior is normal, be quick on the draw LD to external world
50Can't calculate.Long term toxicity test is divided into high, medium and low dosage group and carries out animal experiment, and no animal dead is not also found the abnormal change of nervous system, cardiovascular system, respiratory system etc.
3. the special toxicity test of hepatocyte growth-promoting factor injection:
In order to observe hepatocyte growth-promoting factors whether carcinogenesis, distortion, utilize sister chromosomes exchange (SCE), test such as chromosomal aberration and cell transformation, detect hepatocyte growth-promoting factor injection whether aberration inducing and carcinogenesis from chromosome and cellular level respectively, if three dosage groups and matched group, establish parallel pipe simultaneously, the chromosomal variation of observation of cell, no matter observe and interval, two centromeres, polycentromere from the SCE cell number, big or small ring and interchromosomal exchange are with the relatively more equal no difference of science of statistics (P＞0.05) of matched group.
The carcinogenesis test, adopt the 3T3 cell culture, on soft agar, cultivate and observe, three dosage groups of hepatocyte growth-promoting factor injection are all acellular, and the clone forms, the positive controls cell clone form the positive, blank group feminine gender illustrates not carcinogenesis of hepatocyte growth-promoting factor injection, also unlikely prominent, distortion.
4. the reproductive toxicity test of hepatocyte growth-promoting factor injection:
About optional ripe healthy mice, body weight 20 grams, divide administration and matched group, treatment component high and low dose group, respectively in prefecundation, trimester of pregnancy and medication lactation period, experimental result shows, fetus form, outward appearance, normal, the no monster discovery of allelotaxis, new born animal is all right behind the spontaneous labor, with the equal zero difference of matched group.
Above-mentioned test shows injection safety non-toxic of the present invention.
Two, the pharmacokinetics of hepatocyte growth-promoting factor injection test:
Will
125The hepatocyte growth-promoting factor injection of I labelling, the injection intravenous rabbit, injection back 1,2,4,6,12,24,33,48,72 hour difference vein haemospasia is surveyed the high, medium and low dosage group of radioactivity concentration in the blood, the result injects and reached maximum concentration in the blood in back 4 hours, substantially disappear after 33 hours, half-life is 10 hours, and it is the highest to distribute with liver, mainly passes through renal excretion.
Observe by 8 routine normal person's pharmacokineticss, drug dose 120 μ g/8ml/ time, 5 ', 10 ', 30 ', 45 ', 1 °, 2 °, 3 °, 4 °, 6 °, 8 °, 12 °, 24 ° cryopreservation behind totally 13 this separation of deutero-albumose serum before the medication, after the medication, detect with the biological activity assay method, liver function, electrocardiogram, renal function did not all have change before and after experimental result was tested the experimenter.Beginning to occur high activity in the blood is 0.84 hour, and the half-life is 6.96 hours.
Three, the test of pesticide effectiveness
A. promote hepatocyte DNA anabolic effect
(1) material and method
Animal: healthy pure lines SD rat, body weight 200-250g.
The HepG2 cell.
Reagent:
3H-TdR (0.5mci/ml), other common agents of 1640 culture medium and cell culture.Hepatocyte growth-promoting factor injection, protein concentration 15 μ g/ml.
(2) method:
1. intracorporal method
Select the healthy SD rat, about body weight 200g.If five dosage groups, administration 2.5 μ g/kg body weight/day, 1.25 μ g/kg body weight/day, 0.63 μ g/kg body weight/day, 0.32 μ g/kg body weight/day, 0.16 μ g/kg body weight/day respectively.And establish a matched group, use the physiologic saline for substitute hepatocyte growth-promoting factor injection.Each dosage group and matched group distribute 5 of rat.At first rat is used etherization, surgical method excised 1/3rd of its liver, gives normal diet drinking-water, presses the such scheme intraperitoneal administration once after six hours, after venter posterior injection in 18 hours
3H-TdR50 μ Ci, put to death after three hours and get hepatic tissue 1 gram, after adding the abundant homogenate of 0.1mol/L NaCl-sodium citrate, 3000 rev/mins centrifugal 20 minutes, remove supernatant, precipitation adds 1mol/L NaCl homogenate, after 4 ℃ of standing over night, 3000 rev/mins centrifugal 10 minutes, get supernatant, add dehydrated alcohol to going out to see precipitation, low-speed centrifugal 10 minutes, collecting precipitation adds 2ml1mol/L NaCl, stir accelerate dissolution rapidly, add equal-volume chloroform-isoamyl alcohol (24: 1) thermal agitation 15 minutes, 3000 rev/mins centrifugal 10 minutes, get the upper strata and add 2 times of dehydrated alcohol, the centrifuging and taking precipitation adds the 2ml dissolved in distilled water, gets 1ml and drops on the glass fibre membrane, add the scintillation solution radiocounting after drying, trap is surveyed in 1ml dilution back at 260nm wavelength place in addition.Be equivalent to the concentration that 0.01mg/ml DNA is converted into DNA by 0.200, calculate and respectively organize 1 minute radiocounting value cpm/mgDNA.
2. in vitro method:
With adding the hepatocyte growth-promoting factor injection of three kinds of various dose in the HepG2 culture fluid of In vitro culture, make the hepatocyte growth-promoting factors final concentration be respectively 16.6 μ g/ml, 3.3 μ g/ml and 0.66 μ g/ml.Other establishes the blank group, uses the physiologic saline for substitute hepatocyte growth-promoting factors.Each dosage group and blank group are respectively established four parallel holes again.Cultivate in 37 ℃, 5% CO2 gas incubator that every hole adds after 24 hours
3H-TdR5 μ Ci, continue to cultivate 6 hours, culture fluid is removed in suction, uses pancreatin---and EDTA makes cell take off wall, centrifugal collecting cell, cell is drawn on the glass fibre membrane, on film, use the distilled water broken cell, fix with 5% trichloroacetic acid Denatured protein, 95% ethanol then, put into liquid after diaphragm is dried and dodge cup in 37 ℃ of baking boxs, add scintillation solution (POP-POPOP) 5ml, with liquid glimmer instrument survey one minute put envelope property count value (cpm value).
(3) result:
1. intracorporal method measurement result
Table 1 intracorporal method is measured the active result of hepatocyte growth factor injection
Annotate: X ± SD (* 10
4) represent the mean+SD of each dosage group parallel pipe cpm/mgDNA counting.
Quiet notes ED
50It is 0.60 μ g/kg body weight/day.
The effective dosage ranges of quiet notes is more than the 0.32 μ g/kg body weight/day.
2. cell in vitro culture method measurement result.
Table 2 in vitro method is measured (radiocounting) result
Annotate: X ± SD represents respectively to organize the means standard deviation of parallel hole cpm counting.
(4) conclusion
Learn that through the intracorporal method determination of activity hepatocyte growth-promoting factors is annotated sealing liquid the synthetic effect of obvious promotion hepatocyte DNA,
3Mixing of H-TdR obviously is better than matched group.By studies show that the various dose hepatocyte growth-promoting factor injection is active, the effective dose of quiet notes is more than the 0.32 μ g/kg body weight; The median effective dose of quiet notes is 0.60 μ g/kg body weight; The drug effect of quiet notes increases with dosage and raises, but reaches the state of a kind of similar " stablizing " when reaching 1.25 μ g/kg body weight/day.Viable count was compared also significant difference with radiocounting during cell in vitro was cultivated with matched group.Radiocounting cpm value has been represented in the hepatocyte
3The incorporation of H-TdR, thereby also reflected the aggregate velocity of cell DNA.No matter this shows that hepatocyte growth-promoting factors in vivo or external the synthetic effect of obvious promotion hepatocyte DNA arranged all.Think that hepatocyte growth-promoting factors is the synthetic medicine of cell cultured supernatant DNA of a kind of active height, strong drug action.
Liver injury animals test due to B.D-galactosamine or the carbon tetrachloride
Experiment is with being sheerly each 50 of healthy big white mice, and rat body weight restrains at 132-214, mice body weight 50-100 gram, random packet: 10 of (1) hepatic injury groups; (2) 30 (hepatocyte growth-promoting factors groups: 2.5 μ g/kg, 1.25 μ g/kg, 0.63 μ g/kg of treatment group; Each 10); (3) the normal saline group is 10.The hepatic injury group causes hepatic injury by 500 milligrams/kg body weight lumbar injection or carbon tetrachloride by 0.5ml/100 gram body weight with D-galactosamine, and abdominal cavity or intravenous injection 1ml normal saline were put to death after 72 hours and got hepatic tissue and carry out pathologic finding every day.The treatment treated animal such as before cause hepatic injury, the hepatocyte growth-promoting factors group is pressed 2.5 μ g/kg, 1.25 μ g/kg, 0.63 μ g/kg body weight abdominal cavity and intravenous injection totally three days (once a day) respectively with hepatocyte growth-promoting factor injection every day, and the treatment treated animal is all got liver execution in 72 hours and carried out pathologic finding.The rat of normal saline group is caused hepatic injury without D-galactosamine, and white mice is caused hepatic injury without carbon tetrachloride, only uses 5 milliliters of abdominal cavities of physiological saline solution and intravenous injection once every day, and totally three times, execution in 72 hours is got liver and sent pathologic finding (table 3).
Illustrate: in the table+, ++, +++represent that respectively liver histological is observed, be center, the visual field with central vein and portal vein under the light microscopic, hepatic necrosis, degeneration, infiltration and cloudy swelling degree be light, in, weight; The virus-free change of 0 expression.Numeral produces the number of animals of each pathological change degree in the bracket.
The hepatocyte growth-promoting factor injection of table 3 various dose is to the observation of curative effect of hepatic injury due to different animals, different approaches, the different pharmaceutical
Brief summary:
Improve significantly hepatic necrosis, degeneration, inflammation of hepatocyte growth-promoting factor injection treatment group invaded profit, cloudy swelling effect, and pathological changes is recovered near normal.Normal saline injection group, there were significant differences with the pathological changes of pathologic group, and card has shown that hepatocyte growth-promoting factors has the ability of regenerated liver cell, has proved theoretically that hepatocyte growth-promoting factors has to impel the synthetic of DNA and support metabolism, immunoregulatory effect.
Four, clinical trial
(1) administrated method, dosage and the course of treatment
Divide hepatitis gravis group and severe chronic hepatitis group, two groups all on the basic Comprehensive Treatment basis of routine, add and use hepatocyte growth-promoting factor injection.
Dosage is during use; 120 μ g add 10% glucose 100-250ml iv drip, and once a day, in 4 weeks of the course of treatment, the responder can extend to 6-8 week.
Conventional basic Comprehensive Treatment comprises: quiet glucose injection, Yinzhihuang Injection, vitamin, fat milk, branched-chain amino acid, an amount of fresh blood, fresh plasma, albumin and hemorrhage etc., but during treating, the hepatitis gravis group can not be used medicines such as 17-hydroxy-11-dehydrocorticosterone, Zadaxin, prostaglandin, tire liver injection, tire liver and other newborn hepars; Severe chronic hepatitis group can not be used medicines such as 17-hydroxy-11-dehydrocorticosterone, antiviral drugs, Zadaxin.
(2) observed content
1. symptom and sign (comprise weak, appetite, feel sick, vomiting, abdominal distention, mind, urine amount, hemorrhage situation, ascites volume, jaundice, liver dullness circle), each observed and recorded once before treatment, when per 2 weeks of treatment back and treatment end.
2. routine blood test, routine urinalysis, blood urea nitrogen and creatinine, blood ammonia, alpha-fetoprotein (AFP), electrolyte, ascites routine and antibacterial culturing, each observed and recorded is once before treatment, when per 2 weeks of treatment back and treatment end.
3. liver function: serum alanine transaminase (ALT), aspartic transaminase (AST), total bilirubin (TBIL), bilirubin direct (DBIL), thrombinogen mobility (PTA), total protein (TP), albumin (ALB), before treatment, each observed and recorded is once during 2 weeks and during the treatment end in treatment.
4. serum-virus mark: measure anti--HAVIgM, HBsAg, HbeAg, anti--HBs, anti--HBe, anti--HBc, HBsAg is or/and HBeAg positive person surveys HBVDNA, anti-HCV positive person measures HCVRNA, HBsAg positive person surveys anti--HDV, HDVAg, in addition, also measure anti--HEV, above index writes down once before treatment.
5. write down the adverse events of medicine in application process.
(3) curative effect determinate standard
Curative effect is divided into produce effects, effective, invalid three standards.
Hepatitis gravis and severe chronic hepatitis curative effect determinate standard:
1. produce effects: clinical symptoms is recovered, and serum bilirubin, ALT are normal again.
2. effective; Level was below 50% before serum bilirubin, ALT were reduced to treatment.
3. invalid: finishing the course of treatment afterwards, clinical and liver function does not reach above-mentioned standard or death.
Produce effects example number and effective routine number merging statistics are total effective routine number.
(4) statistical method
1. the variation X of patient symptom, sign
2Statistical analysis is carried out in check.
2. patient's biochemical indicator, represent with the change rate as the variation of ALT, AST, TBIL, ALB, PTA and AFP:
Distribute because of the change rate is skewness, therefore represent it with median ,+representative is risen, and-representative descends.Carry out statistical analysis with rank test.
(5) result
A. hepatocyte growth-promoting factors (Wei Jia) injection is to the therapeutic effect of severe chronic hepatitis
1. the case fatality rate and the cause of death: after using hepatocyte growth-promoting factors, in the 333 routine severe chronic hepatitis patients, dead 2 examples.Wherein 1 example is died from digestive tract hemorrhage, and another example is died from hepatorenal syndrome.331 examples of surviving.Medication viewing duration case fatality rate is 0.6%.
2. hepatocyte growth factor (Wei Jia) injection is to the curative effect of severe chronic hepatitis patient symptom, sign: table 4
2 weeks after the medication, patient's weak, poor appetite, feel sick, vomiting, abdominal distention, epigastric discomfort, jaundice and hemorrhage improvement rate be respectively 73.0%, 77.5%, 85.2%, 83.8%, 71.3%, 58.3%, 54.7% and 63.1%; Improvement rate was respectively 88.1%, 92.2%, 95.5%, 97.5%, 90.6%, 82.0%, 80.1% and 83.3% when medication to 4 was all; Medication is respectively 88.5%, 94.2%, 95.7%, 100%, 89.2%, 86.4%, 87.1% and 94.4% during 6 weeks.These change to learn by statistics handles, and the difference highly significant has statistical significance, P＜0.01.
3. hepatocyte growth-promoting factors (Wei Jia) injection is to the curative effect of severe chronic hepatitis patient biochemical indicator:
Table 5
N: case load, M: the median of change rate, Min: change rate minima, Max, change rate maximum.The severe chronic hepatitis patient with hepatocyte growth-promoting factors after ALT, AST, TBIL change obviously and descend, the variation of PTA, ALB is risen gradually, AFP also has rising, but ascensional range reduces gradually.These variations are learned by statistics and are handled, and the difference of highly significant are arranged, P＜0.01.
4. hepatocyte growth-promoting factors (Wei Jia) injection is to total clinical effectiveness of severe chronic hepatitis: table 6
With hepatocyte growth-promoting factors (Wei Jia) injection for treating severe chronic hepatitis total effective rate is 88.9%.
B. hepatocyte growth-promoting factors is to the therapeutic effect of hepatitis gravis
1. hepatocyte growth-promoting factors (Wei Jia) injection is to the influence of heavy hepatitis patient symptom, sign:
Table 7
2 weeks after the medication, patient's weak, poor appetite, feel sick, vomiting, abdominal distention, epigastric discomfort, jaundice and hemorrhage improvement rate be respectively 56.9%, 64.3%, 71.2%, 76.4%, 58.1%, 51.4%, 40.9% and 50.7%; Improvement rate was respectively 83.5%, 86.3%, 89.5%, 92.8%, 85.2%, 79.2%, 69.7% and 71.9% when medication to 4 was all; Medication is respectively 86.7%, 93.8%, 95.2%, 92.1%, 88.5%, 85.7%, 77.3% and 87.0% during 6 weeks, and these change to learn by statistics handles, and the difference highly significant has statistical significance, P＜0.01.
2. use of the influence of hepatocyte growth-promoting factors (Wei Jia) injection: table 8 to heavy hepatitis patient biochemical indicator
The hepatitis gravis patient uses ALT, AST behind the hepatocyte growth-promoting factors, TBIL, ALB and PTA all tangible change, and along with the prolongation of treatment time, ALT, AST, TBIL descend gradually, ALB and PTA rise, the change rate is learned processing by statistics, and the difference of highly significant is arranged, P＜0.01.
3. hepatocyte growth-promoting factors (Wei Jia) injection is to the clinical effectiveness of hepatitis gravis:
1. hepatocyte growth-promoting factors is to total clinical effectiveness of hepatitis gravis: table 9
With hepatocyte growth-promoting factors (Wei Jia) injection for treating hepatitis gravis total effective rate is 78.4%.
2. hepatocyte growth-promoting factors is to different patient's the clinical observation results by stages of hepatitis gravis (subacute, chronic): table 10
Subacute with hepatocyte growth-promoting factors treatment, chronic severe hepatitis early stage, the total effective rate in mid-term and late period is respectively 89.9%, 84.8% and 27.5%.
*The total effective rate comparing difference not statistically significant in hepatitis gravis early stage and mid-term, X
2=1.59, P=0.21; The total effective rate and the early and middle portion comparing difference in hepatitis gravis late period are remarkable, the height statistical significance are arranged, X
2=89.08, P＜0.001.
Conclusion:
Adding with hepatocyte growth-promoting factors on basic Comprehensive Treatment is 88.9% to the total effective rate of severe chronic hepatitis, is 78.4% to the total effective rate of hepatitis gravis.It is lower to show as patient's case fatality rate, and the improvement of symptom, sign and biochemical indicator is more obvious.This and hepatocyte growth-promoting factor injection can promote hepatocellular regeneration, reduce tumor necrosis factor (TNF) level, and have certain repair relevant to liver plasma membrane.
Above-mentioned result of the test shows that hepatocyte growth-promoting factor injection of the present invention has the ability of significant regenerated liver cell, impels the synthetic of DNA and supports metabolism, immunoregulatory effect, and be ideal treatment liver disease drug.
The specific embodiment
The preparation of embodiment 1 hepatocyte growth-promoting factor injection
Raw material: healthy piglets, pig 15-20 in age days, body weight 7-7.5kg/ only, in the morning during 6-8 sacrificed by exsanguination get liver, clean-20 ℃ of preservations behind the blood.
1. homogenate:
The piglets liver of learning from else's experience after washing tentatively blends on meat mincer earlier, and the reuse milling treatment of colloid obtains the homogenate of piglets liver.
2. stir:
Homogenate after smashing to pieces places in the extraction pot, and in 1: the ratio of 3W/V adds extracting solution, and low speed under room temperature (60 rev/mins) stirred one hour, with the required composition of abundant extraction.
3. heating:
Mode with water-bath heats to extraction pot, stops heating when feed temperature reaches 95 ℃, and keeps 15-20 minute on this temperature spot.
4. filter and cooling:
After heating is finished, earlier feed liquid is cooled to room temperature, carries out solid-liquid separation with filtering mode then, keep filtrate.
5. purify:
5.1.DEAE-Sepharose dress post and the processing of FF:
Ethanol among the DEAE-Sepharose FF that newly buys is outwelled, added 3 times of PBS with upper volume, shake up, with " natural sedimentation " dress post, install no bubble in the rear pillar, no layering is seen and is resembled.Wash 1 column volume with the sodium chloride solution of 2M then, with 3 column volumes of distillation washing, reuse PBS balance is identical with PBS to the pH value of effluent.
5.2, go up sample:
The filtrate that obtains through solid-liquid separation with 10 liters/time flow velocity give installed and counter-balanced DEAE Sepharose FF post on sample, applied sample amount is 6 times of column volume.
5.3.A liquid eluting:
After last sample finishes, earlier with A liquid eluting, flow velocity 15000ml/ hour, with the trap of UV spectrophotometer measuring eluent, less than the inactive A liquid eluting in 0.5 back at 280nm.
5.4.B liquid eluting:
When the A280nm of A liquid eluent less than 0.5 the time, be changed to B liquid eluting, flow velocity 8000ml/ hour, with the A280nm monitoring, when A280nm begins to rise, collect this part eluent equally, stopped to collect less than 0.5 o'clock to A280nm.
5.5. desalination:
Adopt import ultrafilter membrane ultrafilter desalination, behind desalination, be the semi-finished product hepatocyte growth-promoting factors.
6. semi-finished product calibrating:
6.1. protein content: measure protein content with the Lowry method, adjust its content, make it g/ml into 15-25 μ with water for injection.
6.2.PH value is adjusted:
Measure pH value with pH meter, the adjusting pH value is 7.0-7.8 (6.0-8.0).
6.3. chloride ion inspection:
Measure with " silver nitrate titration method ", require chlorine ion concentration less than 1.0mmol/L.
6.4, endotoxin detects:
Detect half-finished endotoxin with tachypleus amebocyte lysate, make protein content when 15-25 μ g/ml, its endotoxin value should be less than 5.25EU/ml.
7. degerming
Carry out aseptic filtration with 0.2 μ micropore filter.
8. packing:
Ampoule, 2ml/ props up, and protein content is 15 μ g/ml, is stored in below 4 ℃.
Embodiment 2 hepatocyte growth-promoting factors protein content determinations
The preparation of reference substance solution is got in phosphorus pentoxide desiccator drying under reduced pressure to the about 15mg of bovine serum albumin reference substance of constant weight, accurately claims surely, puts in the 100ml measuring bottle, adds water and makes dissolving and be diluted to scale, shakes up, promptly.
The preparation of standard curve: precision is measured reference substance solution 0.0,0.2,0.4,0.6,0.8,1.0ml put respectively in the tool plug test tube, all add water and make into 4.0ml, the accurate respectively forint phenol test solution first 6ml that adds, shake up, placed 10 minutes, the accurate respectively again forint phenol test solution second 1ml that adds, shake up immediately, to 35 ℃ of water-baths, be incubated 35 minutes, put and be chilled to room temperature, with first part is blank, according to spectrophotography (24 pages of two appendix of Chinese Pharmacopoeia nineteen ninety version), measure trap at the wavelength place of 660nm, be ordinate with the trap, concentration is abscissa, the drawing standard curve.
Algoscopy: precision is measured this product 4ml, and the method under the sighting target directrix curve preparation from " the accurate respectively forint phenol test solution first 6ml that adds ", is measured trap in accordance with the law, reads Protein content the need testing solution (μ g) from standard curve, calculates, promptly.
Get the hepatocyte growth-promoting factor injection of 8 lot numbers, get 5 samples, record its protein content for every batch according to said method.The results are shown in Table 11.The protein content of hepatocyte growth-promoting factor injection is 15.0-25.0 μ g/ml.
The protein content of the different lot number hepatocyte growth-promoting factor injections of table 11 (μ g/ml)
The amino acid composition analysis of embodiment 3 hepatocyte growth-promoting factor injections:
Get the hepatocyte growth-promoting factor injection of 10 different lot numbers, every lot number is got 10 samples, through the salt acid treatment, 110 ℃ of heating, the centrifuging and taking supernatant is formed with its aminoacid of o-phthalaldehyde(OPA) column front derivation high-performance liquid chromatogram determination, composes relatively with the high-efficient liquid phase chromatogram of aminoacid standard substance.
Get hepatocyte growth-promoting factor injection 2.5ml and add 12N hydrochloric acid 2.5ml, after 110 ℃ of hydrolysis, get supernatant 1ml and drain, add the distilled water dissolving, add last HPLC gained collection of illustrative plates such as o-phthalaldehyde(OPA), mercaptoethanol and standard relatively, calculate the area at each peak.Its aminoacid composition has 15 kinds (seeing Table 12), and total amino acid content is 135.21 ± 10.23nmol/L.
The aminoacid of table 12 hepatocyte growth-promoting factor injection is formed and concentration determination
The polyacrylamide of embodiment 4 hepatocyte growth-promoting factor injections is doubted gel electrophoresis and molecular weight determination:
Hepatocyte growth-promoting factor injection is shown that through 10% polyacrylamide gel electrophoresis (showing) three protein compositions are arranged in the product, through cell culture TdR
3The H method of mixing detects with β-liquid glimmer instrument learns that a main district band A who wherein runs up front is an activated district band, and all the other two bands are the secondary sections band of non-activity.Through UV scanning detect and the high-efficient liquid phase chromatogram area when calculating master tape and accounting for total protein more than 60% (Folin-phenol method mensuration protein content).
Molecular weight determination: get 1 of this product, after the lyophilizing, adding distil water 20 μ l make dissolving, add hydrolyzed solution (get sodium lauryl sulphate 0.1g, thioglycol 100 μ l, glycerol 1g, the bromophenol blue indicator solution is an amount of), put in the boiling water bath hydrolysis 3 minutes, and are standby.Standard protein (SIGM company, molecular weight 14400-94000), the same test sample of method for hydrolysis.Gel strength is 10%.Measure (61 pages of first 1989 appendix of ministry standard biochemical drug) according to the SDS-polyacrylamide gel electrophoresis, to move rate (R ' m) be abscissa with suitable relatively, the logarithm of known molecular amount standard protein is an ordinate, on semilogarithmic paper, draw, from standard curve, find the molecular weight of test sample.The active ingredient of hepatocyte growth-promoting factor injection is the A band, about 2.1 ten thousand dalton of molecular weight.
The determination of activity of embodiment 5 hepatocyte growth-promoting factor injections
Hepatocyte growth-promoting factors impels hepatocyte DNA synthetic, utilizes this principle to set up interior, the external measuring method for activity of body.(HepG
2Cell culture
3H-TdR mixes method)
Material and method:
1. hepatocyte growth-promoting factor injection (self-control protein content 15 μ g/ml).
2. reagent: sodium citrate, chloroform, isoamyl alcohol, dehydrated alcohol, PPO-POPOP scintillation solution, 1640 culture medium, sodium chloride etc.
3. instrument: 754 ultraviolet spectrophotometers, liquid glimmer instrument, CO2 gas incubator.
4. in the activity of hepatocyte auxin assay method one
3H-TdR mixes method.
(1) zoopery: SD rat body weight 180-230g, in 8:00-10:00 etherization in the morning, excision 1/3 (cutting left back leaf), postoperative six hours be in the intraperitoneal injection hepatocyte growth-promoting factor injection, and postoperative 23 hours injects 50uCi in the abdominal cavity
3H-TdR (Chinese Research Institute of Atomic Energy Sciences), postoperative was put to death Mus in 25 hours and is got liver.The matched group rat is used equal-volume physiologic saline for substitute hepatocyte growth-promoting factor injection lumbar injection, all the other same experimental grouies.
(2) hepatocyte DNA extraction and mensuration:
Hepatic tissue 1 gram, adding 0.1mol/L NaCL-0.05mol/L citric acid receives 4ml and makes homogenate, 3000rpm/ divides centrifugal 20 ', precipitation reuse 0.1mol/L NaCl-0.05mol/L sodium citrate washing secondary, precipitation adds 5ml 5M NaCl and grinds, viscosity increases gradually, placed 24-48 hour for 4 ℃, get supernatant and add 95% ethanol and stop, getting jelly and add the 5ml1mol/LNaCl dissolving, add equal amounts of chloroform-different the eleventh of the twelve Earthly Branches alcohol (24: 1) to gel state occurring, vibrated 15 minutes, 3000rpm/ divides three layers of centrifugal 10 minutes branches, gets supernatant and adds 2 times of volume 95% ethanol and fibrous material occurs, gets this thing and washes secondary with 75% ethanol, add the 2ml dissolved in distilled water, wherein 1ml drips in 49 type glass fibre membranes, and radiocounting (cpm value) is surveyed with the FJ-2107 liquid scintillation in dry back, calculates cpm value/mgDNA at last.
5, in vitro method is measured the hepatocyte growth-promoting factor injection biological activity:
Because intracorporal method is surveyed specific activity than very complicated, and differs greatly between the animal individual, has certain experimental error.Cultivate the survey activity methods with reference to the cell in vitro that LaBreque and Romero etc. recommend, the present invention has set up the bioactive method of external test hepatocyte growth-promoting factors, repeatedly easy, accurate, the good reproducibility of experiment confirm the method.So we are with the conventional active inspection method of the method as the hepatocyte growth-promoting factor injection product.Now method is reported as follows.
HepG
2Cell, transferring to cell concentration is 2.5 * 10
5/ ml/ hole adds the hepatocyte growth-promoting factor injection of doses, 5%CO with every hole, the adherent back of 1640 culture medium culturings
2After the continuous culture 24 hours, add 5uCi
3H-TdR/ every hole, 6 hours with pancreas enzyme-EDTA peptic cell and all being collected on the 49 type glass fibre membranes, with trichloroacetic acid, Ethanol Treatment behind the distilled water broken cell, film is dried, be put in the scintillation solution, count 60 seconds radiocounting (cpm) value, be cpm value/2.5 * 10
5Cells/well.For the person more than 1.7 times of matched group is qualified products.
The result:
1. intracorporal method measurement result (table 13)
Table 13 intracorporal method is measured the active result of hepatocyte growth-promoting factor injection
2. in vitro method measurement result (table 14)
Table 14 in vitro method is measured the active result of hepatocyte growth-promoting factor injection
Claims (6)
1. a hepatocyte growth-promoting factor injection is characterized in that a kind of reactive protein of wherein said hepatocyte growth factor for the acquisition of extraction separation from the piglets liver, is made by following method:
Get the piglets liver and tentatively blend on meat mincer earlier, the reuse milling treatment of colloid obtains the homogenate of piglets liver; In 1: the ratio of 3W/V adds extracting solution, and low speed stirred one hour for 60 rev/mins under room temperature; Be heated in the mode of water-bath and stop heating when feed temperature reaches 95 ℃, and kept 15-20 minute in this temperature; Be cooled to room temperature then, carry out solid-liquid separation, keep filtrate with filtering mode; Filtrate is gone up the absorption of DEAE Sepharose FF post, use the PBS eluant solution of sodium chloride-containing 0.28M and 0.65M respectively; Eluent ultrafilter membrane desalination, 0.2 μ micropore filter aseptic filtration, promptly;
This protein is made up of 15 seed amino acids, and molecular weight is 2.1 ten thousand dalton, accounts for more than 60% of total protein content.
2. hepatocyte growth-promoting factor injection according to claim 1 is characterized in that this injection is every milliliter of liquid drugs injection or freeze-dried powder that contains protein 15-25 μ g, pH 6.0-8.0.
3. hepatocyte growth-promoting factor injection according to claim 1 is characterized in that 15 seed amino acids of wherein said constitutive protein matter are: Asp, Glu, Ser, Thr, Gly, His, Tyr, Arg, Ala, Met, Val, Ile, Leu, Lys, Phe.
4. the preparation method of hepatocyte growth-promoting factor injection according to claim 1 is characterized in that this injection is by obtaining piglets liver homogenate, stirring, heating, filtration, absorption, eluting, desalination, degerming, packing.
5. the preparation method of hepatocyte growth-promoting factor injection according to claim 4 is characterized in that the preparation of this injection comprises the following steps:
1) homogenate:
The piglets liver of learning from else's experience after washing tentatively blends on meat mincer earlier, and the reuse milling treatment of colloid obtains the homogenate of piglets liver;
2) stir:
Homogenate after smashing to pieces places in the extraction pot, and in 1: the ratio of 3W/V adds extracting solution, and low speed stirred one hour for 60 rev/mins under room temperature, with the required composition of abundant extraction;
3) heating:
Mode with water-bath heats to extraction pot, stops heating when feed temperature reaches 95 ℃, and keeps 15-20 minute on this temperature spot;
4) filter:
After heating is finished, earlier feed liquid is cooled to room temperature, carries out solid-liquid separation with filtering mode then, keep filtrate;
5) purify:
(5.1) go up sample:
The filtrate that obtains through solid-liquid separation with 10 liters/time flow velocity on sample to installed and counter-balanced DEAE Sepharose FF post in, applied sample amount is 6 times of column volume;
(5.2) eluting:
Use the PBS eluant solution of sodium chloride-containing 0.28M and 0.65M respectively;
(5.3) desalination:
With ultrafilter membrane ultrafilter desalination;
6) semi-finished product calibrating:
Measure protein content with the Lowry method, adjust its content, make it g/ml into 15-25 μ with water for injection;
Decide pH value with the PH instrumentation, the adjusting pH value is 6.0-8.0;
The control chlorine ion concentration is less than 1.0mmol/L;
Detect half-finished endotoxin with tachypleus amebocyte lysate, make protein content when 15-25 μ g/ml, its endotoxin value should be less than 5.25EU/ml;
7) degerming
Carry out aseptic filtration with 0.2 μ micropore filter;
8) packing promptly gets aqueous injection or lyophilization gets freeze-dried powder.
6. the application of hepatocyte growth-promoting factor injection according to claim 1 in preparation treatment liver disease drug is characterized in that described medicine can be used for chronic hepatitis, hepatitis gravis, viral hepatitis, the hardened treatment of toxic hepatitis regulating liver-QI.
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| CNB031160522A CN100417412C (en) | 2003-03-28 | 2003-03-28 | Liver cell growing promotor injection and its preparation method and application |
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| CN100417412C true CN100417412C (en) | 2008-09-10 |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1327851C (en) * | 2005-10-12 | 2007-07-25 | 郭斌阁 | Preparation process of hepatic cell growth promotion protein and its injection preparation method |
| CN100464735C (en) * | 2006-08-02 | 2009-03-04 | 沈阳斯佳科技发展有限公司 | Technology for preparing liquid drugs injection of liver cell growth promoting factor |
| CN101172116B (en) * | 2006-11-02 | 2010-05-12 | 北京同仁堂股份有限公司 | Composition, preparing method and application of the same |
| CN102675451A (en) * | 2011-07-14 | 2012-09-19 | 长春富春制药有限公司 | Method for preparing hepatocyte growth-promoting factors for injection |
| CN102294013B (en) * | 2011-09-08 | 2014-01-29 | 中国人民解放军第四五八医院 | Hepatocyte growth-promoting factor and application thereof |
| CN102294014B (en) * | 2011-09-08 | 2014-01-29 | 中国人民解放军第四五八医院 | Hepatocyte growth-promoting factor and preparation and application thereof |
| CN104056247B (en) * | 2013-03-20 | 2016-03-02 | 长春海悦药业有限公司 | A kind of pharmaceutical composition and preparation thereof containing hepatocyte growth-promoting factors |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990010651A1 (en) * | 1989-03-10 | 1990-09-20 | Snow Brand Milk Products Co., Ltd. | Glycoprotein of human origin, physiologically active factor comprising the same, and pharmaceutical preparation containing the same as active ingredient |
| CN1096053A (en) * | 1993-12-16 | 1994-12-07 | 重庆医科大学病毒性肝炎研究所 | The preparation method of low molecular weight hepatic cells growth hormone |
-
2003
- 2003-03-28 CN CNB031160522A patent/CN100417412C/en not_active Expired - Lifetime
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990010651A1 (en) * | 1989-03-10 | 1990-09-20 | Snow Brand Milk Products Co., Ltd. | Glycoprotein of human origin, physiologically active factor comprising the same, and pharmaceutical preparation containing the same as active ingredient |
| CN1096053A (en) * | 1993-12-16 | 1994-12-07 | 重庆医科大学病毒性肝炎研究所 | The preparation method of low molecular weight hepatic cells growth hormone |
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