CN100398149C - Application of human sDR5 protein as medicine to treat virus hepatitis B - Google Patents
Application of human sDR5 protein as medicine to treat virus hepatitis B Download PDFInfo
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- CN100398149C CN100398149C CNB2006100446728A CN200610044672A CN100398149C CN 100398149 C CN100398149 C CN 100398149C CN B2006100446728 A CNB2006100446728 A CN B2006100446728A CN 200610044672 A CN200610044672 A CN 200610044672A CN 100398149 C CN100398149 C CN 100398149C
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Abstract
The present invention discloses a new purpose of human sDR5 protein, particularly application of human sDR5 protein used as medicine for treating viral hepatitis B particularly for acute grave viral hepatitis B and protecting a liver and application of human sDR5 protein in preparing medicine for blocking viral hepatitis B particularly for acute grave viral hepatitis B hepatocyte apoptosis, wherein the effective dose of human sDR5 protein is from 2 to 64 mg/kg; the human sDR5 protein reduces the hepatocyte apoptosis and relieves liver cell damages through the effect of blocking or sealing TRAIL to achieve the effects of resisting hepatitis and protecting the liver. The human sDR5 protein of the present invention has the new purpose of reducing the hepatocyte apoptosis of hepatitis B and relieving the liver cell damage. After improvement, the present invention also provides a method for treating acute grave viral hepatitis B with a good application prospect and provides bases for the development of the novel medicine.
Description
(1) technical field
The present invention relates to the proteic purposes of people sDR5 (solubility death receptor 5, soluble DR5 are called for short sDR5), relate in particular to of the application of people sDR5 albumen as the particularly acute serious symptom hepatitis B of hepatitis B medicine.
(2) background technology
Hepatitis B is a kind of worldwide disease that is caused by hepatitis B virus (HBV), particularly common in China, the sickness rate height, and be and continue high epidemic status, it not only directly influences crowd's health, even the life security that also jeopardizes the people, having had a strong impact on people's production, life, working and learning, the direct and indirect economic loss that is caused is difficult to estimate.
In the infectious disease of China's statutory report, the morbidity number of hepatitis B and sickness rate are high always for many years occupies the forefront, and countries in 2005 classify hepatitis B as the infectious disease of four big keypoint controls.On February 13rd, 2006, Ministry of Public Health was issued Eleventh Five-Year Plan whole nation hepatitis B control program, this planning application the popular present situation of hepatitis B in China: 1992-1995 whole nation viral hepatitis seroepidemiological survey show, population of China hepatitis B virus infection rate is 57.6%, the hepatitis B virus carrying rate is 9.75%, calculate that in view of the above the whole nation has 6.9 hundred million people once to infect hepatitis B virus, wherein 1.2 hundred million people carry hepatitis B virus for a long time.Estimate that according to the expert present national chronic viral hepatitis B patient has 2,000 ten thousand people approximately, 350,000 people that have an appointment every year die from the chronic viral hepatitis B relevant disease.
Hepatitis B causes heavy financial burden for patient, family, society, bring the influence that can not be ignored to socio-economic development, it is the major reason that many families drive into poverty by medical crises, back into poverty by medical crises, simultaneously also causing a series of social problems, is China present stage one of the most outstanding public health problem.
The clinical manifestation variation of hepatitis B, the most outstanding with serious symptom type hepatitis and chronic hepatitis.Hepatitis B gravis comprises acute serious symptom hepatitis (acute severe hepatitis), subacute serious symptom hepatitis (subacute severe hepatitis) and chronic serious symptom hepatitis, very easily develops into the gangrenosum acne liver cirrhosis, the prognosis extreme difference, and case fatality rate is up to more than the 60-70%.
Hepatitis B is different with common hepatitis, and the kind of common hepatitis is a lot, comprises drug induced hepatitis or toxic hepatitis that medicine or chemical toxicant cause; The alcoholic hepatitis that excessive drinking causes; The abnormal liver function that other systemic diseases and infectious disease cause; Autoimmune hepatitis.Because common hepatitis clinical manifestation is very similar to hepatitis B, in life and the treatment often and hepatitis B obscure, but their pathogenesis is far different, therefore, both treatment measures are also obviously different.
The morbidity of hepatitis B is a results of interaction between hepatitis B virus and the body.Hepatitis B virus belongs to hepadnavirus, and optionally infected liver cell brings out hepatitis.The amount of hepatitis B virus, virulence and route of entry and morbidity have certain relation.Hepatitis B virus often only causes inapparent infection in a small amount, and a large amount of viruses then can cause serious pathological changes.The lesion degree of hepatitis and type and modulation on immune status also have substantial connection.
The hepatic injury and the pathogenesis of hepatitis B are summarized as follows:
(1) mechanism of hepatocyte injury: enter behind the hepatitis B virus intrusion body and duplicate breeding in the hepatocyte, disengage into blood from hepatocyte with " germination " form then.Then stay the virus antigen composition in surface of hepatocytes, do not cause tangible hepatocyte injury this moment.After virus is gone into blood, stimulate body immune system to produce cellular immunization and humoral immunization.The former is by the direct effect of killer T cell and NK cell etc. and the cytotoxicity of antibody dependent, and the latter is by producing each strain specific antibodies, all can react and is killed virus in the blood.But also can attack simultaneously, hepatocyte is damaged necrose the hepatocyte (containing the virus antigen composition on the film) that is infected by the virus.It is generally acknowledged that the cell-mediated cell immune response of T causes that target cell is killed and wounded and be apoptosis-induced is the principal element that causes hepatocyte injury behind the viral infection.
(2) pathogenesis of hepatitis B: above-mentioned hepatocyte immunologic injury mechanism can cause dissimilar hepatitis B: 1. the T cell function is normal, the infective virus amount is many, the hepatocyte of infected and immunologic injury was many and weigh when virulence was strong, showed as acute severe hepatitis; 2. the T cell function is normal, and virus quantity is less, and virulence is more weak, and acute plain edition hepatitis then takes place; 3. the T cell function is normal, and virus quantity is very few, and virulence is very weak then to show as light-duty or subclinical type hepatitis; 4. T cell function deficiency, immunoreation only can be removed part virus and the infected hepatocyte of damaged portion, and the virus of Qing Chuing can not continue to breed and infection, and the part hepatocyte injury takes place repeatedly, and the result shows as chronic hepatitis; 5. body's immunity defective, the T cell is immune tolerance state, this moment virus with host's symbiosis.Virus continues to duplicate in hepatocyte, and the hepatocyte of infection is not subjected to immunologic injury yet, then shows as asymptomatic virus carrier this moment.
The treatment of hepatitis B comprises that mainly antiviral duplicates [medicine commonly used: interferon (Interferons; IFN), acycloguanosine (Acyclovir; ACV, homemade acyclovir), vidarabine (Ara-A) and vidarabine phosphate (Ara-AMP), polyinosini (PolyI:C)], human body immunity improving function [medicine commonly used: thymosin, interleukin II (IL-2)], protection hepatocyte and promote liver cell regeneration measures such as [medicines commonly used: silybin, potenlin, oleanolic acid tablet].But, after these treatment measures are used, though can suppress hbv replication, but this inhibitory action disappears after the drug withdrawal, make former repressed index return to former level again, its reason is that medicine fails to suppress the covalence closed ring-like DNA of superhelix (ccc DNA) of HBV, so cccDNA becomes the template of transcribing in the virus replication again after the drug withdrawal, makes virus again to duplicate.Some drug effect is slower in the treatment, needs the long period just can see effect.Some medicine also is accompanied by side effect such as fear of cold, heating, headache, systemic pain, weak, leukopenia, thrombocytopenia.Therefore, good, the high specificity of exploitation therapeutic effect, effect hepatitis B medicine fast, few side effects are extremely urgent.
Report is arranged recently: death receptor 5 (Death receptor, DR5) be tumor necrosin relative death inducing ligand (TNF-related apoptosis-inducing ligand, TRAIL) one of specificity, high-affinity receptor, with can effectively activate the intracellular signal transduction approach, inducing cell generation apoptotic response after TRAIL combines.DR5 mainly is distributed in peripheral blood lymphocyte and Skeletal Muscle Cell, combines the back with TRAIL and transmits information by linkers FADD/MOTR1, and depend on the cascade reaction cell death inducing of caspase family.The DR5 of total length contains 411 aminoacid, belongs to I type transmembrane glycoprotein.The solubility death receptor 5 (soluble DR5 sDR5) does not contain the soluble form of striding diaphragm area for DR5, stride for want of that diaphragm area can not be expressed on cell membrane and by secretion to the extracellular.SDR5 has low expression level in normal person's peripheral blood.Because of it has the extracellular fragment complete structure that combines with the TRAIL part, can combine the TRAIL molecule with membranous type death receptor competitiveness, thus the inductive apoptosis reaction of blocking-up TRAIL.
Intravital DR5 of mice and human DR5 height homology (79%).
People sDR5 albumen at first utilized engineered method expression and purification to come out by Song etc. in 2000, and be applied to the arthritic research of autoimmunity, the recombinant adenovirus (TRAIL-Ad) that discovery carries trail dna can alleviate the experimental arthritis of mice, personnel selection sDR5 protein blocking TRAIL then can increase the weight of arthritic symptom [referring to SongK, Chen Y, Goke R, et al.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle progression.JExp Med, 2000; 191 (7): 1095-1104.].In June, 2002, Zhang etc. are with adenovirus vector-mediated trail dna transfection BALB/c mouse, the overexpression of TRAIL in hepatocyte can be induced a large amount of hepatocellular death, can effectively weaken TRAIL inductive hepatocyte death effector [referring to Zhang HG if give adenovirus sDR5 (AdsDR5), Xie J, Xu L, et al.Hepatic DR5 induces apoptosis and limits adenovirus genetherapy product expression in the liver.J Virol, 2002; 76 (11): 5692-5700.].November in the same year, overexpression DR5 in patient's hepatic tissue of the acute hepatic failure that discovery such as Mundt HBV is relevant, set up the nonspecific hepatitis model with adenovirus Ad-LacZ inducing mouse, the up-regulated that found that the inductive hepatitis mice hepatic tissue of adenovirus DR5 is [referring to Mundt B, Kuhnel F, Zender L, et al.Involvement of TRAIL andits receptors in viral hepatitis.FASEB J, 2003; 17 (1): 94-96.].
In all research of above-mentioned report to sDR5, just the situation that sDR5 is participated in autoimmunity arthritis and adenovirus mediated nonspecific hepatitis is studied, though be to utilize in the AdsDR5 gene and the effect of TRAIL in adenovirus mediated mice hepatitis research, but only be only limited to nonspecific hepatitis, the pathogenesis and the therapeutic scheme of the liver specificity hepatitis B of adenovirus mediated nonspecific hepatitis and HBV mediation are all different, and above-mentioned report is not seen the prompting of people sDR5 albumen to the liver specificity hepatitis B effect of HBV mediation.
The generation of acute serious symptom hepatitis B (acute severe hepatitis) and hepatocellular excessive apoptosis are closely related, signal transduction pathway at the apoptosis generation, utilize sDR5 to combine TRAIL with membranous type DR5 competition, make TRAIL can not start the generation of apoptosis, thereby can alleviate the prognosis that the hepatocyte injury degree is improved patient.
Look into newly through authoritative institution retrieval, utilize people sDR5 albumen as the hepatitis B medicine, by blocking-up or sealing TRAIL be used for reducing hepatocellular apoptosis, the research that alleviates hepatocyte injury does not appear in the newspapers at present both at home and abroad as yet.
(3) summary of the invention
The object of the present invention is to provide the proteic new purposes of people sDR5, promptly people sDR5 is as the application of the particularly acute serious symptom hepatitis B of hepatitis B liver protecting therapy medicine.
The application of the people sDR5 albumen that the present invention relates in preparation treatment hepatitis B medicine.
Wherein: described people sDR5 albumen is the application in the acute serious symptom hepatitis B medicine of preparation treatment preferably.
In above-mentioned application: the people sDR5 albumen consumption that alleviates hepatocyte injury is 2-64mg/kg.
Wherein: the people sDR5 albumen consumption that obviously alleviates hepatocyte injury is 4-32mg/kg, and optimum amount is 16mg/kg.
The application of the people sDR5 albumen that the present invention relates in preparation blocking-up hepatitis B hepatocellular apoptosis medicine.
Wherein: described people sDR5 albumen is the application in the acute serious symptom hepatitis B hepatocellular apoptosis medicine of preparation blocking-up preferably.
In above-mentioned application: the people sDR5 albumen consumption of blocking-up hepatocellular apoptosis is 2-64mg/kg.
Wherein: the people sDR5 albumen consumption of obviously blocking hepatocellular apoptosis is 4-32mg/kg, and optimum amount is 16mg/kg.
Above-mentioned people sDR5 albumen is used for reducing hepatocellular apoptosis by blocking-up or sealing TRAIL's, alleviates hepatocyte injury, to reach the antiinflammatory hepatoprotective effect.
The proteic new purposes of people sDR5 of the present invention is by the hepatocellular apoptosis that reduces hepatitis B, alleviates hepatocyte injury, improve prognosis, provide the approach of the tempting treatment acute serious symptom hepatitis B of application prospect again, for the exploitation of newtype drug provides foundation.
In order to understand essence of the present invention and the proteic action effect of people sDR5 of the present invention better,, further set forth it in blocking-up hepatocellular apoptosis, the effect that alleviates the hepatocyte injury aspect below with proteic zoopery of people sDR5 and result.
The proteic preparation of people sDR5:
The conventional method activation contains pGAPZ
αA-sDR5 and empty carrier pGAPZ
αThe Pichi strain of A, and a large amount of amplifications contain the Pichi strain GS115 bacterial strain of sDR5 (referring to Song K, Chen Y, Goke R, et al.Tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmuneinflammation and cell cycle progression.J Exp Med, 2000; 191 (7): 1095-1104), collect culture supernatant, utilize ProBond
TMAffinity column purification of Recombinant sDR5 albumen.Obtain the recombined human sDR5 albumen of purification, SDS-PAGE (seeing accompanying drawing 1), Western Blot (seeing accompanying drawing 2) detect the purity of purified product; The proteic concentration of Folin-phenol colorimetric method for determining purification descendant sDR5 (seeing accompanying drawing 3); With the people sDR5 albumen of mtt assay detection purification TRAIL being induced human cervical carcinoma cell is the blocking effect (seeing accompanying drawing 4) of HeLa apoptosis effect.
But transforming male Pichia sp. GS115 bacterial strain successful expression molecular weight size is the sDR5 albumen of 26 kD, and the sDR5 albumen behind the purification is through identifying, the result shows and obtained highly purified recombined human sDR5 albumen that protein yield reaches 20 μ g/ml; Detect through external activity, the result shows that the people sDR5 albumen of purification can partly block the inductive HeLa apoptosis of TRAIL, and the blocking-up rate is up to 53.6%.After above-mentioned checking, the people sDR5 albumen-80 ℃ preservation of purification, standby.
The preparation of acute hepatitis b mouse model:
Extract test kit purification of Recombinant plasmid pcDNA3-HBV1.1 in a large number (referring to Zhang Qiu, Sun Wensheng, Gao Lifen etc. through qiagen plasmid.TRAIL induces HBV transfection hepatoma carcinoma cell BEL-7402 effect of apoptosis and Mechanism Study.China's microbiology and Journal of Immunology, 2005; 25 (5): 357-362.), adjusting concentration with normal saline is 20～50 μ g/ml.
Laboratory mice is a SPF level BALB/c mouse, and in 4～6 ages in week, male, body weight 18～22g is provided by Shandong University's Experimental Animal Center, and the raising condition is carried out according to SPF level animal standard.
Tail vein high-pressure injection: earlier described mice is placed hot environment, make the tail venectasia; Use etherization before the injection, observe the mice reflection case; In 5 seconds with 1.4～2.0ml recombiant plasmid pcDNA3-HBV1.1 liquid in the tail vein at the uniform velocity is injected into the mice body, mice places 25～28 ℃ of environment to observe.
Success is behind tail vein high-pressure injection recombiant plasmid pcDNA3-HBV1.1; Menses diarrhea with indigested food pyruvic transaminase (ALT) (seeing accompanying drawing 5), hepatitis B surface antigen (HBsAg), e antigen (HBeAg) are measured (seeing accompanying drawing 6), real-time quantitative PCR detects HBV DNA (seeing accompanying drawing 7) and hepatic pathology experiments such as (seeing accompanying drawing 8), the result shows: the mice serum ALT behind the tail vein high-pressure injection pcDNA3-HBV1.1 reached 800u at 8 hours, 24 hours is 350u; HBsAg, HBeAg the 1st day are the highest, and main HBsAg expression can reach 1.5, descends gradually later on, disappears after 7 days; Detect HBV DNA in the serum; Liver tissue slices shows the viral hepatitis pathological change.Above result confirms: chmice acute hepatitis B animal model is set up successfully.
Utilize the method for Celluar and Molecular Biology, carry out following experiment: research people sDR5 albumen is to the influence of hepatitis B mouse liver damage.
People sDR5 albumen is dissolved in the normal saline, with reference to the using dosage of this albumen in mouse experiment arthritis [referring to Song K, Chen Y, Goke R, et al.Tumor necrosis factor-relatedapoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammationand cell cycle progression.J Exp Med, 2000; 191 (7): 1095-1104.], designed the serial gradient of 1-64mg/kg: 1mg/kg among the present invention, 2mg/kg, 4mg/kg, 8mg/kg, 16mg/kg, 32mg/kg, 64mg/kg, respectively with people sDR5 albumen before the preparation of above-mentioned animal model in 24 hours or the model preparation lumbar injection give mice, duplicate injection is once again after 24 hours, and set up the people sDR5 albumen treatment group in advance of hepatitis B model group and various dose, the people sDR5 albumen treatment group simultaneously of various dose, U.S. energy treatment group [positive drug matched group, manufacturer: the Japanese minot MinophagenPharmaceutical Co.Tokyo of pharmaceutical Co. Ltd (former Japanese U.S. can rise drugmaker) that rises, Japan; Lot number: 90902B], the people of the people sDR5 matched group of various dose, various dose is from albumen matched group, empty carrier matched group, normal BALB/c mouse matched group a series of experiments and matched groups such as (normal saline matched groups), in different time points blood sampling and regular execution mice, detect the expression of mice serum ALT, HBV surface antigen, e antigen and antibody, HBV dna content, hepatic tissue HBV cAg, TRAIL and DR5, expression and the liver tissue injury situation of splenic T RAIL respectively.
The result shows: the ALT of model group reached in 8 hours the highest (820 ± 138u), reduce gradually later on, reduce to normal range after 10 days; People sDR5 albumen treatment group in advance: 8 hours is 452 ± 13.8u, and 24 hours is 178 ± 9.2u, and 4 days is 151 ± 7.8u, significantly is lower than model group at above time point people sDR5 protein for treatment group ALT, promptly reduces to normal range after 10 days; U.S. energy treatment group: 8 hours is 389 ± 15.2u, 24 hours is 137 ± 11.6u, 4 days is 142 ± 14.5u, ALT significantly be lower than model group in the U.S. energy of above time point treatment group, promptly reduce to normal range after 10 days, beautiful can the treatment group at above time point ALT a little less than people sDR5 protein for treatment group, but there was no significant difference between two groups, only be administered twice in the experiment, after the administration second time, still can significantly reduce the level of ALT the 4th day the time, but the variation tendency that causes ALT is identical with model group because medicine is decayed in vivo the 7th day, 10 days the time; People sDR5 albumen treatment group simultaneously: measured ALT result and (the obviously decline (345 ± 58u) in 800 ± 129u), 24 hours of model group there was no significant difference in 8 hours; Human albumin matched group variation tendency is identical with model group, and people sDR5 albumen matched group, empty carrier matched group, normal saline matched group and normal mouse do not have significant difference (seeing accompanying drawing 9).Liver tissue slices HE dyeing also shows people sDR5 protein for treatment group, people sDR5 albumen treatment group in advance particularly, can obviously alleviate hepatocellular damage and inflammatory reaction in the oxyhepatitis model: model group shows as hepatic tissue diffusivity lymphocytic infiltration, part has neutrophil infiltration, hepatocyte is the balloon sample and becomes, and the part cell has downright bad performance; People sDR5 albumen treatment group in advance and the treatment of U.S. energy are organized rarely seen part of hepatocytes and are acidophilia's change, do not have obvious hepatic tissue inflammatory and change there was no significant difference between two groups (seeing accompanying drawing 10).But people sDR5 albumen is to the expression and the HBV dna content did not influence of HBV antigen, antibody.By SABC and Flow cytometry, show that the expression of people sDR5 protein for treatment group liver, splenic T RAIL is lower than model group, and liver DR5 be expressed in treatment group and model group there was no significant difference.By TUNEL dyeing, organize hepatocellular apoptosis rate through the treatment in advance of flow cytometer detection demonstration people's sDR5 albumen and significantly be lower than model group (seeing accompanying drawing 11).This experimental selection the people sDR5 albumen of 1-64mg/kg (1mg/kg, 2mg/kg, 4mg/kg, 8mg/kg, 16mg/kg, 32mg/kg, 64mg/kg) as test dose, experimental result explanation people sDR5 albumen is through twice lumbar injection, when the experimental amount of 2-64mg/kg, especially during the amount of 4-64mg/kg, the hepatocellular apoptosis that people sDR5 albumen TRAIL capable of blocking causes, thus hepar damnification in the hepatitis B alleviated.Experimental result shows, people sDR5 albumen can significantly alleviate the hepar damnification in the hepatitis B when 16-64mg/kg, but there was no significant difference between each group, the proteic dosage of the people sDR5 state that reached capacity when 16-64mg/kg is described, increase the proteic dosage of people sDR5 again and also can not increase its effect, may cause side effect owing to dosage increases on the contrary, according to above experimental result, the proteic dose concentration of people sDR5 of 4-32mg/kg is comparatively suitable as the preferred effective dose of treatment hepatitis B.
By above-mentioned experiment and result thereof, can draw as drawing a conclusion:
People sDR5 albumen can obviously alleviate the liver inflammation of acute hepatitis b mice, is in particular in that ALT reduces, the no obvious inflammation performance of hepatic pathology section, and shift to an earlier date injection for curing group better effects if.Find that by Mechanism Study people sDR5 protein for treatment group hepatocellular apoptosis rate is starkly lower than model group, and SABC and flow cytometry result show that the expression of TRAIL in treatment group hepatic tissue and the spleen reduces than model group.U.S. energy is as the best hepatic of the hepatitis B of generally acknowledging at present, and the proteic therapeutic effect of people sDR5 can be compared there was no significant difference with U.S..This result shows in the hepatocyte injury process that the inductive hepatocellular apoptosis of TRAIL causes after HBV infects and plays an important role; people sDR5 albumen can shield to hepatocyte by the effect of sealing or blocking-up TRAIL; and it is without any side effects in the therapeutic dose scope; therefore; people sDR5 albumen will have very big using value in the medicine of the particularly acute serious symptom hepatitis B of preparation treatment hepatitis B, have extremely tempting development prospect.
(4) description of drawings
Each gradient product S DS-PAGE electrophoretogram of Fig. 1
Wherein: 1 is low molecular weight protein (LMWP) Marker, and 2 is the 350mmol/L eluent, and 3 is the 200mmol/L eluent, and 4 is the 100mmol/L eluent, and 5 is the 50mmol/L eluent
Each gradient Western Blot colour developing result of Fig. 2
Wherein: 1 is the 50mmol/L eluent, and 2 is the 100mmol/L eluent, and 3 is the 200mmol/L eluent, and 4 is the 350mmol/L eluent
Fig. 3 Folin-phenol colorimetry drawing standard protein concentration curve
The people sDR5 albumen of Fig. 4 purification is to the blocking effect of TRAIL induced Hcla cell apoptosis
Fig. 5 serum glutamic pyruvic transminase (ALT) measurement result
Fig. 6 ELISA detects HBV antigen (HBsAg, HBeAg)
Fig. 7 real-time quantitative PCR detects the HBV dna content
Pathological change is observed in Fig. 8 liver tissue slices HE dyeing
As seen hepatic tissue diffusivity lymphocytic infiltration partly has neutrophil infiltration; Hepatocyte is the balloon sample and becomes, and the part cell has downright bad performance
Fig. 9 respectively organizes mice ALT measurement result
Wherein: model is a model group, and albumin is a 16mg/kg albumin matched group, and sDR5 is a 16mg/kg people sDR5 albumen treatment group in advance, and U.S. can be the U.S. energy of 10mg/kg treatment group
Figure 10 respectively organizes mouse liver pathological section HE dyeing
Model group shows as hepatic tissue diffusivity lymphocytic infiltration, and part has neutrophil infiltration, hepatocyte to be the change of balloon sample, and the part cell has downright bad performance; The people sDR5 albumen treatment in advance of 16mg/kg is organized rarely seen part of hepatocytes and is acidophilia's change, does not have obvious hepatic tissue inflammatory and changes; 10mg/kg is beautiful not to have obvious inflammation change by treatment group hepatic tissue.
Wherein: A1 is the empty carrier matched group, and A2 is a model group, and A3 is a 16mg/kg people sDR5 albumen treatment group in advance, and A4 is the U.S. energy of a 10mg/kg treatment group
Figure 11 TUNEL dyeing detects the level of apoptosis of respectively organizing mouse liver cell
Wherein: A1, A2 are model group, and B1, B2 are 16mg/kg people sDR5 albumen treatment group in advance, and C1, C2 are normal BALB/c mouse matched group
(5) specific embodiment
Embodiment 1: the proteic purification of people sDR5
The conventional method activation contains pGAPZ
αA-sDR5 and empty carrier pGAPZ
αThe Pichi strain of A, and a large amount of amplifications contain the Pichi strain GS115 bacterial strain of sDR5 (referring to Song K, Chen Y, Goke R, et al.Tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmuneinflammation and cell cycle progression.J Exp Med, 2000; 191 (7): 1095-1104), get pGAPZ respectively
αA-sDR5 and empty carrier pGAPZ
αA transforms male yeast, cultivates 24 hours, gets supernatant 3L, through Beckman centrifuge low-temperature and high-speed centrifugal (4 ℃, 8000rpm, 15min) after, supernatant is through 20 times of hollow fiber membrane ultrafiltration device (the ultrafilter membrane amount of damming is 3kDa) ultrafiltration and concentration.
ProBond
TMAffinity column is through 20mmol/L Na
3PO
4After 500mmol/L NaCl (pH7.2～7.6) the binding buffer liquid balance, to cross post with 7 times of blended above-mentioned samples of volume binding buffer liquid, cleaning buffer solution (pH6.0) is washed post and is removed unconjugated foreign protein composition, at last respectively with 50mmol/L, 100mmol/L, 200mmol/L, 350mmol/L contains the elution buffer gradient elution of imidazoles, collects each eluted product.
The proteic concentration of purification descendant sDR5 is 4mg/ml, and 3L yeast culture supernatant can obtain destination protein 60mg altogether, and this yeast protein yield is about 20 μ g/ml.
Embodiment 2: preparation hepatitis B mouse model, and the method for employing Celluar and Molecular Biology, carry out following experiment: research people sDR5 albumen is to the influence of hepatitis B mouse liver damage.
(1) sets up normal BALB/c mouse matched group (normal saline matched group) respectively, the human albumin matched group of seven dosage, the people sDR5 albumen matched group of seven dosage, the empty carrier matched group, the hepatitis B model group, the treatment group of U.S. energy was used in modelling in preceding 24 hours, the people sDR5 protein for treatment group of seven dosage was used in modelling in preceding 24 hours, use the people sDR5 protein for treatment group of seven dosage in the time of modelling, with reference to the using dosage of sDR5 in mouse experiment arthritis [referring to Song K, Chen Y, Goke R, et al.Tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmuneinflammation and cell cycle progression.J Exp Med, 2000; 191 (7): 1095-1104.], experimental design the serial gradient of 1-64mg/kg: 1mg/kg, 2mg/kg, 4mg/kg, 8mg/kg, 16mg/kg, 32mg/kg, 64mg/kg.
Every group of number of mice is 15, tests equal triplicate, and concrete experimental technique and result see following narration for details.
Normal BALB/c mouse matched group is tail vein high-pressure injection normal saline 1.4～2.0ml; The human albumin matched group is lumbar injection human albumin 1-64mg/kg (1mg/kg, 2mg/kg, 4mg/kg, 8mg/kg, 16mg/kg, 32mg/kg, 64mg/kg), repeats once after 24 hours; People sDR5 albumen matched group is lumbar injection people sDR5 albumen 1-64mg/kg (1mg/kg, 2mg/kg, 4mg/kg, 8mg/kg, 16mg/kg, 32mg/kg, 64mg/kg), repeats once after 24 hours; The empty carrier matched group is empty carrier plasmid DNA pcDNA3 1.4～2.0ml of tail vein high-pressure injection 20～50 μ g/ml; The hepatitis B model group is recombinant plasmid dna pcDNA3-HBV1.1 1.4～2.0ml of tail vein high-pressure injection 20～50 μ g/ml; Preceding 24 hours of modelling use beautiful can the treatment group be that at first lumbar injection is beautiful can 10mg/kg[manufacturer: the Japanese minot Minophagen Pharmaceutical Co.Tokyo of pharmaceutical Co. Ltd (former Japanese U.S. can rise drugmaker) that rises, Japan; Lot number: 90902B], U.S. of duplicate injection corresponding dosage can be also simultaneously through tail vein high-pressure injection recombinant plasmid dna pcDNA3-HBV1.1 after 24 hours; Should choose in the preceding 24 hours treatment group of seven dosage of sDR5 albumen of modelling is lumbar injection people sDR5 albumen 1-64mg/kg (1mg/kg, 2mg/kg, 4mg/kg, 8mg/kg, 16mg/kg, 32mg/kg, 64mg/kg) at first respectively, the people sDR5 albumen of duplicate injection corresponding dosage and simultaneously through tail vein high-pressure injection recombinant plasmid dna pcDNA3-HBV1.1 after 24 hours; The treatment group of seven dosage of sDR5 albumen of should choosing in the time of modelling is lumbar injection people sDR5 albumen 1-64mg/kg (1mg/kg, 2mg/kg, 4mg/kg, 8mg/kg, 16mg/kg, 32mg/kg, 64mg/kg) at first respectively, and simultaneously through tail vein high-pressure injection recombinant plasmid dna pcDNA3-HBV1.1, a people sDR5 of duplicate injection albumen again after 24 hours.
In the present invention, the contrast experiment selects for use U.S. can be as the proteic positive control medicine of people sDR5.U.S. can be the trade name (English name: StrongerNeo-MinophagenC of compound glycyrrhizin; SNMC); the beautiful complex that can form by glycyrrhizin one amine, cysteine and glycine etc.; have antiinflammatory, regulate immune, as to protect liver plasma membrane, the effect of steroid sample and inhibition virus multiplication, inactivation of viruses effect; obvious transaminase lowering; the protection hepatocyte there is no obvious toxic-side effects.U.S. can be at first by Japan in 1948 the exploitation be the vein and the oral formulations of main component with glycyrrhizic acid (glycyrrhizin).Because SNMC has antiallergic action, antiinflammatory action, mainly is used in the department of dermatologry field at that time and treats multiple allergic skin illness.1958, Japanese doctor Yamamoto husband was used for the treatment of chronic hepatitis patient with SNMC trial property ground, and liver function index BSP (bromine sulfonephthalein) value has obtained obvious improvement the (when fashion does not have transaminase determination) as a result.After this, medication comes into one's own SNMC as hepatopathy in Japan, and people have done a large amount of bases and clinical research to it.1977, the grand professor of Japanese hepatopathy expert's Suzuki adopted strict randomized controlled method to verify the curative effect of SNMC to chronic hepatitis, confirmed that SNMC can effectively reduce ALT, AST and γ-GTP.In 1986, the man of virtue and ability of nation of Hino verified the definite curative effect of SNMC treatment chronic hepatitis again from hepatopathy reason histology subsequently.2002, Bears Tian Boguang reported the late result result of prolonged application SNMC to the treatment of 500 many cases chronic hepatitis Cs again, find medication after 15 years SNMC can make the hepatocarcinoma incidence rate of chronic hepatitis C reduce more than 50%.Because the curative effect of SNMC obtains the checking support of liver pathology, and the late result research that reaches 22 years arranged, we can say that SNMC is a kind of that curative effect is the most sure in all hepatoprotectives, research is the most deep, at home and abroad used for many years clinically, obtained good efficacy as the active drug of chronic hepatitis.At present, U.S. can be clinically just as the best hepatoprotective extensive use of hepatitis B.Beautiful can the maximum clinical consumption be 2mg/kg, the serial gradient of 1-20mg/kg has been set up in intravenous injection in preliminary experiment of the present invention, overall merit finds that the best use of dosage is 10mg/kg, through intraperitoneal injection.
(2) get blood through the angular vein hole:
Respectively at the 1st, 4,7,10 day of modelling mice being used etherization, use the 1ml syringe to get blood 0.2～0.5ml by the angular vein hole, conventional centrifugal, separation of serum ,-20 ℃ of preservations are used for the detection of every index.
(3) put to death mice:
Respectively at the 1st, 4,7,10 day of modelling mice plucked that eyeball is got blood, the cervical vertebra dislocation is put to death.Get above-mentioned liver, spleen, kidney, heart, lungs and the thymus of respectively organizing mice respectively ,-80 ℃ of preservations, standby.
(4) serum glutamic pyruvic transminase (ALT) is measured:
Utilize alanine aminotransferase test kit (ALT/GPT, reitman-frankel method, Weifang 3V biological engineering company limited) to set up standard curve and carry out sample determination.The sample OD value curve ranges that is above standard then will be measured behind the diluted sample again.
The ALT testing result shows: the ALT of model group reached in 8 hours the highest (820 ± 138u), reduce gradually later on, reduce to normal range after 10 days; Preceding 24 hours proteic treatment groups of the sDR5 that should choose of modelling, people sDR5 albumen does not have obvious influence to ALT when 1mg/kg, people sDR5 albumen ALT level when 2mg/kg slightly reduces, but compare there was no significant difference with model group, people sDR5 albumen can significantly reduce the level of ALT when 4-64mg/kg, and along with people sDR5 albumen test dose increases, the ALT level has reduction trend, but the horizontal there was no significant difference of ALT between each group when the amount of 16-64mg/kg, the prompter sDR5 albumen state that when the amount of 16-64mg/kg, reached capacity, increase the proteic effect dosage of people sDR5 again and also can not improve the effect that it reduces ALT, therefore, should be the best use of dosage during near the amount of people sDR5 albumen 16mg/kg, ALT 8 hours was 452 ± 13.8u, and 24 hours is 178 ± 9.2u, and 4 days is 151 ± 7.8u, significantly be lower than model group at above time point people sDR5 protein for treatment group ALT, promptly reduce to normal range after 10 days; U.S. energy treatment group: 8 hours is 389 ± 15.2u, 24 hours is 137 ± 11.6u, 4 days is 142 ± 14.5u, ALT significantly be lower than model group in the U.S. energy of above time point treatment group, promptly reduce to normal range after 10 days, beautiful can the treatment group at above time point ALT a little less than people sDR5 protein for treatment group, but there was no significant difference between two groups, only be administered twice in the experiment, after the administration second time, still can significantly reduce the level of ALT the 4th day the time, but the variation tendency that causes ALT is identical with model group because medicine is decayed in vivo the 7th day, 10 days the time; The proteic treatment group of sDR5 of should choosing in the time of modelling: measured ALT result in 8 hours and compare there was no significant difference with model group and (obviously descended (345 ± 58u) in 800 ± 129u), 24 hours; Human albumin matched group variation tendency is identical with model group, illustrates that the proteic effect of people sDR5 is specific; Empty carrier matched group, normal saline matched group and normal mouse do not have significant difference.Each is organized mice ALT measurement result and sees Fig. 9.
(5) pathological change is observed in liver HE dyeing:
Each is organized the mouse liver tissue and carries out frozen section, conventional H E dyeing, and microscopically is observed: the visible hepatic tissue diffusivity of model group lymphocytic infiltration, part have neutrophil infiltration, hepatocyte to be the change of balloon sample, and the part cell has downright bad performance; People sDR5 albumen treatment group in advance when 1mg/kg to liver inflammatory reaction do not have obvious influence, people sDR5 albumen is when 2mg/kg, liver inflammatory reaction and hepatocyte injury alleviate to some extent, but compare there was no significant difference with model group, people sDR5 albumen when 4-64mg/kg in the hepatic tissue inflammatory cell infiltration and hepatocyte injury degree significantly alleviate, people sDR5 albumen reaches the best use of effect when the therapeutic dose of 16mg/kg, show as hepatic tissue and do not have obvious inflammatory change, rarely seen part of hepatocytes is the acidophilia and becomes, hepatic tissue pathology changed there was no significant difference between people sDR5 albumen was respectively organized when 16-64mg/kg, this result is the prompter sDR5 albumen state that reaches capacity when the effect dosage of 16-64mg/kg also, use the above people sDR5 albumen of 64mg/kg can not reach better antiinflammatory hepatoprotective effect, may cause on the contrary liver or systemic toxic side effect, but in this tests used test dose scope, not see significant side effects; U.S. energy treatment group is not seen tangible inflammatory reaction, compares there was no significant difference with people sDR5 albumen treatment group in advance; And people sDR5 albumen treatment group is simultaneously compared no significant difference with model group.Each is organized the mice pathological section and the results are shown in Figure 10.
(6) ELISA detects HBV antigen, antibody expression level:
Utilize the ELISA test kit of HBsAg, HBeAg and corresponding antibodies to detect,, survey the OD value in the 450nm place with microplate reader in strict accordance with the description operation.
ELISA result shows: model group mice serum HBsAg, HBeAg the 1st day are the highest, and HBsAg can reach 1.5; Descend gradually later on, disappear after 7 days, the expression of HBsAb is measured positive after 7 days.The expression of model group and people sDR5 protein for treatment group mice serum HBV antigen, antibody does not have significant difference.
(7) the liver SABC detects HBV cAg, TRAIL expression:
The above-mentioned mouse liver organizational routine of respectively organizing carries out frozen section, utilizes two anti-dyeing of one anti-, the horseradish peroxidase-labeled of HBcAg and TRAIL respectively, and substrate is OPD, and positive staining is pale brown color.
Showed by immune group result: 1-4 days about (8 ± 1) % of HBcAg positive cells of model group, fade away afterwards; The cell of model group TRAIL stained positive is obviously more than matched group and people sDR5 protein for treatment group.
(8) real-time quantitative PCR detects the HBV dna content:
Model group and experimental mice serum specimen utilize hepatitis B virus (HBV) PCR kit for fluorescence quantitative (Shenyou Biotechnology LLC, Shanghai) to detect HBV DNA.
At first specimen cracking (directly cracking process): after getting 20 μ l lysis buffers adding 0.5ml centrifuge tube, add 20 μ l serum specimens, negative control, critical contrast, positive control more respectively, mixing.Boiling water bath 10 minutes, centrifugal 10 minutes of 14000rpm gets supernatant 2 μ l and does the PCR reaction.
Be set as follows program:
Collect fluorescence signal in the time of 55 ℃, detect fluorescence (Bio Rad Laboratories) with BIO-RAD iCycler.Standard curve is all done in each test, and curve correlation coefficient is controlled at more than 0.99.
The real-time quantitative PCR testing result shows: the model group mice in the copy number of the 1st day HBV DNA up to 2.67 * 10
5, obviously dropped to 3.85 * 10 on the 4th day
3, be significantly higher than HBV transgenic mice levels of replication (P＜0.05).The levels of replication of model group and treatment group HBV DNA does not have significant difference.
(9) expression of Flow cytometry splenocyte TRAIL and hepatocyte DR5:
Above-mentionedly respectively organize mouse liver and spleen places the RPMI-1640 culture fluid that contains 10% calf serum respectively, utilize 200 order copper mesh to grind, and filter, counting 1 * 10 with 400 order copper mesh
6Individual cell washes twice with PBS, adopts the indirect labelling method to detect the expression of surface of hepatocytes DR5 and the expression of spleen cell surface TRAIL respectively.
Flow cytometry result shows: each organizes the proteic expression there was no significant difference of mouse liver histiocyte DR5, and the model group (12.1%) that is expressed in of splenocyte surface TRAIL is respectively organized (1.8-5.5%) apparently higher than other.
(10) TUNEL fluorescence staining in situ detection hepatic tissue cell apoptosis situation, Flow cytometry hepatocellular apoptosis rate:
Utilize the TUNEL labelling kit, respectively each group murine liver tissue specimens paraffin embedding slices and fresh hepatocyte are carried out TUNEL dyeing, fluorescence microscope hepatic tissue cell apoptosis situation, cells were tested by flow cytometry hepatocellular apoptosis rate.
The apoptosis testing result shows: people sDR5 albumen treatment group does not in advance have influence to hepatocellular apoptosis when 1mg/kg, people sDR5 albumen can reduce the hepatocellular apoptosis rate when 2mg/kg, but compare there was no significant difference with model group, people sDR5 albumen can significantly reduce hepatocellular apoptosis when 4-64mg/kg, and along with the proteic dosage of people sDR5 increases, hepatocellular apoptosis alleviates, but hepatocellular apoptosis rate there was no significant difference between people sDR5 albumen is respectively organized when 16-64mg/kg, the people sDR5 albumen state that reaches capacity when 16-64mg/kg is described, increase effect dosage can not strengthen the effect that people sDR5 albumen reduces hepatocellular apoptosis yet again, therefore, the therapeutic dose of 16mg/kg should be the best use of dosage; The hepatocellular apoptosis rate of model group is 15.5 ± 2.8%; 16mg/kg people sDR5 albumen treatment group in advance is 4.5 ± 2.3%; The normal control group is 1.2 ± 0.6%.
TUNEL dyeing detects the Figure 11 that the results are shown in that respectively organizes the mouse liver cell apoptosis.
(11) experimental data statistical procedures:
Above-mentioned experimental data is represented with mean+/-standard error, checks through t: P＜0.05 expression has notable difference.
By above-mentioned experiment and result thereof, can draw as drawing a conclusion:
People sDR5 albumen consumption is when 2-64mg/kg, can alleviate the liver inflammation of acute hepatitis b mice, people sDR5 albumen consumption particularly when the 16mg/kg consumption, can obviously alleviate the liver inflammation of acute hepatitis b mice when 4-64mg/kg.Be in particular in that the ALT level reduces, hepatic pathology section inflammatory reaction and hepatocyte injury alleviate, and hepatocellular apoptosis reduces.And injection for curing group better effects if in advance.The treatment in advance in effective dosage ranges of people sDR5 albumen can significantly improve hepar damnification and the inflammatory reaction of hepatitis B mice, U.S.'s energy there was no significant difference of its therapeutic effect and the best use of dosage, and people sDR5 albumen is not found any toxic and side effects in the therapeutic dose scope.
Find by Mechanism Study; people sDR5 protein for treatment group hepatocellular apoptosis rate is starkly lower than model group; and SABC and flow cytometry result show that the expression of TRAIL in treatment group hepatic tissue and the spleen reduces than model group; this result shows in the hepatocyte injury process that the inductive hepatocellular apoptosis of TRAIL causes after HBV infects and plays an important role; people sDR5 albumen can shield to hepatocyte by the effect of sealing or blocking-up TRAIL, and without any side effects in the therapeutic dose scope.Carry out Comprehensive Assessment according to above result; people sDR5 albumen is when 2-64mg/kg; particularly has tangible hepatoprotective effect during 4-64mg/kg; but hepatoprotective effect there was no significant difference when 16-64mg/kg; the prompter sDR5 albumen state that when the amount of 16-64mg/kg, reached capacity; increasing the proteic effect dosage of people sDR5 can not improve it more yet and protect the liver effect; therefore select the people sDR5 albumen of 4-32mg/kg comparatively economical as effective protection dosage of treatment hepatitis B; wherein, the people sDR5 protein concentration amount of 16mg/kg should be the optimal dose of treatment hepatitis B.By contrasting discovery with the positive control medicine is beautiful, people sDR5 albumen can significantly alleviate hepar damnification and inflammatory reaction in the therapeutic dose scope, and the hepatoprotective effect when 16mg/kg and U.S. of optimal therapeutic dosage can there was no significant differences.Yet, beautiful can still can causing as shock, irritated sample symptom as the hepatic safe, effective, most worthy few in number of generally acknowledging at present, increase dose or long-term use continuously, the severe hypokalemia can occur, increase the hypokalemia incidence rate, toxic and side effects such as pseudohyperaldosteronism shape such as increased blood pressure, sodium and fluid retention, edema, weight increase, and do not find any toxic and side effects in the used in the present invention test dose scope of people sDR5 albumen.And U.S. can be the import medicine, costing an arm and a leg, (20ml:40mg/ props up, about 27 yuan of retail prices, chronic hepatopathy suggestion consumption 40-60ml/ day), need administration every day, this will inevitably increase the financial burden of hepatitis B patient family, and can cause in various degree complication, and people sDR5 albumen is as gene engineering product, production technology is simple, and low price has no side effect, can obviously alleviate the financial burden of patient family and society, solve the serious public health problem that China exists at present.
In a word, the people sDR5 albumen that the present invention relates to will have very big using value in the medicine of the particularly acute serious symptom hepatitis B of preparation treatment hepatitis B, have extremely tempting development prospect.
Claims (9)
1. the application of people sDR5 albumen in preparation treatment hepatitis B medicine.
2. application as claimed in claim 1 is characterized in that: the application of described people sDR5 albumen in the acute serious symptom hepatitis B medicine of preparation treatment.
3. application as claimed in claim 1 or 2 is characterized in that: the people sDR5 albumen consumption that alleviates hepatocyte injury is 2-64mg/kg.
4. application as claimed in claim 3 is characterized in that: the people sDR5 albumen consumption that obviously alleviates hepatocyte injury is 4-32mg/kg.
5. the application of people sDR5 albumen in preparation blocking-up hepatitis B hepatocellular apoptosis medicine.
6. application as claimed in claim 5 is characterized in that: the application of described people sDR5 albumen in the acute serious symptom hepatitis B hepatocellular apoptosis medicine of preparation blocking-up.
7. as claim 5 or 6 described application, it is characterized in that: the people sDR5 albumen consumption of blocking-up hepatocellular apoptosis is 2-64mg/kg.
8. application as claimed in claim 7 is characterized in that: the people sDR5 albumen consumption of obviously blocking hepatocellular apoptosis is 4-32mg/kg.
9. as claim 5 or 6 described application, it is characterized in that: described people sDR5 albumen is used for reducing hepatocellular apoptosis by blocking-up or sealing TRAIL's, alleviates hepatocyte injury, to reach the antiinflammatory hepatoprotective effect.
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| sTRAIL与HBV感染临床转归的研究. 岳风娥等.中华实验和临床病毒学杂志，第19卷第2期. 2005 |
| sTRAIL与HBV感染临床转归的研究. 岳风娥等.中华实验和临床病毒学杂志，第19卷第2期. 2005 * |
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