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Objective To investigate difference of NKG2D receptor expression level on the surface of natural killer (NK) cells in the patients with hepatitis B and its clinical significance.Methods This was a four-arm study with different types of subjects,including patients with chronic hepatitis B (CHB,n =22),HBV carriers (HBVC,n=10),patients with acute hepatitis B (AHB,n=18) and healthy donors (HD,n=18).NKG2D protein and mRNA levels on the surface NK cells in the peripheral blood were examined by reverse transcription-polymerase chain reaction assay.The relationship between NKG 2D expression and serum hepatitis B virus (HBV) DNA level was analyzed.The data were compared by analysis of variance and linear regression.Results NKG2D mRNA expression levels in groups of HBVC, HD, AHB and CHB were 0.96±0.17, 1.03±0.12,1.53±0.30 and 1.51 ± 0.35,respectively; the differences among groups were statistically significant (q=7.586,7.485,7.920 and 7.880,respectively; all P＜0.01).NKG2D protein expression levels in groups of AHB,HD,CHB and HBVC were 0.87±0.14,0.89±0.17,0.67±0.09 and 0.59±0.13,respectively; the differences among groups were statistically significant (q=6.92,7.67,7.53and 8.16,respectively; all P＜0.01).The NKG2D mRNA expression levels on NK cells were negatively correlated with serum HBV DNA viral loads in patients with CHB,AHB or HBVC (r=-0.75,-0.66 and-0.69,respectively; all P＜0.01).The NKG2D protein levels on NK cells from patients with AHB and CHB were negatively correlated with serum HBV DNA levels (r=-0.47 and -0.45,respectively; both P＜0.05).Conclusion NKG2D mediated NK cytotoxicity may play a role in viral clearance in hepatitis B.
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Objective To establish a method to determine the concentration of Danshensu in rats sera by HPLC after rats were fed with Kangxianerhao(KXEH) extract.Metheds Rats were given 0.45g or 0.55g KXEH extract 7 times in 4 days by gavage,and blood were collected from heart of rats at different time.Condition of HPLC: C18A column was used,the mobile phase was consist of a mixture of acetonitrile-H_3PO_4,the detection wavelength was 205nm.Result Concentration of KXEH reached the highest level one hour after administration of KXEH in different doses,which showed that high dose of drug got higher concentration of drug Danshensu in serum than the medium dose.Linear correlation was obtained over the concentration of 76～1520mg/L,r=0.9932.Average recoveries of samples were 98.3%,101.5%,99.3% respectively at different dose of 76mg/L、304mg/Land 1520mg/L,with RSD was 5.9%、2.5%、2.0% respectively.It was proved indirectly that the concentration of KXEH in rat serum after administraion of KXEH extract reached effective concentration in blood.Conclusion It shows that large dose KXEH get higher concentration of Danshensu in blood than the medium dose.The concentration of drug serum is quantitated by HPLC assay.
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Objective To investigate the influence of HCV core protein on cell apoptosis, cell cycles and cell telomerase activities of HepG2 cells. Methods A eukaryotic expression plasmid containing HCV C gene was constructed by DNA recombinant technique and the recombinant plasmid was transfected into HepG2. Thereafter, HepG2 cells transfected with recombinant eukaryotic expression plasmid were obtained. The HCV C mRNA and protein in HepG2 cells transfected with recombinant plasmid were verified by RT PCR and indirect immunofluorescence assay. The HepG2 cells were studied as follows: (1) The cell proliferation ratio of three groups cells(HepG2 cells transfected with the recombinant plasmid, HepG2 cells transfected with blank plasmid and HepG2 cells without transfection) was evaluated by MTT assay; the cell cycles were also examined by FACS. (2) The apoptotic ratio of three groups cells were examined by FACS. (3) The cell telomerase activities of all three group cells were examined by TRAP ELISA assay. Results (1) The cell proliferation ratio in the group of HepG2 cells transfected with recombinant plasmid was higher than that of the group of HepG2 cells transfected with blank plasmid or the group of HepG2 cells without transfection; The proportion of phase S in the group of HepG2 transfected with the recombinant plasmid was significantly higher than that of the group of HepG2 without transfection. (2) The apoptotic ratio in the group of HepG2 cells transfected with recombinant plasmid was significantly lower than that of the group of HepG2 cells transfected with blank plasmid or the group of HepG2 cells without transfection. (3) There were no significant differences among the three group cell telomerase activities. Conclusions (1) HCV C protein had the potential role in inhibiting cell apoptosis. (2)HCV C protein could induce HepG2 cells from phase G 0/1 to phase S, and might promote cell proliferation, inhibit cell apoptosis. (3) HCV C protein had no influence on cell telomerase activities of HepG2 cells, thus HCV C protein regulated cell cycle, promoted cell proliferation and inhibited cell apoptosis not by enhancing cell telomerase activities.
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Objective On the basis of the lasted clinical experience,our group will discuss the treatmented mechanism of Chinese herb kang-xian-er-hao(KXEH) ameliorate hepatic cirrhosis. Methods Male wistar rats were divided into five groups,excepted for normal group N,the remnant four groups were all given intraperitoneal injection of porcine serum((0.5) ml/time,2 times/week,total 12 weeks).In KXEH early treatment group B,the rats were fed with KXEH by gavage,1 g/100 g,once a day at the third week.In KXRH late treatment group C,the rats were fed with KXEH by gavage,1 g/100 g,once a day at the ninth week.In ?-interferon treated group D the rats were subcutaneous injection ?-interferon((0.1) million) every day at the ninth week.The model group A and normal group N were fed with the same amount of saline by gavage.The rats were killed at the end of the twelfth weeks,the formation of liver fibrosis was observed with HE stain and Masson stain.The expression of Smooth muscle actin(SMA) was observed by immunohistochemistry.As well as SMA,collagen Ⅰ、Ⅲ mRNA and Smad3 mRNA,which is TGF-?1 downstream signal,were detected in liver samples with RT-PCR assay. Results In KXEH treated group B and C,the body weight was heavier,the size of liver and spleen was smaller and the ratio of liver weight/body weight and spleen weight/body weight was decreased compared with the model group A(P
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Objective To study the complexity of S region qusispecies in various disease stages of chronic hepatitis B virus(HBV) infection and its relation to disease activity. Methods Serum samples were obtained from 112 patients with chronic hepatitis B virus infection;22 with chronic carries(ASC),30 with chronic mild or moderate hepatitis(CH),60 with fulminant hepatitis failure(FHF). HBV qusispecies populations were separated by the single strand conformation polymorphism (SSCP) method targeted the S region and DNA sequencing analysis. Results The number of SSCP bands detected in the patients with ASC、CH and FHF was 1.45?0.13,3.70?0.22 and 5.93?0.24, respectively. There was a statistically significant difference in the number of quasispecies among various disease stages ( P
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Objective To observe the effects of truncated type Ⅱ receptor of TGF ? 1 expressing plasmid on rat liver fibrosis. Methods An eukaryotic expression plasmid of a truncated type Ⅱ receptor of TGF ? 1 was constructed by recombinant DNA technique. Sequence analysis of this recombinant plasmid revealed that it contained the complete extracellular and transmembrane domains of TGF ? 1 Ⅱ receptor but lacked most of the cytoplasmic kinase domain. The recombinant plasmid encapsulated by liposome was transferred into the rats by intraperitoneal injection and its effects on the grade of the pathologic fibrosis and the levels of HA,LN,PCⅢ,Ⅳ C were observed. Results The levels of serum HA,LN,PCⅢ,Ⅳ C were lower in the early treatment group than those in the model control group ( P 0.05). Either in the early or later treatment group, the grade of liver fibrosis was decreased apparently compared with the model control group ( P
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Objective To construct the recombinant plasmid of human MMP 1 clone and study its antigenecity of MMP 1 fusion protein. Methods The total RNA was extracted from human liver and used as a template for reverse transcription. After PCR amplification, a 1 432 bp fragment was obtained and cloned into T vector. After digested with restriction enzyme, the target fragment was subcloned into plasmid pMAL c2x. The recombinant plasmid was transferred into JM109 which can express a fusion protein. We analyze the protein with SDS PAGE and Western blot. Results We obtained human MMP 1 gene and its recombinant plasmid clone. The expressed protein can be recognized by MMP 1 polyclonal antibody in Western blot.Conclusions We obtained the MMP 1 protein with antigenicity. The fusion protein can be used to prepare polyclonal antibody against MMP 1.
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In order to study the affection of neutralizing antibody on the therapeutic effect of IFN? -2b,NBA was used to detect the neutralizing antibody to against IFN in the sera from 27 patients with chronic hepatitis B.The results indicated that neutralizing antibody against IFN was present in 15 of 27 patients who had been treated with IFN?-2b,and the overall frequency of neutralizing antibody was 55.6%(15/27).At the end of IFN therapy,HBV DNA was undetectable in 4 of the 15 patients(4/15, 26.7%)with neutralizing antibody.By contrast.HBV DNA became undetectable in 10 of the 12 patients (10/12,83.3 % ) without neutralizing antibody (P
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Objective:To investigate the role of FASL and TRAIL in the AICD (activation induced cell death) of PBLs in patients with chronic hepatitis B.Methods:The PBLs of 20 nonnal control,24 patients with chronic hepatitis B and 24 patients with chronic severe hepatitis B were isolated and cultured with or without phytohemagglutinin( 10 pig/ml) for 48 hours in vitro. After incubation,the cells were harvested by centrifugation and the expression of FASL.,TRAIL in PBLs was assayed by reverse transcriptase-polymerase chain reaction (RT-PCR) and im-munohistochemical staining (SABC Method) .Results-.The expression of FASL mRNANTRAIL mRNA was undetectable in the resting PBLs of three investigated groups, but it was obviously increased after PHA stimulation in vitro. In comparison with the group of normal controls, the expression of FASL mRNA,TRAIL mRNA in PBLs was significantly higher in the group of patients with chronic hepatitis B( P