Objective To probe into the effect of the endotoxin(ET) on cellular immunity function of patients wih chronic hepatitis B.Methods 98 patients and 15 normal persons as control group were studied.Measurements of endotoxin,interleukin-2 and peripheral blood T lymphocyte subset were done.Results The level of endotoxin in patients was higher than that in controls(P

Objective To construct prokaryotic expression vector of XTP3 gene and induce the expression of fusion protein in E.coli,and prepare XTP3-specific rabbit polyclonal antibody,detect the specificity of the antibody in hepatic carcinoma tissue and normal liver tissue.Methods DNA fragment of XTP3 amplified by PCR was inserted into pET-32a(+) to construct prokaryotic expression vector pET-32a(+)-XTP3.After identified by sequencing,pET-32a(+)-XTP3 was transformed into E.coli BL21 and induced with IPTG.After analyzed by SDS-PAGE and Western blotting,the induced expression product was purified and renatured by Ni+ affinity column chromatography.The purified protein was used to immunize New Zealand rabbits to gain polyclonal antibody,and the polyclonal antibody was then detected by ELISA,immunohistochemistry and Western blotting.Results Prokaryotic expression vector pET-32a(+)-XTP3 was successfully constructed,and the XTP3 fusion protein of about 52kD was highly expressed in E.coli.DS-PAGE showed that the protein product was mainly in inclusion body.The purified protein and polyclonal antibody were obtained successfully.It was manifested by ELISA that the titer of polyclonal antibody was over 1∶128 000.Immunohistochemistry showed that XTP3 antibody presented membrane-positive in hepatic carcinomous tissue.Conclusions The recombinant XTP3 protein and polyclonal antibody have been obtained successfully.These results lay a foundation for studying the immuneogenicity and bionomics of XTP3 protein.

AIM: To study the roles of endotoxemia in the occurence and development of cirrhosis. METHODS: The cirrhosis of rats were made by oral intake of thioacetamide (TAA-treated rats). A separate group of rats were subjected to a injection of small dosage of lipopolisaccharride (LPS) in the period of thiocetamide treatment in order to observe the possible effect of LPS on the forming of cirrhosis. RESULTS:Both plasma endotoxin levels and hepatic collagen contents were increased in TAA-treated rats,and there was a linear correlation between them ;Plasma and hepatic tumor necrosis factor (TNF?)、endothelin-1(ET-1)、nitric oxide(NO)、and MDA levels were all increased,and plasma endotoxin was correlated with all of their plasma contents;Only TNF? levels and hepatic collagen contents of TAA+LPS group were increased significantly compared with TAA group. CONCLUSION: These results suggest that endotoxemia (ETM) could play an important role in the development of cirrhosis mainly through activating Kupffer cells, which secrete TNF?, ET-1, NO and free radicals could be involved in the process.

AIM: To explore the effect of endotoxemia on hepatocarcinoma course induced by TAA. METHODS: The rat cirrhosis was induced by intaking TAA (0.03%) for four months, hepatocarcinom for six months. Lipoplysaccharide (100 ?g/kg) was administered subcutaneously every two days from the fifth month to the end of sixth month in TAA-treated rats. The plasma endotoxin content, ?-glutamyl transpeptidase(?-GT) activity, DNA index and aneuploidy index were determined, the point mutation of N-ras, p53 gene was detected in hepatocarcinoma rats. RESULTS: Endotoxin increased protein overexpression of p53, bcl-2 and N-ras , p53 gene mutation point and free radical production, reduced antioxidase activity and aggravated DNA damage in hepatocarcinoma rats. CONCLUSION: Endotoxin could accelerate hepatocarcinogenesis promoted by TAA.

Objective To screen promoter binding protein of cyclin B2 by using human liver cDNA library, and investigate the expression and regulation mechanism of cyclin B2 gene. Methods By using cyclin B2 biotinylated promoter DNA as the selective molecule, the T7select human liver cDNA library was biopanned and positive clones were selected. After screening, positive plaques were performed to amplify for inserted DNA fragment, and the DNA fragment was cloned into pGEM-Teasy vector. Results Sequence analysis was performed in 20 positive plaques, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 6 coding sequences were obtained, all of which were known ones. Conclusion Cyclin B2 promoter binding proteins were screened. The results will be useful for further study the expression and regulation mechanism of cyclin B2.

Objective To clone and identify human genes transactivated by homo sapiens HCV FTP2 by constructing a cDNA subtractive library with suppression subtractive hybridization tech- nique.Methods Suppression subtractive hybridization(SSH)and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HCV FTP2.The mRNA was iso- lated from HepG2 cells transfected pcDNA3.1(-)-HCV FTP2 and pcDNA3.1(-)empty vector re- spectively,and SSH method was employed to analyze the differentially expressed DNA sequence be- tween the two groups.After digestion with restriction enzyme Rsa I,small-size cDNAs were ob- tained.Then tester cDNA was divided into two groups and ligated to the specific adaptor I and adap- tor 2 respectively.After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR and then was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5?.Futhermore,the cDNA was se- quenced and analyzed in GenBank with Blast search after PCR.Results The subtractive library of genes transactivated by HCV FTP2 was constructed successfully.The amplified library contains 71 positive clones.Colony PCR shows that 56 clones contain 200～1000 hp inserts.Sequence analysis was performed in 24 clones randomly,and the full length sequences were obtained with bioinformatics method.Altogether 20 coding sequences in total were obtained,consisting of 19 known and 1 un- known.Conclusion The obtained sequences may be target genes transactivated by HCV FTP2,and some genes coding proteins involved in cell cycle regulation,metabolism and cell apoptosis.

Objective To understand Severe Acute Respiratory Syndrome(SARS)better and accumulate more experience of preventing and treating this disease. Methods 63 cases of SARS patients diagnosed during March and May of 2003 were studied by retrospective. Results The average age of patients was(35.1?14.4) years old. Among them, 36 patients had the histories of closing contact with diagonosed patients and 22 patients showed infectious links. There were 3 medical staff patients infected. The average incubations period was (6.2?3.1) days and the SARS epidemic pick in Taiyuan is on April of 2003. People between 20～40 years old had the highest incidence. The most common symptom is fever. The second is dry cough. Most of the patients with SARS had normal or lower white blood counts than normal. The chest radiographs showed infiltration signs such as ground-glass opacities, focal consolidation or pathy consolidation within one week after being ill. The Average period of beginning absorption of the infiltration on chest radiograph is (11.6?5.9) days and the average period of completely resolution is (22.9?6.7) days after being admitted in hospital. There were 14 cases of severe type and 6 patients died of ARDS among 63 patients. Conclusions SARS patients showed obvious infectious link. Therefore it supports the view that SARS is mainly conmmunicated by close air droplets. The main characteristics of SARS are fever, normal or lower white blood cells counts and abnormal chest radiographs.

Objecfive To investigate the effects of endotoxin on nuclear factor-κB p65(NF-κB p65)mRNA expression and ahtosteron secretion in rat hepatic stellate cells(HSCs).Methods Cultured rat HSCs(HSC-T6)were divided into endotoxin-treated group and control group.Cells in endotoxin-treated group were exposure to 1 mg/ml.endotoxin.Aldosteron secretions of HSCs were determined by radioimmunoassay,and NF-κB p65 mRNA expressions of HSCs were detected by one-step RT-PCR.Results At 6,12,24 and 48 h,aldosteron secretions in endotoxin-treated group were significantly hisher than those in the control group(t=3.063,4.577,6.847 and 9.317,P<0.05),and the expressions of NF-κB p65 mRNA in endotoxin-treated group were also higher than those in control group(t=5.155,6.095,7.875 and 9.313,P<0.01).Aldosteron secretions and NF-κB p65 mRNA expressions in HSCs displayed a positive correlation(r=0.886,P<0.01).Conclusion Endotoxin can up-regulate the aldosteron secretion and NF-κB p65 mRNA expression in rat HSCs,which may be one of the mechanisms of liver fibrosis induced by endotoxin.

Objective To investigate the correlation between dose and effect of thioacetamide (TAA) on rat model of intestinal endotoxemia. Methods The models of intestinal endotoxemia were induced by three different doses of TAA by gavage administration of TAA 200, 400, 600mg/kg respectively once per day for two days.The doses were given at same time point every day. Each group included 10 rats. The rats in the control group were administrated with 2 mL 0.9% NaCl saline gavage. The death of the rats was observed at 24 hours and 48 hours after administration. The blood samples of the living rats were drawn from abdominal aorta for determining the plasma endotoxin levels, serum alanine aminotransferase(ALT)and aspartated transaminase (AST) levels. The histopathological changes of liver were examined. Single factor analysis of variance was performed and comparision between groups was done using t test. Results No rat in the control group died. Two rats of 200 mg/kg TAA group, five rats of 400 mg/kg TAA group and eight rats of 600 mg/kg TAA group died during the experiment. The mean serum ALT levels of TAA model groups [(305.09±116.78)U/L,(901.67±274.31)U/L,(1454.84±473.49)U/L] were all significantly higher than that of the control group(47.81±22.61)U/L(t=14.583, 25.896 and 20.596, respectively; all P＜0.05). The mean serum AST levels of TAA model groups [(465.88±139.96)U/L, (884.37±250.90)U/L,(1889.23±159.67)U/L] were all significantly higher than that of the control group (69.33±22.04)U/L(t=12.988,18. 455 and 13.542, respectively; all P＜0.05). The mean plasma endotoxin levels of TAA model groups [(0.436±0.110)EU/mL, (0.550±0.095) EU/mL, (0.620±0.057)EU/mL] were all significantly higher than that of the control group (0.103±0.056)EU/mL(t=7.335, 5.260 and 8.191, respectively; all P＜0.05). The histological results of TAA model groups showed hepatic cell degeneration and necrosis in different degrees. Conclusions TAA with 200-600mg/kg is proper to establish the rat model of intestinal endotoxemia. The death rate of rats in the 200mg/kg TAA group is lower than those of other model groups, which suggests that 200mg/kg TAA may be the best dosage for establishing rat model for further studies.

Objective To study the effects of endotoxin on aldosteron secretion and nuclear factor-κB P65(NF-κB P65)mRNA expression in rat hepatic stellate cell(HSC).Methods Cultured rat HSC were treated with endotoxin of different concentrations. Aldosteron secretion of HSC was detected by radio-immunoassay. NF-κB P65 mRNA expression of HSC was determined by semiquantitative reverse transcription-polymerase chain reaction(RT-PCR).The data were analyzed by variance analysis,t-test and Pearson linear regression analysis. Results Aldosteron secretions of HSC groups treated with 0.01,0.1,1.0 and 10.0 mg/L endotoxin[(4.95±0.35),(5.52±0.32),(6.04±0.60)and(5.16±0.46)μg/L, respectively] were all significantly higher than that in control group(3.655±0.51)μtg/L(t=2.9745,5.8725,6.8465 and 3.2065,respectively;all P＜0.05).NF-κB P65 mRNA expressions of HSC groups treated with 0.01,0.1,1.0 and 10.01 mg/L endotoxin (0.82±0.06、1.07±0.07,1.23±0.06 and 0.96±0.05.respectively)were also significantly higher than that in control group 0.43±0.04(t=5.4776,6.8084,7.9382 and 7.5136,respectively;all P＜0.01).Both aldosteron secretion and NF-κB P65 mRNA expression in HSC groud treated with 10.0 mg/L endotoxin were significantly lower than those in HSC group treated with 1.0 mg/L endotoxin(t=4.3865,3.7246;both P＜0.05).In these treated HSC,aldosteron secretion was positively correlated with NF-κB P65 mRNA expression(r=0.886,P＜0.01).Conclusions Aldosteron secretion and NF-κB P65 mRNA expression in rat HSC could be up-regulated by stimulation with endotoxin, which shows a certain dose-response relationship. This may be one of the important factors of hepatic fibrosis development.