| ObjectiveHepatic cirrhosis is common disease. It has a high death rate. But we still have no specific treatment for it. Chronic liver disease must transform hepatic fibrosis before it transforms cirrhosis. So to block the formation of hepatic fibrosis plays important role in improving prognosis of hepatic cirrhosis. Prevention and cure in hepatic fibrosis are hot spot and nodus in clinical research. Transforming growth factor beta 1(TGF β 1) is cytokine which have many kinds of biological functions such as accommodating cell growth, death, apoptosis, cell differentiation and formation of extracellular matrix, et al. It plays important role in forming, depositing and degrading extracellular matrix in liver and is closely related with pathogenesis of hepatic fibrosis. We have found that it had any individual difference in formation of HBV-induced hepatic fibrosis. It may be association with genetic background. The levels of serum TGF β 1 serum may be due to the effect of polymorphisms of its gene. We observed the effects of TGF β 1 gene polymorphisms on the levels of serum TGF β 1 and analyzed the relationship between polymorphism of TGF β 1 gene and its level in serum as well as hepatic fibrosis in patients with HBV infection in order toinvestigate how TGF P 1 gene polymorphism plays role in the pathogenesis of hepatic fibrosis, so as to look for new theoretical basis for gene therapy in patients with hepatic fibrosis in future.Method1. Subject(1) Chronic hepatitis group: There are 65 patients with chronic hepatitis B, 50 patients were males, and 15 patients were females. The range of age was from 18 to 68, the average age was 36.22+11.67. Among them 12 patient's conditions are mild, 37 conditions are moderate, and 16 conditions are severe. The range of course from 6 months to 20 years, the average was (3.36+4.91 ) years. All of them there are 36 patients that have HBV infection's family histories.(2) Hepatic cirrhosis group: There are 56 patients with HBV-induced hepatic cirrhosis, 50 patients were males, and 6 patients were females. The range of age was from 30 to 77, the average age was 50.88+11.33 . Among them 26 patients are in compensated stages, others are in decompensated stages. The range of course from 7 months to 32 years, the average was (4.15+5.61) years. All of them there are 45 patients that have HBV infection's family histories.Two groups cases were selected from patients with HBV infection who were in hospital in infection ward of our hospital from July 2004 to December 2005. All patients were diagnosed by schemes of prevention and cure in viral hepatitis which were established in the nationwide liver disease's meeting in 2000.(3) Control group: There were 50 health examination people whose markers of HBV were negative. 35 cases were males, and 15 cases were females. The rang of age was from 18 to 65, the average age was 48.72± 12.90.Subject who had taken medicine such as virus-fighting drug or anti-hepatic fibrosis drug was excluded. Subject with HAV, HCV, HDV or HEV infection, other liver diseases such as alcoholic liver disease, drug liver disease, fibrosclerosis diseases such as glomerular sclerosis or hematonosis and so on was also excluded. In addition,6subject who had infection or tissue damage in recent one month was excluded too. 2. Method(l)Samples gather: Samples of 6ml blood were collected on an empty stomachs, 3ml blood samples were anticoagulated by EDTA and were preserved in -80 °C refrigerator.Polymorphisms of TGF P 1 gene were measured from these bloods samples. Other 3ml blood samples weren't anticoagulated but centrifugalized in order to separate serums and serum samples were preserved in -80 °C refrigerator. The levels of TGF P 1 and markers of hepatic fibrosis were measured from these serum samples.(2)The polymorphisms at position + 869 and + 915 of TGF 3 1 gene determination: The polymorphisms of TGF P 1 gene were determined by PCR-DNA sequence analysis. First, genomes DNA in blood were extracted. Second, the purpose genes were amplified by PCR.Primer series:? 869T/C1 5' —AGCAGCGGTAGCAGCAGCA—3'(D 869T/C2 5' —OCAGCGGTAGCAGCAGCG—3'(D 869T/C3 5' —GTCACCAGAGAAAGAGGAC—3'? 915G/C1 5' —GTGCTGACGCCTGGCCG—3'? 915G/C2 5' —GTGCTGACGCCTGGCCC—3'? 915G/C3 5' —GGCTCCGGTTCTGCACTC—3Third, productions of PCR were separated by electrophoresis of 1.5% Agarose II(voltage:180V,40mA). Last, allelotype of each sample was determined by comparing specific amplification analysis with sequence analysis. (3)TGF P 1 measurement: The levels of TGF P 1 in serum were measured by ELISA.(4)The markers of hepatic fibrosis in serum measurement: Four markers of hepatic fibrosis are hyaluronic acid(HA), laminin(LN), collagon type IV(CIV) and procolagen type III(PCIII). All markers of hepatic fibrosis were measured by radio immunoassay.3. Statiscal treatmentAll data were analyzed in statistics by spss 10.0 ststistical software.Resultsl.Frequencies of genotype and allele both T/C gene at position +869 and G/C gene at position +915 of TGF P 1 of three groups: Three genotypes (TT, TC, CQwere found at position +869 and three genotypes (GG, GC, CC)were found at position +915. The frequencies of genotype CC and allele C at position +869 of chronic hepatitis group and hepatic cirrhosis group (0.277 and 0.577 respectively in chronic hepatitis group, 0.250 and 0.545 respectively in hepatic cirrhosis group ) were both significantly higher than those of control group (0.100 and 0.400 respectively), both PO.05. But it didin't show any difference between chronic hepatitis group and hepatic cirrhosis group. At position +915, The frequencies of genotype GC and CC and allele C of chronic hepatitis group and hepatic cirrhosis group were higher than those of control group, but there was no difference.2.The relationship between the level of TGF ￡ 1 in serum and genotype +869T/C as well as blood platelet count: By covariance analysis, both genotype and platelet count had influenced the levels of TGF P 1 in serum, both PO.05. Regression coefficient of influence on the levels of TGF P 1 by CC genotype,TC genotype and platelet count were 12.034, 4.027, 0.042 respectively. So CC genotype was play a leading role in the influence on serum levels of TGF P 1.3.The relationship between the level of TGF P 1 in serum and genotype +915G/C: The levels of TGF ￡ 1 in serum didn't show any difference among three groups that were GG genotype group, GC genotype group and CC genotype group. So there was no statistically significant difference, P>0.05.4.The relationship between the frequencies of genotype and allele of TGF P 1 +869 T/C and +915G/C and family history of chronic hepatitis or HBV-induced hepatic cirrhosis: In the chronic hepatitis group and hepatic cirrhosis group, the frequencies of genotype CC and allele C at position +869T/C in the patients withfamily history of chronic hepatitis or HBV-induced hepatic cirrhosis (0.346 and 0.611 respectively) were significantly higher than those patients without family history (0.100 and 0.462 respectively), both P<0.05. But no significant difference between with and without family history of chronic hepatitis or HBV-induced hepatic cirrhosis for position +915G/C was found.5.Comparison of the levels of TGF ￡ 1 (ng/ml) in serum among chronic hepatitis group, hepatic cirrhosis group and control group: The levels of TGF P 1 in serum in chronic hepatitis group (21.55 + 12.62) were significantly higher than those in control group( 11.05 + 8.90), PO.05. The levels of TGF P 1 in hepatic cirrhosis group (9.77+9.04) were similar with those in control group, there was no significant difference between them. What's more, the levels of TGF ￡ 1 in decompensated hepatic cirrhosis group (6.89 + 6.76) were significantly lower than those in compensated hepatic cirrhosis group (13.08+11.85) , P<0.05. 6.Comparison of the marks of hepatic fibrosis (ng/ml) among chronic hepatitis group, hepatic cirrhosis group and control group: Four marks of hepatic fibrosis (HA, LN, CIV and PC EH) in serum in the chronic hepatitis group and hepatic cirrhosis group (313.49 + 285.71, 109.51+35.31, 111.77+102.29 and 233.82 + 131.36 respectively in the chronic hepatitis group, 712.38+265.80, 127.20+44.35, 155.80+ 74.83 and 237.48 + 126.46 respectively in the hepatic cirrhosis group) were all significantly higher than those in the control group (63.16+ 24.89,72.08 + 24.75,48.72± 18.12 and 62.14+ 24.23 respectively) , all PO.05.Conclusions1. On this empirical study, polymorphisms was found at position +869T/C of TGF P 1 gene. The frequencies of genotype CC and allele C at position +869 of chronic hepatitis group and hepatic cirrhosis group in patients with hepatitis B were both significantly higher than those of control group. Genotype CC has positive correlation with the levels of TGF P 1 in serum.2. On this empirical study, the frequencies of genotype CC and allele C of TGF P 1 +869 T/C in the patients with family history of chronic type B hepatitis orHBV-induced hepatic cirrhosis were significantly higher than those patients without family history.3. Base on the results, It may be no relationship between +915G/C polymorphisms of TGF P 1 gene and the onset and development of HBV-induced hepatic fibrosis.4. In these subjects, four marks of hepatic fibrosis (HA,LN, CIV and PCHI) in the chronic type B hepatitis group and HBV-induced hepatic cirrhosis group were all significantly higher than those in the control group. The levels of TGF ￡ 1 in serum in chronic type B hepatitis group were significantly higher than those in control group.
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