| Transforming growth factor β1 (TGF P 1) is a strongly fibrosis-induced cytokine, which could play an important role in liver fibrosis by stimulating the synthesis and deposition of a broad spectrum of excellular matrix (ECM) molecules of liver and inhibiting the degradation of these matrix molecules. It needs to be comprehensively studied about the influence of polymorphisms of TGF β1 gene on TGF β1 plasma concentration. Many studies showed that eight polymorphisms of TGF β1 gene at least have been identified: three of them are localized in the promoter region at positions-988 C>A, -800 G>A and -509C>T upstream of 1 exon, and three were detected in codons 10(CTGXXG), 25(CGG>CCG) and 263(ACOATC), which leads to amino acid substitutions LeulOPro, Arg25Pro and Thr263Ile, respectively. Among the polymorphisms, -509 C>T and codons 10(CTG>CCG) were found to be closely related to a large number of diseases. Based on all above, we detected alleles of six SNPs at 5' -flanking region and coding region of TGFP 1 gene and further studied the association of the SNPs with liver cirrhosis in Chinese populations.Part I The study of association between SNPs in regulating regionof TGFβ1 gene and liver cirrhosis induced by HBVUsing ARMS-PCR combined with sequencing, the three SNPs in 5' -flanking region of TGFP 1 gene were identified in 134 patients with HBV-induced liver cirrhosis and 92 healthy blood donors, anddistributions of genotype and allelic frequency were confirmed. By means of statistical analysis, We explored correlation between the three polymorphisms and liver cirrhosis. The results showed that there was no polymorphism at positions - 800 and - 988 of TGF P 1 gene, and For position -509 of TGFP 1 gene, there was no heterozygous CT and no significant difference was found at frequency of genotype CC or TT and allelic C or T between patients and controls. According to Child-Pugh classification, the patients with liver cirrhosis were divided into three groups, namely group A, B and C. In group Child-Pugh C, the frequency of genotype -509CC and allelic C of TGFP 1 gene was significantly higher than genotype TT and allelic T. Similarly, the frequency of genotype -5O9CC and allelic C was statistically higher in group Child-Pugh C than that in group Child-Pugh B.Part II The study of association between SNPs in 5' -flankingregion of TGF |3 1 gene and liver cirrhosis induced by HBVWe developed a novel method for detection of the three TGFP 1 gene polymorphisms based on real-time PCR followed by detection of variable sites using oligonucleotide hybridization and melting curve analysis, The assay was conducted in a LightCycler?(LC) system and specially distinguished the different alleles by use of the fluorescence energy transfer(FRET) principle. Our results indicated that there is no SNP expected both in codon 25 and codon 263. On the other hand, the polymorphism in coding 10 was found and heterozygous codonlOCT genotype was the highest at distribution frequency in either patients with liver cirrhosis or control group. The distribution frequency of homozygous genotype TT and allelic T in codonlO was significantly higher in patients than in controls(P<0. 05), and for genotype CC and allelic C in codonlO, thedistribution frequency was increasing gradually with Child-Pugh A to C, however, the difference was not significant. By ARLEQUN Ver2. 0 software analysis, we found that the - 509OT and codonlODC polymorphisms were in tight linkage disequillibrium(D' =0.8957, P<0. 05) and that the major haplotypes for these two loci were OT and T~C which accounted for more than 90%. Furthermore, the frequency of haplotype OT was statistically higher in patients with liver cirrhosis than in controls (P<0. 05) .Part III The study of the association of the TGFP1 gene polymorphismswith TGFP1 in plasmaIn this part, the concentrations of total TGF PI, collagen type IV (IVC) ,hyaluronic acid(HA) and N-terminal type III procollagen peptide (PIIINP) were measured in plasma, the former two by ELISA, the latter two by RIA. The results showed that compared with the control group, the plasma concentrations of TGF P 1, IVC, HA, PIIINP were significantly increasing in diseased group (P<0. 05 or P<0. 001) . The concentration of TGF P 1 was not closely related to IVC, HA and PIIINP, however, there was significant relationship between IVC and HA(r=0. 4016, P<0. 01), HA and PIIINP(r=0. 5435, P<0. 001) or FVC and PIIINP ( r=0. 4535 , P<0. 01) . The TGF P 1 plasma concentrations were statistically lower in populations with TT genotype than in those with CC genotype in diseased group (p<0.001) , but the concentrations did not show any difference between genotypes - 509CC and TT in control group. For polymorphism in codonlO, there was no difference between any genotype both in diseased and control groups. The plasma concentration of TGF P 1 was significantly increasing in populations with haplotype C-T.
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