Function of TTG1A in hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the biological functions of TTG1A in liver fibrosis.</p><p><b>METHODS</b>Yeast two-hybrid system was used to screen proteins associated with TTG1A. Briefly, the coding sequence of TTG1A was cloned into pGBKT7 vector, and the recombinant plasmid was transformed into yeast cells AH109 ( a type), then these cells were mated with yeast cells Y187 (a type) transformed with human leukocyte cDNA library plasmid pACT2. The obtained diploid yeast cells were plated on synthetic dropout nutrient medium containing X-alpha-gal for double selection. The plasmids from positive colonies were transformed into E.coli and sequenced.</p><p><b>RESULTS</b>The recombinant yeast expression vector pGBKT7-TTG1A was successfully constructed. Nineteen TTG1A binding proteins, including Homo sapiens major histocompatibility complex, class II DP beta 1 (HLA-DPb1), Homo sapiens ribosomal protein L30 (RPL30), Homo sapiens nucleophosmin Homo sapiens nucleobindin 2 (NUCB2), Homo sapiens ash2, variant Gaucher disease and variant metachromatic leukodystrophy, MORF4L1, Homo sapiens ubiquitin-conjugating enzyme E2L3 (UBE2L3), APOA1, Homo sapiens lectin, and galectin 1, were identified.</p><p><b>CONCLUSIONS</b>This study may help to elucidate the molecular function of TTG1A.</p>

Screening and cloning genes transactivated by TGF beta 1 in hepatic stellate cells using suppression subtractive hybridization technique / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To construct a cDNA subtractive library of genes transactivated by TGF beta 1 in LX02 hepatic stellate cells (HSC); to screen and to clone the regulated genes transactivated by TGF beta 1; and to elucidate the molecular biological mechanism of hepatic fibrosis mediated by TGF beta 1.</p><p><b>METHODS</b>mRNA was isolated from HSC treated with TGF beta 1 or with PBS (as controls). Suppression subtractive hybridization (SSH) technique was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent polymerase chain reaction twice it then was subcloned into pGEM-Teasy plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5a. The cDNA was sequenced and analyzed in GenBank with Blast search.</p><p><b>RESULTS</b>The subtractive cDNA library of genes transactivated by TGF beta 1 in HSC was constructed successfully. The amplified library contained 146 positive clones, which contained 200-1000 bp of inserts. Randomly, thirty clones were analyzed by sequencing and bioinformatics, consisting of 28 known genes and 2 unknown genes.</p><p><b>CONCLUSIONS</b>The subtractive cDNA library of genes transactivated by TGF beta 1 in HSC using SSH technique was constructed successfully. Some gene coding proteins are those involved in cell growth regulation, protein synthesis, signal transduction, extracellular matrix metabolism, and anti-lipid peroxidative, which gives us some new clues for the study of the mechanism of liver fibrosis.</p>

The impact of HBeAg positivity/negativity and HBV DNA loads on the prognosis of chronic severe hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the impact of HBeAg positivity/negativity and HBV DNA loads on the prognosis of chronic severe hepatitis B.</p><p><b>METHODS</b>206 patients with chronic severe hepatitis B hospitalized in Beijing Ditan Hospital from July 2002 to Dec. 2004 were analyzed. HBeAg positivity/negativity, HBV DNA loads and other factors relating to the prognosis of the patients were studied with univariate and multivariate analyses.</p><p><b>RESULTS</b>Chi2 univariate analysis showed that there was no significant difference in the prognosis between different HBeAg groups (chi2 = 0.440, OR = 0.777, 95% CI 0.424-1.425, P = 0.50). But there was a significant difference in the prognosis between different HBV DNA load groups: the prognosis of patients with lower HBV DNA loads was better than those with higher loads (chi2 = 9.806, OR = 3.055, 95% CI 1.554-6.007, P = 0.002), and the improving rates of the two groups were 53.1% and 27.0% respectively. Using multivariate logistic regression analysis, 9 screened factors showed great impact on the prognosis of chronic severe hepatitis B. Cirrhosis, hepatorenal syndrome, hepatic encephalopathy, PTA < 20%, TBil > 513 mmol/L, Alb < 30 g/L, CHO < 1.6 mmol/L, PLT < 5 x 10(9)/L, and higher HBV DNA loads (HBV DNA > 3 x 10(4) copies/ml in HBeAg negative patients and > 1 x 10(5) copies/ml in HBeAg positive patients) were shown to be associated with a poor prognosis. Coefficients of regression of the above factors were 1.539, 21.356, 1.398, 1.650, 2.440, 2.266, 1.738, 2.631 and 2.656 respectively. The coefficients of regression of HBV DNA loads were: B = 2.656, Wald = 7.768, P = 0.005, EXP(B) = 14.235, and 95.0% CI for EXP(B) = 2.199-92.133.</p><p><b>CONCLUSIONS</b>Our results indicate that the HBV DNA loads were one of the most important factors influencing the prognosis of the chronic severe hepatitis B patients, the importance is only next to hepatorenal syndrome and over grade II hepatic encephalopathy. HBeAg positivity/negativity has no influence on the prognosis, but HBV DNA loads are important; the lower the viral loads, the better the prognosis.</p>