Objective:To investigate the prognosis and outcome of patients with chronic hepatitis C (CHC) related cirrhosis after achieved sustained virologic response (SVR) treated with direct-acting antiviral agent (DAA).Methods:Ninety-five patients diagnosed with CHC related cirrhosis who had complete data in Tianjin Second People′s Hospital from January 2014 to June 2017 were retrospectively followed up. Among them, 72 patients were treated with DAA and all of them achieved SVR, and the other 23 patients did not receive any antiviral therapy. The differences of mortality and incidence of hepatocellular carcinoma (HCC) between DAA treatment group and non-antiviral treatment group were compared. Statistical analysis was performed by independent sample t test, Mann-Whitney U test and chi-square test. Results:At the end of follow-up for three to 71 months, patients in DAA treatment group had a significant improvements in alanine aminotransferase, aspartate aminotransferase, albumin and liver stiffness measurement compared with those before treatment (42(23, 61) U/L vs 18(13, 28) U/L, 54(37, 75) U/L vs 23(18, 28) U/L, 39(33, 42) g/L vs 45(41, 48) g/L, 26(18, 37) kPa vs 15(11, 26) kPa, respectively, Z=-6.005, -7.008, -6.057 and -3.162, respectively, all P<0.01). However, there were no significant differences in incidence of HCC (12%(9/72) vs 17%(4/23)) and mortality (3%(2/72) vs 13%(3/23)) between the DAA treatment group and non-antiviral treatment group (both P>0.05). There was no significant difference of cumulative incidence of HCC in DAA treatment group compared with non-antiviral treatment group ( P=0.609). The age of patients progressed to HCC was older than those without HCC ((60.3±3.6) years vs (54.4±9.9) years, t=-3.948, P<0.01). In subgroup analysis, among the six patients with HCC, four had diabetes, the prevalence of diabetes in the patients without HCC was 17%(7/42); the level of fasting blood glucose (FBG) ((7.3±1.9) mmol/L vs (5.9±1.1) mmol/L) were higher in patients progressed to HCC than those without HCC in DAA treatment group with compensated cirrhosis ( χ2=7.430 and t=-2.442, respectively, both P=0.019). Conclusions:DAA treatment could notably improve liver function and alleviate liver fibrosis, but could not reduce the mortality and incidence of HCC in patients with CHC related cirrhosis significantly. Diabetes and high level FBG may be the risk factors for occurrence of HCC in patients with CHC related compensated cirrhosis.

Cloning of flanking sequences of double-copy gene is a challenge in molecular biology. We developed a method to solve this problem by combining an optimized inverse PCR (iPCR) with TAIL-PCR. First, Southern blotting analysis was used to determine a proper restriction enzyme that could obtain proper-length restriction fragments that contained the target gene. Then optimized iPCR was performed to amplify the restriction fragments that contained the separated copies of the gene. Based on the obtained sequences, TAIL-PCR was performed to amplify further flanking regions of the gene. With this method, we obtained all of the EcoR I restriction fragments (2.2-5.1 kb) and Hind III restriction fragments (8.5-11.7 kb) of mitochondrial atpA gene in cytoplasmic male sterile (CMS) line and maintainer line of Upland cotton. The results showed that this method was an efficient approach to clone flanking sequences of double-copy gene.
Chromosome Walking ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Genes, Mitochondrial ; Genes, Plant ; genetics ; Gossypium ; genetics ; Plant Proteins ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; Terminal Repeat Sequences