| Backgrounds:Liver is a hormone-sensitive organ that can be affected by androgen and estrogen.For Chronic-hepatitis-B(CHB),the gender and sex hormone levels could affect the morbidity,natural history and progression towards end-stage liver diseases.And the level of sex hormone differs in the process of cirrhosis and hepatocellular carcinoma(HCC),however,there are few studies concerning effect of sex hormone on acute-on-chronic liver failure(ACLF).HBV-ACLF is a clinical manifestation with high mortality,progressing from CHB and no one knows clearly how it occurs.Some researchers hold the view that HBV mutation –induced immune imbanlance may account for HBV-ACLF’s occurrence.However,the HBV quasipecies studies designed for HBV-ACLF are rare and mostly based on clone-based sequencing(CBS)which is time-consuming and incapable to detect novel varations.Some are based on next-generation sequencing(NGS)which needs to reconstruct the sequence because of the short reading length(shorter than 600 base pairs,bp).Here,we chose the single molecular realtime(SMRT)detection technology,which belongs to third-generationsequencing(TGS),to analyze the HBV quasispecies patterns of HBV-ACLF.Aims:The study was based on a prospective cohort of HBV-ACLF in China,attempting to explore the association between sex hormone and the progression and prognosis of ACLF.Moreover,it aimed to screen out ACLF-related quasispecies mutations with TGS and then compare the HBV kinetics in male and female mice.It will help to understand the potential mechanism of ACLF considering the gender-based interaction between virus and host immunity.Material and methods:Part 1 Testosterone and estradiol as novel prognostic indicators for HBV-ACLF1 A prospective cohort of 300 chronic hepatitis B(CHB)patients was enrolled,among which 108 were diagnosed with HBV-ACLF at admission and 20 developed to HBV-ACLF during hospitalization.2 We compared the serum testosterone and estradiol levels of patients with varied disease severity and cirrhosis conditions and analyzed the predictive ability of short-term prognosis.3 A novel prognostic model involving testosterone was developed and evaluated.Part 2 Screening on ACLF-related quasispecies mututions1 Serum samples of patients in HBV-ACLF cohort were collected.2 We extracted the HBV-DNA and amplified the full-length of virus.3 The full-length sequencees of HBV were obtained with TGS technology.4 We detected the mutations that were associated with ACLF.Part 3 Effects of gender on HBV kinetics in vivo1 We constructed the animal models through transfection of p AAV-HBV1.2 plasmid.2 We changed of sex hormone level with ovariectomy and testectomy.3 We monitored the HBV kinetics(HBV DNA and HBs Ag)dynamically.Results:1、The baseline estradiol level of HBV-ACLF group was significantly higher while testosterone was lower than that of non-ACLF group.The estradiol level increased while the testosterone level decreased as the number of organ failures increased.Testosterone had high accuracy in predicting the short-term mortality in HBV-ACLF and estradiol did better in predicting the occurrence of ACLF during hospitalization.2、Quasipecies diversity(amino acid level)was lower in pre S/S and P regions among ACLFgroup and we detected the frequnency of several novel muatioins was significantly higher in ACLF group: N45D(A2439G,nt)in P region、S50T(T1961A,nt)、L113S(T2151C,nt)、E209G(A2439G,nt)in pre C/C region、T1047A in Enhancer1 region and G1677A、G1721A in Enhancer2 region.3、The titers of HBV-DNA and HBs Ag were significantly higher in male mice and testectomy could derease the HBV titer while ovx helped to keep the HBs Ag higher.Conclusions:We developed the prospective HBV-ACLF cohort and confirmed that testosterone and estradiol can be utilized as novel prognostic indicators for HBV-ACLF;then we detected novel ACLF-related quasispecies mutations with TGS and finally verified the regulatory function of sex hormones on HBV amplification and protein expression. |
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