Genotypes and drug-resistance mutation of hepatitis B virus in chronic hepatitis B patients in West Guangxi: Analysis of 869 cases / 医学研究生学报

ObjectiveThe genotypes and drug-resistance mutation of hepatitis B virus (HBV) are closely related to the progression of chronic hepatitis B (CHB) and effects of medication. This study was to identify the genotypes and drug-resistance mutation genes of HBV in West Guangxi and provide some laboratory evidence for anti-HBV treatment.MethodsWe collected serum samples from 869 CHB patients with HBV DNA >1.0×103 IU/mL, all from West Guangxi, between January 2016 and March 2018. We detected the genotypes and drug-resistance mutation genes in the patients by PCR and reverse dot blot (PCR-RDB).ResultsA total of 7 genotypes were identified in the patients, including genotypes B in 304 cases (34.98%), C in 268 (30.84%), D in 147 (16.92%), B+C in 28 (3.22%), B+D in 22 (2.53%), C+D in 1 (0.12%) and B+C+D in 1 (0.12%). Drug-resistance mutation was observed in 63 cases (7.25%), including 31 cases with genotype C (49.21%) and 19 with genotype B (30.16%). Fourteen gene mutation patterns were found in all, including rt204I, rt181V, rt236T, rt180M+rt204V, and rt180M+rt204V+rt204I, among which, rt180M+rt204V exhibited the highest mutation frequency (17.46%). There was a statistically significant difference between the mutation rates of genotypes B and C (19% vs 31%, P < 0.05).ConclusionCHB patients in West Guangxi have a wide variety of genotypes of HBV, mainly including genotypes B and C, with a high rate and complicated patterns of drug-resistance mutation, which has provided some reference for anti-HBV treatment in West Guangxi.

Inhibition of hepatitis B virus S gene expression by anti-gene locked nucleic acid in vivo / 医学研究生学报

Objective There is still a lack of effective treatment for hepatitis B. The article aimed to investigate the inhibito-ry and antiviral effects of hepatitis B virus(HBV)S gene expression by anti-gene locked nucleic acid(LNA)in vivo in transgenic mice. Methods The HBV transgenic mice were divided into 5 groups by random number,6 in each group. They were blank group, irrelevant sequence group,lamivudine group,antisense locked nucleic acid group,and anti-gene locked nucleic acid group respective-ly. The lamivudine group was treated by oral gastric lavage,with 2mg/kg dose 2 times per day for continuous 7d,and the rest groups were injected with 500 mL by tail vein injection at 1,3,5 d after ad-ministration. HBV DNA was tested by real-time quantitative polymer-ase chain reaction(qPCR);HBsAg was tested by ELISA;mRNA of HBV S gene was detected by reverse transcription PCR(RT-PCR);the positive rate of HBsAg in liver cells was detected by immunohisto-chemistry. Results After the treatment of anti-gene locked nucleic acid on 3rd,5th,7thday,the inhibition rate of HBV DNA were 37.18%,50.27%,61.46%,and HBsAg were 30.17%,44%,57.76%. The inhibitory effect on 7thday was more obvious than those of the blank group,the unrelated sequence group,the lamivudine group and the antisense lock nucleic acid group,and the difference was statistically significant(P<0.05).The expression of mRNA S gene HBV(0.33)and liver cells HBsAg positive rate(31%)was signif-icantly reduced compared with control group(P<0.05).No abnormal change was found in the function of liver and kidney tests and tis-sue HE staining. Conclusion Anti-gene lock nucleic acid targeting to S gene has strong inhibitory effects on HBV replication and expression in transgenic mice,which provides theoretical and experimental knowledge for HBV gene therapy.

Prevalence of a high non-high-density lipoprotein cholesterol level in children aged 9-11 years in Mianyang Science City in Sichuan Province, China / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the non-high-density lipoprotein cholesterol (non-HDL-C) level and the prevalence rate of a high non-HDL-C level in children aged 9-11 years in the Mianyang Science City area in Sichuan Province, China.</p><p><b>METHODS</b>From September to October, 2015, a field investigation was performed for the students from three primary schools in the Mianyang Science City area by cluster sampling. Fasting venous blood was collected for blood lipid tests. The cut-off value of serum non-HDL-C level and prevalence rate of a high non-HDL-C level in children aged 9-11 years in this area were calculated, as well as the prevalence rate of a high non-HDL-C level in obese children.</p><p><b>RESULTS</b>In the children aged 9-11 years in this area, the cut-off value of non-HDL-C level was 3.67 mmol/L, and the prevalence rate of a high non-HDL-C level was 3.7% (22/589). Compared with the non-obese children, the obese children had a significantly higher serum non-HDL-C level (P<0.01) and a significantly higher prevalence rate of a high non-HDL-C level (10.0% vs 2.9%; P<0.01).</p><p><b>CONCLUSIONS</b>The cut-off value of serum non-HDL-C level in children aged 9-11 years in the Mianyang Science City area is established. Obesity is associated with an increased prevalence rate of a high non-HDL-C level in children aged 9-11 years.</p>

Inhibition of hepatitis B virus (HBV) replication using antisense LNA targeting to both S and C genes in HBV / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory effect on HBV replication of antisense locked nucleic acid (LNA) targeting to both S and C genes in HBV transgenic mice.</p><p><b>METHODS</b>Thirty HBV transgenic mice were randomly divided into five groups (n = 6): glucose control group were treated with 5% glucose solution, liposome control group were treated with liposome alone, S group were treated with LNA targeting to S gene, C group were treated with LNA targeting to C gene, and dual-target group were treated with LNA targeting to both S and C genes. Antisense LNA was injected into mice via the tail vein. Serum HBsAg was quantified by TRFIA. Serum HBV DNA was quantified by real-time PCR. The expression of HBV C-mRNA in the liver was detected by RT-PCR. The expression of HBsAg and HBcAg in the liver was detected by immunohistochemistry. Serum ALB, ALT, BUN and CR were measured using an automatic biochemical analyzer. The effects of antisense LNA on mouse organs were investigated by HE staining.</p><p><b>RESULTS</b>5 days after LNA injection, serum HBsAg levels in the dual-target group were reduced by 72.8%, and serum HBV DNA levels were decreased by 52.9%. These values were significantly higher than those in the control groups (all P < 0.05). No significant differences were noted in serum ALB, ALT, BUN and CR between the experiment groups and the control groups (all P > 0.05). The expression levels of HBsAg and HBcAg in the liver of dual-target group were significantly lower than those in the control groups. No significant histopathological abnormality was found in liver and kidney tissues in all groups.</p><p><b>CONCLUSION</b>Antisense LNA targeting to both S and C genes can significantly inhibit HBV replication in transgenic mice.</p>

Bcl-xl blocks tumor necrosis factor alpha-induced caspase 8 activation and apoptosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the effect of Bcl-xl on tumor necrosis factor-alpha (TNFalpha)-induced apoptosis signal pathway and apoptosis.</p><p><b>METHODS</b>A dominant negative mutant of ikB (pmi kappaB) and Green Fluorescent Protein (GFP) expression plasmid pEGFP-C1, pmi kappab and pEGFP-C1 and Bcl-xl expression construct pBcl-xl/HA, were co-transfected into HeLa cells. Expression plasmid pBcl-xl/HA was introduced into p65-/-MEF cells in which nuclear factor-kappaB (NF-kappaB)/p65 was deficient, to establish cell line p65-/-Bcl-xl expressing Bcl-xl by selection with puromycin. These cells were treated with TNFalpha at a concentration of 10 ng/ml, and apoptotic cell death was examined microscopically with trypan blue staining. The proteins were abstracted from treated cells, and caspase 8 activation and cleavage of poly (ADP-ribose) polymerase (PARP) were examined by western blot using a specific antibody that recognized cleaved caspase 8 and cleaved PARP, respectively.</p><p><b>RESULTS</b>HeLa cells transfected with pmi kappaB, TNFalpha showed significant cell death as they became rounded, shrank, and detached. However in HeLa cells co-transfected with pBcl-xl and pmi kappaB, no cell death was observed after treatment with TNFalpha. In p65-/- MEF cells; cell death was observed at 4 hours after treatment with TNFalpha, and cell death reached 90% at 12 hours after the treatment. However, in p65-/-Bcl-xl/HA cells expressing Bcl-xl, no cell death was seen even when treated with TNFa for 24 hours. Meanwhile, in pmikB/HeLa cells transfected with pmi kappaB, TNFalpha induced caspase 8 activation and PARP cleavage, but in the HeLa cells co-transfected with pBcl-xl and pmi kappaB, no activated caspase 8 and cleaved PARP were observed after treatment with TNFalpha.</p><p><b>CONCLUSION</b>In the experimental system in which NF-kB was inhibited, Bcl-xl blocked TNFalpha-induced apoptosis signal pathway and apoptosis. These results bring to light that further studies of the pathogenesis and therapy of TNFa-related diseases are needed.</p>