Objective To investigate the effect of hepatitis C virus (HCV) F protein on proliferation and collagen expression of hepatic stellate cells. Methods After pcDNA3.1-f plasmid containing HCV f gene or empty pcDNA3.1 plasmid was transfected hepatic stellate cells LX2 by liposome, LX-f or LX-p cells were obtained by G418 screening. The proliferation of LX-f or LX-p cells was analyzed by MTT, and the contents of collagen type Ⅰand Ⅲ secreted by LX-f or LX-p cells were detected by ELISA. Results After 24 h cultivation, the proliferation rate of LX-f cells was higher than that of LX-p cells at each time point （Plt;0.01）. After 48 h cultivation, the contents of collagen typeⅠand Ⅲ secreted by LX-f were (25.89±0.42) ng/ml and (18.21±0.49) ng/ml, which was significantly higher than those of LX-p cells 〔(22.65±0.49) ng/ml and (15.29±0.62) ng/ml〕, Plt;0.01. Conclusion HCV F protein is able to promote proliferation of hepatic stellate cells, and up-regulate the excretion of collagen type Ⅰand Ⅲ in those cells, which induces hepatic fibrosis.
【摘要】 目的 探讨丙型肝炎病毒非结构蛋白NS4B对肝细胞内p53表达的影响，以及在肝癌发生中的作用与机制。 方法 设置空白对照组、空白载体组、转染NS4B组、转染p53组、共转染NS4B及p53组。使用脂质体介导转染法，转染丙型肝炎病毒非结构蛋白重组质粒PCXN2-NS4B及突变型p53基因重组质粒pC53-CX22AN3进入Chang肝细胞内，并用G418筛选获得稳定表达细胞。采用免疫细胞化学法检测p53表达率。 结果 空白对照组无p53表达，空白载体组及转染NS4B组呈弱阳性表达，转染p53组及共转染组呈阳性表达；转染p53组、共转染组分别与空白对照组、空白载体组及转染NS4B组比较，差异均有统计学意义 （Plt;0.05）。 结论 NS4B可能抑制p53表达，也可能阻止其进入细胞核，但NS4B与突变型p53关系不明确。NS4B导致肝细胞异常增生，诱导肝癌发生可能不依赖p53的异常表达及突变。【Abstract】 Objective To investigate the effect of hepatitis C Virus on-structural protein 4B(HCV NS4B) on expression of p53 in hepatic cell, and to study the role and mechanism in development of hepatocellular carcinoma. Methods The experiment was divided into negative control, pure vector PCXN2, PCXN2-NS4B, PC53-cx22AN3, and co-transfection group. Recombinant plasmid PCXN2-NS4B and mutant p53 gene--PC53-cx22AN3, PC53-cx22AN3 with PCXN2-NS4B, blank vectors were transfected into Chang liver cell by liposome-mediated transfection respectively. Positive cells were screened by G418. The expression rate of p53 was measured by immunocytochemistry. Result No expression rate of p53 gene in control group was found, lower positive expression in group PCXN2 and PCXN2-NS4B. The expression of p53 gene in group PC53-CX22AN3 and co-transfection was ber than the others (Plt;0.005). Conclusion HCV-NS4B may inhibit the expression of p53 gene, and it may play a crucial role in inhibiting p53 transfered to hepatic cells nuclear. But it isn’t clear that the. HCV-NS4B can enhance the role of mutant p53 gene. It suggested that HCV-NS4B induce proliferation of hepatic cell not through regulating the expression of p53.
目的:探讨丙型肝炎病毒非结构蛋白NS4B在原发性肝癌发生中的作用,及其发生机制。方法:设置对照组、空白载体PCXN2组、转染NS4B组。使用脂质体介导法,转染丙型肝炎病毒非结构蛋白重组质粒NS4B进入Chang肝细胞内,并用G418筛选。绘制生长曲线,分别用流式细胞仪检测瞬时表达及稳定表达时肝细胞凋亡率,用均数±标准差表示,统计学分析采用Dunnett t检验及q检验。结果:瞬时表达各组(PCXN2,NS4B)凋亡率分别为:(918±060)%,(445±053)%。稳定表达各组(PCXN2,NS4B)凋亡率分别为:(1575±209)%,(366±034)%。与PCXN2组比较Plt;001。结论:NS4B抑制肝细胞凋亡率,可能导致肝细胞异常增殖,诱导肝癌发生。
Hepatitis C virus (HCV) do harm to people's health. The present study aims to establish a simple HCV detection method by reverse transcription-loop mediated isothermal amplification technique (RT-LAMP). A total of 75 clinical samples were collected and pre-detected by fluorescence quantitative-polymerase chain reaction (FQ-PCR), which was considered as the gold standard. Firstly, four common primers were designed according to the conservative 5'UTR region of HCV on the NCBI website to establish an integrated RT-LAMP reaction system. Then, the reaction efficiency was evaluated by adding Taq DNA polymerase to the conventional system. The specificity of RT-LAMP was evaluated by observing the length of fragment after endonuclease digestion and by a templates exchange assay, the sensitivity of RT-LAMP was evaluated by detection of diluted clinical templates. The results were compared with that of reverse transcription polymerase chain reaction (RT-PCR). At the same time, the performance was judged using calcein and hydroxynaphthol blue (HNB) stain methods, The two results were compared with that of electrophoresis method. At last, 75 clinical samples were detected by both RT-LAMP and RT-PCR methods. Results showed that the reaction efficiency was increased 20 minutes after adding Taq DNA polymerase to the normal RT-LAMP system. RT-LAMP showed good specificity, the digestion length was consistent with our expectation (216 bp) after restriction endonuclease cleavage assay, and only the templates of HCV were amplified using the common RT-LAMP primers. After detection of diluted temples, the sensitivity of RT-LAMP was 10 IU/tube, which was 10 fold higher than that of PCR. In addition, the results using calcein and HNB stain methods were the same with that of electrophoresis method. After detection of all 75 clinical samples, the results indicated that RT-LAMP had worse consistency with RT-PCR (P < 0.05, Kappa=0.375). However, RT-LAMP, on the contrary, showed good consistency with FQ-PCR (P > 0.05, Kappa=0.762). In conclusion, RT-LAMP has characteristics of simplicity, specificity and sensitivity, and this technique is suitable for the primary care hospitals.
ObjectiveTo explore the association between viral hepatitis and extrahepatic cholangiocarcinoma (ECC). MethodsDatabase of Medline, Embase, PubMed, CNKI, and Wanfang were searched for the articles which were related to the relationship between viral hepatitis and ECC. After the quality evaluation and the data extraction of the literatures, statistical software of RevMan 5.0 was used to perform Meta analysis. ResultsAccording to the inclusion criteria and exclusion criteria, 9 articles were enrolled, 8 articles of them were related to hepatitis B virus(HBV) and 6 articles of them were related to hepatitis C virus(HCV). Meta analysis results showed that the HBV infection may be the risk factor for ECC(OR=1.69, 95% CI:1.32-2.17, P<0.000 1). In the United States, HCV infection may be the risk factor for ECC(OR=5.53, 95% CI:2.21-13.82, P=0.000 3), but the relationship was not found in China(OR=0.82, 95% CI:0.44-1.52, P=0.520 0). ConclusionsThe present studies suggest that HBV infection may be a high risk factor for ECC. HCV in the United States can increase the incidence of ECC, but the situation can not be found in China.
Objective To investigate HCV genotypes in HCV patients in West China Hospital of Sichuan University, and to analyze the major genotypes and clinical characteristics. Methods From March 2011 to September 2016, 4 520 HCV patients who were successfully genotyped HCV genotypes were enrolled in West China Hospital of Sichuan University. The genotypes distributions and the characteristics of laboratory characteristics of liver function, the viral loading were all analyzed. In addition, the genotypes in HCC patients, liver cirrhosis, HBC/HCV co-infection were also analyzed. Results HCV genotypes of HCV patients were divided into five genotypes of 1, 2, 3, 4, 6 and 23 subtypes, including predominant genotypes/subtypes 1b, 1*, 3b, 2a, 3a and 6a, accounting for 66.42%, 8.01%, 6.57%, 4.54%, 4.29%, and 3.41%, respectively. Subtype 1b was the predominant subtype for both sex. In male patients, the levels of ALT were highest in 6a subtype, while in female, the levels of ALT were highest in 3a subtype. For the 94 liver cirrhosis patients, 42 patients were 1b subtypes; as for the 6 HCC patients, 1b and 3b subtypes were the only detected. Conclusion HCV genotypes/subtypes of HCV patients in West China Hospital of Sichuan University have unique characteristics of distribution, while the predominant genotype/subtypes are 1b,1*, 3b, 2a, 3a, 6a.