目的:探討丙型肝炎病毒非結構蛋白NS4B在原發性肝癌發生中的作用,及其發生機制。方法:設置對照組、空白載體PCXN2組、轉染NS4B組。使用脂質體介導法,轉染丙型肝炎病毒非結構蛋白重組質粒NS4B進入Chang肝細胞內,并用G418篩選。繪制生長曲線,分別用流式細胞儀檢測瞬時表達及穩定表達時肝細胞凋亡率,用均數±標準差表示,統計學分析采用Dunnett t檢驗及q檢驗。結果:瞬時表達各組(PCXN2,NS4B)凋亡率分別為:(918±060)%,(445±053)%。穩定表達各組(PCXN2,NS4B)凋亡率分別為:(1575±209)%,(366±034)%。與PCXN2組比較Plt;001。結論:NS4B抑制肝細胞凋亡率,可能導致肝細胞異常增殖,誘導肝癌發生。
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.
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.
【摘要】 目的 探討丙型肝炎病毒非結構蛋白NS4B對肝細胞內p53表達的影響，以及在肝癌發生中的作用與機制。 方法 設置空白對照組、空白載體組、轉染NS4B組、轉染p53組、共轉染NS4B及p53組。使用脂質體介導轉染法，轉染丙型肝炎病毒非結構蛋白重組質粒PCXN2-NS4B及突變型p53基因重組質粒pC53-CX22AN3進入Chang肝細胞內，并用G418篩選獲得穩定表達細胞。采用免疫細胞化學法檢測p53表達率。 結果 空白對照組無p53表達，空白載體組及轉染NS4B組呈弱陽性表達，轉染p53組及共轉染組呈陽性表達；轉染p53組、共轉染組分別與空白對照組、空白載體組及轉染NS4B組比較，差異均有統計學意義 （Plt;0.05）。 結論 NS4B可能抑制p53表達，也可能阻止其進入細胞核，但NS4B與突變型p53關系不明確。NS4B導致肝細胞異常增生，誘導肝癌發生可能不依賴p53的異常表達及突變。【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.
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.
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.