乙型丙型肝炎病毒合并感染细胞模型的建立
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摘要
目的:利用慢病毒载体在可感染丙型肝炎病毒(hepatitis C virus,HCV)的Huh7.5.1细胞中过表达人肝细胞乙型肝炎病毒(hepatitis B virus,HBV)受体钠离子牛磺胆酸共转运蛋白(sodium taurocholate cotransporting polypeptide,NTCP)编码基因,建立Huh7.5.1-hNTCP细胞模型,使其可以被HBV、HCV共同感染。方法:使用携带人NTCP编码基因、绿色荧光蛋白(green fluorescent protein,GFP)基因、嘌呤霉素抗性基因的GV358重组慢病毒载体,以感染复数(MOI)50感染Huh7.5.1细胞。经过嘌呤霉素筛选获得稳定表达人NTCP基因的Huh7.5.1-hNTCP细胞株,并通过Western blot验证NTCP蛋白的表达。使用HepAD38细胞来源的HBV感染Huh7.5.1-hNTCP细胞,通过ELISA及qPCR等方法检测HBV感染后不同时间点乙肝病毒表面抗原(HBsAg)、e抗原(HBeAg)的表达以及HBV DNA的复制。HBV感染后24 h予以HCV-JFH1感染,检测合并感染状态下HBV DNA以及HCV RNA的复制。结果:Western blot结果表明Huh7.5.1-hNTCP细胞株可稳定表达HBV受体NTCP。HBV感染Huh7.5.1-hNTCP细胞后ELISA、qPCR结果显示,HBsAg、HBeAg、HBV DNA合成逐渐增加,并在第9天达峰。HBV/HCV合并感染Huh7.5.1-hNTCP细胞后,qPCR检测结果显示不同时间点HBV DNA、HCV RNA复制逐渐增加。结论:建立了Huh7.5.1-hNTCP细胞模型,该模型可被HBV/HCV合并感染。
Objective:To establish a sodium taurocholate cotransporting polypeptide(NTCP)-complemented Huh7.5.1 cell line(Huh7.5.1-hNTCP)that supports hepatitis B virus(HBV)and hepatitis C virus(HCV)co-infection. Methods:Huh7.5.1 cells were infected with GV358 recombinant lentiviral vector carrying human NTCP encoding gene,green fluorescent protein(GFP)gene and puromycin resistance gene with the multiplicity of infection(MOI)of 50. NTCP positive cells were obtained through the puromycin screening and NTCP expression was identified by Western blotting. Huh7.5.1-h NTCP cells were infected with HBV derived from HepAD38 cells. The expression of HBsAg,HBeAg and HBV DNA at different time points were detected by ELISA and qPCR. HCVJFH1 was added 24 hours after HBV infection,and then HBV DNA and HCV RNA were detected in co-infected cells. Results:Western blotting results indicated that Huh7.5.1-hNTCP cell line stably expressed the HBV receptor NTCP. ELASA and q PCR results showed that HBsAg,HBeAg and HBV DNA secretion increased gradually and peaked on the 9 thday in HBV infected Huh7.5.1-hNTCP cells. The results of qPCR suggested that the replication of HBV DNA and HCV RNA increased gradually at different time points in HBV and HCV co-infected cells. Conclusion:Huh7.5.1-hNTCP cell line was established,and it can be co-infected by HBV and HCV.
Objective:To establish a sodium taurocholate cotransporting polypeptide(NTCP)-complemented Huh7.5.1 cell line(Huh7.5.1-hNTCP)that supports hepatitis B virus(HBV)and hepatitis C virus(HCV)co-infection. Methods:Huh7.5.1 cells were infected with GV358 recombinant lentiviral vector carrying human NTCP encoding gene,green fluorescent protein(GFP)gene and puromycin resistance gene with the multiplicity of infection(MOI)of 50. NTCP positive cells were obtained through the puromycin screening and NTCP expression was identified by Western blotting. Huh7.5.1-h NTCP cells were infected with HBV derived from HepAD38 cells. The expression of HBsAg,HBeAg and HBV DNA at different time points were detected by ELISA and qPCR. HCVJFH1 was added 24 hours after HBV infection,and then HBV DNA and HCV RNA were detected in co-infected cells. Results:Western blotting results indicated that Huh7.5.1-hNTCP cell line stably expressed the HBV receptor NTCP. ELASA and q PCR results showed that HBsAg,HBeAg and HBV DNA secretion increased gradually and peaked on the 9 thday in HBV infected Huh7.5.1-hNTCP cells. The results of qPCR suggested that the replication of HBV DNA and HCV RNA increased gradually at different time points in HBV and HCV co-infected cells. Conclusion:Huh7.5.1-hNTCP cell line was established,and it can be co-infected by HBV and HCV.
引文
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[16] Yao WL,Ikeda S,Tsukamoto Y,et al. Establishment of a human hepatocellular cell line capable of maintaining long-term replication of hepatitis B virus[J]. Int Immunol,2017,29(3):109-120
[17] Ni Y,Lempp FA,Mehrle S,et al. Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes[J]. Gastroenterology,2014,146(4):1070-1083
[18] Yan R,Zhang Y,Cai D,et al. Spinoculation enhances HBV infection in NTCP-reconstituted hepatocytes[J].PLoS One,2015,10:e0129889
[19] Zhu C,Xiao F,Hong J,et al. EFTUD2 is a novel innate immune regulator restricting hepatitis C virus infection through the RIG-I/MDA5 pathway[J]. J Virol,2015,89(13):6608-6618
[20] Potthoff A,Manns MP,Wedemeyer H. Treatment of HBV/HCV co-infection[J]. Expert Opin Pharmacother,2010,11(6):919-928
[21]张若洋,范恩勇,王明元,等.基于PCR-化学发光的HBV/HCV/HIV并行核酸检测试剂盒的评价[J].南京医科大学学报(自然科学版),2017,37(3):335-339
[22] Sagnelli E,Coppola N,Marrocco C,et al. Hepatitis C virus superinfection in hepatitis B virus chronic carriers:a reciprocal viral interaction and a variable clinical course[J]. J Clin Virol,2006,35(3):317-320
[23]Liaw YF. Hepatitis C virus superinfection in patients with chronic hepatitis B virus infection[J]. J Gastreenterol,2002,37(Suppl 13):65-68
[24] Schuttler G,Fiedler N,Schmidt K,et al. Suppression of hepatitis B virus enhancer l and 2 by hepatitis C virus core protein[J]. J Hepatol,2002,37(6):855-862
[2] Caccamo G,Saffioti F,Raimondo G. Hepatitis B virus and hepatitis C virus dual infection[J]. World J Gastroenter-ol,2014,20(40):14559-14567
[3] Ramboer E,Craene BD,Kock JD,et al. Strategies for im-mortalization of primary hepatocytes[J]. J Hepatol,2014,61(4):925-943
[4] Wu Q,Li Z,Liu Q. An important role of SREBP-1 in HBVand HCV co-replication inhibition by PTEN[J]. Virology,2018,520:94-102
[5] Wakita T,Pietschmann T,Kato T,et al. Production of in-fectious hepatitis C virus in tissue culture from a clonedviral genome[J]. Nat Med,2005,11(7):791-796
[6] Zhong J,Gastaminza P,Cheng G,et al. Robust hepatitis Cvirus infection in vitro[J]. Proc Natl Acad Sci U S A,2005,102(26):9294-9299
[7] Yan H,Zhong G,Xu G,et al. Sodium taurocholate cotrans-porting polypeptide is a functional receptor for humanhepatitis B and D virus[J]. eLife,2012,1(1):e00049
[8] Hayes CN,Chayama K. HBV culture and infectious sys-tems[J]. Hepatol Int,2016,10(4):559-566
[9] Masashi I,Koichi W,Senko T,et al. Evaluation and iden-tification of hepatitis B virus entry inhibitors usingHepG2 cells overexpressing a membrane transporterNTCP[J]. Biochem Biophys Res Commun,2014,443(3):808-813
[10] Sun Y,Qi Y,Peng B,et al. NTCP-reconstituted in vitro HBV infection system[M]//Guo HT,Cuconati A. Hepati-tis B virus,methods and protocols. New York:HumanaPress,2017,1540:1-14
[11] Ren JH,Hu JL,Cheng ST,et al. SIRT3 restricts HBVtranscription and replication via epigenetic regulation ofcccDNA involving SUV39H1 and SETD1A histone meth-yltransferases[J]. Hepatology,2018,68(4):1260-1276
[12] Ladner SK,Otto MJ,Barker CS,et al. Inducible expres-sion of human hepatitis B virus(HBV)in stably transfect-ed hepatoblastoma cells:a novel system for screening po-tential inhibitors of HBV replication[J]. AntimicrobAgents Chemother,1997,41(8):1715-1720
[13]王坤,朱甜甜,李毓雯,等. c-met慢病毒载体的构建及稳定转染骨髓间充质干细胞[J].安徽医科大学学报,2017,52(4):597-601
[14]朱甜甜,李毓雯,胡平平,等.功能性受体钠离子牛磺胆酸共转运蛋白-Huh7细胞株的构建及其HBV易感性[J].医学研究生学报,2018,31(11):1142-1147
[15] Okuyamadobashi K,Kasai H,Tanaka T,et al. Hepatitis B virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide[J]. Sci Rep,2015,5:17047
[16] Yao WL,Ikeda S,Tsukamoto Y,et al. Establishment of a human hepatocellular cell line capable of maintaining long-term replication of hepatitis B virus[J]. Int Immunol,2017,29(3):109-120
[17] Ni Y,Lempp FA,Mehrle S,et al. Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes[J]. Gastroenterology,2014,146(4):1070-1083
[18] Yan R,Zhang Y,Cai D,et al. Spinoculation enhances HBV infection in NTCP-reconstituted hepatocytes[J].PLoS One,2015,10:e0129889
[19] Zhu C,Xiao F,Hong J,et al. EFTUD2 is a novel innate immune regulator restricting hepatitis C virus infection through the RIG-I/MDA5 pathway[J]. J Virol,2015,89(13):6608-6618
[20] Potthoff A,Manns MP,Wedemeyer H. Treatment of HBV/HCV co-infection[J]. Expert Opin Pharmacother,2010,11(6):919-928
[21]张若洋,范恩勇,王明元,等.基于PCR-化学发光的HBV/HCV/HIV并行核酸检测试剂盒的评价[J].南京医科大学学报(自然科学版),2017,37(3):335-339
[22] Sagnelli E,Coppola N,Marrocco C,et al. Hepatitis C virus superinfection in hepatitis B virus chronic carriers:a reciprocal viral interaction and a variable clinical course[J]. J Clin Virol,2006,35(3):317-320
[23]Liaw YF. Hepatitis C virus superinfection in patients with chronic hepatitis B virus infection[J]. J Gastreenterol,2002,37(Suppl 13):65-68
[24] Schuttler G,Fiedler N,Schmidt K,et al. Suppression of hepatitis B virus enhancer l and 2 by hepatitis C virus core protein[J]. J Hepatol,2002,37(6):855-862