DNA识别受体AIM2和cGAS相关的HBV感染体外模型建立
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摘要
目的构建黑色素瘤缺乏因子2(AIM2)和环鸟苷酸-腺苷酸合成酶(c GAS)相关的HBV感染肝细胞模型,为研究DNA识别受体在肝细胞内固有免疫的炎症作用提供媒介。方法从4种细胞系中筛选出AIM2和cGAS相对表达高的细胞系。通过慢病毒转染构建稳定过表达钠离子-牛磺胆酸共转运蛋白(NTCP)的该细胞系。超离和纯化的方法获取高纯度高HBV病毒量的血清液,用该血清培养细胞系,3 d后通过罗氏PCR方法测定细胞上清HBV病毒量,ELISA方法测定细胞上清液中乙型肝炎表面抗原(HBsAg)和e抗原(HBeAg)。结果 Western blot及rtPCR结果显示4种细胞系中7721细胞系的AIM2和cGAS均高表达,其中AIM2的mRNA在各细胞系组间差异有统计学意义(P<0.05),cGAS的差异无统计学意义(P>0.05)。成功构建稳定过表达NTCP的7721细胞系后,感染病毒3 d后细胞上清可检测出HBV病毒和HBsAg阳性,HBeAg阴性。结论 HBV感染的7721细胞系构建成功,可利用该细胞系进行AIM2和cGAS在肝细胞内固有免疫相关研究。
Objective To establish an in vitro model for HBV infection associated with DNA recognition receptors absent in melanoma 2(AIM2)and cyclic GMP-AMP synthase(c GAS),and for the study of role of DNA recognition receptors in the innate immunity of hepatocytes. Methods Cells expressing high levels of both AIM2 and cGAS were secreened from four liver cell lines. Lentivirus carrying the sodium taurocholate cotransporting polypeptide(NTCP)gene was used to infect the screened cells and the infected cells with stable expression of NTCP were selected. High purity and high HBV virus quantity serum obtained by super separation and purification was used to infect the selected cells. After 3 days of infection,the HBV virus quantity of the culture supernatant was determined by Roche PCR. Hepatitis B surface antigen(HBsAg)and hepatitis Be antigen(HBeAg)in supernatant were detected by ELISA. Results The results showed that among these 4 cell lines,7721 is the one in which both AIM2 and cGAS highly express.And AIM2 mRNA expression in 7721 was significant higher than that in other cell lines(P<0.05),while there was no significance difference of c GAS mRNA among 4 cell lines(P>0.05). The transfection of lentivirus with NTCP gene successfully made the NTCP stably express of in 7721. The model in vitro of HBVinfection was also successfully established in 7721 finally. And in the third day after HBV infecting,the HBV and HBsAg could be detected in the cellular supernatant,but not HBeAg. Conclusion the cell model 7721 with HBV infection was successfully established. This cell can be used for the study of the innate immunity triggered by DNA receptor in hepatocytes.
Objective To establish an in vitro model for HBV infection associated with DNA recognition receptors absent in melanoma 2(AIM2)and cyclic GMP-AMP synthase(c GAS),and for the study of role of DNA recognition receptors in the innate immunity of hepatocytes. Methods Cells expressing high levels of both AIM2 and cGAS were secreened from four liver cell lines. Lentivirus carrying the sodium taurocholate cotransporting polypeptide(NTCP)gene was used to infect the screened cells and the infected cells with stable expression of NTCP were selected. High purity and high HBV virus quantity serum obtained by super separation and purification was used to infect the selected cells. After 3 days of infection,the HBV virus quantity of the culture supernatant was determined by Roche PCR. Hepatitis B surface antigen(HBsAg)and hepatitis Be antigen(HBeAg)in supernatant were detected by ELISA. Results The results showed that among these 4 cell lines,7721 is the one in which both AIM2 and cGAS highly express.And AIM2 mRNA expression in 7721 was significant higher than that in other cell lines(P<0.05),while there was no significance difference of c GAS mRNA among 4 cell lines(P>0.05). The transfection of lentivirus with NTCP gene successfully made the NTCP stably express of in 7721. The model in vitro of HBVinfection was also successfully established in 7721 finally. And in the third day after HBV infecting,the HBV and HBsAg could be detected in the cellular supernatant,but not HBeAg. Conclusion the cell model 7721 with HBV infection was successfully established. This cell can be used for the study of the innate immunity triggered by DNA receptor in hepatocytes.
引文
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[14]Chen C,Zhu X,Xu W,et al.IFNA2 p.Ala120Thr impairs the inhibitory activity of Interferon-alpha2 against the hepatitis B virus through altering its binding to the receptor[J].Antiviral Res,2017,147:11-18.
[15]Zhu X,Xie C,Li YM,et al.TMEM2 inhibits hepatitis B virus infection in Hep G2 and Hep G2.2.15 cells by activating the JAKSTAT signaling pathway[J].Cell Death Dis,2016,7(6):e2239.
[16]Peng L,Zhao Q,Li Q,et al.The p.Ser267Phe variant in SLC10A1 is associated with resistance to chronic hepatitis B[J].Hepatology,2015,61(4):1251-1260.
[2]冯玉奎.乙型肝炎免疫发病机制研究现状[J].热带医学杂志,2013,13(9):1176-1178.Feng YK.The state of immune pathogenesis research in Hepatitis B[J].J Trop Med,2013,13(9):1176-1178.
[3]Pan X,Xu H,Zheng C,et al.Human hepatocytes express absent in melanoma 2 and respond to hepatitis B virus with interleukin-18 expression[J].Virus Genes,2016,52(4):445-452.
[4]Dansako H,Ueda Y,Okumura N,et al.The cyclic GMP-AMP synthetase-STING signaling pathway is required for both the innate immune response against HBV and the suppression of HBV assembly[J].FEBS J,2016,283(1):144-156.
[5]Thomsen MK,Nandakumar R,Stadler D,et al.Lack of immunological DNA sensing in hepatocytes facilitates hepatitis B virus infection[J].Hepatology,2016,64(3):746-759.
[6]Karimi-Googheri M,Daneshvar H,Khaleghinia M,et al.Decreased expressions of STING but not IRF3 molecules in chronic HBV infected patients[J].Arch Iran Med,2015,18(6):351-354.
[7]Schulze-Bergkamen H,Untergasser A,Dax A,et al.Primary human hepatocytes--a valuable tool for investigation of apoptosis and hepatitis B virus infection[J].J Hepatol,2003,38(6):736-744.
[8]Hantz O,Parent R,Durantel D,et al.Persistence of the hepatitis B virus covalently closed circular DNA in Hepa RG human hepatocyte-like cells[J].J Gen Virol,2009,90(Pt 1):127-135.
[9]Yan H,Zhong G,Xu G,et al.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus[J].Elife,2012,1:e00049.
[10]Ni Y,Urban S.Hepatitis B virus infection of Hepa RG cells,Hepa RG-h NTCP cells,and primary human hepatocytes[J].Methods Mol Biol,2017,1540:15-25.
[11]Gonzalez SA,Keeffe EB.Chronic viral hepatitis:epidemiology,molecular biology,and antiviral therapy[J].Front Biosci(Landmark Ed),2011,16:225-250.
[12]黄劲,庞秀青,谢冬英.DNA识别受体在乙型肝炎发病机制中的作用[J].临床肝胆病杂志,2017,33(9):1794-1797.Huang J,Pang XQ,Xie DY.Role of DNA recognition receptors in pathogenic mechanism of hepatitis B[J].J Clin Hepatol,2017,33(9):1794-1797.
[13]Sun L,Wu J,Du F,et al.Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway[J].Science,2013,339(6121):786-791.
[14]Chen C,Zhu X,Xu W,et al.IFNA2 p.Ala120Thr impairs the inhibitory activity of Interferon-alpha2 against the hepatitis B virus through altering its binding to the receptor[J].Antiviral Res,2017,147:11-18.
[15]Zhu X,Xie C,Li YM,et al.TMEM2 inhibits hepatitis B virus infection in Hep G2 and Hep G2.2.15 cells by activating the JAKSTAT signaling pathway[J].Cell Death Dis,2016,7(6):e2239.
[16]Peng L,Zhao Q,Li Q,et al.The p.Ser267Phe variant in SLC10A1 is associated with resistance to chronic hepatitis B[J].Hepatology,2015,61(4):1251-1260.