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  

Construction of NTCP-Huh7 cell lines and their susceptibility to HBV

作　　者：朱甜甜 李毓雯[2] 胡平平 李军[3] 朱传龙 ZHU Tian-tian;LI Yu-wen;HU Ping-ping;LI Jun;ZHU Chuan-long(Department of Infectious Diseases,Provincial Hospital Affiliated of Anhui Medical University,Hefei 230001,Anhui,China;Department of Pediatrics;Department of Infectious Diseases,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,Jiangsu,China)

机构地区：[1]安徽医科大学附属省立医院感染科,合肥医学硕士研究生230001 [2]南京医科大学第一附属医院儿科,南京210029 [3]南京医科大学第一附属医院感染科,南京210029

出　　处：《医学研究生学报》2018年第11期1142-1147,共6页Journal of Medical Postgraduates

基　　金：国家自然科学基金(81770591)

摘　　要：目的功能性受体钠离子牛磺胆酸共转运蛋白(NTCP)表达短暂,导致乙型肝炎病毒(HBV)感染肝癌细胞模型的效率低下且不稳定。文中旨在将GV358-NTCP转染Huh7细胞构建NTCP-Huh7细胞,并鉴定其是否具有HBV易感性。方法使用GV358作为慢病毒载体,和NTCP编码基因连接后获得重组质粒GV358-NTCP,构建NTCP基因重组慢病毒,将GV358-NTCP转染Huh7细胞构建NTCP-Huh7细胞。采用HBV感染NTCP-Huh7细胞(NTCP-Huh7组)和Huh7细胞(Huh7组)。将HBV分别与NTCP-Huh7组和Huh7组细胞共孵育18 h,换液后继续培养,并在第2、4、6、8、10、12天做相关检测。检测上清中HBsAg、HBeAg、HBc Ag、HBV DNA以及细胞内HBV ccc DNA含量,确定NTCP-Huh7重组细胞是否具有HBV易感性。结果 Western blot结果表明NTCP-Huh7细胞株可稳定表达NTCP蛋白。其孵育第8天,NTCP-Huh7组HBsAg、HBeAg(0.866±0.040、0.603±0.053)明显高于Huh7组(0.237±0.063、0.209±0.112),且在第8天达到最高峰,第4、6、10天亦明显高于Huh7组(P<0.05)。NTCP-Huh7组第4、6、8、10、12天上清中HBV DNA、HBV ccc DNA明显高于Huh7组(P<0.05)。免疫荧光法显示NTCP-Huh7细胞内检测到核心抗原,Huh7细胞内未检测到。结论构建GV358-NTCP重组质粒转染Huh7后,获得的NTCP-Huh7细胞具有HBV易感性。Objective The transient expression of the functional receptor sodium taurocholate cotransporting polypeptide （NTCP） leads to the inefficiency and instability of the hepatitis B virus （HBV）-associated hepatocellular carcinoma cell model. The aim of this study was to construct NTCP-Huh7 cell lines by transfecting GV358-NTCP into Huh7 cells and identify their susceptibility to HBV. Methods The recombinant plasmid GV358-NTCP was obtained by ligation of GV358 and NTCP, and then transfected into Huh7 cells for the construction of NTCP-Huh7 cells. The NTCP-Huh7 cells （NTCP-Huh7 group） and Huh7 cells （Huh7 group） were infected with HBV and co-incubated with HBV for 18 hours, and the co-incubation was continued after change of the culture medium. At 2, 4, 6, 8, 10, and 12 days of incubation, the supernatant and cells were collected for measurement of the contents of HBsAg, HBeAg, HBcAg and HBV DNA in the supernatant and HBV cccDNA in the cells as well as for determination of HBV susceptibility of the NTCP -Huh7 recombinant cells. Results Western blot showed stably expressed NTCP proteins in the NTCP-Huh7 cells. At 8 days of incubation, the levels of HBsAg and HBeAg were significantly higher in the NTCP-Huh7 group （0.866±0.040 and 0.603±0.053） than in the Huh7 group （0.237±0.063 and 0.209±0.112） （ P 〈0.05）, reaching the peak at 8 days and also remarkably higher in the former than in the latter group at 4, 6 and 10 days （ P 〈 0.05）. So were the levels of HBV DNA and HBV cccDNA in the NTCP-Huh7 than in the Huh7 group at 4, 6, 8, 10 and 12 days （ P 〈 0.05）. Immunofluorescence assay revealed the core antigen of HBV in the NTCP-Huh7 but not in Huh7 cells. Conclusion NTCP-Huh7 cells obtained by transfection of the GV358-NTCP recombinant plasmid into Huh7 cells are susceptible to HBV infection.

关 键 词：乙型肝炎病毒 钠离子牛磺胆酸共转运蛋白 易感性 

分 类 号：R512.32[医药卫生—内科学]