Ad-HBV4.1在人肝癌细胞株HepG2中的复制情况及在BalB/C小鼠肝细胞中的表达观察  

Replication of Ad-HBV4.1 in human hepatoma cell line HepG2 and its expression in liver cells of BalB/C mice

作　　者：张媛媛 ZHANG Yuanyuan(Sichuan Integrative Medicine Hospital, Chengdu 610041, China)

机构地区：[1]四川省中西医结合医院,成都610041

出　　处：《山东医药》2018年第31期26-29,共4页Shandong Medical Journal

摘　　要：目的观察复制型HBV重组腺病毒Ad-HBV4.1在人肝癌细胞株HepG2中的复制情况及在Bal B/C小鼠肝细胞内的表达情况。方法用HBV4.1片段与重组腺病毒同源重组构建Ad-HBV4.1,测算Ad-HBV4.1的感染复数(MOI)。取MOI为100的Ad-HBV4.1感染HepG2细胞,采用免疫组化法检测Ad-HBV4.1感染的HepG2细胞HBs Ag、HBcAg,采用Southern吸印杂交法检测细胞HBV DNA复制中间体HBV DNA复制中间体。取Bal B/C小鼠30,分为5组,每组6只。一组1 m L的Ad-HBV4.1(约108efu/m L)尾静脉普通注射,二组1 m L的Ad-HBV4.1腹腔注射。三组10μg的pHBV4.1片段用1.8 m L生理盐水稀释后,尾静脉高压5~8 s内完成注射。四、五组分别生理盐水1.8 m L尾静脉、腹腔注射。注射第3 d处死三组小鼠,注射第1、3、5 d处死其余各组小鼠,取出肝组织,采用免疫组化法检测各组小鼠肝组织HBcAg。结果 Ad-HBV4.1感染的HepG2胞质中存在HBcAg表达,阳性率达90%以上,HepG2细胞胞质中未见HBcAg表达。转染72 h时,HepG2细胞中未能检测到HBV DNA复制中间体;Ad-HBV4.1感染的HepG2细胞中存在HBV DNA复制中间体。第1、3 d时一组小鼠肝组织中未见HBcAg,第5 d小鼠肝组织中可见HBcAg;第3 d三组肝组织中可见HBcAg;各时段二、三、四组小鼠肝组织均未见HBcAg表达。结论 Ad-HBV4.1感染HepG2细胞成功建立HBV复制与表达的体外模型,感染效率达90%以上,可见HBs Ag、HBcAg及HBV DNA复制中间体表达。Ad-HBV4.1尾静脉注射Bal B/C小鼠肝组织存在HBV表达。Objective To observe the replication of replicative HBV recombinant adenovirus Ad-HBV4. 1 in the human hepatoma cell line HepG2 and its expression in liver cells of Bal B/C mice. Methods Ad-HBV4. 1 was constructed by homologous recombination with HBV4. 1 fragment and recombinant adenovirus,and the multiplicity of infection（MOI）of Ad-HBV4. 1 was measured. HepG2 cells were infected with Ad-HBV4. 1 with an MOI of 100. HBs Ag and HBcAg were detected by immunohistochemistry in HepG2 cells infected with Ad-HBV4. 1,and HBV DNA replication intermediate HBV DNA replication was detected by Southern blot hybridization. Thirty Bal B/C mice were divided into 5 groups with 6 in each. Mice in the first group were given 1 m L Ad-HBV4. 1（about 108 efu/m L） through caudal vein injection. Mice in the second group were given 1 m L Ad-HBV4. 1 through intraperitoneal injection. In the third group,after 10 μg of pHBV4. 1 fragment were diluted with 1. 8 m L of physiological saline,we completed the injection within 5-8 s through the caudal vein.The mice in the fourth and fifth groups were injected with 1. 8 m L of normal saline through caudal vein and intraperitoneal injection. Three groups of mice were sacrificed on the 3 rd day after injection. The mice of the other groups were sacrificed on the 1 st,3 rd and 5 th day after injection. The liver tissues were taken out and the liver tissue HBcAg was detected by immunohistochemistry. Results The expression of HBs Ag was observed in the cytoplasm of HepG2 infected by Ad-HBV4.1,and the positive rate was over 90%. No expression of HBs Ag was found in the cytoplasm of HepG2 cells. The expression of HBcAg was observed in the cytoplasm of HepG2 infected with Ad-HBV4. 1,and the positive rate was over 90%.HBcAg expression was not found in the cytoplasm of HepG2 cells. At 72 h after transfection,HBV DNA replication intermediates were not detected in HepG2 cells; HBV DNA replication intermediates were present in Ad-HBV4. 1-infected HepG2 cells. On the 1 st and 3 rd day,HBcAg was not found

关 键 词：乙型肝炎 乙肝病毒 重组腺病毒 复制型HBV重组腺病毒Ad-HBV4.1 病毒复制 

分 类 号：R735.7[医药卫生—肿瘤]