改良Alu-PCR技术检测HepG2.2.15细胞HBV整合的研究  

Detection of hepatitis B viral DNA integration in HepG2.2.15 cells by a modified Alu-PCR method

作　　者：阮鹏[1] 何春萍 黄超 周瑞 RUAN Peng;HE Chun-ping;HUANG Chao;ZHOU Rui(Department of Gastroenterology,Renmin Hospital of Wuhan University,Hubei 430060,China)

机构地区：[1]武汉大学人民医院消化内科,湖北430060

出　　处：《肝脏》2022年第7期785-788,共4页Chinese Hepatology

基　　金：十堰市科学技术研究与开发项目(15Y37);湖北省自然科学基金面上项目(2020CFB608)。

摘　　要：目的采用改良Alu-PCR技术检测HepG2.2.15细胞中HBV整合位点。方法对传统检测HBV整合的Alu-PCR技术进行改良简化,检测HepG2.2.15细胞中的HBV整合位点,HepG2细胞进行对照。RT-PCR定量检测HepG2.2.15细胞中检测到的HBV整合位点和cccDNA水平。结果在HepG2.2.15细胞中检测到一插入Alu重复序列的HBV整合位点,病毒结合端为1228 nt,嵌合片段有3bp的同源序列(CTG)。加入双氧水(H_(2)O_(2))的HepG2.2.15细胞中该整合位点水平为(-1.13±0.07)lg拷贝/细胞高于未加入H_(2)O_(2)的(-2.10±0.82)lg拷贝/细胞,差异有统计学意义(P<0.01)。加入H_(2)O_(2)的HepG2.2.15细胞中cccDNA水平为(-1.94±1.45)lg拷贝/细胞,未加入H_(2)O_(2)的为(-1.79±1.40)lg拷贝/细胞,差异无统计学意义(P=0.915)。该整合位点和cccDNA水平无显著相关性(P=0.463)。结论采用简易、经济的改良Alu-PCR技术检测HBV整合位点有效简化了实验步骤和节省实验费用,大大降低了HBV整合研究的门槛。对整合位点的定量检测显示肝细胞持续损伤可导致HBV整合细胞的优势克隆扩增。Objective To investigate the integration site of hepatitis B viral DNA(HBV DNA)in HepG2.2.15 cells using a modified Alu-PCR method.Methods This study detected the HBV integration site in HepG2.2.15 cells using a simplified Alu-PCR method,followed by a quantitative analysis of the integration site that was found in HepG2.2.15 cells with and without H_(2)O_(2)treatment by RT-qPCR.Results One HBV integration site was found.The binding junction of the inserted viral fragment was at 1,228nt of HBV DNA with 3 bp(CTG)of microhomology.The viral fragment was inserted into Alu repeats of host DNA in HepG2.2.15 cells.Logarithmically transformed analysis(log10copies/cell)showed that the average copy numbers of this integration site in the cells with H_(2)O_(2)treatment(-1.13±0.07)were significant higher than those without H_(2)O_(2)treatment(-2.10±0.82,P<0.001).No significant difference was found between the HBV cccDNA levels in cells with and without H_(2)O_(2)treatment(-1.94±1.45 and-1.79±1.40,P=0.915).No correlation was found between cccDNA level and the integration site in the cells(P=0.463).Conclusion This study provided a cost-effective,simplified method for the detection of HBV DNA integration by a modified Alu-PCR method.

关 键 词：乙型肝炎病毒 HBV整合 Alu-PCR技术 HEPG2.2.15细胞 

分 类 号：R512.62[医药卫生—内科学]