【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1 228 nt，嵌合片段有3bp的同源序列（CTG）。加入双氧水（H₂O₂）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H₂O₂的（-2.10±0.82）lg拷贝/细胞，差异有统计学意义（P<0.01）。加入H₂O₂的HepG2.2.15细胞中cccDNA水平为（-1.94±1.45）lg拷贝/细胞，未加入H₂O₂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。更多还原

【Abstract】 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 aquantitative analysis of the integration site that was found in HepG2.2.15 cells with and without H₂O₂ 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 with3 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 H₂O₂ treatment（-1.13±0.07）were significant higher than those without H₂O₂ treatment（-2.10±0.82,P<0.001）.No significant difference was found between the HBV cccDNA levels in cells with and without H₂O₂ 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 costeffective,simplified method for the detection of HBV DNA integration by a modified Alu-PCR method.更多还原