改良Alu-PCR技术检测HepG2.2.15细胞HBV整合的研究

肝脏 ›› 2022, Vol. 27 ›› Issue (7): 785-788.

改良Alu-PCR技术检测HepG2.2.15细胞HBV整合的研究

阮鹏, 何春萍, 黄超, 周瑞

430060 湖北武汉大学人民医院消化内科

收稿日期:2021-08-18 出版日期:2022-07-31 发布日期:2022-08-25
通讯作者: 周瑞,Email:1020524175@qq.com
基金资助:
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)

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

RUAN Peng, HE Chun-ping, HUANG Chao, ZHOU Rui

Department of Gastroenterology, Renmin Hospital of Wuhan University, Hubei 430060, China

Received:2021-08-18 Online:2022-07-31 Published:2022-08-25
Contact: ZHOU Rui, Email: 1020524175@qq.com

摘要/Abstract

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。

关键词: 乙型肝炎病毒, HBV整合, Alu-PCR技术, HepG2.2.15 细胞

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 a quantitative 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 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 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 cost-effective, simplified method for the detection of HBV DNA integration by a modified Alu-PCR method.

Key words: Hepatitis B virus, HBV integration, Alu-PCR, HepG2.2.15 cell

阮鹏, 何春萍, 黄超, 周瑞. 改良Alu-PCR技术检测HepG2.2.15细胞HBV整合的研究[J]. 肝脏, 2022, 27(7): 785-788.

RUAN Peng, HE Chun-ping, HUANG Chao, ZHOU Rui. Detection of hepatitis B viral DNA integration in HepG2.2.15 cells by a modified Alu-PCR method[J]. Chinese Hepatolgy, 2022, 27(7): 785-788.

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