摘要
目的针对乙型肝炎病毒(hepatitis B virus,HBV)前S1 dsDNA同聚嘌呤区设计反基因寡核苷酸药物-锁核酸(locked nucleic acid,LNA),并在体外观察抑制HBV复制的效果。方法针对HBV前S1 dsDNA的3023~3037 nt、3094~3109 nt两个同聚嘌呤区,利用RNA structure软件分别设计并合成LNA,以阳离子脂质体为载体,将药物转染入HepG2.2.15细胞内,采用荧光定量PCR技术和化学发光免疫技术分别检测3 d、6 d和9 d细胞培养上清液中HBV DNA和HBsAg的含量;CCK8法检测锁核酸对HepG2.2.15细胞代谢的影响。结果LNA对HepG2.2.15细胞的HBV DNA转录和HBsAg表达有显著的抑制作用,并且抑制效果随着时间进一步加强,在第9天的抑制率分别为45.37%和52.09%。两个同聚嘌呤组与空白对照组比较差异均有统计学意义(均P<0.05),而封闭3023~3037nt同聚嘌呤区的LNA抑制作用较强,且最适序列长度为15~25 bp。CCK8实验显示LNA对HepG2.2.15细胞无任何明显的代谢影响。结论针对前S1 dsDNA同聚嘌呤区的反基因锁核酸分子在体外能够有效地抑制HBV的复制,以封闭3023~3037 nt靶位效果较强,且合适序列长度为15~25 bp。
Objective To design an antisense oligodeoxynucleotide,a locked nucleic acid,against the pre-S1 dsDNA homopolyfluoride region of hepatitis B virus(HBV),and observe the inhibition effect of HBV replication in HepG2.2.15 cells.Methods The two polymorphic regions of 3023-3037 nt and 3094-3109 nt of pre-S1 dsDNA of hepatitis B virus were designed and synthesized by RNA structure software.The transfected HepG2.2.15 cells were mediated by cationic liposome.Fluorescence quantitative polymerase chain reaction(PCR)and chemiluminescence immunoassay were used to detect the content of HBV DNA and HBsAg in cell culture supernatants at 3,6 and 9 days,respectively.The effect of LNA on the metabolism of HepG2.2.15 cells was detected by CCK8 method.Results LNA significantly inhibited HBV DNA replication and HBsAg expression in HepG2.2.15 cells,and the inhibition rate increased with time.After 9 days,the inhibition rates were 45.37%and 52.09%,respectively.The difference between the experimental group and the control group was statistically significant(both P<0.05),while the blocking effect of LNA in the 3023-3037 nt polypyrogenic region was stronger,and the optimal sequence length was 15-25 bp.LNA has no significant metabolic effect on cells.Conclusion The anti-gene-locked nucleic acid molecule targeting the pre-S1 dsDNA homomeric region can effectively inhibit the replication of HBV in vitro,and the effect of blocking the target of 3023-3037 nt is strong,and the suitable sequence length is 15-25 bp.
作者
彭彬
许桂丹
韦武均
农顺强
陈晓昊
肖树荣
潘柳叶
邓益斌
PENG Bin;XU Guidan;WEI Wujun;NONG Shunqiang;CHEN Xiaohao;XIAO Shurong;PAN Liuye;DENG Yibin(Department of Clinical Laboratory,Affiliated Hospital of Youjiang Medical University for Nationalities;Department of Laboratory,Yangjiang People’s Hospital,Yangjiang,529500;Guangxi Clinic Medicine Research Center of Hepatobiliary Disease,Baise 533000,China)
出处
《右江医学》
2020年第2期91-95,共5页
Chinese Youjiang Medical Journal
基金
国家自然科学基金(81460123)
广西科技计划项目基地与人才专项(桂科AD17129025)
广西自然科学基金(2018GXNSFAA281187)
广西肝胆疾病临床医学研究中心(桂科AD17129025-24)
关键词
反基因寡核苷酸药物
乙型肝炎病毒
锁核酸
反基因治疗
antisense oligodexymucleotide
hepatitis B virus
locked nucleic acid
antigene therapy
