靶向La的外源性microRNA抑制HBV复制和表达的实验研究  

Inhibition of hepatitis B virus gene replication and expression by artificial microRNA targeted La gene

作　　者：郜玉峰[1] 叶珺[1] 邹桂舟[1] 余莉[2] 沈继龙[2] 李家斌[3] 李旭[3] 

机构地区：[1]安徽医科大学第二附属医院肝病科,合肥230601 [2]安徽省人兽共患病实验室教育部省部共建重点实验室,合肥230032 [3]安徽医科大学第一附属医院感染病科,合肥230032

出　　处：《安徽医科大学学报》2011年第1期11-15,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:30700698)

摘　　要：目的设计并构建靶向La的microRNA表达载体,观察其对La基因的抑制作用,探讨其在HBV感染基因治疗中的应用价值。方法以La为靶基因,设计并构建3个针对La不同位点的microRNA表达载体,通过脂质体方法转染HepG2.2.15细胞,RT-PCR和Western blot检测其对La mR-NA和蛋白的抑制作用,乙肝五项定量和HBV DNA检测其对HBV的抑制作用。结果 3个外源性miRNA(amiRNA)均能明显降低La mRNA和蛋白的表达,其中amiRNA-La-1130抑制效应最强,转染后72h La mRNA和蛋白的水平平均下降了58.6%和50.6%(P<0.01);但仅有amiRNA-La-1130质粒能显著抑制相应细胞株中HBsAg和HBeAg的分泌,抑制率为23.3%和25.8%(P<0.01),HBV DNA的平均抑制率为22.3%(P<0.01)。结论靶向La的amiRNA能显著抑制靶基因的表达,进而抑制HBV的复制和表达。La基因可作为慢性HBV感染联合基因治疗的候选靶点之一。Objective To investigate the inhibitory effects of hepatitis B virus（HBV） replication and expression by transfeeting artificial microRNA targeted La gene into HepG2.2. 15 cells. Methods Three amiRNA-HBV plasmids were constructed and transfected into HepG2.2. 15 cells via LipofectamineTM 2000 reagent. The level of La mRNA and La protein was measured by semi-quantitative RT-PCR and Western blot respectively . HBV antigen secretion was detected in the cells with transient and stable transfection by time-resolved fluoroimmunoassays（TRFIA）. HBV DNA replication Was examined by fluorescence quantitative PCR. Results Three amiRNA could significantly reduce the La mRNA and protein expression, the greatest reduction was amiRNA-La-610 transfected group, the expression of La mRNA and protein levels were down-regulated by 57.3% and 49.8% at 72 h（P 〈0.01 ）. In the virus level, three amiRNA-La plasmids could inhibit the secretion of HBsAg and HBeAg, the greatest reduction group was amiRNA-La-610 transfected group, the expression levels of HBsAg and HBeAg were down-regulation by 23.3% % and 25.8% after 72 h（P 〈0.01 ） ,HBV DNA level was down-regulated by 22.3% at 72 h（P 〈0.01 ）. Conclusion In HepG2.2. 15 cells ,HBV replication and expression can be inhibited by artificial microRNA targeted La. La gene can be a promising therapy approach for chronic HBV infection.

关 键 词：肝炎病毒 乙型 RNA干扰 

分 类 号：R575.1[医药卫生—消化系统] R34[医药卫生—内科学]