EphrinA1基因小干扰RNA的构建及其在Huh-7细胞中的沉默效应【荐】.doc

EphrinA1基因小干扰RNA的构建及其在Huh-7细胞中的沉默效应 　　作者：王怡，陈钢，陈必成，刘佳明，黄陈平 作者单位：1.温州医学院 环境与公共卫生学院，浙江 温州 325035;2.温州医学院附属第一医院 肝胆外科，浙江 温州 325027 　　【摘要】 目的 ：构建针对EphrinA1基因的发夹状RNA(ShRNA)表达载体pSilencer2.1-EphrinA1-SiRNA，观察其对人肝癌细胞Huh-7中EphrinA1基因表达的特异性抑制效应。方法 ：设计合成针对EphrinA1 mRNA的RNAi核苷酸片段，并定向克隆到pSilencer2.1-U6载体中，构建重组质粒pSilencer2.1-EphrinA1- SiRNA，同时构建不针对任何序列的重组质粒pSilencer2.1-EphrinA1-ScrRNA，通过脂质体介导转染入Huh-7细胞，应用逆转录聚合酶链反应检测小干扰RNA的沉默效应。结果：酶切鉴定证实pSilencer2.1-EphrinA1-SiRNA和pSilencer2.1-EphrinA1-ScrRNA重组质粒构建成功，转染Huh-7细胞后，pSilencer2.1-EphrinA1-SiRNA组EphrinA1 mRNA表达较pSilencer2.1-EphrinA1-ScrRNA组和空白对照组明显下调，差异有统计学意义(P0.05)。结论：pSilencer2.1-EphrinA1-SiRNA载体的成功构建为下一步研究以EphrinA1基因为靶位点的肝细胞癌基因治疗奠定了重要的实验基础。 　　【关键词】 EphrinA1;小干扰RNA;肝细胞癌 　　Construction of EphrinA1 SiRNA and analysis of its silenced effect in hepatoma cell line 　　Huh-7 WANG Yi， CHEN Gang， CHEN Bi-cheng， LIU Jia-ming， HUANG Chen-ping. 　　Environmental Science and Public Health School，Wenzhou Medical College，Wenzhou，325035 　　Abstract: Objective: To observe the inhibition effect of ShRNA on EphrinA1 expression in hepatoma cell line Huh-7 through construction of the expressed carrier pSilencer2.1-EphrinA1-ScrRNA of EphrinAI. Methods: Two pairs of SiRNA were designed and synthesized according to the EphrinA1 mRNA sequence in GenBank，and then were inserted into pSilencer2.1-U6 plasmids to create recombinant plasmids pSilencer2.1-EphrinA1-SiRNA and pSilencer2.1-EphrinA1-ScrRNA (control). After being transfected into Huh-7 cells，EphrinA1 expressed by ShRNAs was detected to be down regulated by RT-PCR. Results: All plasmids were constructed successfully. Expression of EphrinA1 in Huh-7 cells infected with pSilencer2.1-EphrinA1-SiRNA plasmids was significantly down regulated compared with that is infected with pSilencer2.1-EphrinA1-ScrRNA plasmids (P0.05). Conclusion: Con-struction of pSilencer2.1-EphrinA1-SiRNA lays an important experimental foundation for gene therapy of hepatocellular carcinoma (HCC) by targeting the Ephr