文档介绍：EphrinA1基因小干扰RNA的构建及其在Huh-7细胞中的沉默效应
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作者:王怡,陈钢,陈必成,刘佳明,黄陈平
【摘要】目的:构建针对EphrinA1基因的发夹状RNA(ShRNA)-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,-U6载体中,-EphrinA1- SiRNA,-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:-EphrinA1--EphrinA1-ScrRNA重组质粒构建成功,转染Huh-7细胞后,-EphrinA1-SiRNA组EphrinA1 -EphrinA1-ScrRNA组和空白对照组明显下调,差异有统计学意义(P<)。结论:-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
Abstract: Objective: To observe the inhibition effect of ShRNA on EphrinA1 expression in hepatoma cell line Huh-7 through construction of the expressed carrier -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 -U6 plasmids to create binant plasmids -EphrinA1-SiRNA and -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 essfully. Expression of EphrinA1 in Huh-7 cells infected with -EphrinA1-SiRNA plasmid