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作 者:匡军秀[1] 王卫星[1] 孙圣荣[1] 王万荣[1]
出 处:《中华实验外科杂志》2009年第2期174-176,共3页Chinese Journal of Experimental Surgery
基 金:湖北省卫生厅基金资助项目(JX3A14)
摘 要:目的构建人CCL5基因RNA干扰(RNAi)慢病毒载体。方法根据人CCL5基因信息,构建4个携带RNAi序列的pGCSIL—GFP质粒,与pHelper1.0、Helper2.0质粒一起利用293T细胞进行慢病毒包装。用CCL5RNAi慢病毒载体感染人宫颈癌细胞(Hela),使用RT—PCR方法验证其干扰效率。结果4个靶点中有3个靶点(a1、a2、a3)在Hela细胞上对CCL5基因的表达都有非常显著的敲减效果,敲减效率均达到95%以上。结论构建的CCL5RNAi慢病毒载体在Hela细胞中有较高的敲减效率,提示RNAi技术能够使细胞CCL5基因沉默。Objective To construct a lentiviral vector of RNA interference of chemokine CC motif ligand-5 (CCLS) gene and identify its knockout effectiveness in Hela cells. Methods The pGCSIL-GFP plasmids which expressed four RNAi sequences ( al, a2, a3, a4) respectively for human CCL5 gene togeth- er with pHelper 1.0 and Helper 2.0 plasmids were transfected into 293T cells in order to construct lentiviral vectors of RNA interference of CCL5 gene. Then the lentiviral vectors were transfected into Hela cells, and RT-PCR was used to evaluate its knockout effectiveness for CCLS. Results The knockout effective- ness of al, a2 or a3 was beyond 95% excepting a4,whose knockout effectiveness was beyond 90% in Hela cells. Conclusion The knockout effectiveness of the constructed lentviral vectors for human CCI.5 was satisfactory. Our results demonstrated that RNAi is able to silence the potential key gene CCL5 in human cancer cells.
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