SiRNA真核表达载体阻断HeLa细胞stathmin基因表达探究.docVIP

SiRNA真核表达载体阻断HeLa细胞stathmin基因表达探究 　 作者：王燕，吴冰，苏海川，刘丽，林芳，张惠中 【关键词】 真核表达载体 Blocking effect of vectorbased siRNA on stathmin gene expression in human HeLa cells 　　【Abstract】 AIM: To explore the blocking effect of siRNA on the expression of stathmin gene in HeLa cell line using siRNA eukaryotic expression vector. METHODS: Two stathmin siRNA cDNAs were synthesized according to the stathmin gene sequence and cloned into the vector pSilencer4.1CMVneo and named pSilencerS1 and pSilencerS2 respectively, which were further identified by restriction endonuclease digestion analysis and DNA sequencing. HeLa cells were then transfected with pSilencerS1 and pSilencerS2. After G418 selection, the cells were selected and the interfering effect was detected by RTPCR. RESULTS: Restriction endonuclease digestion analysis and DNA sequencing results showed that the 2 target segments were cloned into pSilencer4.1CMV neo vector respectively. The results of RTPCR indicated that both siRNA vectors could successfully knock down stathmin gene expression. CONCLUSION: The vectorbased siRNA on stathmin gene can effectively knock down stathmin gene expression, which has the potential of being applied in gene therapy against malignant tumors. 　　【Keywords】 stathmin; RNA, small interfering; eukaryotic expression vector; HeLa cells 　　【摘要】 目的： 构建人stathmin基因的siRNA真核表达载体，探讨其对HeLa细胞中stathmin基因表达的干涉作用. 方法： 将合成的siRNA寡核苷酸链退火形成双链，连接入经BamHⅠ和HindⅢ双酶切后的pSilencer4.1CMV neo真核表达载体，酶切及测序鉴定. 脂质体法转染重组质粒入HeLa细胞，G418筛选后RTPCR检测其对stathmin基因mRNA的干涉效果. 结果： 经酶切及测序鉴定，成功构建siRNA真核表达载体. 经脂质体转染HeLa细胞后，RTPCR显示所构建的干涉stathmin基因的真核表达载体成功地抑制了目的基因的表达，HeLa细胞中stathmin基因的mRNA表达水平明显降低. 结论： 成功构建了人stathmin基因的RNA干涉真核表达载体pSilencerS1和pSilencerS2，并在HeLa细胞中有效地发挥了对stathmin基因表达的干涉作用. 　　【关键词】 stathmin； RNA，小分子干扰；真核表达载体；HeLa细胞 　　0引言 　　Stathmin为一种高度保守的细胞内蛋白，在多种恶性肿瘤细胞中高表达，研究表明［1，2］通过应用反义核酸、单克隆抗体、核酶、stathmin蛋白抑制剂及丝氨酸位点突变体等方法封闭该基因表达均证实，抑制其表达可使细胞受阻于G2/M期，同时还可以使恶性肿瘤表型发生逆转. 因此，stathmin是一个极具吸引力的恶性肿瘤生物治疗新靶点. RNA干涉是最新的基因敲除技术，具有高效性和特异性，能够降解与之序列相对应的细胞内mRNA, 使基