稳定整合靶向性Survivin基因RNA干涉载体胶质瘤细胞系的建立

Establishment of a glioma cell line stably transfected with survivin specific targeting RNA interference vector

目的构建针对细胞凋亡抑制蛋白survivin(SVV)基因的特异性RNA干涉载体,并建立稳定转染该干涉载体的人胶质瘤细胞系。方法设计合成针对SVV基因和针对荧火虫荧光素酶(FLF)基因特异性RNA干涉片断,并分别克隆入pSilencer3.1-H1neo载体中,成功构建SVV基因RNA干涉载体pSilencer3.1-SVV和无关序列载体pSilencer3.1-FLF。RNA干涉载体、无关序列载体和空白载体经脂质体法转染胶质瘤细胞系U251,经G418筛选,采用实时定量反转录聚合酶链反应(real-timeRT-PCR)、免疫蛋白印迹(Westernblot)和免疫细胞化学方法分别检测SVV基因在转染细胞中的表达情况。结果酶切鉴定和核苷酸序列分析证实,成功构建了SVV基因RNA干涉载体pSilencer3.1-SVV。获得了稳定转染干涉载体、无关序列载体和空白载体的细胞系分别命名为U251-S、U251-F和U251-P。U251-S细胞中SVV的mRNA和蛋白表达受到明显抑制,U251-F和U251-P细胞中SVV基因表达水平无明显变化。结论SVV基因特异性RNA干涉载体能够明显抑制SVV基因在U251细胞中的表达,这为进一步研究SVV在胶质瘤细胞系U251中的生物学功能和作用机制奠定了实验基础。

Objective To construct the specific RNA interference vector targeting human survivin gene and to establish a human glioma cell line stably transfected with the vector. Methods The specific RNA interference （RNAi） fragments targeting SVV and firefly luciferase gene were designed and synthesized, which were cloned into pSilencer 3. 1-H1 neo plasmid vector, and the specific RNA interference vector pSilencer 3. 1-SVV targeting SVV gene and non-specfic sequence vector pSilencer 3. 1-FLF were constructed. The pSilencer 3.1-SVV vector, pSilencer 3.1-FLF and blank pSilencer 3.1-HI neo vector were transfected respectively into U251 cells by lipofectamine 2000, and then the transfected cells were selected by G418. Expression of mRNA and protein of survivin were respectively investigated by real-time reverse transcription polymerase chain reaction, Western blot and immunocytochemistry methods. Results The specific RNA interference vector pSilencer3.1-SVV targeting survivin gene was constructed successfully. The stable transfectants U251-S, U251-F and U251-P were obtained. Expression of mRNA and protein of survivin were inhibited significantly in U251-S cells, whereas survivin gene expression levels were hardly changed in U251-F and U251-P ceils. Conclusion Survivin gene expression can be suppressed markedly by specific RNA interference vector in U251 cells, and the current results have established the experimental foundation for the further study on the biological functions and its mechanisms of survivin in U251 cells.