摘要
目的:构建存活素(survivin)基因短发夹RNA(shR-NA)的表达质粒,为乳腺癌RNAi介导的基因治疗打下基础.方法:设计并合成有小发夹结构的8条survivinshRNA对应模板序列,退火并与pShuttle 1.0-CMV载体重组质粒;将重组质粒转染人乳腺癌细胞MCF-7;MTT实验筛选出最有效质粒及其有效浓度,流式细胞术检测细胞周期的变化,经Hoechst染色观察凋亡情况,RT-PCR和Western Blot检测survivin基因的表达情况.结果:成功构建重组质粒并筛选出最有效质粒及其有效浓度;细胞受阻于G2/M期,并通过荧光染色发现有凋亡细胞;survivin基因表达受到抑制.结论:构建的重组质粒能抑制survivin基因的表达,这为研究乳腺癌RNAi介导的基因治疗打下基础.
AIM： To construct an expression vector with short hairpin RNA of survivin, which paves the way for RNAi-mediated breast cancer therapy. METHODS： Eight strands of DNA fragment with the structure of survivin shRNA were designed and synthesized. After annealed, the strands were cloned into pShuttle vector to construct the recombinant plasmids. After the plasmids were transfected into MCF-7 cells, MTY assay was used to screen out the most powerful one and the effective concentration. Cell cycle and apoptosis were obversed by flow cytometry and Hoechst staining. RT-PCR and Western Blot were done to detect the expression of survivin. RESULTS： The recombinat plasmids were constructed successfully. MCF-7 cells were arrested in the period of G2/M, moreover, some cells began to die. Survivin expression was depressed. CONCLUSION： The constructed recombinants can knockout survivin expression in cell MCF-7, which offers a basis for the research on the RNAi-mediated gene therapy of hu- man breast cancer.
出处
《第四军医大学学报》
北大核心
2008年第12期1104-1107,共4页
Journal of the Fourth Military Medical University
