摘要
目的了解慢病毒载体对胶质瘤干细胞的亲嗜性及对靶基因表达的干扰效率,初步探索干扰STAT3基因对胶质瘤干细胞增殖的影响。方法构建STAT3基因shRNA的慢病毒表达载体,经293T细胞包装后,获得可表达STAT3基因shRNA的慢病毒颗粒;活性载体病毒感染人原代胶质瘤干细胞后流式细胞分析细胞感染效率;实时定量多聚酶链反应(PCR)和Western blot检测细胞STAT3基因mRNA和蛋白表达及活化水平;增殖分析试剂盒测定细胞生长曲线,流式细胞分析细胞周期分布。结果在病毒感染比率值20:1时慢病毒载体对人原代胶质瘤干细胞的感染效率为98.6%;细胞感染可表达STAT3基因shRNA的载体慢病毒后,STAT3基因mRNA显著下降,抑制率为84.3%,STAT3蛋白表达下降81.5%,活化的pSTAT3下降97.9%;胶质瘤干细胞STAT3表达、活化受抑后细胞生长显著变慢,G1期细胞比例显著增高。结论慢病毒载体对人原代胶质瘤干细胞有着很高的感染效率,介导的RNAi可显著抑制靶基因的表达与活化,是对人胶质瘤干细胞基因功能研究的理想工具。人胶质瘤干细胞STAT3基因受抑后细胞生长显著变慢,出现G1期阻滞。
Objective To determine the efficiency of lentivirus shRNA vector infected glioblastoma stem cells and inhibited expression and activation of STAT3 gene in glioblastoma stem cells and observe the effect of suppressed expression of STAT3 on the proliferation of glioblastoma stern cells. Methods A lentivirus shRNA expression vector targeting STAT3 gene was constructed and packaged. The rate of glioblastoma stem cells infected lentivirus shRNA vector were analyzed by fluorescence activated cell sorter （FACS）. Expression and activation of STAT3 in glioblastoma stem cells were analyzed by realtime Q-PCR and Western blot. Cell proliferation assays were performed using the Cell Counting kit 8. Cell cycle analysis of glioblastoma stem cells was determined by flow cytometry analysis. Results The rate of glioblastoma stem cells infected lentivirus shRNA vector was 98. 6%. The expression and activation of STAT3 gene significantly decreased in glioblastoma stem cells after infected the lentivirns shRNA vector targeting STAT3 gene. The proliferation of cells was inhibited and the cell proportion of G0/G1 phase warkedly increased after knockdown of STAT3 expression in glioblastoma stem cells. Conclusion The lentivirns shRNA vector targeting STAT3 gene shows high efficiency on infecting glioblastoma stem cells and significantly inhibits the expression and activation of STAT3 in cells. STAT3 silencing by lentivirus vector significantly suppresses proliferation and induces G1 arrest in human glioblastoma stem cells. Lentivirus vector-mediated RNAi may serve as a novel therapeutic strategy for treatment of glioblastoma expressing constitutively activated Stat3.
出处
《中华神经外科疾病研究杂志》
CAS
2008年第6期498-501,共4页
Chinese Journal of Neurosurgical Disease Research
