稳定整合靶向性MSP58基因RNA干涉载体的胶质瘤细胞系建立

Establishment of glioma cell line with stable RNA interference vector targeting MSP58 gene

目的探讨经载体介导的RNA干涉法建立抑制癌基因微小球蛋白(58-kDa microspherulep rotein,MSP58)表达的稳转细胞系的可行性。方法根据MSP58 cDNA编码序列,设计并合成针对MSP58基因的特异性RNA干涉片断,并将其克隆入pSilencer3.1-H1 neo干涉载体中,构建MSP58基因shRNA真核表达载体pSilencer-MSP58。重组载体pSilencer-MSP58和pSilencer3.1-H1 neo阴性对照载体pSilencer3.1-H1neoNegtive经脂质体LipofectamineTM2000介导法转染胶质瘤细胞株U251;转染细胞经过G418筛选,采用逆转录酶-多聚酶链反应(Reverse transcriptase-polymerase chain reaction,RT-PCR)、Western blot方法分别在mRNA水平和蛋白水平检测、筛选G418抗性的克隆细胞。结果成功构建了MSP58基因shRNA真核表达载体pSilencer-MSP58,经酶切、测序鉴定证实克隆正确。获得了稳定转染pSilencer-MSP58载体的6个胶质瘤细胞株。结论特异性shRNA能够明显抑制MSP58基因在U251细胞中的表达,并建立了稳定整合有靶向MSP58基因RNA干涉载体的胶质瘤细胞系,这为进一步研究MSP58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 explore the establishment of glioma cell line with stable RNA interference vector targeting MSP58 gene. Method According to MSP58 cDNA coding sequence, the specific RNA interference （RNAi） fragments targeting MSP58 gene were designed and synthesized, which were cloned into pSilencer3.1- Hlneo plasmid vector, and the shRNA eukaryotic expression vector pSilencer-MSP58 targeting MSP58 gene was constructed. The pSilencer-MSP58 vector and negative pSilencer-MSP58 vector were transfected respectively into 13251 cells by Lipofectamine^TM2000, and the transfected cells were selected by G418. Anti-G418 clones were isolated and Reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blot methods were carried out for the identification of integration of RNAi vectors in mRNA and protein levels. Results The specific shRNA eukaryotic expression vector pSilencer-MSP58 targeting MSP58 gene was constructed successfully, which was identified by restriction endonuclease digestion and sequencing. Six cell clones sorted out with G418 selection which had stably integrated RNAi vector targeting MSP58 gene were obtained. Conclusion MSP58 gene expression can be suppressed markedly by specific shRNA in 13251 cells.