Osteopontin特异性siRNA慢病毒表达载体的构建及其在U251胶质瘤细胞中OPN的表达

Construction of lentiviral vector of siRNA specific for osteopontin and its expression in U251 glioma cell line

目的:观察慢病毒表达载体介导的RNA干扰(RNAinterference,RNAi)对人胶质瘤细胞U251骨桥蛋白(osteopon-tin,OPN)表达的影响,为后续的以OPN基因为靶点的神经胶质瘤基因治疗研究奠定基础.方法:应用基因工程技术,筛选出2条针对OPN基因的RNAi靶序列,分别与pGCL-GFP载体连接,构建2个重组慢病毒表达载体LV-OPNshRNA1,LV-OPNshRNA2;将连接产物转化到DH5α感受态细胞,经PCR筛选阳性克隆、测序鉴定.将LV-OPNshRNA,pHelper 1.0,pHelper 2.0共转染293T细胞,包装产生慢病毒颗粒并测定病毒滴度.将包装产生的2种重组慢病毒分别感染U251细胞,实时定量PCR和Western印迹检测U251细胞OPN mRNA和蛋白的表达,并与未转染及空转染细胞进行比较.结果:2个慢病毒载体PCR和测序结果与预期结果一致,经包装产生的病毒滴度为8×1010TU/L和5×1010TU/L.感染U251细胞后,OPN基因mRNA和蛋白的表达量与未感染慢病毒的细胞组及空载体感染组相比均明显下降.结论:成功构建针对OPN基因的2个慢病毒载体OPN shRNA,体外感染U251细胞后可有效抑制OPN基因和蛋白的表达.

AIM： To observe the influence of lentiviral vector- mediated RNA interference on expression of human osteopontin （OPN） gene in human glioma cell line U251 so as to pave a way for OPN gene-targeted gene therapy of glioma. METHODS ： Gene engineering technique was used to screen 2 RNA interference sequences targeting OPN gene. The sequences were separately cloned into the pGCL-GFP vector to construct LV-OPNshRNA1 and LV-OPNshRNA2, which were subsequently confirmed by PCR and DNA sequencing analysis. The titer of lentivirus was determined after 293T cells were cotransfected with LV- OPNshRNA, pHelper 1.0 and pHelper 2.0. The 2 kinds of recombinant l entiviruses were injected into U251 cells, the OPN mRNA and protein expression were examined by real-time PCR and Western blotting and the results were compared with those of the non-transfected and blank vector transfected U251 cells. RESULTS： PCR analysis and DNA sequencing confirmed that the 20PN shRNA sequences were successfully inserted into the lentiviral vectors. The titers of concentrated virus were 8×10^10 and 5×10^10TU/L. After transfectiou with LV-OPNshRNA, OPN expression in U251 cells was significantly inhibited at both mRNA and protein levels compared with that in non-transfected and empty vector transfected U251 cells. CONCLUSION： Two lentiviral RNAi vectors of OPN gene have been successfully constructed and they can effectively inhibit the expression of OPN gene in U251 cells in vitro.