【摘要】 目的观察慢病毒表达载体介导的RNA干扰(RNAi)对人肺腺癌细胞株A549细胞Toll样受体3(Toll-likereceptor 3,TLR3)表达的影响,为后续的以TLR3基因为靶点的肺癌研究和治疗奠定基础。方法应用基因工程技术,筛选出4条针对TLR3基因的RNAi靶序列,分别与pCCL-GFP载体连接,构建4个重组慢病毒表达载体TLR3-RNAi-LV 1#,TLR3-RNAi-LV 2#,TLR3-RNAi-LV 3#,TLR3-RNAi-LV 4#;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TLR3-RNAi-LV、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4种重组慢病毒分别感染A549细胞,实时定量PCR和Western印迹检测A549细胞TLR3mRNA和蛋白的表达。根据筛选的结果,选取最有效的载体进行病毒的大量包装。结果 4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分别为2×108、2×108、2×108、1×108 TU/mL。感染A549细胞后,TLR3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(均P<0.05),其中TLR3-RNAi-LV 4#作用较明显,使mRNA表达下降80%,蛋白表达下降68%(均P<0.05)。4#病毒载体大量包装后其滴度为1×109 TU/mL。结论成功构建针对TLR3基因的4个慢病毒载体TLR3-RNAi-LV,体外感染A549细胞后可有效抑制TLR3基因和蛋白的表达。更多还原

【Abstract】 Objective To examine the effect of the lentiviral vector-mediated RNA interference(RNAi)on the expression of human Toll-like receptor 3(TLR3)gene in human pulmonary adenocarcinoma cell line A549 in an attempt to lay a foundation for TLR3 gene-targeted gene therapy of pulmonary adenocarcinoma.Methods Gene engineering technique was used to screen four RNAi sequences targeting TLR3 gene.The sequences were separately cloned into the pCCL-GFP vector to construct 4 lentiviral vectors(i.e.TLR3-RNAi-LV 1#,TLR3-RNAi-LV 2#,TLR3-RNAi-LV 3# and TLR3-RNAi-LV 4#),which were subsequently confirmed by PCR and DNA sequencing.The titer of lentiviruses was determined after 293T cells were co-transfected with TLR3-RNAi-LV,pHelper 1.0 and pHelper 2.0.The four kinds of recombinant lentiviruses were used to infect A549 cells and the expression levels of TLR3 mRNA and protein were detected by real-time RT-PCR and Western blotting,respectively.Results PCR analysis and DNA sequencing confirmed that the four TLR3 lentiviral shRNA sequences were successfully inserted into the lentiviral vectors.The titer of the concentrated virus was 2×108TU/mL,2×108TU/mL,2×108TU/mL and 1×108TU/mL,respectively.The TLR3 expression in A549 cells infected with lentiviral vectors was significantly inhibited at both mRNA and protein levels when compared with that in the non-transfected and empty vector-transfected A549 cells(P<0.05).The effect was most significant in A549 cells infected with TLR3-RNAi-LV 4#.The TLR3 mRNA expression was decreased by 80% and the TLR3 protein expression by 68% after TLR3-RNAi-LV 4# infection(P<0.05).Conclusion Four lentiviral RNAi vectors of TLR3 gene were successfully constructed,and they could effectively inhibit the expression of TLR3 gene in A549 cells in vitro.更多还原