整合素β1 shRNA重组慢病毒载体的构建及其对Sombati癫痫细胞模型整合素β1基因表达的影响

Construction of recombinant lentiviral vector containing integrin β1 shRNA and its effects on integrin β1 gene expression in Sombati cell culture model of refractory epilepsy

目的 构建大鼠整合素β1（integrin β1）基因RNA干扰慢病毒载体,为研究Sombati癫痫细胞模型integrin β1的作用机制提供载体.方法 构建integrin β1基因的4个shRNA慢病毒干扰载体,利用HEK 293T细胞进行慢病毒包装,感染靶细胞PC12,采用Western印迹验证干扰效果,筛选出沉默效果最好的慢病毒shRNA,感染新生大鼠海马神经元细胞和Sombati癫痫细胞,通过Western印迹检测其对海马神经元和Sombati癫痫细胞integrin β1的基因沉默效果.结果 经测序证实RNAi慢病毒载体构建成功,病毒达到有效滴度,PC12细胞病毒转染率达95％以上；Western印迹证实,重组慢病毒integrin β1 shRNA感染海马神经元细胞和Sombati癫痫细胞后integrin β1蛋白表达下降,integrinβ1 shRNA2重组慢病毒载体对海马神经元和Sombati癫痫细胞的沉默效率分别为70％和90％.结论 成功构建integrin β1 RNA干扰慢病毒载体,并在新生大鼠海马神经元和Sombati癫痫细胞中实现有效的基因沉默效应.

Objective To construct a recombinant lentiviral vector containing integrin β1 shRNA to provide an effective tool for integrin β1 gene effect and a possible mechanism of Sombati cell of clinical refractory epilepsy. Methods Four lentiviral vectors containing integrin β1 shRNA were constructed and transfected into 293T ceils. PC12 cells were infected by concentrated lentivirus and the gene-silcncing efficiency was verified. And the most effective lentivirus containing shRNA was selected with Western blot. Then neonatal rat hippocampal neurons and Sombati cells were infected by lentivirus containing shRNA and the gene-silencing efficiency was also monitored by Western blot. Results RNAi lcntivirus expression vectors targeting rat integrin β1 gcne were successfully constructed and confirmed by DNA sequencing. The recombinant lentivirus particles were packaged successfully to produce a sufficient titer for subsequent experiments. The expression of protein significantly decreased in rat hippocampal neurons and rat Sombati cells after vector transfection. Conclusion The recombinant lentiviral vector containing integrin β1 shRNA is constructed successfully. And the gene-silencing effects are effective and stable in neonatal rat hippocampal neurons and Sombati cells.