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目的制备针对黏着斑激酶(focal adhesion kinase,FAK)的siRNA慢病毒载体,转染入原代培养的大鼠海马神经元细胞,筛选最佳的感染条件和沉默靶点序列。方法根据siRNA原理设计、构建、酶切、转化、PCR鉴定、阳性克隆测序并病毒包装3种靶向大鼠FAK的siRNA(T1,T2,T3),并转染至原代培养的大鼠海马神经元细胞,荧光显微镜观察转染效率,实时荧光定量PCR检测神经元细胞FAK基因表达,Western blot检测FAK蛋白表达。结果成功制备3种FAK siRNA慢病毒载体,并成功转染到神经元细胞中,在感染复数(MOI)为10并加入5μg/mL polybrene条件下转染率达到90%以上。T1、T2、T3均可抑制FAK基因及蛋白的表达,但T1沉默效果最好,敲除效率达到65%,与正常对照组比较差异有统计学意义(P<0.05)。结论筛选出最佳的转染条件及最佳沉默靶向性序列T1,为后续研究整合素在阿尔茨海默病发病中的机制奠定了实验基础。
Objective To prepare a siRNA lentiviral vector targeting focal adhesion kinase (FAK) and transfected into primary cultured rat hippocampal neurons to screen for optimal infection conditions and target sequences. Methods According to the principle of siRNA design, construct, digest, transform and identify PCR products. The positive clones were sequenced and packaged into three kinds of siRNA targeting FAK (T1, T2 and T3). The siRNAs were transfected into primary cultured hippocampus Transfection efficiency was observed by fluorescence microscopy, FAK gene expression in neurons was detected by real-time fluorescence quantitative PCR, and FAK protein expression was detected by Western blot. Results Three FAK siRNA lentivirus vectors were successfully prepared and successfully transfected into neuronal cells. The transfection rate was over 90% at a MOI of 10 and 5μg / mL polybrene. T1, T2 and T3 inhibited the expression of FAK gene and protein, but the effect of T1 silencing was the best, with a knockdown efficiency of 65%, which was significantly different from the normal control group (P <0.05). Conclusion The optimal transfection conditions and optimal silencing targeting sequence T1 were screened, which laid the experimental foundation for the subsequent study on the mechanism of integrin in the pathogenesis of Alzheimer’s disease.