慢病毒载体靶向沉默原代大鼠海马神经元黏着斑激酶表达siRNA的筛选  

Construction and Screening of Effective anti-FAK of Hippocampal Neurons siRNA Lentivirus Vector

作　　者：张金平[1] 唐荣华[1] 吴军[1] 刘红星[1] 焦莉[1] 许峰[1] 康慧聪[1] 朱遂强[1] 薛峥[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院神经内科,武汉430030

出　　处：《华中科技大学学报（医学版）》2011年第1期1-4,8,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.30700244)

摘　　要：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 construct FAK gene-targeted small interfering RNA（siRNA）and its lentivirus vector,and to screen out the optimal efficient pairs of siRNA following transfection of the primary hippocampal neurons with the vector.Methods Three pairs of siRNA sequences（T1,T2,T3）anti-FAK of hippocampal neurons were designed,synthesized,digested with restriction enzyme,transformed,identified by PCR,DNA sequenced and then packed by lentivirus.After transfecting the neurons by the lentivirus,the transfection efficiency was measured under the fluorescence microscopy and the expression of FAK gene and protein was detected by real-time PCR and Western blot,respectively.Results Three siRNA sequences were constructed and transfected into hippocampal neurons.The transfection efficiency was over 90% when the MOI was 10 and 5 μg/mL polybrene was added.Compared to T2 and T3,T1 sequence had more significantly efficiency and could silence 65% of FAK gene.Conclusion The optimal efficient transfecting condition and the siRNA sequence T1 were screened out,which will provide experimental basis for researching the role of integrin in the progress of Alzheimer＇s disease.

关 键 词：小干扰RNA 黏着斑激酶 海马神经元 

分 类 号：Q781[生物学—分子生物学]