作　　者： ; ; ; ; ;

机构地区： 广州医学院第二附属医院

出　　处： 《南华大学学报（医学版）》 2009年第3期257-260,共4页

摘　　要： 目的采用人SCN1A基因非保守区DNA构建短发夹RNA(shRNA)稳定表达的重组质粒,在HEK293细胞中抑制SCN1A基因表达。方法PCR分别正反向扩增SCN1A基因特异序列,构建成一个绿色光蛋白(GFP)与发夹结构RNA(shRNA)融合表达的重组质粒pEGFP-SCN1A-shRNA;该质粒和对照质粒(pCMV-EGFP)分别与SCN1A基因表达质粒(pCMV-SCN1A)共转染HEK293细胞株,采用半定量RT-PCR及双荧光共定位法鉴定SCN1A基因的表达情况。结果与对照相比较,shRNA表达质粒转染的培养细胞中SCN1A基因mR-NA表达量下调90%;双荧光共定位显示实验组阳性细胞中只检测到GFP蛋白,未能检测到Nav1.1,而对照组阳性细胞中能同时检测到GFP蛋白和Nav1.1。结论shRNA表达质粒在培养细胞中能有效地抑制SCN1A基因的表达。该质粒可进一步改造成腺病毒表达载体,在动物脑组织中进行永久性表达,建立癫痫等神经系统疾病的动物模型,可望成为致病机理研究和开发新治疗途径的工具。 Objective To construct the vector expressing SCN1A short -hairpin RNA （shRNA） and investigate the silencing effect of shRNA in HEK293 cells. Methods PCR was applied to amplify the SCNI A - specific sense and antisense sequences to construct a recombinant vector pEGFP- SCN1A -shRNA expressing GFP and SCN1A -shRNA fusion transcript. The vector pEGFP - SCN1A - shRNA and pCMV - EGFP co - transfected with pCMV - SCN1A respectively into cultured HEK293 cells. Semi - quantitative RT - PCR and dual fluorescent in situ hybridization were used to investigate the expression level of SCN1A. Results Compared with the control, SCN1A mRNA transcripts in those cells expressing shRNA decreased about 90%. Fluorescem -labeled experiments showed that beth GFP and Na,1.1 were detected in pCMV - EGFP and pCMV - SCN1A co - transfected ceils, but only GFP were detected in pEGFP - SCNI A - shRNA and pCMV - SCN1A co - transfected cells. Conclusion The recombinant vector expressing shRNA could restrict the expression of SCN1A in cultured HEK293 cell. The vector can be reconstructed into a recombinant adenoviral vector to silencing SCN1A expression in brain tissue. This study will lay the foundation for constructing animal models of neurological disorders and further investigating into the molecular mechanisms of diseases and their treatment means.

关 键 词： 干扰 钠通道 癫痫

领　　域： [生物学]