靶向大鼠β-catenin基因的shRNA真核表达质粒的构建与筛选

Construction and Screening of Eukaryotic Expression Plasmids Containing shRNA Targeting β-catenin Gene

目的构建靶向β-连环蛋白（β-catenin）基因的shRNA的真核表达质粒，并筛选出沉默β-catenin基因效果最明显的shRNA表达质粒。方法设计3对针对β-catenin基因不同位点的shRNA片段，构建携带此shRNA片段的真核表达质粒（shRNA1—3）并行测序分析，电穿孔转染神经干细胞（neural stem cells NSCs），测定转染率，并分别采用RT—PCR及Western印迹检测β-catenin mRNA和蛋白表达情况，免疫组化方法检测shRNA对神经干细胞分化的影响。结果构建靶向β-catenin基因的shRNA真核表达重组质粒shRNA1，2，3。经筛选，NSCs转染shRNA3后，B—catenin RNA水平和蛋白水平均明显低于阴性对照组和空白对照组（0．08±0．00 vs 0．75±0．01，0．74±0．02；0．12±0．10，vs 0．56±0．02，0．65±0．01，P均〈0．01；与空白组和阴性对照组相比，shRNA3组NSCs分化为GFAP阳性细胞的百分率明显增加，分化为NSE阳性细胞的百分率明显减少（P〈0．05）。结论β—catenin RNA3对神经干细胞的β—catenin表达具有明显抑制作用，可影响细胞的分化。为研究wnt／β-catenin信号途径在NSCs生长分化中的作用奠定基础。

Objective To construct eukaryotic expression plasmids containing short hairpin RNA （shRNA） that targets β-catenin gene in neural stem cells （NSCs）, and to select plasmids that silence β-catenin gene most efficiently. Methods Three pairs of shRNAs targeting β-catenin gene were designed. The eukaryotic expression plasmids, named shRNA1, 2, and 3, were constructed and identified by sequencing analysis. The plasmids were then transfected into NSCs by electroporation. Transfection efficiency was measured 48 h after transfection. β-catenin mRNA and protein expression were determined by RT-PCR and Western blotting respectively. Results Sequencing analysis confirmed the construction of shRNA1, 2 and 3. Forty-eight hours after transfection, β-catenin mRNA and protein levels were reduced by shRNA1, 2 and 3 respectively, significantly lower than that of negative control group and blank control group （P 〈0.01 ） . Compared with shR- NA1 and shRNA2, shRNA3 had the strongest inhibitory effect on mRNA and protein expression of β-catenin Conclusions The shRNA eukaryotic expression plasmids targeting β-catenin gene in NSCs was constructed and selected successfully. These obtained plasmids suppressed significantly β- catenin mRNA and protein expression in NSCs, which can be applied to studying the role of wnt/β- catenin signaling in development of NSCs.