pSITITIN载体的构建及表达

Construction and expression of pSITITIN plasmid vector

目的构建能阻断Wistar大鼠肌联蛋白表达的siRNA载体,为研究骨髓间充质干细胞(mesenchymal stemcells,MSCs)向心肌细胞分化的过程中肌小节结构蛋白之间的相互关系打下基础。方法针对Wistar大鼠TITIN N2B区设计并构建重组质粒pSITITIN,针对人β-ACTIN设计并构建重组质粒pSIACTIN,利用阳离子脂质体将其分别转染入体外培养1周的新生大鼠心肌细胞和经5-氮杂胞苷(5-azacytidine,5-aza)诱导后2周的MSCs中,免疫荧光检测肌联蛋白的表达。结果重组质粒pSITITIN转染入正常心肌细胞和经5-aza处理后的MSCs后,肌联蛋白的表达均减弱(P<0.01);而转染pSIACTIN后不影响肌联蛋白的表达。结论pSITITIN具有确切阻断肌联蛋白表达的效果。

Objective To construct small RNAi plasmid vectors which can suppress the expression of TITIN in Wistar rat. Methods A recombinant plasmid vector involving shRNA, which matched the base pair of rat TITIN N2B region mRNA perfectly, was constructed and transfected into normal neonatal cardiomyocytes and mesenchymal stem cells （MSCs） induced by 5-aza cytidine respectively. The expression of TITIN Z band was investigated by immunofluorescence. Results The expression of TITIN was weakened after recombinant plasmid was transfected into neonatal cardiomyocytes and MSCs induced by 5-aza cytidine. Conclusion The recombinant plasmid was constructed successfully, and it actually can block the expression of TITIN.