INTU-siRNA质粒的构建及在人甲状腺Nthy-ori-3-1细胞中的筛选

Construction of the INTU-siRNA Plasmid and Screening in Human Thyroid Nthy-ori-3-1 Cells

目的:构建人源靶向特异INTU-si RNA质粒,转染人源性甲状腺Nthy-ori-3-1细胞以构建INTU基因沉默的Nthy-ori-3-1细胞模型,为探索Graves病的发病机制提供合适的细胞模型。方法:构建四对人源INTU序列(编号分别为INTU-29、INTU-31、INTU-33、INTU-35)si RNA质粒和空载体质粒,将空载体和四对INTU-si RNA质粒分别转染Nthy-ori-3-1细胞培养24 h,通过倒置荧光显微镜观察质粒转染细胞后的荧光表达,使用多功能酶标仪检测荧光强度,通过荧光定量PCR法验证INTU基因的沉默效率,通过蛋白质免疫印迹法验证各组细胞内INTU蛋白的表达水平,筛选沉默INTU基因效率最佳的质粒。结果:空载体和INTU-si RNA质粒转染入Nthy-ori-3-1细胞24 h后,倒置荧光显微镜和荧光酶标仪检测结果显示空载体和四组INTU-si RNA组呈现绿色的荧光表达,与正常组相比,空载体与四组INTU-si RNA组的荧光强度均显著高于正常组(P<0.01),提示质粒均转染成功。荧光定量PCR结果显示,与正常组和空载体组相比,四组INTU-si RNA组的INTU基因m RNA相对表达量均显著降低(P<0.01),其中INTU-35组表达降低最显著,INTU的m RNA表达降低了61.02%。蛋白质免疫印迹法结果显示,与正常组和空载体相比,INTU-29组和INTU-31组并无显著降低,INTU-33和INTU-35组较正常组和空载体组明显下降(P<0.05,P<0.01),其中INTU-35组最佳,INTU的蛋白表达降低了44.54%。以上结果提示INTU-35组沉默INTU基因效果最佳。结论:本研究成功构建INTU-si RNA质粒,并将该质粒成功转染人源甲状腺Nthy-ori-3-1的细胞模型。INTU-si RNA甲状腺细胞模型的构建,为进一步探索Graves病的发病机制奠定了实验基础。

Objective:The target-specific INTU-si RNA plasmids were constructed and transfected into Human thyroid Nthy-ori-3-1 cells to establish the Nthy-ori-3-1 cell model of silenced-INTU.This model provides a suitable cell model for exploring the pathogenesis of Graves'disease.Methods:Four pairs of human INTU sequences(numbered INTU-29,INTU-31,INTU-33,INTU-35)si RNA plasmids and an empty vector plasmid were constructed and individually transfected into Nthy-ori-3-1 cells,which were then cultured for 24 hours.The fluorescence expression of each group was observed using a Fluorescent Inverted Microscope,and the fluorescence intensity was detected by a Multi-function Measuring Instrument.The silencing efficiency of the INTU gene was determined by fluorescence quantitative PCR and Western blotting.Finally,the most effective silenced-INTU plasmid was identified through screening.Results:After the empty vector and the four INTU-si RNA plasmids were transfected into Nthy-ori-3-1 cells for 24 hours,the results of Fluorescent Inverted Microscope and Multi-function Measuring Instrument indicated that the four INTU-si RNA groups showed green fluorescence expression,and their fluorescence intensity was significantly higher than the normal group(P<0.01),suggesting that the plasmids were successfully transfected.The results of fluorescence quantitative PCR demonstrated that the relative m RNA expression of the INTU gene in four INTU-si RNA groups was significantly reduced compared with the normal group and the empty vector group(P<0.01),with the most significant reduction of the INTU-35 group decreased by 61.02%.The results of Western blotting showed that compared to the normal group and the empty vector group,the INTU-29 and the INTU-31 group did not exhibit a significant decrease,while the INTU-33 and INTU-35 groups showed a remarkable decline(P<0.05,P<0.01),with the protein expression of the INTU-35 group reduced by44.54%.These results suggest that the INTU-35 group achieved the most effective silencing of the INTU gene.Conclusions:In this study,the INTU-si RNA plasmids were successfully constructed and transfected into human thyroid Nthy-ori-3-1 cells.The construction of the INTU-si RNA thyroid cell model lays the experimental foundation for further exploring the pathogenesis of Graves'disease.