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
闫淑鑫 1张淑静 1袁慧敏 1孙逸卓 1骈雪 1孙燕 1汤阳1
作者信息
- 1. 北京中医药大学中医学院 北京 102488
- 折叠
摘要
目的:构建人源靶向特异INTU-siRNA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、IN-TU-33、INTU-35)siRNA质粒和空载体质粒,将空载体和四对INTU-siRNA质粒分别转染Nthy-ori-3-1细胞培养24h,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IN-TU-siRNA 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-siRNA组呈现绿色的荧光表达,与正常组相比,空载体与四组INTU-siRNA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-siRNA组的INTU基因mRNA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mRNA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IN-TU-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-siRNA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-siRNA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.
Abstract
Objective:The target-specific INTU-siRNA 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)siRNA plasmids and an empty vector plasmid were constructed and individually transfected into Nthy-ori-3-1 cells,which were then cul-tured for 24 hours.The fluorescence expression of each group was observed using a Fluorescent Inverted Microscope,and the fluores-cence 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-siRNA 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-siRNA groups showed green flu-orescence expression,and their fluorescence intensity was significantly higher than the normal group(P＜0.01),suggesting that the plas-mids were successfully transfected.The results of fluorescence quantitative PCR demonstrated that the relative mRNA expression of the INTU gene in four INTU-siRNA 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 IN-TU-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 bv 44.54％.These results suggest that the INTU-35 group achieved the most effective silencing of the INTU gene.Conclusions:In this study,the INTU-siRNA plasmids were successfully constructed and transfected into human thyroid Nthy-ori-3-1 cells.The construction of the INTU-siRNA thyroid cell model lays the experimental foundation for further exploring the pathogenesis of Graves'disease.
关键词
INTU-siRNA质粒/人甲状腺Nthy-ori-3-1细胞/Graves病Key words
INTU-siRNA plasmid/Human thyroid Nthy-ori-3-1 cells/Graves'disease引用本文复制引用
基金项目
国家自然科学基金(82004337)
中央高校基本科研业务费专项自主选题项目(2020-JYB-XJSJJ-002)
出版年
2024
NETL
NSTL
万方数据