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作者	单位	E-mail
闫淑鑫	北京中医药大学中医学院北京 102488	yanshuxin528@bucm.edu.cn
张淑静	北京中医药大学中医学院北京 102488
袁慧敏	北京中医药大学中医学院北京 102488
孙逸卓	北京中医药大学中医学院北京 102488
骈雪	北京中医药大学中医学院北京 102488
孙燕	北京中医药大学中医学院北京 102488
汤阳	北京中医药大学中医学院北京 102488

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中文摘要:

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、INTU-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-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-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。

英文摘要:

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 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-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 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 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 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 by 44.54 %. These results suggest that the INTU-35 group achieved the most effective silencing of the INTU gene. Conclusion: 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.

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