小窝蛋白-1敲减对人甲状腺上皮细胞趋化因子mRNA表达的影响

Effect of Caveolin-1 RNA knockdown by interference lentivirus on chemokines mRNA expression in human thyroid epithelial cells

目的:探讨小窝蛋白-1(Caveolin-1)RNA干扰慢病毒对人甲状腺上皮细胞Nthy-ori3-1趋化因子mRNA表达的影响。方法:采用分子克隆技术,根据Caveolin-1的短发夹RNA序列,构建pLKO.1-Caveolin-1重组质粒,将其或空载质粒与慢病毒包膜质粒pMD2.G和包装质粒psPAX2共转染人胚肾上皮293T细胞。48 h后收集病毒液并感染Nthy-ori 3-1细胞,用嘌呤霉素筛选出阳性细胞,于倒置显微镜下观察Nthy-ori 3-1细胞的生长情况;荧光实时定量PCR和蛋白质印迹检测Caveolin-1的敲减效果;荧光实时定量PCR检测CXC趋化因子配体10(CXCL10)、CC趋化因子配体20(CCL20)mRNA表达水平。结果:慢病毒感染后的Nthy-ori 3-1细胞生长状态良好,Caveolin-1的表达显著降低。抑制Nthy-ori 3-1细胞中Caveolin-1表达后,其CXCL10、CCL20的mRNA表达水平显著上调。结论:成功构建Caveolin-1 RNA干扰慢病毒,Caveolin-1可能通过影响某些趋化因子的表达,参与桥本甲状腺炎的发生。

Objective： To construct Caveolin-1 gene RNA interference lentiviral vector, and investigate the change of chemokines mRNA expression following Caveolin-1 knockdown in human thyroid epithelial cells. Methods ： Molecular cloning technology was adopted to design short hairpin RNA sequence targeting Caveolin-1 sequences and construct the recombinant plasmid pLKO. 1-Caveolin-1. The target plasmid Caveolin-1, or the control empty vector pLKO. 1 with the envelope plasmid pMD2. G and the packaging plasmid psPAX2 were co-transfected into human embryonic kidney epithelial 293 T cells. The filtered culture media was collected at 48 h and the lentiviral particles were packaged and applied to infect Nthy-ori3-1 cells. By adding puromycin into the media, infected cells were selected,and cells were observed under inverted microscope. The efficiency of depressing Caveolin-1 gene expression was determined by quantitative real time PCR and Western blotting analysis. CXC chemokine ligand 10（CXCL10）and CC chemokine ligand 20（CCL20） gene expression was determined by quantitative real time PCR.Results ： The virus infected Nthy-ori 3-1 cells grew well,and Caveolin-1 mRNA and protein expression was efficiently inhibited. Furthermore,CXCL10 and CCL20 mRNA levels were significantly enhanced after inhibition of Caveolin-1 expression in Nthy-ori 3-1 cells. Conclusion ： The Caveolin-1 RNA interference lentivirus was successfully constructed. Caveolin-1 may participate in the pathogenesis of Hashimoto＇s thyroiditis by regulating the expression of chemokines.