Abstract：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.
［Key words］Caveolin-1； RNA interference； chemokines； Hashimoto′s thyroiditis
2008, Vol. 28 
