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目的建立CASK下调表达细胞株,研究钙/钙调蛋白依赖性丝氨酸蛋白激酶(calcium,calmodulin-associated serine/threonine kinase,CASK)下调表达对ECV-304细胞增殖能力的影响。方法将包含针对CASK基因干扰siRNA片段的pGenesil-1-hCASK重组质粒转染人ECV-304细胞株,经G418加压筛选,成功筛选出CASK下调表达的细胞株。用蛋白免疫印迹法鉴定转染细胞中CASK基因的表达水平。采用流式细胞技术、MTT法观察CASK下调表达对ECV-304细胞增殖能力的变化。结果成功筛选出CASK下调表达ECV-304细胞株(siCASK细胞株),这些细胞在传代过程中绿色荧光蛋白能维持表达,细胞株中转染阳性率在90%以上;经蛋白免疫印迹法检测CASK表达明显降低;siCASK细胞株中G0/G1细胞比率下降,G2M期细胞比率明显上升,增殖指数明显提高,生长曲线左移。结论成功建立了CASK下调表达的ECV-304细胞株,CASK下调表达可导致细胞增殖能力增强。
OBJECTIVE: To establish a CASK down-regulated cell line to investigate the effect of down-regulation of calcium and calmodulin-associated serine / threonine kinase (CASK) on the proliferation of ECV-304 cells. Methods pGenesil-1-hCASK recombinant plasmids containing siRNA fragment targeting CASK gene were transfected into human ECV-304 cell line, and were screened by G418. The cell lines with CASK down-regulated were selected successfully. The expression level of CASK gene in transfected cells was identified by Western blotting. Flow cytometry was used to observe the change of CASK expression on ECV-304 cell proliferation by MTT assay. Results CASK down-regulated expression of ECV-304 cell line (siCASK cell line) was successfully screened. The expression of green fluorescent protein (EGFP) was maintained in these cells during passage, and the positive rate of transfection was over 90% in cell lines. CASK The percentage of G0 / G1 cells in siCASK cells decreased, the percentage of cells in G2M phase increased significantly, the proliferation index increased obviously, and the growth curve shifted to the left. Conclusion The CASK down-regulated ECV-304 cell line was established successfully. CASK down-regulated expression resulted in enhanced cell proliferation.