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[目的]为深入研究hCASK功能,构建Id1真核表达载体并转染ECV-304细胞,筛选出Id1强制表达细胞株,观察Id1上调表达对ECV-304细胞增殖能力的影响。[方法]采用RT-PCR方法获取编码Id1的cDNA序列,并将其导入真核表达载体成功构建pIRES2–EGFP-Id1重组表达质粒。将重组质粒转染人ECV-304细胞株,经G418加压筛选,成功筛选出Id1强制表达的细胞株。用蛋白免疫印迹法鉴定转染细胞中Id1基因的表达水平。采用流式细胞技术、细胞计数的方法观察筛选出的细胞株增殖能力的变化。[结果]成功建立了Id1强制表达的ECV-304细胞株。同未转染细胞相比,Id1强制表达细胞株中G0/G1细胞比率下降约4.8%,G2M期细胞比率上升约26.3%,增殖指数明显增高,细胞增殖能力增强。[结论]Id1强制表达细胞株的成功建立和鉴定为CASK、Id1基因功能研究提供了有效手段,Id1的上调表达可导致ECV-304细胞增殖能力增强。
[Objective] To investigate the function of hCASK in vitro and to construct Id1 eukaryotic expression vector and transfected into ECV-304 cells. Id1 forced expression cell line was screened to observe the effect of Id1 up-regulation on the proliferation of ECV-304 cells. [Method] The cDNA encoding Id1 was obtained by RT-PCR and inserted into the eukaryotic expression vector to construct the recombinant plasmid pIRES2-EGFP-Id1. The recombinant plasmids were transfected into human ECV-304 cell line and screened by G418 for selection. The cell lines with forced expression of Id1 were successfully screened. Id1 gene expression in transfected cells was identified by Western blotting. Flow cytometry, cell counting method was used to observe the changes of cell proliferation. [Results] The ECV-304 cell line that was forcedly expressed by Id1 was successfully established. Compared with untransfected cells, the ratio of G0 / G1 cells in Id1 forced expression cells decreased by 4.8%, the percentage of cells in G2M phase increased by 26.3%, the proliferation index was significantly increased and the cell proliferation ability was enhanced. [Conclusion] The successful establishment and identification of Id1 forced expression cell line has provided an effective method for the study of the function of CASK and Id1 gene. The up-regulated expression of Id1 can lead to the enhancement of ECV-304 cell proliferation.