下调TSG101基因对人结肠癌细胞LOVO生长的调节作用

目的:探讨TSG101基因对人结肠癌细胞生长的调节作用。方法:构建TSG101基因的小干扰RNA载体并将其转导入LOVO细胞,获得稳定转染的阳性克隆后,应用RT-PCR和western blot进行鉴定;MTT法和流式细胞仪检测细胞转染前后生长速度和细胞周期的变化;Western blot检测细胞转染前后细胞周期相关蛋白Cyclin D1、CDK4、CDK6、p21和p27的表达变化。结果:成功构建了TSG101的小干扰RNA载体;筛选到稳定的TSG101低表达的结肠癌细胞模型;转染TSG101小干扰RNA后的细胞生长速度显著减慢,出现G1期阻滞,且Cyclin D1的表达明显降低,p21的表达明显增高。结论:下调TSG101基因能抑制结肠癌细胞生长,提示该基因可能具有临床应用前景。

Effects of downregulating TSG101 on growth regulationg activity of LOVO cells

Objects:To investigate the effects of TSG101 siRNA on the growth and drug sensitivity of human colon cancer cell line LOVO.Methods:The small interfering RNA eukaryotic expression vector specific to human TSG101 gene was constructed by gene recombination,then transfected into LOVO cells.Stable transfectants were obtained by G418 screening and further identified by RT-PCR and Western blot analysis.The growth curve was made using MTT assay.Cell cycle distribution of the transfected cells was studied by flow cytometry.The expressions of Cyclin D1,CDK4,CDK6,p21 and p27 were analyzed by western blot.Results:mU6pro-TSG101 siRNA was successfully constructed and transfected into LOVO cells.Down-regulation of TSG101 could significantly suppress the proliferation of LOVO cells with a G1 cell cycle arrest.The expressions of Cyclin D1 in transfected cells were decreased as compared with that of the control,while p21 increased.Conclusion:Down-regulation of TSG101 could suppress the proliferation of LOVO cells to a degree,suggesting TSG101 could be used for gene therapy in future.