摘要
目的：使用RNA干扰技术阻断人结肠癌细胞cyclin D1的表达，检测其对细胞生长增殖的影响和机制。方法：将人结肠癌细胞株HT-29分成3组，antisense组（转染反义链组）、sense组（转染正义链组）和对照组，免疫沉淀和蛋白质印迹法观察cyclin D1 siRNA对细胞cyclin D1蛋白质表达的影响，并对蛋白质电泳图像进行分析，MTT比色法绘制细胞生长曲线，^3H-TdR技术和流式细胞技术观测细胞周期变化。结果：反义siRNA有效抑制了cyclin Dl蛋白质表达，MTT实验显示，转染反义siRNA的细胞，生长代谢减慢，与对照组细胞差异显著（P〈0．01），而antisense组细胞24h的^3H-TdR渗入量，G0／G1期和S期细胞较对照组和sense组明显减少（^3H-TdR：1181.8±117.97 VS 1798.4±55.36，1851.4±83.46；P〈0.01;G0／G1期：79.31％ VS 60.87％，59.14％；S期：13.67％ VS 26.42％．27.93％：P〈0.01）。结论：RNA干扰技术能有效减弱cyclin D1蛋白质表达，使细胞周期受阻，并抑制结肠癌细胞的生长增殖。
AIM： To inhibit the expression of cyclin D1 in a human colorectal cancer cell line using RNA interference, and to examine its effect on the growth and proliferation of colorectal cancer cells. METHODS： Human colorectal cancer HT-29 cells were divided into three groups： antisense group transfected with antisense siRNA against cyclin D1, sense group transfected with sense siRNA against cyclin D1, and control group. The inhibitory effect of antisense siRNA on cyclin D1 protein expression was detected by immuno- precipitation and Western blotting. MTT assay was used to detect the effect of antisense siRNA on the growth and metabolism of HT-29 cells. 3H-TdR incorporation assay and flow cytometry were used to test the metabolism and cell cycle changes in HT-29 cells transfected with antisense siRNA against cyclin D1. RESULTS： The expression level of cyclin D1 was clearly decreased in HT-29 cells in the antisense group. MTT assay showed a significant decrease in the metabolism of HT-29 cells in the antisense group, between 12 h and 72 h, compared with that in the control and sense groups （P 〈 0.01）. 3H-TdR incorporation was lower in the antisense group than that in the control and sense groups （^3H-TdR： 1181.8±117.97 vs 1798.4±55.36, 1851.4±83.46; P 〈 0.01; G0/G1： 79.31% vs 60.87%, 59.14%; S： 13.67% vs 26.42%, 27.93%; P 〈 0.01）. CONCLUSION： RNA interference can effectively inhibit the expression of cyclin D1, and inhibition of cyclin D1 expression causes a decrease in the growth, metabolism and proliferation of colorectal cancer cells, due to a delay in the progression of the cell cycle from G1 to S phase.
出处
《世界华人消化杂志》
CAS
北大核心
2007年第34期3572-3576,共5页
World Chinese Journal of Digestology
基金
福建省科技厅青年人才创新基金赞助项目
No 2006F3035
福建省卫生厅青年基金赞助项
No.2006-1-10~~
