干扰ski基因对视网膜色素上皮细胞增生和周期的影响

The effects of silencing of ski gene expression by small interfering RNA on proliferation and cell cycle of human retinal pigment epithelial cells

目的观察siRNA干扰c—ski对视网膜色素上皮（RPE）细胞增生和周期的影响。方法RPE细胞分为3组：空白对照组（转染时加脂质体）、阴性对照组（无关序列非特异性siRNA转染）和ski—siRNA转染组，另设阳性对照转染组（采用B—actinsiRNA转染）。采用蛋白电泳检测转染24h和48h时的c．ski和CyclinD1蛋白表达变化；比色法检测转染1、2、3、4d时细胞的增生速率；流式细胞术检测转染24h和48h细胞周期的变化。结果与空白对照组和阴性对照组相比，siRNA转染hRPE细胞增生在各个时间点均受到明显抑制（P〈0．05）；3组间RPE细胞c—ski和CyclinD1蛋白表达比较，差异有统计学意义（P〈0．05），与空白对照组和阴性对照组相比，c—skisiRNA转染组c—ski、CyclinD1蛋白表达下降（P〈0．05）。各组GO／G1、S和G2／M期RPE细胞比例比较，差异有统计学意义（P〈0．01），c-skisiRNA组GO／G1期细胞比例高于空白对照组和阴性对照组siRNA组（P〈0．05），S期细胞比例低于空白对照组和阴性对照组siRNA组（P〈0．05）。结论c—ski基因干扰可以有效地抑制RPE细胞增生，该分子机制可能是抑制RPE细胞G1期的调节子cyclinD，造成G0／G1期阻滞，不能进入S期。

Objective To investigate the effect of silencing c-ski gene with small interfering RNA （siRNA） on cell proliferation and cell cycle of human retinal pigment epithelial （RPE） cells. Methods The RPE cells were divided into 3 groups： blank control group, negative control group and ski siRNA group. Lipofectamine 2000 was utilized to transfect RPE cells in Ski siRNA and control siRNA group. Western blotting was used to detect the protein expressions of ski and Cyclin D1 on 24 h and 48 h after transfected. MTT colorimetric assay and flow cytometry were used to examine cell proliferation on 1 d, 2 d, 3 d and 4 d after transfected and cell cycle on 24 h and 48 h after transfected, respectively. Results The proliferation of RPE cells transfected with ski-siRNA was significantly inhibited on each time points and were significantly lower than that in blank group and in negative control group （P 〈 0.01 ）. The protein expressions of ski and Cyclin D1 among the 3 groups had statistically significant differences （ P 〈 0.05 ）. The expressions of ski and Cyclin D1 decreased in ski siRNA group （P 〈 0.05 ）. The percentage of G0/G1, S and G2/M phases of RPE cells among the 3 groups had statistically significant differences （P 〈 0.01 ）. The percentage of G0/G1 phase of RPE cells in ski siRNA group was higher than those in blank control group and negative control group （P 〈 0.05 ）, whereas S and G2/M phases appeared the opposite result （P 〈 0.05 ）. Conclusion Silencing ski by siRNA significantly inhibited the proliferation of RPE cells. The ski siRNA might inhibit the G1 phase modulator Cyclin D1 and induced cell cycle arrest at G0/G1 phase from S phase.