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目的:观察Chk1/2基因沉默对人胃癌MGC803细胞增殖及周期的影响。方法:采用RNAi技术在MGC803细胞中分别将Chk1和Chk2基因沉默,运用蛋白质印变法验证,然后采用MTT实验、流式细胞术分析Chk1和Chk2基因沉默对人胃癌MGC803细胞增殖及周期的影响。结果:蛋白质印迹法结果显示,转染靶向Chk1和Chk2siRNA24h的Chk1、Chk2蛋白相对灰度值分别为0.09±0.04和0.12±0.01,明显弱于未转染对照组(0.39±0.09)和脂质体转染组(0.38±0.17),P均<0.05,而未转染对照组和脂质体转染组之间比较差异无统计学意义,P=0.458。转染Chk1、Chk2siRNA的MGC803细胞Chk1和Chk2蛋白表达分别下降85.0%和83.4%。MTT实验证明,Chk1蛋白表达缺失导致MGC803细胞增殖活性下降,其增殖抑制率为38.0%(t=26.797,P<0.05);Chk2蛋白表达缺失对细胞增殖无明显抑制作用(t=6.098,P>0.05)。Chk1基因沉默后细胞周期在G0/G1期发生停滞,但并不引发凋亡,而Chk2基因沉默后对细胞周期影响不明显。结论:Chk1基因沉默可明显抑制人胃癌细胞增殖,且其抑制增殖作用与诱导G0/G1期阻滞有关。
Objective: To observe the effects of Chk1 / 2 gene silencing on proliferation and cell cycle of human gastric cancer cell line MGC803. Methods: Chk1 and Chk2 genes were silenced in MGC803 cells by RNA interference (RNAi), and then verified by Western blotting. The effects of Chk1 and Chk2 gene silencing on the proliferation and cell cycle of human gastric cancer cell line MGC803 were analyzed by MTT assay. Results: Western blot results showed that the relative gray values of Chk1 and Chk2 proteins transfected into Chk1 and Chk2 siRNA24h were 0.09 ± 0.04 and 0.12 ± 0.01, respectively, which were significantly lower than those of untransfected control (0.39 ± 0.09) and lipid (0.38 ± 0.17), P <0.05, while there was no significant difference between untransfected control group and lipofection group, P = 0.458. The expression of Chk1 and Chk2 in MGC803 cells transfected with Chk1 and Chk2 siRNA decreased by 85.0% and 83.4%, respectively. MTT assay showed that the loss of Chk1 protein expression resulted in a decrease of proliferation of MGC803 cells, and the inhibition rate of proliferation was 38.0% (t = 26.797, P <0.05). The lack of Chk2 protein expression had no significant effect on cell proliferation (t = 6.098, P> 0.05). After the Chk1 gene silenced, the cell cycle stagnated at G0 / G1 phase but did not induce apoptosis. However, the effect of Chk2 gene silencing on the cell cycle was insignificant. Conclusion: Chk1 gene silencing can significantly inhibit the proliferation of human gastric cancer cells, and its inhibitory effect on proliferation is related to the induction of G0 / G1 arrest.