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在细胞周期检测点信号传导通路中,Chkl和Chk2起着重要作用,主要参与G2/M期细胞周期检测点信号传导.首先采用RNAi技术在BGC823细胞中将Chk1、Chk2基因沉默,Chk1、Chk2 siRNA转染BGC823细胞后24h加入15mg/L二烯丙基二硫(diallyl disulfide,DADS),接着通过Real-timePCR、Western blot分析Chk1、Chk2基因在转染前后的表达差异,然后运用流式细胞术和Western blot分析Chk1、Chk2基因沉默后对DADS诱导的细胞周期G2/M期阻滞作用及相关周期蛋白CDC25C和cyclinB1表达的影响.实验结果表明,与未转染对照组相比,转染Chk1、Chk2siRNA后BGC823细胞中Chk1、Chk2表达明显被抑制,二者的mRNA表达分别下降84.7%和69.0%,蛋白质水平分别下降73.4%和78.5%.流式细胞术分析结果发现,ChklsiRNA转染的BGC823细胞在15mg/LDADS处理24h后,G2/M期比例由单纯DADS处理组的58.1%降至10.4%(P<0.05).但Chk2 siRNA转染后加入15mg/LDADS,与单独15mg/LDADS作用相比,细胞周期G2/M期比例差异不明显(P>0.05).Chk1和Chk2下游分子的改变显示,DADS可抑制BGC823细胞中CDC25C和cyclinB1表达(P<0.05),但Chk1基因沉默后加入DADS,CDC25C和cyclinB1表达却不被抑制(P>0.05),Chk2基因沉默后对DADS抑制CDC25C和cyclinB1表达的作用无影响.由此得出结论,Chkl基因沉默可消除DADS诱导BGC823细胞G2/M期阻滞作用,DADS阻滞G2/M期可能是通过Chk1/CDC25C/cyclinB1信号通路起作用.
Chkl and Chk2 play an important role in the signal transduction pathway of cell cycle checkpoint, which is mainly involved in G2 / M cell cycle checkpoint signaling.Firstly, RNAi was used to silence Chk1 and Chk2 genes in BGC823 cells and Chk1, Chk2 siRNA The expression of Chk1 and Chk2 genes before and after transfection was analyzed by Real-time PCR and Western blot respectively after addition of 15 mg / L diallyl disulfide (DADS) for 24 h after transfected into BGC823 cells. Flow cytometry And Western blot analysis of the Chk1, Chk2 gene silencing on DADS-induced cell cycle G2 / M phase arrest and related cyclins CDC25C and cyclinB1 expression.The experimental results show that compared with the untransfected control group transfected Chk1 The expression of Chk1 and Chk2 in BGC823 cells was significantly inhibited after Chk2 siRNA treatment, the mRNA and protein levels of Chk2 and Chk2 decreased by 84.7% and 69.0%, 73.4% and 78.5%, respectively.Flow cytometry analysis showed that ChklsiRNA transfected BGC823 After treated with 15 mg / L LDH for 24 h, the proportion of G2 / M phase decreased from 58.1% in the DADS-treated group to 10.4% (P <0.05), but 15 mg / LDADS was added after the Chk2 siRNA transfection, Ratio, cell cycle (P> 0.05) .The changes of Chk1 and Chk2 downstream molecules showed that DADS could inhibit the expression of CDC25C and cyclinB1 in BGC823 cells (P <0.05), but the expression of DADS, CDC25C and cyclinB1 (P> 0.05). Chk2 silencing had no effect on DADS inhibiting CDC25C and cyclinB1 expression.Conclusion: Chkl gene silencing can eliminate G2 / M arrest induced by DADS in BGC823 cells, DADS block G2 / M phase may be through the Chk1 / CDC25C / cyclinB1 signaling pathway.