中文摘要：

目的：以细胞周期作为抗癌药物新靶点的研究，可能是很有前途的。笔者的前期工作发现，二烯丙基二硫化物（diallyl disulfide，DADS）可抑制人胃癌BGC823细胞增殖，其增殖抑制与细胞周期G2/M期阻滞有关；DADS可能是通过抑制细胞分裂周期蛋白25C（Cell division cycle protein25C，Cdc25C）、cyclinB1表达使部分BGC823细胞停滞在G2/M期，但G2/M期阻滞的机制还未完全阐明。本研究进一步探讨DADS诱导人胃癌BGC823细胞周期G2/M期阻滞的可能机制。方法：RT—PCR检测Chk1和Chk2在mRNA水平的改变；Western blot检测DADS处理BGC823细胞前后细胞周期相关蛋白ATM—RAD3相关基因（ATM—RAD3-related gene，ATR）、细胞周期检查点蛋白激酶1（checkpoint kinase1，Chk1）、细胞周期检查点蛋白激酶2（checkpoint kinase2，Chk2）表达和ATR、Chk1、Chk2的磷酸化程度；免疫共沉淀检测Chk1、Chk2与Cdc25C结合情况。结果：RT—PCR检测显示，Chk1和Chk2的mRNA水平在处理组与未处理组之间无显著性差异（P〉0．05）。Western blot检测显示，总Chk1和Chk2蛋白表达在细胞处理前后均无明显改变。但15mg／LDADS刺激BGC823细胞2h后，处理组细胞Chk1磷酸化程度明显增加，并呈时间依赖性（P〈0．05）。而Chk2磷酸化程度在处理组与未处理组之间无显著性差异（19〉0．05）。15mg／LDADS作用15～120min，ATR磷酸化程度明显增加，呈时间依赖性（P〈0．05），而ATR表达无改变。免疫共沉淀分析表明，DADS能促进BGC823细胞Chk1与Cdc25C结合。而对Chk2与Cdc25C结合无影响。结论：DAD诱导人胃癌BGC823细胞G2/M期阻滞与Chk1的活化有关，DADS可能是通过激活ATR、Chk1，调节Cde25C的表达引起人胃癌BGC823细胞G2/M期阻滞。

英文摘要：

Objective： Cell cycle has recently become more appealing as a new target of anti-carcinogenic agent. Diallyl disulfide （DADS） inhibits growth and induces cell cycle G2/M arrest in human gastric cancer BGC823 cells. Cell division cycle protein 25C （Cdc25C） and CyclinB1 expression are involved in G2/M arrest. However, mechanisms of G2/M arrest are not yet fully understood. The aim of this study was to elucidate the mechanism of cell cycle G2/M arrest in human gastric cancer BGC823 cells induced by DADS. Methods： The expression of Chk1 and Chk2 mRNA associated with cell cycle arrest of BGC823 cells after the induction with DADS for 1 or 2 days was detected by RT-PCR. The protein expression of cycle-related proteins ATM-RAD3-related gene （ATR）, checkpoint kinasel （Chk1）, checkpoint kinase 2 （Chk2）, P-ATR, P-Chk1 and P-Chk2 was measured by Western blot. Interaction between Chk1/2 and Cdc25C was analyzed by immunoprecipitation. Results： After the cells were treated with 15 mg/L DADS for 1 or 2 days, the expression of Chk1 and Chk2 mRNA was not significantly different from that in untreated cells （P〉0.05）. Western blot analysis showed that the expression of total Chk1 and Chk2 treated with 15 mg/L DADS was not significantly different from that in untreated cells. But phospho-Chk1 showed a significant increase after stimulation with 15 mg/L DADS for 2h to 12h and continued to increase gradually as time went on （P〈0.05）. Phospho-Chk2 showed a weak expression and a weaker expression after stimulation with DADS, but the changes were not statistically significant （P〉0.05）. Addition of 15 mg/L DADS to BGC823 cells for 15 min to 120 min resulted in an increase in phospho-ATR expression, whereas no changes were found in ATR expression （P〈0.05）. The Chk1 Ab increasingly precipitated Cdc25C in BGC823 cells treated with DADS （P〈0.05）. In contrast, Chk2 Ab failed to change precipitation with Cdc25C by DADS （P＇〉0.05）. Conclusion： Activation of Chk1 was involved in cel