目的:考察抗胆碱药(R,R’)-富马酸戊乙奎醚[(R,R’)-penehyclidine fumarate,R2PHF]对非小细胞肺癌H157细胞增殖的影响,探讨其作用机制.方法:应用RT-PCR检测H157上M受体的表达；磺酰罗丹明B(sulforhodamine B,SRB)法测定R2PHF作用于H157后细胞活力改变；流式细胞术(FCM)检测细胞周期和细胞凋亡；Western Blot分析细胞周期蛋白cyclin D1表达水平的变化.结果:M受体五种亚型在H157上有不同的表达,R2PHF在体外可时间及浓度依赖性抑制H157细胞增殖并诱导其发生G0/G1期阻滞.FCM检测结果显示,20 μmol·L-1 R2PHF作用48 h后,H157细胞G0/G1期比率显著增高,S期比率显著下降,而细胞凋亡未发生明显变化.Western Blot分析表明R2PHF可时间及浓度依赖性下调细胞周期蛋白cyclin D1的表达.结论:R2PHF抑制非小细胞肺癌H157细胞增殖与其下调cyclin D1的表达水平并将细胞阻滞在G0/G1期密切相关.%Objective.- To investigate the effect of cholinergic antagonist (R,R' )-penehyclidine fumarate (R2PHF) on proliferation of human non-small cell lung cancer HI57 cells and explore the potential mechanism. Methods; The muscarinic receptors expressed in H157 cells were detected by RT-PCR. H157 cells were treated with different concentration of R2PHF for various time, and cell viability was detected by sulforhodamine B( SRB) . Flow cytometry( FCM) was used to analyze cell cycle and apoptosis after treatment with R2PHF. The expression of cyclin Dl was analyzed by Western Blot. Results-. Expressions of the five muscarinic receptor subtypes were different in H157 cells. The proliferation of H157 cells was inhibited by R2PHF in a concentration-and time-dependent manner in vitro and the cell cycle was arrested in Go/G1. The percentage of Go/G1 phase cells increased while the S phase cells decreased after treated with 20 u,mol-L~ R2PHF for 48 h. There was no change observed in cell apoptosis. R2PHF significantly down regulated the expression of cyclin Dl in a concentration- and time-dependent manner. Conclusion; These results reveal that R2PHF could inhibit proliferation of non-small cell lung cancer H157 cells in vitro by down-regulating expression of cyclin Dl and arresting the cell cycle in Go/G1 phase.
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