烟碱型乙酰胆碱受体对人肺腺癌细胞生长和凋亡的影响

Effect of nicotinic acetylcholine receptors on the growth and apoptosis of human lung adenocarcinoma A549 cell line

目的:研究烟碱型乙酰胆碱受体(nicotinic acetylcholine receptor,n AChR)激动剂和拮抗剂对人肺腺癌A549细胞生长和凋亡的影响。方法:将A549细胞随机分成对照组、激动剂组和拮抗剂组,分别加入PBS、激动剂(10-3,10-4,10-5mol/L)、拮抗剂(10-3,10-4,10-5mol/L)作用细胞24,48,72 h;MTT筛选出n AChR激动剂和拮抗剂作用细胞的最佳浓度和时间点;RT-PCR法检测α7n AChR基因的表达,Tunel检测细胞凋亡,蛋白印迹法检测Caspase-9,Bax和Bcl-2表达。结果:A549细胞株稳定表达α7 n AChR,与对照组相比,拮抗剂组α7n AChR基因表达明显减少,激动剂组增强;与对照组相比,拮抗剂各浓度组48 h细胞的增殖明显抑制(P<0.01),且10-3mol/L细胞增殖抑制明显强于10-5mol/L(P<0.05);10-3mol/L激动剂作用A549细胞48 h后细胞增殖明显增强(P<0.01)。Tunel检测结果显示,拮抗剂组细胞凋亡明显多于激动剂组和对照组(P<0.01),而激动剂组与对照组比较差异无统计学意义;与对照组相比,拮抗剂组α7n AChR表达降低,促凋亡蛋白Caspase-9,Bax表达增加,Bcl-2表达降低;激动剂组反之。结论:α7n AChR激动剂和拮抗剂分别与肺腺癌A549细胞增殖及凋亡密切相关,其中细胞凋亡可能与拮抗剂激活机体的Bax/Bcl-2和Caspase家族有关。

Objective: To explore the effect of nicotinic acetylcholine receptor( α7n AChR) agonist and antagonist on the growth and apoptosis of lung adenocarcinoma A549 cell line. Methods: Human lung adenocarcinoma cell line A549 was divided randomly into control group,agonist group and antagonist group,A549 cell line was infected with PBS,agonist,and antagonist following 24,48 and 72 h,respectively. And the optimum concentration and time of n AChR agonist and antagonist were selected by MTT; the expression of Caspase-9,Bax and Bcl-2 were detected by Western blotting,α7n AChR expression was determined by real-time PCR and cell apoptosis was examined by Tunel method. Results: A549 cells stably expressed α7n AChR. 10- 3mol / L and 48 h were the optimum concentration and time for n AChR agonist and antagonist.At the concentration( 10- 3mol / L) and time( 48 h),compared with control group,α7n AChR in antagonist group was decreased,while increased in agonist group; but the expression of Caspase-9 and Bax / Bcl-2 and cell apoptosis levels were increased in antagonist group( P < 0. 01). Conclusion: α7n AChR agonist and antagonist was closely related to the growth and apoptosis of lung adenocarcinoma A549 cell line,respectively,the cell apoptosis of which might correlated with activation of Bax / Bcl-2 and Caspase family induced by α7n AChR antagonist.