摘要目的 探讨尼古丁促进人支气管平滑肌细胞收缩的作用机制.方法 体外培养原代人支气管平滑肌细胞,根据细胞是否经10-5mol/L尼古丁处理48 h分为对照组和尼古丁组,根据转染α7烟碱样受体(nAChR) siRNA 序列或 siNC 序列分为 siNC 组、siα7nAChR 组、siNC +尼古丁组和 siα7nAChR +尼古丁组.胶原凝胶收缩实验检测尼古丁对细胞收缩功能的影响;蛋白免疫印迹法检测尼古丁对人支气管平滑肌细胞 α7nAChR 和 TRPC6 蛋白表达;钙离子成像系统检测尼古丁对细胞内钙离子水平的改变.采用t检验或单因素方差分析进行统计分析.结果 尼古丁组胶原凝胶面积为(24 ±8)%,明显低于对照组的(59±14)%,差异有统计学意义(t=3.78,P<0.05);尼古丁组α7nAChR 蛋白表达为(173±16)%,明显高于对照组的(100±0)%,差异有统计学意义(t=-6.848, P<0.05).siNC +尼古丁组胶原凝胶面积为(37±10)%,明显低于 siNC 组的(72±10)%,siNC +尼古丁组细胞内基础钙水平(1.04±0.02)、钙库调控钙内流(SOCE)水平(0.68±0.03)及受体调控钙内流(ROCE)水平(0.36±0.02)明显高于 siNC 组(分别为0.79±0.07、0.41±0.04、0.17±0.02),差异有统计学意义(均P<0.05).siα7nAChR +尼古丁组胶原凝胶面积为(62±10)%,明显高于siNC +尼古丁组,siα7nAChR+尼古丁组细胞内基础钙水平(0.78±0.06)、钙库调控钙内流(SOCE)水平(0.39±0.05)及受体调控钙内流(ROCE)水平(0.15±0.02)明显低于 siNC +尼古丁组(均P<0.05).结论 尼古丁可通过上调α7nAChR 蛋白的表达,从而增加 TRPC6 蛋白的表达以及 SOCE 和 ROCE 水平,增加细胞内钙浓度,进而促进人支气管平滑肌细胞收缩.

abstractsObjective To investigate the molecular mechanism of contractility dysfunction of human bronchial smooth muscle cells induced by nicotine.Methods Primary human bronchial smooth muscle cells were cultured in vitro.The cells were divided into a control group and a nicotine group which was treated with 10 -5mol/L nicotine for 48 h and transfected with or without α7nAChR-siRNA (The siNC group,siNC + nicotine group and siα7nAChR + nicotine group).The effects of nicotine on the cell contractile function were examined by collagen gel shrinkage assay.The expressions of α7nAChR and TRPC6 protein in nicotine-treated human bronchial smooth muscle cells were detected by Western blotting.The change of intracellular calcium concentration by nicotine was detected by calcium ion imaging system.Data were analyzed by t test or single factor analysis of variance.Results The area of collagen gel in the nicotine group (24±8)% was significantly lower than that in the control group (59±14)% (t=3.78, P<0.05).Compared with the control group,the expression of α7nAChR protein in nicotine-induced group (173 ±16)% was significantly higher than that of controls 100±0)%, t=-6.848, P<0.05.Compared with the siNC group [(72±10)%,(0.79±0.07),(0.41±0.04) and (0.17 ±0.02) respectively],the collagen gel area of siNC + nicotine group was significantly reduced by (37±10)%.However,the basal calcium level (1.04±0.02), store operated calcium entry level(SOCE,0.68±0.03) and receptor operated calcium entry level (ROCE, 0.36±0.02) were remarkably elevated in the nicotine treated group (all P<0.05).Furthermore, compared with siNC + nicotine group, the area of collagen gel in siα7nAChR + nicotine group was significantly increased (62±10)%, and the basal calcium level (0.78±0.06), SOCE level (0.39±0.05) and ROCE level (0.15±0.02) were significantly reduced (all P<0.05).Conclusions Nicotine can increase the expression of TRPC6 protein,SOCE and ROCE level,and increase the intracellular calcium concentration by upregulating the expression of α7nAChR protein, thereby promoting smooth muscle cell contraction.