d-尼古丁对血管平滑肌细胞迁移的影响

Effect of d-Nicotine on Migration of Vascular Smooth Muscle Cells

为了在分子水平上揭示吸烟导致动脉粥样硬化的机制,探讨了烟草致病的主要成分d-尼古丁对豚鼠大脑基底动脉血管平滑肌细胞GbaSM-4迁移作用的影响。应用Boyden小室实验发现,d-尼古丁具有促进GbaSM-4细胞迁移的作用。免疫荧光染色显示,在d-尼古丁作用下有GbaSM-4细胞伪足内肌动蛋白表达和分布增加的现象。为了进一步阐明d-尼古丁促进平滑肌细胞迁移作用的分子机制,应用RT-PCR方法检测到在GbaSM-4细胞内有α7型烟碱乙酰胆碱受体的表达。应用烟碱乙酰胆碱受体的特异性抑制剂甲基牛扁碱和肌肉收缩的关键酶——肌球蛋白轻链激酶(myosinlightchainkinase,MLCK)抑制剂ML-9作用GbaSM-4细胞后,发现d-尼古丁对GbaSM-4细胞的诱导迁移作用被明显的抑制。采用RNA干扰技术,成功地使GbaSM-4细胞内MLCK的表达水平下调,观察到d-尼古丁对GbaSM-4细胞的诱导迁移作用也被明显的抑制。上述研究结果表明,d-尼古丁以趋化因子的作用促进血管平滑肌细胞迁移,其分子机制可能与α7型烟碱乙酰胆碱受体和MLCK等因素有关,这一发现为揭示吸烟导致动脉粥样硬化提供了实验依据。

In order to demonstrate the relationship between smoking and arteriosclerosis the effect of d-nicotine on migration of vascular smooth muscle GbaSM-4 cells, a cell line of vascular smooth muscle cells （VSMCs） derived from guinea pig brain basilar arteries, was investigated. It was found that d-nicotine was able to promote the migration of the cells by Boyden＇s chamber assay. The expression and distribution of actin were increased in the body and lamellipodia of GbaSM-4 cells by immunofluorescent staining. The acetylcholine receptor （α7-nAChR） was detectable in GbaSM-4 cells by RT-PCR. The effect of inducing migration was inhibited by an α7-nAChR antagonist （methyllycaconitine） and an antagonist （ML-9） of myosin light chain kinase （MLCK） in the cells. And the effect of inducing migration was also depressed by down-regulating MLCK through RNA interference. The data demonstrated that the d-nicotine promotes the migration of vascular smooth muscle as chemotaxis, in which α7-nAChR and MLCK are involved in the molecular mechanism. The finding provides experimental evidence for smoking resulting in arteriosclerosis.