尼古丁对多种干细胞中烟碱型乙酰胆碱受体表达的影响

Effects of nicotine on expression of nicotinic acetylcholine receptors in several kinds of stem cells

目的:明确干细胞中烟碱型乙酰胆碱受体(nAChRs)亚基的表达以及尼古丁处理后受体改变的情况。方法:选择胚胎、胚胎样干细胞和成体干细胞作为探究对象。采用无饲养层细胞的方法培养人胚胎干细胞系H9和人诱导多能干细胞(IPSC);成体干细胞选择人脐带间充质干细胞(UCMSC)和人骨髓间充质干细胞(BMMSC)。4种细胞传代培养后分成对照组和处理组,处理组培养基中加入尼古丁,分别提取总RNA和蛋白。采用RT-PCR、RT-qPCR和Western blot检测nAChRs亚基的mRNA和蛋白表达水平,采用细胞免疫荧光染色检测受体亚基的表达位置。结果:RT-PCR和Western blot结果显示,H9细胞、IPSC和UCMSC中均检测到nAChRs的α2、α3、α4、α7、α9和β1亚基,而BMMSC仅α4、α7、α9和β1表达明显。尼古丁处理后,RT-qPCR结果显示α2在H9细胞和UCMSC中显著下调,而在IPSC中发生上调;β2在BMMSC和IPSC中显著上调,而在UCMSC中显著下调。结论:胚胎干细胞和成体干细胞中表达的nAChRs亚基不同,且分化潜能越高,表达种类越多。尼古丁对nAChRs亚基的表达调控也存在细胞差异,其中H9细胞对尼古丁的作用表现出高敏感性。

AIM:To detect the subunits of nicotinic acetylcholine receptors(nAChRs)in stem cells and the changes of the receptors after nicotine treatment.METHODS:Human embryonic stem cell line H9 and human induced pluripotent stem cells(IPSC)were cultured without feeder layer cells.Human umbilical cord mesenchymal stem cells(UCMSC)and human bone marrow mesenchymal stem cells(BMMSC)were also selected.Total RNA and protein were extracted from 4 kinds of cells after subculture,and total RNA was extracted after nicotine treatment.The mRNA and protein expression levels of the subunits of nAChRs were determined by RT-PCR,RT-qPCR and Western blot,and the expression location of the receptors was observed by cellular immunofluorescence.RESULTS:The results of RT-PCR and Western blot showed thatα2,α3,α4,α7,α9 andβ1 subunits of nAChRs were detected in H9 cells,IPSC and UCMSC,while onlyα4,α7,α9 andβ1 were expressed in BMMSC.After nicotine treatment,RT-qPCR results showed thatα2 was significantly down-regulated in H9 and UCMSC but up-regulated in IPSC,whileβ2 was significantly up-regulated in BMMSC and IPSC but down-regulated in UCMSC.CONCLUSION:The subunits of nAChRs expressed in embryonic stem cells and adult stem cells are different,and with the higher differentiation potential,the cells express the more types of subunits.The regulatory effect of nicotine on nAChR subunit expression in various stem cells is different,and H9 cells show high sensitivity to the effect of nicotine.