乙酰胆碱受体对HaCaT细胞黏附功能的影响及作用机制

Effect and Mechanism of Acetylcholine Receptors on the Adhesion Function of Ha Ca T cells

目的研究乙酰胆碱途径药物对Ha Ca T细胞黏附功能的影响及其作用机制。方法培养Ha Ca T细胞至融合状态后,分别加入适当浓度的乙酰胆碱途径药物(M受体抑制剂阿托品,N受体抑制剂筒箭毒碱,乙酰胆碱受体激动剂卡巴胆碱),用细胞黏附检测方法检测细胞黏附的变化情况,结果以细胞碎片数表示;分别用Triton X-100和RIPA裂解细胞,得到胞浆蛋白和总蛋白,用蛋白免疫印迹测定细胞表面与黏附相关的蛋白桥粒芯蛋白1、桥粒芯蛋白3、桥斑珠蛋白、E-钙黏素的变化;用定量多聚酶链反应(q PCR)检测上述细胞表面蛋白在mRNA水平的变化。结果对照组、阿托品、筒箭毒碱、卡巴胆碱作用后细胞碎片数目分别为6.33±1.15,35.00±2.65,6.33±1.53,6.00±1.00;阿托品在蛋白质和mRNA水平减少桥粒芯蛋白3表达,并使桥粒芯蛋白3从胞膜脱离,卡巴胆碱可增加桥粒芯蛋白1、桥粒芯蛋白3、桥斑珠蛋白表达,并促使桥粒芯蛋白1、桥斑珠蛋白黏附于胞膜。筒箭毒碱对各种细胞表面蛋白均无明显影响。结论 Ha Ca T细胞表面的乙酰胆碱受体可以调节细胞间黏附分子,尤其是桥粒分子的表达,并影响这些分子在细胞膜上的稳定性,从而介导细胞间黏附,这种作用主要通过M受体实现。

Objective To study the effect and mechanism of cholinergic drugs on adhesion function of HaCaT cells.Methods HaCaT cells were cultured to fusion,and then proper concentration of cholinergic drug（ m AChR antagonist atropine,n AChR antagonist tubocurarine or AChR agonist carbachol） was added to the culture. Adhesion assay was conducted to detect the adhesion function of HaCaT cells,and the result was expressed as the number of cellular debris. Triton X-100 and RIPA were used to lyse the Ha Ca T cells to obtain the cytoplasm and total protein. Western blot was conducted to detect protein level of adhesion molecules such as desmoglein1,desmoglein 3,plakoglobin and E-cadherin. QPCR was conducted to detect the mRNA level of these molecules. Results The cellular debris number of control,atropine,tubocurarine and carbachol group was respectively 6. 33 ± 1. 15,35. 00 ± 2. 65,6. 33 ± 1. 53,6. 00 ± 1. 00. Atopine reduced the expression of desmoglein 3 at protein and mRNA level,and made the desmolgein 3 detach from the membrane; carbachol increased the expression of desmoglein 1,and desmoglein 3,plakoglobin and promote the adhesion of desmoglein1 and plakoglobin to the membrane. Tubocurarine had no effect on these adhesion molecules. Conclusion Acetylcholine receptor on the membrane of HaCaT cells can mediate adhesion between HaCaT cells through regulating the expression of the adhesion molecules（ especially the desmosomes molecules） and affecting the stability of these molecules on the membrane,and these effects are mainly mediated by the muscarinic receptor.