3T3-L1前脂肪细胞中烟碱样乙酰胆碱受体α7对脂肪因子肾上腺髓质素mRNA表达的影响

Effect of Nicotinic Acetylcholine Receptor α7 on mRNA Expression of Adrenomedullin in 3T3-L1 Preadipocytes

目的研究3T3-L1前脂肪细胞和成熟脂肪细胞中是否有胆碱能系统物质mRNA的表达,及烟碱样乙酰胆碱受体(nAChR)α7功能状态对脂肪因子肾上腺髓质素(AM)mRNA表达的影响,初步阐明脂肪细胞中非神经胆碱能系统与肥胖、胰岛素抵抗及其他脂代谢性疾病的关系。方法以3T3-L1前脂肪细胞和诱导分化成熟的3T3-L1脂肪细胞为研究对象,采用反转录聚合酶链反应(RT-PCR)方法检测前脂肪细胞和成熟脂肪细胞中是否有乙酰胆碱酯酶(AChE)、胆碱乙酰转移酶(ChAT)和nAChR α7三种胆碱能系统主要组分的mRNA表达;对前脂肪细胞及成熟脂肪细胞分别予不同水平的广谱nAChR激动剂尼古丁、特异性nAChR α7激动剂氯化胆碱及特异性nAChRα7拮抗剂甲基牛扁亭碱处理12、24、36h,并设立相应处理时间的空白对照组,应用RT-PCR方法检测前脂肪细胞和成熟脂肪细胞中AM mRNA的表达。结果1.RT-PCR方法证实在3T3-L1前脂肪细胞和成熟脂肪细胞中均有AChE、ChAT、AChR α7的mRNA表达。2.给予不同剂量的尼古丁和氯化胆碱,干预作用不同时间,3T3-L1前脂肪细胞和成熟脂肪细胞中AM mRNA表达水平与相应的空白对照组比较均呈剂量依赖性升高(P<0.05或P<0.01),但随干预作用时间延长,这种剂量依赖性上调效应减弱。二种激动剂以氯化胆碱的剂量依赖性上调效应更明显。而相同剂量下不同作用时间的处理组间无统计学差异(Pa>0.05)。3.拮抗剂甲基牛扁亭碱对3T3-L1前脂肪细胞和成熟脂肪细胞中AM mRNA表达则呈明显的剂量依赖性抑制作用(P<0.05或P<0.01)。结论3T3-L1前脂肪细胞和成熟脂肪细胞中存在非神经元型胆碱能系统,其中nAChR α7调节脂肪因子AM mRNA的表达,脂肪组织中非神经元型胆碱能系统的重要组分nAChR α7可能在肥胖、胰岛素抵抗及其他脂代谢相关疾病的病理生理过程中发挥重要作用。

Objective To study the expression of non - neuronal cholinergic system mRNA and the effect of nicotinic acetylcholine receptor（nAChR） α7 on mRNA expression of adrenomedullin （AM）in 3T3 -L1 preadipocytes and adipocytes,and provide functional implication about the non - neuronal cholinergic system in adipocytes and preadipocytes on obesity,insulin resistance and other related diseases. Methods The research objects were 3T3 - L1 preadipocytes and adipocytes, reverse transcription - polymerase chain reaction （ RT - PCR） were used to detect the mRNA expression of acetylcholinesterase （ACHE）, choline acetyhransferase （CHAT）and nAChR α7, the 3 principal composers of non - neuronal cholinergic system in 3T3 - L1 preadipocytes and adipocytes. After expose to different doses of non - selective nAChR agonist nicotine,selective nAChR α7 agonist choline chloride or selective nAChR α7 antagonist methyllycaconitine for 12,24 and 36 h respectively,serum- free Dulbecco＇s Modified Eagle Media：Nutrient Mixture F- 12 （DMEM/F12） were included as control in the same time,the mRNA expression of AM was detected by RT - PCR. Results 1. The existence of ACHE, CHAT, AChR α7 in 3T3 - L1 preadipocytes and adipocytes were confirmed by RT - PCR. 2. After exposed to different doses of nicotine and choline chloride for different time respectively,resulted in the significantly increase of AM mRNA level in a dose- dependent manner（P 〈0.05 or P 〈0.01 ） compared with control group, but this effect reduced accompanying with the prolonged time of exposure. The increase was more obviously in choline chloride treatment group than in nicotine treatment group. While the time - dependent change in same dose had not significant difference （ P 〉 0.05 ）. 3. Methyllycaconitine inhibited adrenomedullin mRNA expression with dose - dependent （P 〈 0.05 or P 〈 0. 01 ）. Conclusions These data demonstrated that non - neuronal cholinergic system exists in 3T3 - L1 preadipocytes and adipocytes,and nAChR α7 is responsible for regulation of AM mRNA expression. In conclusion,nAChR α7,one of principal composers of non - neuronal cholinergic system may play an important role in the nathophvsiology of obesity,insulin resistance and other related disease.