MASH1介导肾上腺髓质嗜铬细胞发生神经元转分化  

MASH1 induces neuron transdifferentiation of adrenal medulla chromaffin cells

作　　者：彭婀敏 胡成平[2,3,4] 冯俊涛 贺若曦[2,3,4] PENG Emin;HU Chengping;FENG Juntao;HE Ruoxi(Outpatient Clinic of Xiangya Hospital International Medical Center,Central South University,Changsha 410008;Department of Respiratory Medicine,National Clinical Key Specialty of Respiratory Disease,Branch of National Clinical Research Center for Respiratory Disease,Xiangya Hospital,Central South University,Changsha 410008;Center of Respiratory Medicine,Xiangya Hospital,Central South University,Changsha 410008;Clinical Research Center for Respiratory Diseases in Hunan Province,Changsha 410008,China)

机构地区：[1]中南大学湘雅医院国际医疗部门诊,长沙410008 [2]中南大学湘雅医院呼吸内科,国家呼吸疾病临床重点专科,国家呼吸系统疾病临床医学研究中心分中心,长沙410008 [3]中南大学湘雅医院呼吸中心,长沙410008 [4]湖南省呼吸疾病临床医学研究中心,长沙410008

出　　处：《中南大学学报（医学版）》2023年第4期526-537,共12页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(81400022);湖南省自然科学基金(2020JJ5897,2020JJ4904)。

摘　　要：目的:肾上腺髓质嗜铬细胞(adrenal medulla chromaffin cells,AMCCs)在神经生长因子(nerve growth factor,NGF)诱导下向神经元转分化,引起肾上腺素分泌减少,其可能参与了支气管哮喘的发病。神经分化发育的关键因子哺乳动物无刚毛-鳞甲同源物(mammalian achaete scute-homologous 1,MASH1)在神经元转分化的AMCCs中升高。本研究拟探讨MASH1对AMCCs神经元转分化的影响及机制。方法:分离并培养大鼠AMCCs,用siMASH1、MASH1过表达质粒转染,再用NGF和/或地塞米松、MAPK激酶-1抑制剂PD98059刺激48 h。在光镜及电镜下观察AMCCs形态。用免疫荧光法检测酪氨酸羟化酶和肾上腺素合成关键酶苯乙醇胺-N-甲基转移酶(phenylethanolamine-N-methyltransferase,PNMT)水平,蛋白质印迹法检测AMCCs中PNMT、MASH1、外周蛋白、细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)及磷酸化的细胞外调节蛋白激酶(phosphorylated extracellular regulated protein kinases,pERK)、JMJD3的蛋白质水平,Real-time RT-PCR法检测MASH1、JMJD3的mRNA水平,ELISA法检测细胞上清液中肾上腺素水平。结果:培养的细胞经免疫荧光染色示酪氨酸羟化酶和PNMT均为阳性,鉴定为AMCCs。NGF处理后AMCCs出现细胞突起,pERK、外周蛋白、MASH1蛋白表达量均显著上升(均P<0.05);PNMT蛋白表达和肾上腺素分泌水平下降(均P<0.01)。MASH1干扰逆转了NGF的作用,使AMCCs中PNMT蛋白和肾上腺素水平升高,外周蛋白及细胞突起数量减少(P<0.01)。过表达MASH1可使细胞突起、外周蛋白表达量显著增加(均P<0.01),PNMT蛋白和肾上腺素水平下降(均P<0.01)。与NGF组对比,NGF+PD98059组AMCCs中MASH1、JMJD3蛋白及mRNA水平均下降(均P<0.05)。经过PD98059和地塞米松处理后,NGF促进AMCCs转分化的作用受到抑制,细胞突起数量及肾上腺素水平均下降(均P<0.05);此外,NGF激活的pERK/MASH1通路活性也受到抑制。结论:MASH1是AMCCs发生神经元转分化的关键因子Objective:Nerve growth factor(NGF)induces neuron transdifferentiation of adrenal medulla chromaffin cells(AMCCs)and consequently downregulates the secretion of epinephrine(EPI),which may be involved in the pathogenesis of bronchial asthma.Mammalian achaete scute-homologous 1(MASH1),a key regulator of neurogenesis in the nervous system,has been proved to be elevated in AMCCs with neuron transdifferentiation in vivo.This study aims to explore the role of MASH1 in the process of neuron transdifferentiation of AMCCs and the mechanisms.Methods:Rat AMCCs were isolated and cultured.AMCCs were transfected with siMASH1 or MASH1 overexpression plasmid,then were stimulated with NGF and/or dexamethasone,PD98059(a MAPK kinase-1 inhibitor)for 48 hours.Morphological changes were observed using light and electron microscope.Phenylethanolamine-Nmethyltransferase(PNMT,the key enzyme for epinephrine synthesis)and tyrosine hydroxylase were detected by immunofluorescence.Western blotting was used to test the protein levels of PNMT,MASH1,peripherin(neuronal markers),extracellular regulated protein kinases(ERK),phosphorylated extracellular regulated protein kinases(pERK),and JMJD3.Real-time RT-PCR was applied to analyze the mRNA levels of MASH1 and JMJD3.EPI levels in the cellular supernatant were measured using ELISA.Results:Cells with both tyrosine hydroxylase and PNMT positive by immunofluorescence were proved to be AMCCs.Exposure to NGF,AMCCs exhibited neurite-like processes concomitant with increases in pERK/ERK,peripherin,and MASH1 levels(all P<0.05).Additionally,impairment of endocrine phenotype was proved by a signifcant decrease in the PNMT level and the secretion of EPI from AMCCs(all P<0.01).MASH1 interference reversed the effect of NGF,causing increases in the levels of PNMT and EPI,conversely reduced the peripherin level and cell processes(all P<0.01).MASH1 overexpression significantly increased the number of cell processes and peripherin level,while decreased the levels of PNMT and EPI(all P<0.01).Compared with the NGF gr

关 键 词：MASH1 嗜铬细胞 神经元 神经生长因子 地塞米松 转分化 

分 类 号：R586[医药卫生—内分泌]