基于小鼠P815与N2a细胞体外共培养构建模拟肠易激综合征内脏高敏神经增生细胞模型  

Established cell model and mechanism of visceral hypersensitivity and nerve hyperplasia in IBS using P815 and N2a co-culture

作　　者：李鸿斌 甘春莉[1,2] 谢翔雨 刘珊 卢琴[4] 柯威 齐诗语 黄育生 唐洪梅[6] LI Hongbin;GAN Chunli;XIE Xiangyu;LIU Shan;LU Qin;KE Wei;QI Shi-yu;HUANG Yusheng;TANG Hongmei(The First Clinical Medical College,Guangzhou University of Traditional Chinese Medicine,Guangzhou 510405,China;Lingnan Medical Research Center,Guangzhou University of Chinese Medicine,Guangzhou 510405,China;Science and Technology Innovation Center,Guangzhou University of Chinese Medicine,Guangzhou 510405,China;Chongqing Hospi-tal of the First Affiliated Hospital of Guangzhou University of Chinese Medicine(Beibei District Hospital of Traditional Chi-nese Medicine of Chongqing City),Chongqing,400700,China;Foshan Hospital of Traditional Chinese Medicine,Foshan 528099,China;The First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510405,China)

机构地区：[1]广州中医药大学第一临床医学院,广东广州510405 [2]广州中医药大学岭南医学研究中心,广东广州510405 [3]广州中医药大学科技创新中心,广东广州510405 [4]广州中医药大学第一附属医院重庆医院(重庆市北碚区中医院),重庆400700 [5]佛山市中医院,广东佛山528099 [6]广州中医药大学第一附属医院,广东广州510405

出　　处：《中国病理生理杂志》2025年第4期825-832,共8页Chinese Journal of Pathophysiology

基　　金：广东省基础与应用基础研究基金项目(No.2023A1515220155);国家中医药传承创新中心科研专项(No.2023ZJ03);广东省医院协会药学科研专项(No.2021YXZD02);广东省普通高校重点领域专项(No.2023ZDZX2019)。

摘　　要：目的:利用小鼠P815肥大细胞和N2a神经细胞共培养建立模拟肠易激综合征内脏高敏神经增生的细胞模型,探究其可能的作用机制。方法:使用ELISA法确认C48/80刺激P815脱颗粒的方法。明场观察神经突触长度确认神经分化细胞数以选择视黄酸(retinoic acid,RA)刺激N2a细胞分化浓度。用Transwell小室建立P815和N2a共培养体系,分别设置单独培养N2a细胞组、N2a细胞与P815细胞共培养组、N2a细胞与P815细胞共培养+C48/80组和N2a细胞+RA组。明场观察共培养后N2a细胞分化细胞数,Western blot和PCR检测N2a细胞神经生长因子(nerve growth factor,NGF)、酪氨酸激酶受体A(tyrosine kinase receptor A,TRKA)、生长相关蛋白43(growth-associated protein-43,GAP43)、神经元特异烯醇化酶(neuron specific enolase,NSE)、突触素(synapsin,SYN)、突触后致密蛋白95(postsynaptic density protein-95,PSD-95)的蛋白及基因的表达。结果:10μmol/L的RA刺激24 h是N2a分化的最佳条件。20 mg/L的C48/80刺激P815细胞24 h是使其脱颗粒的最佳条件。与单独培养的N2a细胞相比,N2a+P815+C48/80组及N2a+RA组的分化细胞数显著增高(P<0.01),NGF、TRKA、GAP43、NSE、SYN、PSD-95蛋白及基因表达显著增高(P<0.05)。结论:小鼠肥大细胞神经细胞体外共培养模型结果显示,肥大细胞脱颗粒增强了肠神经细胞的神经增生水平,促进神经结构与功能改变;NGF/TRKA通路关键蛋白表达异常调控的突触重塑参与了肥大细胞脱颗粒诱导的神经增生。AIM:To establish a cell model of visceral hypersensitivity and nerve hyperplasia in irritable bowel syndrome(IBS)by conducting an in vitro co-culture of mouse P815 mast cells and N2a nerve cells and explore its possible mechanism.METHODS:Enzyme-linked immunosorbent assay(ELISA)with three replicates was used to confirm the C48/80-induced P815 degranulation.The length of neurites was observed under bright field microscopy to determine the number of differentiated neurons,thereby selecting the concentration of retinoic acid(RA)for stimulating the differentiation of N2a cells,with four replicates.A co-culture system of P815 and N2a cells was established using Transwell chambers with four replicates.The following groups were established:N2a cells cultured alone,N2a cells co-cultured with P815 cells,N2a cells co-cultured with P815 cells plus C48/80,and N2a cells plus RA group.After co-culturing,the number of differentiated N2a cells was observed under bright field.The expression of nerve growth factor(NGF),tyrosine kinase receptor A(TRKA),growth-associated protein-43(GAP43),neuron-specific enolase(NSE),synapsin(SYN),and postsynaptic density protein-95(PSD-95)at both protein and gene levels in N2a cells was detected using Western blot and polymerase chain reaction(PCR),with four replicates.RESULTS:The best condition for N2a differentiation was stimulation with 10μmol/L RA for 24 hours,whereas the best condition for degranulation was stimulation of P815 cells with 20 mg/L C48/80 for 24 hours.Compared with N2a cells cultured alone,the differentiation ratio of the N2a+P815+C48/80 and N2a+RA groups was significantly increased(P<0.01),and the protein and mRNA expressions of NGF,TRKA,GAP43,NSE,SYN,and PSD-95 were significantly increased(P<0.05).CONCLUSION:Our results revealed that mast cell degranulation enhances the level of nerve hyperplasia in enteric nerve cells and promotes changes in nerve structure and function.Synaptic remodeling regulated by abnormal expression of key proteins such as NGF,TRKA,and GAP43 is involved

关 键 词：肠易激综合征 内脏高敏 肠神经系统 神经可塑性 

分 类 号：R363[医药卫生—病理学] R574.4[医药卫生—基础医学] R749.92