摘要
目的:探讨神经营养因子-3(NT-3)联合全反式维甲酸(RA)与β-巯基乙醇(β-BME)体外诱导SD大鼠骨髓间充质干细胞(BMSCs)向神经元样细胞分化的差异性。方法:SD大鼠全骨髓经贴壁法培养BMSCs并传代,P3代细胞行流式细胞仪检测其表面标志物CD29、CD90及CD45的表达。P3代BMSCs用碱性成纤维细胞生长因子(bFGF)预诱导24h后,再分别加入NT-3+RA和β-BME进行诱导,用免疫荧光法鉴定神经元特异性烯醇化酶(NSE)的表达情况。结果:(1)全骨髓贴壁法可以获得的细胞经流式细胞仪检测,细胞CD29和CD90阳性,CD45阴性,证实细胞为BMSCs,数量多且纯度高。(2)2组NSE均阳性表达,NT-3+RA组7d阳性率高于其他时间点(P<0.01);β-BME组5h阳性率高于1h,NT-3+RA组1h及5h阳性率均低于β-BME组(均P<0.01),但β-BME组24h后细胞大量死亡。结论:全骨髓贴壁筛选法是一种提取BMSCs的合适的方法,2种诱导方法均能在体外诱导BMSCs向神经元样细胞转化,NT-3+RA组细胞生存时间及后期阳性率均优于β-BME组。
Objective： To explore the differences between two methods of inducing BMSCs into neuron-like cells in vitro. Methods： BMSCs were cultured and sub-cultured by the main method of whole bone marrow directly adherent to flasks, and the surface markers CD29, CD90 and CD45 of the third generation were then detected by flow cytometry. The third generation of BMSCs were pre-induced by basic fibroblast growth factor（bFGF）, then neurotrophin-3 （NT-3）＋retinoic acid （RA） and beta-mercaptoethanol （β-BME） were added respectively. Immunofluorescence was used to identify whether cells expressed neuron-specific enolase （NSE）. Results： BMSCs with high quantity and purity could be obtained by the method of whole bone marrow directly adherent to flasks. We observed that the cells expressed CD29 and CD90 but not CD45 through flow cytometric detection, confirming that the cells were BMSCs. Both groups expressed NSE. The positive rate of 7 d in （NT-3＋RA） group was higher than other time points （P〈0.01）, and the positive rate of 5 h in the β-BME group was higher than 1 h, and the positive rate of 1 h and 5 h in （NT-3＋RA） group was lower than the β-BME group （P〈0.01）, but almost all cells in the β-BME group died in 24 h. Conclusion： The method of whole bone marrow directly adherent to flasks is suitable for extracting BMSCs. Both methods can be used to induce BMSCs differentiation into neuron-like cells. The survival time and later-stage positive rate of （NT-3＋RA） group is better than β-BME group.
作者
张杰
秦华丽
蒋明
ZHANG Jie;QIN huali;JIANG Ming(Department of Otolaryngology,the First Affliated Hospital of Wenzhou Medical University,Wenzhou,325015;Department of Otolaryngology,Changsha Hospital for Maternal and Child Health Care,Changsha,410007;Department of Otolaryngology,the Third Xiangya Hospital of Central South University,Changsha,410013)
出处
《温州医科大学学报》
CAS
2018年第9期641-645,共5页
Journal of Wenzhou Medical University
基金
湖南省医药卫生科研计划项目(C2014-13)
关键词
骨髓间充质干细胞
神经元样细胞
分化
大鼠
bone marrow mesenchymal stem cells
neuron-like cells
differentiation
rats
