成人骨髓间质干细胞分化为神经元样细胞及与凋亡的关系  

Relationship Between Apoptosis and Differentiation from Human Mesenchymal Stem Cells into Neuron-like Cells

作　　者：姚晓黎[1] 张成[1] 刘卫彬[1] 冯善伟[1] 柳太云[1] 卢锡林[1] 张为西[1] 黄文[1] 

机构地区：[1]中山大学附属第一医院神经科,广东广州510080

出　　处：《中山大学学报（医学科学版）》2004年第6期516-520,共5页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：卫生部临床学科重点基金资助项目(2001321);广东省科技计划基金资助项目(2KM05501S)

摘　　要：【目的】探讨体外诱导成人骨髓间质干细胞(humanmesenchymalstemcells,hMSC)向神经元样细胞分化过程中分化与凋亡的关系,为进一步研究延长神经元样细胞的存活时间提供基础。【方法】从成人骨髓分离MSC,培养扩增。以β-巯基乙醇和参芪液诱导分化,于诱导后1、3、5h分别进行免疫组化鉴定神经元烯醇化酶(NSE)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)的表达,计数神经元样细胞,计算分化率。采用TUNEL方法分别检测诱导后1、3、5h的细胞凋亡率,并分析两者是否相关。【结果】hMSC经β-巯基乙醇和参芪液诱导后,可见神经元样细胞。免疫组化显示诱导出的神经元样细胞表达NSE、NF阳性、GFAP阴性。神经分化的定量分析显示诱导后1、3、5h,经β-巯基乙醇诱导NSE、NFH阳性细胞分别为43.5%±1.8%,59.3%±2.2%,76.5%±2.3%和42.6%±1.9%,55.1%±2.1%,73.5%±2.3%。参芪液诱导NSE、NFH阳性细胞分别为49.5%±1.9%,62.3%±2.1%,79.5%±2.5%和45.6%±1.9%,59.1%±2.1%,76.5%±2.3%。经两种诱导剂诱导后1、3、5h,TUNEL检测两者的凋亡率分别为1.5%,6.5%,19.5%和0.5%,2.3%,12.5%。【结论】β-巯基乙醇和参芪液均可在体外诱导hMSC分化为神经元样细胞。hMSC向神经元样细胞分化过程中也发生凋亡,分化率与凋亡率相关。ObjectiveTo elongate the lifetime of neuron-like cells differentiated from human mesenchymal stem cells (hMSC), the relationship between apoptosis and differentiation in the process of induction was investigated. MethodshMSC were separated from rib marrow and expanded to five passages in culture medium. hMSC were induced to differentiate with different inducers, β-mercaptoethanol and shenqiye. After 1 h, 3 h, and 5 h of induction, neuron-specific enolase (NSE), neurofilament (NF), and glial fibrillary acidic protein (GFAP) were examined by immunocytochemistry respectively. The ratio of neuron-like cells were counted by phase-contrast microscopy. At the same time, the ratio of apoptotic cells after induction was detected by TUNEL. ResultshMSC were expanded as undifferentiated cells in culture for more than 5 passages. When treated with inducers β-mercaptoethanol and shenqiye, hMSC exhibited neuronal phenotype. The expression of NSE and NF in the neuron-like cells were positive, but the glial astrocyte marker GFAP was not found. After 1 h, 3 h, and 5 h of induction with β-mercaptoethanol, the ratios of NSE positive cells were 43.5%±1.8%, 59.3%±2.2%, and 76.5%±2.3%, respectively, and all that of NF positive cells were 42.6%±1.9%, 55.1%±2.1%, and 73.5%±2.3%, respectively. After 1 h, 3 h, and 5 h of induction with shenqiye, the ratios of NSE positive cells were 49.5%±1.9%,62.3%±2.1%, and 79.5%±2.5%, respectively, and that of NF positive cells were 45.6%±1.9%, 59.1%±2.1%, and 76.5%±2.3%, respectively. After 1h, 3h, and 5h of induction with β-mercaptoethanol and shenqiye, the ratios of apoptotic cells were 1.5%, 6.5%, 19.5%and 0.5%, 2.3%, 12.5%, respectively. Conclusionβ-mercaptoethanol and Shenqiye can induce hMSC to differentiate into neuron-like cells. Apoptosis is observed in the process of differentiation. The ratio of differentiation is correlated with apoptosis. Shenqiye could improve the ratio of differentiation and reduce the ratio of apoptosis.

关 键 词：神经元样细胞 诱导 NSE 凋亡率 间质干细胞 β-巯基乙醇 TUNEL 成人骨髓 分化过程 神经分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学] Q254[医药卫生—基础医学]