摘要
目的:探讨体外诱导大鼠骨髓间充质干细胞(MSCs)向神经元样细胞分化过程中凋亡发生的机制。方法:分离纯化大鼠骨髓间充质干细胞,随机分为3组:对照组为单纯β-巯基乙醇诱导分化组;NEK1/2抑制剂1,4-二氨基-2,3-氰基-1,4-双[2-氨基苯基硫代]丁二烯(U0126)组为自预诱导开始在加入β-巯基乙醇的同时加入10μmol/LU0126;蛋白激酶PKC激动剂氟波酯(PMA)组为自预诱导开始在加入β-巯基乙醇的同时分别加入30nmol/L、120nmol/L与300nmol/LPMA。诱导5h后终止反应,MTT法检测细胞活性,免疫细胞化学检测神经元特异性巢蛋白nestin及凋亡相关蛋白caspase、Bcl-2、Bax的表达情况,TUNEL法检测细胞凋亡指数。结果:诱导后5h与对照组相比,U0126组神经元样细胞的活性、nestin的表达、Bcl-2蛋白阳性表达率均出现增强或升高(P<0.05),细胞凋亡指数降低(P<0.05);300nmol/LPMA组则显著抑制神经元样细胞的活性和nestin的表达(P<0.05),增强Bax并降低Bcl-2的表达(P<0.05),细胞凋亡指数明显升高(P<0.05)。结论:大鼠MSCs诱导分化为神经元样细胞过程中发生凋亡机制涉及MAPK及PKC信号途径。
AIM： To investigate the mechanism of apoptosis during the process of adult rat mesenchymal stem ceils （MSCs） differentiating into neuron- like cells in vitro. METHODS： The MSCs were isolated primarily from adult rats bone marrow by density gradient and then expanded in medium as undifferentiated ceils for 3 -5 generations. The MSCs were divided into three groups at random. The control group was induced with β - mercaptoethanol （ β - ME）. Meanwhile, the U0126 group was given β - ME and U0126 （ 10 μmol/L） added at the beginning of induction. The PMA groups were treated with β - ME and different concentrations of PMA since pre - induction. The effects of U0126 and PMA on the activity of neuron - like cells were observed by MTT. The effects of U0126 and PMA on the expression of neuron specific marker nestin and expression of apoptosis - related protein caspase, Bcl - 2, Bax in neuron - like cells were detected by using immunocytochemistry method. TUNEL technique was used to detect apoptosis index. RESULTS： Compared to control group, neuron viability, nestin and Bcl -2 increased and neuron apoptosis decreased in U0126 group （P 〈 0.05 ）. The activity of neuron - like ceils, the expression of nestin and Bcl - 2 in experiment group treated with 300 nmol/L PMA decreased （ P 〈 0. 05 ）, while Bax protein expression and apoptosis index increased （ P 〈 0. 05 ）. CONCLUSION： Both MAPK and PKC signal pathways may be involved in the differentiation of MSCs into neuron - like cells as well as the apoptotic process in differentiated neuron- like cells.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2009年第10期1931-1935,共5页
Chinese Journal of Pathophysiology
基金
四川省科技厅自然科学基金资助项目(No.2005JY029-144)
关键词
骨髓间充质干细胞
神经元
细胞凋亡
信号转导
Bone marrow mesenchymal stem cells
Neurons
Apoptosis
Signal transduction
