摘要
目的探讨大鼠骨髓间充质干细胞神经分化过程中哺乳动物雷帕霉素靶蛋白(mammalian target of rapamy-cin,mTOR)分布的变化及意义。方法常规体外培养大鼠MSCs,采用β-巯基乙醇诱导MSCs分化为神经细胞。实验分MSCs空白对照组、二甲基亚砜(DMSO)阴性对照组、β-巯基乙醇诱导组、不同浓度雷帕霉素干预组、不同浓度雷帕霉素干预+β-巯基乙醇诱导组。采用免疫荧光法检测诱导前后mTOR在细胞内的分布,激光共聚焦显微镜观察照相;Westernblot法检测诱导前后神经细胞相关蛋白和mTOR通路蛋白的表达。结果①诱导前mTOR主要在MSCs细胞核内点状分布,细胞质也有少量的分布;诱导后核内荧光信号减弱,mTOR向细胞质转移。雷帕霉素处理后,随着雷帕霉素浓度的提高(10～30μmol/L),mTOR向细胞质转移越明显。雷帕霉素浓度超过50μmol/L后,细胞形态变圆,部分细胞脱壁,死亡率显著增加(P<0.05)。②β-巯基乙醇可诱导骨髓间充质干细胞分化为神经细胞,诱导后神经细胞Tau蛋白、MAP-2蛋白表达显著增加。雷帕霉素(10～30μmol/L)干预后,随着雷帕霉素浓度(10～20μmol/L)的提高,Tau蛋白、MAP-2蛋白表达逐渐增加,30μmol/L时Tau蛋白、MAP-2蛋白表达下降(P<0.05)。③Western blot检测结果提示,诱导前后总mTOR表达不变;磷酸化mTOR、磷酸化p70S6K、磷酸化4EBP1诱导后较诱导前表达下降(P<0.05),雷帕霉素干预后下调更加显著(P<0.05)。结论 mTOR在MSCs神经分化过程中由细胞核内向细胞质转移,活性下降,表达下调,提示mTOR在MSCs神经分化过程中可能发挥了重要作用。
Objective To investigate the distribution and significance of mammalian target of rapamy- cin （roTOR） during the differentiation of rat bone marrow mesenchymal stem cells （MSCs） into neurons. Methods Rat MSCs were cultured by conventional method in vitro, and were induced by β-mercaptoethanol （β-ME） to differentiate into neurons. The MSCs were divided into a MSCs blank control group, a dimethyl sulfoxide （DMSO） negative control group, a β-ME induction group, different concentrations of rapamycin inter vention groups, and different concentrations of rapamycin intervention ＋ β-ME induction groups. Immunofluo rescence and laser confocal microscopy were used to observe the intracellular distribution of mTOR before and after induction. Western blotting was used to detect the expression of neuron-related proteins and roTOR before and after induction. Results （ 1 ） Before β-ME induction, mTOR were mainly distributed in the cell nucleus like spots, and a small amount was distributed in the cytoplasm. Afterβ-ME induction, the nuclear fluores cence signal reduced, and roTOR translocated into the cytoplasm. After rapamycin treatment, mTOR transloca tion to the cytoplasm became more obvious along with the increase of rapamycin concentration （10 to 30 p.mol/L）. When the concentration of rapamycin was more than 50 μmol/L, the morphology of MSCs became round, some cells detached, and the cell death rate was significantly increased （P 〈 0.05 ）. （2） MSCs were induced by β-ME to differentiate into neurons and the expression of Tau and MAP-2 increased significantly. After rapamycin treatment, the expression of Tau and MAP-2 gradually increased along with the increase of rapamycinconcentration （10 to 20 μmol/L）. When the concentration reached 30 μmol/L, the expression of Tau and MAP-2 was down-regulated （ P 〈 0.05 ）. （ 3 ） Western blotting results suggested that the total expression of mTOR was the same before and after β-ME induction. The expression of phospho-mTOR, phosp
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2013年第13期1345-1349,共5页
Journal of Third Military Medical University
基金
国家自然科学基金(81071114)~~
