骨髓间充质干细胞诱导分化为神经样细胞的离子通道:是否有电生理特性?  

Whether the ion channel of differentiation from bone marrow mesenchymal stem cells into neural-like cells has electrophysiological characteristics?

作　　者：杨慧民[1] 张苏明[2] 郑荣远[1] 

机构地区：[1]温州医学院附属第一医院神经内科,浙江省温州市325000 [2]华中科技大学同济医学院附属同济医院神经内科,湖北省武汉市430030

出　　处：《中国组织工程研究与临床康复》2009年第40期7881-7884,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30070825)~~

摘　　要：背景:以往研究证实,骨髓间充质干细胞经体外诱导先分化为神经干细胞,然后分化为神经样细胞,但是对于分化的神经样细胞是否具有电生理特性尚不明确。目的:观察骨髓间充质干细胞诱导分化为神经样细胞的离子通道是否具有电生理特性?设计、时间及地点:观察性实验,于2005-03在华中科技大学同济医学院附属同济医院神经内科实验室完成。材料:4周龄Wistar大鼠37只,雌雄不拘,体质量150g左右。方法:取Wistar大鼠股骨和胫骨,进行骨髓间充质干细胞原代培养,传至第15～20代融合状态的间充质干细胞置于预诱导培养基中培养24h后,换用诱导培养基。在倒置显微镜下连续观察间充质干细胞的形态变化,分别采用免疫组织化学和Western Blot对未经诱导分化的间充质干细胞和诱导后第3天的间充质干细胞进行检测。利用电生理膜片箝技术检测50个骨髓间充质干细胞诱导分化后的神经样细胞。主要观察指标:记录到的钾电流、GABA电流。结果:①诱导分化前呈梭形细胞,在加入预诱导培养基后未出现明显的形态变化,换用诱导培养基1h,胞体开始收缩,出现少数较小的卵圆形或纺锤形细胞,诱导24h后,约60%细胞变成双极形、多极形和锥形,出现类似神经元细胞的形态。②免疫细胞化学检测未经诱导分化的间充质干细胞无NeuN、Nestin、GFAP表达,诱导分化后第3天的间充质干细胞NeuN表达明显增强,有Nestin表达,无GFAP表达。③Western Blot检测在未诱导分化时和预诱导24h,无NeuN、Nestin表达,Nestin表达在诱导分化后6h呈强阳性,在诱导分化24h和48h逐渐减弱。NeuN在诱导分化后6h有表达,在诱导分化后24h和48h表达增强。④在检测的50个细胞中,共有25个细胞检测到ATP电流(50%)、GABA电流13个(26%)、总钾电流46(92%)、复极化钾电流46(92%)。结论:在诱导分化的神经样细胞上存在功能性离子通道,具有电生理特性。BACKGROUND：Previous studies have shown that bone marrow mesenchymal stem cells（BMSCs） can differentiate into neural stem cells following induction,and then differentiate into neural-like cells. However,it is still unclear whether neural-like cells have electrophysiological characteristics. OBJECTIVE：To study whether the ion channels of neural-like cells differentiated from BMSCs have electrophysiological characteristics. DESIGN,TIME AND SETTING：The observational study was performed at the Laboratory of the Department of Neurology,Tongji Hospital Affiliated to Tongji Medical College,Huazhong University of Science and Technology in March 2005. MATERIALS：37 Wistar rats aged 4 weeks old,of both genders,weighing approximately 150 g were used in this study. METHODS：Femur and tibia were obtained from Wistar rats for primary culture of BMSCs. MSCs at passage 15-20 were firstly incubated in preinduction medium,for 24 hours,and then incubated in induction medium containing α-MEM,2% dimethyl sulfoxide and 200 μmol/L butylated hydroxyanisole. Under an inverted microscope,morphological changes of MSCs were observed. Non-induced MSCs and MSCs following 3 days of induction underwent immunohistochemistry and Western Blot. Electrophysiological patch clamp technique was used to determine neural-like cells differentiated from 50 BMSCs. MAIN OUTCOME MEASURES：Potassium current and GABA current were recorded. RESULTS：Cells were spindle shape before induction,obviously varied following preinduction,and then the body began contracted after being induced in the medium for 1 hour,with the presence of a few small orbicular-ovate or spindle cells. Following 24 hours of induction,about 60% cells became double pole,multipolar or cone shape,showing neuron-like structure. Using immunocytochemistry,non-induced BMSCs were negative for NeuN,Nestin and GFAP. At day 3 following induction,MSCs were strongly positive for NeuN,weakly for Nestin,but negative for GFAP. Western Blot results demonstrated that cells were negative for Neu

关 键 词：骨髓间充质干细胞 全细胞膜片箝 ATP电流 复极化钾电流 总钾电流 

分 类 号：R394.2[医药卫生—医学遗传学]