hUC-MSC在成神经分化过程中的趋化迁移研究

Chemotactic migration of hUC-MSCs during neurogenic differentiation

目的探究人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSC)向神经样细胞的分化,并对其分化的神经样细胞的趋化作用进行研究。方法检测hUC-MSC细胞表面的标记;无血清的诱导培养基[含2%二甲基亚砜(DMSO)和200μmol/L丁羟基茴香醚(BHA)]诱导其向神经细胞分化,并进行神经细胞特异性标志物神经上皮干细胞蛋白(nestin)和神经元特异性烯醇化酶(NSE)的免疫荧光染色检测,并对分化的成神经诱导过程中的形态变化进行鉴定。此外,不同分化状态的hUC-MSC,在加入500μL含有20 ng/mL浓度的基质细胞衍生因子(stromal Cell-derived Factor-1α,SDF-1α)的不含血清的L-DMEM培养基,利用倒置相差显微镜下拍照,统计迁移至室膜下方的细胞数。结果成功分离出hUC-MSC,行流式细胞仪检测结果显示MSC:CD71+、CD29+,而CD34-、CD45-符合MSC的生物学特征。对不同诱导时间的hUC-MSC免疫荧光结果的统计学分析,nestin的表达呈现出先增高后降低的趋势,NSE在诱导期和维持前期均为阴性,在整个过程中末期才开始表达,到维持48 h时有62.5%的细胞表达,维持72 h时有76.5%的细胞表达。不同成神经分化状态的hUC-MSC迁移能力不同,诱导30 h,细胞向SDF-1α的趋化迁移能力达到最强。结论hUC-MSC具有易分离并扩增培养的生物特性,能诱导分化成为成神经细胞,并且具有一定的迁移能力,该研究能为中枢神经系统细胞的移植提供理论基础。

Objective To investigate the differentiation of human umbilical cord mesenchymal stem cells(hUC-MSCs)into neural like cells,and to study the chemotaxis of the differentiated neural like cells.Methods The surface markers of hUC-MSCs were detected,and serum-free induction medium(with 2% DMSO and 200μmol/L BHA)was used to induce them to differentiate into neural cells.The specific markers nestin and NSE of neural cells were detected by immunofluorescence staining.The morphological changes and identification during the induction of differentiated neurons were made.In addition,hUC-MSCs in different differentiation states were added 500μl of serum-free l-dmem medium with 20 ng/ml SDF-1α,and photographed under the inverted phase contrast microscope,and the number of cells migrating to the lower part of ependyma was counted.Results hUC-MSCs were successfully isolated.The results of flow cytometry showed that MSCs:CD71+,CD29+,and CD34-,CD45-,accorded with the biological characteristics of MSCs.According to the statistical analysis of immunofluorescence results of hUC-MSCs at different induction time,Nestin expression showed the trend of increasing first and then decreasing.NSE was negative during induction and maintenance period,and began to express in the middle and end of the whole process.62.5% of cells expressed in 48 hours and 76.5% in 72 hours.The ability of migration of hUC-MSCs to SDF-1αwas the strongest 30 hours after induction.Conclusion hUC-MSCs has biological characteristics of easy isolation and expansion,can induce differentiation into neural cells,and has a certain migration ability.This study can provide a basis for the transplantation of central nervous system cells.