大鼠骨髓间充质干细胞的培养鉴定以及向神经干细胞分化的实验研究.docVIP

大鼠骨髓间充质干细胞的培养鉴定以及向神经干细胞分化的实验研究 蒲晓姝，果磊 重庆医科大学附属第一医院烧伤整形外科，重庆 400016 【摘要】目的 体外培养并鉴定大鼠骨髓间充质干细胞（BMSCs），研究其生物学特性，初步探索酸性成纤维细胞生长因子（aFGF）诱导其向神经干细胞（NSCs）分化的可行性。方法 全骨髓贴壁培养法分离培养大鼠BMSCs，倒置相差显微镜观察其形态特征，MTT法绘制生长曲线，流式细胞仪（FCM）测定细胞周期并鉴定细胞表面标志物。用80ng/ml aFGF的无血清DMEM/F12培养液诱导大鼠BMSCs向NSCs分化，镜下观察细胞形态特征，免疫荧光染色方法检测神经巢蛋白（nestin）、神经元特异性烯醇化酶（NSE）的表达。结果 大鼠BMSCs贴壁生长，呈梭形，多角形。第1、3、5代BMSCs的生长曲线均呈S形，活性无明显差异。细胞周期显示94.34% BMSCs处于G0/G1期，有较强的分裂增殖能力。FCM检测CD29、CD54、CD90表达阳性，CD45表达阴性。诱导6h后细胞的胞体收缩成椭圆形或球形并伸出细长突起，免疫荧光染色显示nestin表达阳性，继续诱导发现双极和多极细胞增多，诱导6d后相互连接成网状，成典型的神经元样细胞，NSE表达阳性。结论 全骨髓贴壁培养法能培养出高纯度的BMSCs，细胞生长稳定、增殖快、可多次传代，具有干细胞特性。经aFGF诱导后具有向NSCs分化的潜能，NSCs能进一步分化为神经元样细胞。 【关键词】骨髓间充质干细胞；全骨髓贴壁培养法；诱导分化；神经干细胞；酸性成纤维细胞生长因子 The experimental study on cultivation and identification of rat bone marrow mesenchymal stem cells and their differentiation into neural stem cells Pu Xiaoshu, Guo Lei. Department of Burn and Plastic Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China. ［Abstract］ Objective To culture in vitro and identify rat bone marrow mesenchymal stem cells (BMSCs), study their biological characteristics, and initially tentatively explore the HYPERLINK app:ds:feasibility \t feasibility of acidic fibroblast?growth factor?(aFGF) inducing BMSCs to differentiate into neural stem cells (NSCs). Methods Rat BMSCs were isolated and cultured by the whole bone marrow adherent culture method. The morphological characteristics of BMSCs were observed under the inverted phase contrast microscope and the cell growth curves were drawn by MTT method. The cell cycle and surface markers were detected with flow cytometry(FCM). Rat BMSCs were induced to differentiate into NSCs by 80ng/ml aFGF of DMEM/F12 medium. The cell morphological changes were continuously observed after using aFGF. Immunofluorescence staining was used to detect the expressions of nestin and neuron specific enolase (NSE). Results Rat BMSCs showed adherent growth and spindle-shaped or polygonal appearance. The?growt