骨髓间充质干细胞的体外培养及定向诱导分化  

In Vitro Culture of Human Bone Marrow Mesenchymal Stem Cells and Induced Differentiation into Neuron-like Cells

作　　者：汪泱[1,2] 邓志锋[1,2] 赖贤良[1,2] 王共先[1,2] 黄学明[1,2] 

机构地区：[1]江西医学院第一附属医院泌尿外科研究所,南昌330006 [2]江西医学院第二附属医院神经外科,南昌330006

出　　处：《中国临床医学》2004年第2期202-204,共3页Chinese Journal of Clinical Medicine

基　　金：国家自然科学基金资助(30160084);江西省自然科学基金资助(0240047)

摘　　要：目的:探讨人骨髓间充质干细胞(hMSCs)的体外培养生长特性及其在体外定向分化为神经样细胞的条件。方法:采用Ficoll-paque(1.077g/ml)分离液密度梯度分离hMSCs,显微镜下观察其生长特性,测定生长曲线;取传至3～6代的hMSCs,用阿魏酸钠对其进行诱导培养,并以β-巯基乙醇作对照观察诱导培养后细胞的形态变化,分别在6h、1d、3d和7d通过免疫荧光细胞化学染色方法测定诱导后细胞的神经元烯醇化酶(neuron specific enolase,NSE)和胶质纤维酸性蛋白(glia fiber acidprotein,GFAP),进行定量分析。结果:体外培养条件下hMSCs呈长梭形,细胞生长曲线呈S形,细胞倍增时间约为72h。阿魏酸钠诱导培养后6h可见细胞形态明显变化,NSE和GFAP表达阳性。24h后诱导细胞表现为典型的神经细胞样形态。第3天,NSE和GFAP表达最高,分别为67±3.5%和39±1.8%。结论:人骨髓间充质干细胞在体外具有自我更新能力及多分化潜能;阿魏酸钠具有诱导体外培养的人骨髓间充质干细胞向神经细胞分化的作用。Objective: To observe the biological features of human marrow mesenchymal stem cells (hMSCs) and explore the protocols to inducing hMSCs into neuron in vitro. Methods: hMSCs were harvested from residual human ribs and separated by a density gradient centrifuge. biological features of cultured MSCs were observed under phase contrast microscope and their growth curves were investigated. In this study, hMSCs between passeges 3 and 6 were induced by sodium ferulate. others treated with β-mercaptoethand (β-ME) as a positive contrast. The morphological changes of induced hMSCs were observed in different culture period and we monitored expression of neuronal specific enolase(NSE) and glial librillary acidic protein (GFAP) product in hMSC-derived neurons at 5 hr, 1 day, 3day and 7day post-differention by indirect immunocytochemisty staining. Results: hMSCs have spidle shape under experimental cell culture conditions. The growth curve like 's' shape. The double time of MSCs was about 72h. After treated with sodium ferulate in 6 hours, hMSCs exhibit obvious morphological changes and expressed the protein for NSE and GFAP. Twenty-four hours later, the differentiated cells showed typical neuronal morphorlogy. With an optional differentiation protocol, almost 67% of the cells expressed NSE and 39% of the cells expressed GFAP at 3 day. Conclusion: hMSCs have the capacity of self-renewal and multiple potentiality of differentiation in vitro, and Sodium ferulate can induce hMSCs into neurons.

关 键 词：骨髓间充质干细胞 体外培养 定向诱导 神经细胞 细胞分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]