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目的:寻找一种稳定、高效的分离人脐带间充质干细胞(MSCs)的方法,并探讨脐带MSCs向神经细胞方向分化的可能性。方法:分别采用组织块贴壁法和双酶消化法分离人脐带MSCs,对比其培养成功率;BrdU掺入实验检测脐带MSCs的增殖能力;流式细胞仪检测脐带MSCs表面分子标志;采用丹参联合生长因子的方法诱导其向神经细胞分化,免疫荧光方法检测神经元特异性烯醇化酶(NSE)、微管相关蛋白2(MAP2)、胶质纤维酸性蛋白(GFAP)的表达。结果:组织块贴壁法获得脐带MSCs成功率高;BrdU阳性标记率达90%以上;流式细胞仪检测显示细胞表达CD29、CD44和CD90,不表达CD34;脐带MSCs经诱导分化,伸出长突起,呈神经元样细胞改变,且表达神经元标志性蛋白NSE、MAP2。神经胶质细胞标志性蛋白GFAP表达较少。结论:成功建立了高效、稳定的脐带MSCs分离培养方法。脐带MSCs经诱导可向神经细胞方向分化,为临床移植治疗神经系统疾病提供了理想的细胞来源。
OBJECTIVE: To find out a stable and efficient method for isolation of human umbilical cord mesenchymal stem cells (MSCs), and to explore the possibility of differentiation of umbilical cord MSCs to nerve cells. Methods: Human umbilical cord MSCs were isolated by tissue block adhesion assay and double enzyme digestion method, respectively. The success rate of MSCs culture was compared with BrdU incorporation assay. The proliferation of MSCs was detected by flow cytometry. The surface markers of MSCs were detected by flow cytometry. The growth factor was used to induce neuronal differentiation. The expression of neuron specific enolase (NSE), microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence. Results: The success rate of MSCs obtained by tissue block adhesion method was high; the positive rate of BrdU was over 90%; the expression of CD29, CD44 and CD90 was detected by flow cytometry, while CD34 was not expressed; Protuberant neuronal cell-like changes, and expression of neuronal markers NSE, MAP2. Glial cell marker protein GFAP expression less. Conclusion: An efficient and stable method of isolation and culture of umbilical cord MSCs has been successfully established. Umbilical cord MSCs can differentiate into nerve cells after induction, which provides an ideal source of cells for clinical transplantation in the treatment of nervous system diseases.