人脐血干细胞培养及其向神经元样细胞定向诱导分化的研究

目的：通过体外培养脐血干细胞并诱导分化为神经元样细胞，为研究脐血干细胞移植修复脊髓损伤奠定基础。方法：用密度梯度离心法将脐血单个核细胞从脐血中分离出来，置于含20%胎牛血清的DMEM/F12培养液中培养、扩增、传代，取体外扩增5代的脐血间充质干细胞（MSCs），接种于培养板内，将诱导剂3－叔丁基4－羟基茴香醚（BHA）和二甲基亚砜（DMSO）加入条件培养基静置培养，观察细胞的形态学和组织学变化，取神经元样细胞进行免疫组化检测细胞表面标志物神经元烯醇化酶（NSE）、神经丝蛋白（NF）及胶质纤维酸性蛋白（GFAP），然后，在光镜下随机数取10个非重叠视野(×100), 计算NSE、NF及GFAP阳性细胞占总细胞数的比例，实验结果用统计学分析。结果:光镜下hMSC在预诱导液中处理24?h后,加入诱导液,1?h后细胞形态发生变化,原来宽大扁平的hMSCs胞质向核收缩,胞体向胞核收缩成锥形且变得致密并有折光性,有较多的长突起,6?h后多数细胞均转变为类似于神经元的形态，3?d后免疫组化检测NSE、NF及GFAP的阳性率分别为（35.24±3.63）％、（33.46±3.21）％、（15.12±1.65）%。结论:脐血MSC在体外有较强的扩增能力,并且在一定条件下可以诱导成为神经元样细胞。

Umbilical cord blood stem cells: culture and differentiation to neuron-like cells in vitro

To study an experimental technology for repairing injured spine by transplantation of cord blood stem cells. Methods: (Human mesenchymal stem cells, hMSCs) were separated from cord blood by density gradient centrifuging and cultured in DMEM with 20% FBS. Amplified and taken the 5th generation to have an innoculation in the culture plate, they were put into the culture medium added inducer and cultivated at 37?℃, 5% CO2 in satured humidity. The neuron symbol NSE, NF and the astrocyte symbol GFAP were detected by immunohistochemical method, then under the microscope, 10 un wraped eyeshot was counted randomly and calculated the positive percentage of NSE, NF and GFAP denoting as ±s. Result: The hMSCs seemed like neurons and changed into taper cells with many long tuber and cytoplast shrinking to the nucleus which became denser and more refracting. Twenty four hours after hMSCs were incubated in beforehand 3.63）％ of NSE, （33.46±3.21）％ of NF and （15.12±1.65）% of GFAP were found. Conclusion: Cord blood stem cells have a relatively strong ability of proliferation and a potent of differentiating into neuron like cells at a certain condition.