两份人脐血间充质干细胞混合培养及向神经元样细胞的分化

Mixed culture and differentiation of two samples of human umbilical cord blood-derived mesenchymal stem cells into neuron-like cells

背景：脐血间充质干细胞可诱导分化为神经样细胞，但在临床中发现干细胞培养细胞增殖的数量不足。目的：观察两份脐血间充质干细胞混合培养的生长情况，以及经碱性成纤维细胞生长因子诱导脐血间充质干细胞向神经元样细胞的分化。设计、时间及地点：于2007—01/10在郑州大学基础医学院微生物与免疫学实验室完成以细胞为对象的对照观察性实验。材料：健康分娩产妇自愿捐献的脐血60～180mL。方法：应用Ficoll—Hypaque淋巴细胞分离液以密度梯度法获取脐血间充质干细胞，加入碱性成纤维细胞生长因子诱导分化。当细胞传至第3代时，随机取培养的6份脐血间充质干细胞（半量）为A组，6份脐血间充质干细胞（半量）为B组，将A、B组各剩余的半量脐血间充质干细胞混合为C组。3组细胞密度均为1．0×10^4L^-1。主要观察指标：以活细胞计数和MTT比色法比较3组细胞扩增数量，免疫组织化学检测脐血间充质干细胞神经胶质细胞标志物、神经元特异性烯醇化酶和巢蛋白的表达。结果：活细胞计数和MTT比色检测显示，将两份不同脐血间充质干细胞混合后，细胞贴壁比单份脐血间充质干细胞快（P〈0．05），增殖也非常快（P〈0．05）。3组细胞经碱性成纤维细胞生长因子诱导后均表达神经胶质细胞标志物、神经元特异性烯醇化酶和巢蛋白，并且巢蛋白阳性表达率无差别（P〉0．05）。结论：混合脐血间充质干细胞之间有互相促增殖作用，应用碱性成纤维细胞生长因子诱导的脐血间充质干细胞可表达神经胶质细胞标志物、神经元特异性烯醇化酶和巢蛋白。

BACKGROUND： Umbilical cord blood mesenchymal stem cells （UCBMSCs） can differentiate into nerve-like cells, but number of stem cell proliferation was not enough in clinic. OBJECTIVE： To investigate the mixed culture growth of two samples of UCBMSCs, and the differentation of UCBMSCs into neuron-like cells after induced by basic fibroblast growth factor （bFGF）. DESIGN, TIME AND SETTING： A control observational cell study was performed at the Laboratory of Department of Microbiology and Immunology of Basic Medical College of Zhengzhou University from January to October in 2007. MATERIALS： 60-180 mL of umbilical cord blood was collected from healthy voluntary puerperants. METHODS： Human UCBMSCs was harvested with Ficoll-Hypaque lymphocyte isolating medium and by density gradient centrifugation, subsequently, induced by bFGF. To obtain 3rd passage cells, the six samples of UCBMSCs were labelled by random as group A （n=6, half）. Another six samples of UCBMSCs were labelled as group B （n=6, half）. The six mixed UCBMSCs were labelled as group C （n=6, surplus parts of groups A and B）. Cell density in the three groups was adjusted to 1.0× 10^4 L^-1. MAIN OUTCOME MEASURES： Amount of cell proliferation was compared by counting living cells and MTT colorimetry. Nerval marker of glial fibrillary acidic protein （GFAP）, neuron specific enolase （NSE） and nestin expressions in UCBMSCs were detected by immunohistochemistry. RESULTS： Adherence of the mixed UCBMSCs was faster than the single by counting cell number and MTT colorimetry （P 〈 0.05）, so did the proliferation of mixed cells （P 〈 0.05）. UCBMSCs induced by bFGF displayed positive immunoreactivity to nerval marker of GFAP, NSE and nestin. There was no significant difference among three groups in percentage of nestin expression （P 〉 0.05）. CONCLUSION： The mixed MSCs promote proliferation each other. MSCs induced by bFGF express positive immunoreactivity to nerval marker of GFAP, NSE and nestin.