多效生长因子在骨髓间充质干细胞向神经元样细胞分化中的表达变化

The Expression of Pleiotrophin in the Differentiation of Human Bone Marrow Mesenchymal Stem Cells into Neuron-like Cells in Vitro

目的探讨多效生长因子(PTN)在体外诱导成人骨髓间充质干细胞(BMSCs)向神经元样细胞分化中过程不同时间的表达变化,了解多效生长因子是否参与BMSCs向神经元样细胞分化的机制。方法以密度梯度离心加贴壁培养法分离成人BMSCs,进行原代和传代培养,以流式细胞仪鉴定纯度。对照组和实验组对传代第6代的BMSCs进行诱导分化,诱导后30min至3d,观察细胞形态并计数。将分化后细胞采用免疫细胞化学法测定神经细胞特异性表面标志神经元特异性烯醇化酶(NSE)、微管相关蛋白-2(MAP-2)、胶质纤维酸性蛋白(GFAP)表达。对诱导前和诱导后3h、6h、12h、24h、3d细胞以RT-PCR法测定PTN mRNA表达。结果 BMSCs在接种1d后黏附贴壁,呈椭圆形或圆形,3d^5d后呈梭状细胞成簇生长,7d^12d融合。传代至第5代~6代可见较为均一的成纤维细胞样形态。流式细胞仪检测结果显示:细胞表达CD44、CD105,不表达造血干细胞的特异标记CD45,证实其纯度。诱导后细胞胞体开始向细胞胞核收缩,出现双极或多极细胞。12h变形细胞增多,细长突起相互连接。24h后细胞变化不明显。诱导后细胞经免疫化学染色显示:多数表达NSE(64.79±0.06)%、MAP-2(60.05±0.09)%,而未检测到GFAP。实验组诱导后细胞不同时间点PTN mRNA的表达不同(P<0.01)。诱导后12h^24hPTN mRNA表达最高。结论建立成人BMSCs培养增殖体系基础上诱导BMSCs向神经元样细胞分化,诱导过程细胞有外观形态变化,同时表达神经细胞特异性标志物NSE和MAP-2。诱导后BMSCs与形态改变相似,向神经元样细胞分化的不同时间点,有PTN mRNA表达变化,提示PTN可能参与BMSCs向神经元样细胞的分化。

Objective To study the expression of pleiotrophin（PTN）mRNA in the differentiation of human bone marrow mesenchymal stem cells（BMSCs）into neuron-like cells in vitro,and investigate the mechanism.Methods Human BMSCs were isolated primarily from bone marrow of adult volunteers＇ iliac and purified sterilely with gradient centrifugation.The low-density cells including BMSCs were cultivated and purified by passage control.We observed the growth status of BMSCs,and identity the immunological characteristic phenotype of CD45,CD44 and CD105 by using FACS.The 6th passage of BMSCs were planted into plastic culture flask and induced.The control group did not receive any induced treatment.We observed the morphology change of BMSCs,after the induction 6 hours,neuron-specific enolase（NSE）,microtubule-associated protein-2（MAP-2）and glial fibrillary acidic protein（GFAP）,which were neuron marker were detected by using immunocytochemistry method.The express of PTN mRNA was detected by RTPCR before induction and at 3 h,6 h,12 h,24 h,3 d after induction.Results The BMSCs were cultivated primarily and following passaged successfully.The living behavior of BMSCs from passage four to passage six were stable.Flow cytometry analysis showed that the cells were positive for CD44 and CD105 receptor,in contrast,negative for CD45 receptor,which demonstrated the purity of BMSCs.The treatment protocols induced BMSCs to differentiate into neuron-like cells,Which expressed NSE and MAP-2（the express rate were 64.79%±0.06% and 60.05%±0.09%）,but did not express GFAP.PTN mRNA was detected to expresse after induction,and from 12 to 24 hours after induction,the expression were highest.After three days,the expression was difficult to be detected.Conclusion The marrow mesenchymal stem cells could be obtained and purified in vitro,and the amplification was stable in our experimental conditions.After being induced,BMSCs could differentiate into neural like cells with the transformation of morphology and the expression of NSE and MAP-2,the same characters which the true neuron cells have.The expression of PTN mRNA had the same change with morphology after the induction,which indicated PTN maybe take a part of the induction.