多效生长因子诱导人脐血间充质干细胞向多巴胺能神经元样细胞分化

PTN promotes the differentiation of human umbilical cord blood mesenchymal stem cells into dopaminergic neuron-like cells

目的探讨多效生长因子(PTN)诱导人脐血间充质干细胞(CB-MSCs)向多巴胺(DA)能神经元样细胞分化的可能机制。方法密度梯度离心和贴壁筛选法纯化CB-MSCs,加入重组人PTN(rPTN)作为诱导组(rPTN组),以单纯传代培养的CB-MSCs作为对照(CON组),培养48 h后用免疫荧光染色和RT-PCR鉴定CB-MSCs表达PTN及其受体SDC3和ALK,以及诱导分化的细胞表达TH、DAT和NSE的情况。结果随rPTN诱导时间延长,CB-MSCs形态发生变化,TH、DAT和NSE的表达量明显增加,免疫荧光细胞阳性率:TH(CON组)为3.77%±0.42%,(rPTN组)为7.91%±0.55%;DAT(CON组)为1.89%±0.28%,(rPTN组)为6.21%±0.77%;NSE(CON组)为4.35%±0.76%,(rPTN组)为7.67%±0.97%,同对照组相比(P<0.05);诱导的CB-MSCs表达PTN受体SDC3明显高于对照组;rPTN可诱导CB-MSCs自身表达PTN增加,免疫荧光细胞阳性率:PTN(CON组)为14.38%±0.54%,(rPTN组)为20.21%±1.51%;SDC3(CON组)为5.70%±0.78%,(rPTN组)为10.02%±1.45%;ALK(CON组)为4.87%±0.34%,(rPTN组)为5.01%±0.99%(P>0.05)。结论 rPTN能够通过促进CB-MSCs上调PTN及其受体SDC3表达,诱导CB-MSCs向DA能神经元样细胞分化。

Objective To investigate the potential mechanism of CB-MSCs differentiated into DA neuron-like cells induced by PTN.Methods Preparation of the mononuclear cell fraction was performed by the Ficoll gradient technique according to the manufacturer＇s instructions.rPTN was added as inducing group（rPTN group）,passage 1 CB-MSCs with H-DMEM as control group（CON group）.Fourty-eight hours later,RT-PCR and immunofluorescence were employed to indentify the expression of PTN and its receptors SDC3、ALK,also to indentify the expression of TH、DAT and NSE.Results During the induction,cell morphology changed obviously,the expression of TH,DAT,and NSE increased.The ratios of positive cells were： TH（CON group）： 3.77%±0.42%,（rPTN group）： 7.91%±0.55%;DAT（CON group）： 1.89%±0.28%,（rPTN group）： 6.21%±0.77%;NSE（CON group）： 4.35%±0.76%,（rPTN group）： 7.67%±0.97%;The expression of SDC3 also increased comparing with the CON group.Our study discovered PTN can promote the PTN secretion of CB-MSCs.The ratios of positivecells were：PTN（CON group）： 14.38%±0.54%,（rPTN group）： 20.21%±1.51%;SDC3（CON group）： 5.70%±0.78%,（rPTN group）：10.02%±1.45%;ALK（CON group）：4.87%±0.34%,（rPTN group）：5.01%±0.99%.Conclusion PTN may induce CB-MSCs differentiate into DA neuron-like cells by prompting CB-MSCs express PTN and its receptor SDC3.