骨髓间充质干细胞向神经细胞分化后免疫原性研究

目的:通过体外天麻诱导人骨髓间充质干细胞(Human mesenchymal stem cells,hMSCs)向神经元样细胞分化,验证其多能分化特性,检测分化后细胞的免疫原性.方法:通过贴壁法分离hMSCs,体外扩增培养.天麻定向诱导分化为神经元样细胞.光镜下观察细胞形态,免疫细胞化学检测神经细胞特异性抗原标志物神经元烯醇化酶(NSE)、巢蛋白(Nestin)、胶质纤维酸性蛋白(GFAP)的表达.流式细胞术(FCM)检测分化后细胞HLA-DR表达,用单向混合淋巴细胞反应(MLR)的方法检测神经细胞分化的hMSCs是否引起人外周血淋巴细胞增殖反应(Human peripheral blood lymphocytes,hPBLs).结果:hMSCs可通过贴壁法成功分离并可在体外大量扩增.天麻诱导3小时后大部分hMSCs转变为神经元样细胞,出现胞体和突起.免疫细胞化学染色NSE及Nestin呈阳性,GFAP阴性.流式细胞术显示hMSCs向神经细胞分化后HLA-DR表达阴性,混合培养结果也显示分化hMSCs不引起hPBLs增殖反应.结论:天麻可在体外诱导hMSCs分化为神经元样细胞,分化hMSCs不引起人淋巴细胞增殖反应,具有低免疫原性.

Immunogenicity of neuron-like cells differentiated from mesenchymal stem cells

Objective:Marrow mesenchymal stem cells differentiates into neuron-like cells when induced by Gastrodia,which verifies the multipotentiality of the stem cells.Here to detect the immunogenic properties of the neuron-like cells after differentiation.Methods:Human mesenchymal stem cells were isolated from bone marrow by wall sticking method,amplified by in vitro culture,and differentiated into neuron-like cells by oriented induction with Gastrodia.The morphology of cells was observed under light microscopy.Neuron-specific enolase (NSE),nestin and glial fibrillary acidic protein (GFAP) were detected by imnunocytochemistry.The expression of HLA-DR protein after induction was tested by flow cytometry(FCM).The immunogenic properties of the neuron-like cells induced from hMSCs were detected by one-way mixed lymphocyte reaction(MLR).Results:Human mesenchymal stem cells could be separated and amplified in vitro.After being induced by Gastrodia for 3 hours,most of the cells would be differentiated into neuron-like cells,revealing cytodendrite.By immunochemical staining,the cells showed positive of NSE,nestin,and negative of GFAP.HLA-DR was not detectable on differentiated hMSCs by FCM.MLR assays demonstrate that differentiated hMSCs fail to stimulate proliferative response of hPBLs.Conclusion:Human mesenchymal stem cells could be induced to differentiate into neuron-like cells by Gastrodia.MLR assays demonstrate that differentiated hMSCs fail to stimulate proliferative response of hPBLs.They are low immunogenic.