血管紧张素Ⅱ联合生长因子诱导人骨髓间充质干细胞分化为多巴胺能神经元

Differentiation of human bone marrow mesenchymal stem cells into dopaminergic neuron-like cells induced by angiotensinⅡ combined with growth factor

摘要:

目的：观察血管紧张素Ⅱ（Angiotensin Ⅱ，AngⅡ）联合应用多种生长因子体外诱导人骨髓间充质干细胞（Human bone marrow mesenchymal stem cells，hBMSCs）向神经元和多巴胺神经元分化的潜能。方法：从正常人骨髓中分离单个核细胞并对hBMSCs进行纯化、鉴定；hBMSCs先经用碱性成纤维细胞生长因子和表皮细胞生长因子预诱导，再用胶质细胞源性神经营养因子（Glial cell line-derived neurotrophic factor，GDNF）和AngⅡ联合诱导生长良好的hBMSCs向神经元分化。倒置显微镜观察hBMSCs分化过程中细胞形态的变化；RT-PCR方法检测诱导前后hBMSCs神经干细胞的标记物巢蛋白（Nestin）、神经元特异性烯醇化酶（Neuron-specific enolase，NSE）、神经胶质细胞的特异性标记物酸性纤维蛋白（Glial fibrillary acidic protein，GFAP）以及多巴胺能神经元特异性标记物酪氨酸羟化酶（Tyrosinehydroxylase，TH）等标志物的表达情况；免疫荧光法检测诱导前后hBMSCs的NSE、TH和GFAP蛋白表达情况。结果：流式分析获得了高纯度的hBMSCs。在不同浓度的AngⅡ诱导2周后，倒置显微镜观察hBMSCs，具有典型神经元细胞形态，大多呈双极、多极和锥形； Nestin、NSE、TH的基因mRNA表达量，诱导组相较于对照组，有显著性差异（P<0.05）；免疫荧光细胞化学检测结果表明诱导分化后表达NSE、Nestin、TH蛋白的细胞数明显增加（P<0.05），但诱导前后的细胞都不表达GFAP基因。结论：多种生长因子联合AngⅡ可以在体外诱导hBMSCs分化为多巴胺能神经元样细胞。

Abstract:

Objective：To explore the potential of human bone marrow mesenchymal stem cells（hBMSCs） to differentiate into neurons and dopaminergic neuron-like cells in vitro. Methods：The hBMSCs were isolated and purified from normal human bone marrow mononuclear cells in vitro. After the identification and pretreatment with basic fibroblast growth factor（bFGF） and epidermal growth factor（EGF），the hBMSCs were cultured at the medium containing glial cell line-derived neurotrophic factor（GDNF） and Angiotensin Ⅱ（AngⅡ）. The cell morphology of hBMSCs during the differentiation was observed by inverted microscope. The mRNA expressions and proteins of Nestin，neuron-specific enolase（NSE），human glial fibrillary acidic protein（GFAP），tyrosinehydroxylase（TH） of hBMSCs were detected by RT-PCR and immunofluorescence method before and after the induction. Results：Ｔhe hBMSCs with high purity of were collected. The majority of the hBMSCs were transformed into neuron-like cells displaying as bipolar，multipolar and cone in sharp at two weeks after the differentiation of hBMSCs by AngⅡ. The mRNAs expressions of Nestin，NSE and TH by hBMSCs after the induction were significantly more than those before the induction（P<0.05）. Immunofluorescence analysis showed that the number of cells expressing proteins of NSE，Nestin and TH was increased significantly after the induction（P<0.05）. GFAP gene was not expressed by cells before and after the induction. Conclusion：AngⅡcombined with other growth factors can induce hBMSCs to differentiate into dopaminergic neuron-like cells.