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目的:研究大鼠骨髓间充质干细胞(BMSCs)在顺序性化学诱导培养基诱导下向多巴胺(DA)能神经元细胞分化的能力。方法:通过密度梯度离心法获取大鼠骨髓中的单个核细胞,贴壁培养纯化BMSCs。以碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)、神经条件培养基(NCM)、音速波状蛋白(SHH)和成纤维细胞生长因子8(FGF8)联合对第4代BMSCs进行诱导。RT-PCR、Western blot和细胞免疫荧光染色检测DA能神经元分化过程中的标志物酪氨酸羟化酶(TH)、多巴胺转运蛋白(DAT)的表达以及转录因子Nurr1、TUJ1、AADC和VAMT的表达。结果:诱导2周后,RT-PCR结果显示Nurr1、AADC、VAMT、DAT和TH的mRNA均有表达;Western blot显示TH和TUJ1蛋白均有表达,细胞免疫荧光染色结果表明诱导2周后TH阳性细胞占(41.18±3.24)%。结论:按一定顺序给予化学诱导剂可以在体外诱导BMSCs向DA能神经元分化,并具有DA能神经元的功能特征,是临床用于治疗帕金森病这一类神经精神性疾病的理想细胞来源。
AIM: To investigate the ability of rat bone marrow mesenchymal stem cells (BMSCs) to differentiate into dopaminergic neurons under the induction of sequential chemical induction medium. METHODS: Mononuclear cells from bone marrow of rats were obtained by density gradient centrifugation, and purified BMSCs were adherent culture. Generation 4 BMSCs were treated with bFGF, EGF, NCM, SHH and FGF8 Induced. The expression of tyrosine hydroxylase (TH), dopamine transporter (DAT) and the transcription factors Nurr1, TUJ1, AADC and VAMT in DA neurons were detected by RT-PCR, Western blot and immunofluorescence staining expression. Results: The expression of Nurr1, AADC, VAMT, DAT and TH mRNA was detected by RT-PCR after 2 weeks of induction. The expression of TH and TUJ1 protein was detected by Western blot. The results of immunofluorescence staining showed that after 2 weeks of induction, TH positive Cells accounted for (41.18 ± 3.24)%. Conclusion: The chemical inducer can induce the differentiation of BMSCs into DA neurons in vitro and have the functional characteristics of DA neurons in vitro. It is an ideal cell source for the clinical treatment of neuropsychiatric diseases such as Parkinson’s disease .