SOX6调控人多能干细胞源中脑多巴胺能祖细胞的体外异质性分化及其脑内移植的作用及机制研究

As a promising treatment for Parkinson's disease, Stem cell replacement therapy is near the clinical stage. The midbrain dopamine (DA) progenitor cells derived from human pluripotent stem cells (HPSC) were ony differentiated into A9 DA neurons to recover the movement function of patients with PD. However, it was identified that the midbrain DA progenitor cells were transplanted into the host brain to be differentiated into the two subgroups of dopamine neurons A9 and A10, heterogeneously. The transplant patients would be at risk of medical adverse effects induced by the heterogeneity of differentiation. The experiments were shown that sox6 was a determinant of A9 DA neuron development in vivo. The effect of SOX6 on the terminal differentiation of the midbrain DA progenitor cells into A9 DA neurons remains unclear in vitro. Here, we put forward the following hypothesis: the regulation of SOX6 expression could control DA progenitor cells from HPSC to be differentiatedinto A9 DA neurons in vitro. The mechanism might be related to the regulation of WNT1/ -catenin signaling pathway. Four kinds of pluripotent stem cell lines were selected in this research. After using exogenous agonists and inhibitors, gene expression regulation interventions, the proportion of A9 DA neurons were detected in intro and intracerebral transplantation, to identify the relationship of SOX6 and dopaminergic differentation of HPSC. The regulation mechanism of HDAC1/SOX6/β-catenin on WNT signaling pathway were detected by some methods such as Chromatin Immunoprecipitation Assay. The results of this study woud provide new ideas and new targets for the selective production of A9 DA neurons, and promote the clinical applicaiton.

人多能干细胞（HPSC)替代疗法治疗帕金森病(PD)已进入临床前期研究。HPSC源中脑多巴胺能（DA）祖细胞移植后分化为A9DA神经元才能促进患者运动功能恢复。然而目前它在宿主脑内异质性地分化为A9、A10DA神经元两种亚群，给患者带来医疗风险。在体研究证实，SOX6是中脑A9DA神经元发育的决定因素。但体外干预SOX6表达对HPSC源DA祖细胞终末分化为A9DA神经元的影响仍不清楚。因此提出假说：体外干预SOX6表达可调控HPSC源DA祖细胞分化为A9DA神经元，可能与调控WNT1/β-catenin信号通路相关。本研究拟选用四种多能干细胞系，采用外源性激动剂和抑制剂调控SOX6表达、病毒转染调控SOX6基因表达等干预手段，通过体外实验和脑内移植实验检测SOX6表达变化对A9DA神经元分化的影响，初步阐明SOX6调控作用及相关机制，为选择性产生A9DA神经元提供新思路和新靶点。

人胚胎干细胞（human Embryonic Stem Cell, hESCs）源中脑多巴胺能神经祖细胞移植治疗帕金森病已进入临床前期研究。hESCs分化为中脑多巴胺能神经祖细胞过程存在异质性是影响移植安全性的重要因素。巨噬细胞迁移抑制因子（MIF）在几乎所有哺乳动物细胞中都有表达，参与多种功能，包括白细胞募集、炎症、免疫反应、细胞增殖、肿瘤发生和糖皮质激素的反调节等。我们的前期研究表明MIF参与hESCs的神经分化。然而，巨噬细胞迁移抑制因子（MIF）对于人胚胎干细胞的具体作用及机制尚未明确。本项目明确了hESCs自分泌MIF，hESCs上高表达受体CXCR2和CXCR7，弱表达CXCR4、CD44和CD74。并且生理浓度的MIF通过CXCR4受体促进hESCs的增殖，高于生理浓度的MIF通过CXCR4受体促进hESCs的神经分化。在双“SMAD”抑制分化方案的基础上添加MIF，与对照组相比，MIF可促进hESCs向中脑多巴胺能神经祖细胞分化，抑制其分化为其它类型神经祖细胞，有效降低了分化的异质性，提高了移植安全性。