电针联合人脂肪间充质干细胞移植治疗PD恒河猴的线粒体自噬机制研究

The therapeutic efficacy of human adipose-derived stem cells (ADSCs) has been assessed in various animal models with PD. The treatment of PD using electro acupuncture (EA) has long been also effective. Previous studies have found that individuals of PD have mitochondrial dysfunction and the mitophagy. Meanwhile, the mitophagy occurring through activated PINK1/Parkin pathway has long been the focus of many researchers. We proposed to test the hypothesis that EA and ADSCs may have therapeutic potential for PD by regulating the autophagy-lysosome pathway and inhibiting excessive activation of mitophagy and alleviating the damage of mitochondrial structure and function. Therefore, the aim of this study was to use the aging Monkey (Macaca rhesus) model of PD could be created by the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)injection into the subcutaneous. ADSCs is transplanted into the brain of the PD monkey model by bilateral carotid artery respectively. The behavior cold be observed before and after the injection of MPTP in all the animals by Kurlan Scale; the proliferation, differentiation, migration of exogenous and endogenous stem cell will be examined through immunohistochemical method or MRI. Also, metabolites of dopamine (DA)will be examined by high performance liquid chromatography (HPLC), and biochemical analysis of blood for the relevant indicators of oxidative stress level. The mitochondrial ultrastructural changes in dopaminergic neuron(DN) was observed by transmission electron microscope; the content of quantitative enzyme CS and the activities of mitochondrial respiratory chain complex II,IV and COXI were measured by ELISA; the protein expressions of DN mitochondrial α-synuclein,Parkin,Ub,ERK1/2 and LC3 were detected by western blot and their amount of co-localization of mitochondria respectively with Parkin,Ub,p62 and LC3 were measured by immunofluorescence double labeling technique. Overall, this research could be the pre-clinical experimental basis of ADSCs transplantation for the treatment of PD.

人脂肪间充质干细胞（ADSCs）移植在各种帕金森病（PD）动物模型中都得到了有效验证，电针（EA）治疗PD临床疗效确切，但两者均缺少深入的机制研究。前期研究发现PD个体存在线粒体自噬和损伤，而PINK1/Parkin介导的自噬溶酶体通路是线粒体自噬的研究热点。我们推测EA、ADSCs移植以及两者联合可能通过此通路抑制线粒体自噬过度激活，缓解了线粒体结构和功能破坏。为验证此假说，本研究应用MPTP皮下注射制作老年恒河猴PD模型，经颈内动脉移植ADSCs，应用行为学评估、MRI、HPLC、电镜、ELISA以及免疫荧光双标等技术，探究内外源干细胞存活、增殖、分化、迁移和线粒体超微结构的变化；分析泛素接头蛋白P62与线粒体自噬相关蛋白（α-synuclein、Parkin、Ub、ERK1/2、LC3等）、线粒体定量酶CS、呼吸链复合体II、IV和COXI相互作用的调控机制，为治疗PD提供实验依据。

电针（EA）治疗PD临床疗效确切，人脂肪间充质干细胞（ADSCs）移植在各种帕金森病（PD）动物模型中都得到了有效验证，但两者均缺少深入的机制研究。本研究把ADSCs诱导分化为类DA样干细胞，并植入PD食蟹猴模型中且可长期存活发挥疗效，EA可起到促进作用。前期研究发现PD个体存在线粒体自噬和损伤，而PINK1/Parkin介导的自噬溶酶体通路是线粒体自噬的研究热点。我们推测EA、ADSCs移植以及两者联合可能通过此通路抑制线粒体自噬过度激活，缓解了线粒体结构和功能破坏。为验证此假说，本研究应用MPTP脑立体定向注射制作食蟹猴PD模型和移植ADSCs，应用行为学评估、MRI、PETCT、ELISA、免疫荧光双标以及激光共聚焦显微镜等技术，发现内、外源干细胞可存活、增殖、分化、迁移；我们同时应用6-OHDA诱导体内体外PD模型，应用行为学评估、免疫组化、流式细胞术，Western blot、qRT-PCR、ELISA等技术，验证circ_0105596（circ-FTO）竞争性吸附hsa-miR-1287-5p介导EEF2表达加重PD恶化；同时导致多巴胺神经元凋亡、炎症和氧化损伤；结合RIP和双荧光素酶报告实验我们初步证实circ-FTO-hsa-miR-1287-5p-EEF2互作的调控网络。同时发现EA百会穴和双侧舞蹈震颤区在此通路中的作用靶标为EEF2的下游通路且通路已被证明能影响CIRBP和KCNS3的表达，为针灸机理研究补充客观依据。总之，本研究可为同种异体脂肪源间充质干细胞移植治疗PD提供临床前研究证据，并可通过EA改善干细胞的生存环境，以提高疗效。