经鼻通路-帕金森可能致病路径及干细胞移植治疗途径的实验研究

Environmental neurotoxins play an important role in the developing sporadic Parkinson's disease(PD). However, no neurotoxins existing in the environment were proved to induce PD through real route of exposure and meanwhile explain the hyposmia, a common phenomenon occuring in 90% patients with early PD. We hypothesize that intranasal route might be a direct way of exposure of some kinds of PD-inducing neurotoxins to brain and account for the smell loss in early PD, and of delivering therapeutic biologics such as Marrow Mesenchymal Stem Cells(MSCs) to the brain for PD treatment, since it averts clearance by the liver and bypasses the Brain Blood Barrier. Lipopolysaccharide(LPS), a Gram-negative bacteria's endotoxin inducing PD animal model, is ubiquitously distributed in environment and has been studied in the pathogenesis of PD. It activates microglia and enhances inflammatory process in brain. Trichloroethylene(TCE), a ubiquitous compound in the environment as well, has been shown to be a potential neurotoxin causing PD. In our preliminary experiments, we observed that intranasal exposure of LPS or TCE in C57BL/6 mice for 5 months caused behavioral change of PD and selective loss of dopaminergic neurons in the substantia nigra. A number of key questions in this regard, however, remain to be addressed, including the time-effect and dose response of LPS/TCE in inducing motor dysfunction of PD, the non-motor features of these PD models, and the underlying mechanism of the neurotoxicity. Hence, we will chronically apply LPS and TCE exposure respectively on C57BL/6 mice through intranasal route, and then longitudinally evaluate its effect on the motor ability, non-motor function, neuroimagings by MicroPET for assaying dopamine transporter and neuroinflammation, tyrosine hydroxylase(TH) positive cells counting by immunostaining, TH and alpha-synuclein level by Western blot, dopamine and its metabolites level by HPLC. Meanwhile, at baseline,1,3,and 5 months after the exposure, we will investigate the time-effect and dose response of LPS/TCE on the above parameters and the spatial-temporal patterns of those changes and their correlation, including activation of microglia and related cytokines, loss of DA neurons and alpha-synuclein deposits. By using the corresponding approches above, we will also deliver MSCs intranasally to the brains in PD mice model induced by intranasal exposure of LPS/TCE and intraperitoneal injection of MPTP, track down their migration from nose to brain, investigate the efficacy of the MSCs transplants and the underlying mechanism. Taken together, intranasal route might be a key pathway for both the environmental exposure of PD-inducing pathogens and noninvasive and efficient delivery of stem cells for treating PD. The inplementation of the current research proposals might be able to shed light on elucidating the true etiology for those patients with sporadic PD, and to provide a novel and promising stratagy for PD therapy.

大多数帕金森病(PD)患者系散发，提示其病因可能主要由环境因素所致。考虑到高达90%PD患者早期即存在嗅觉减退这一重要临床现象,经鼻通路则可能是环境毒物入脑且解释嗅觉减退的路径。但迄今未见自然界中实际存在的神经毒物经鼻暴露致PD的报道。本课题我们将经鼻给予小鼠空气中广泛存在的神经毒物(脂多糖、三氯乙烯),应用运动和非运动障碍行为学评估、免疫组化和WB检测PD病理变化、MicroPET测定脑多巴胺功能障碍及小胶质细胞炎性激活、HPLC分析纹状体多巴胺等递质等,探讨其经鼻慢性暴露致PD的作用及可能机制。经鼻通路避开了血脑屏障和体循环,也是药物入脑治疗PD的"捷径",我们经鼻给予小鼠PD模型以骨髓基质干细胞,应用上述实验方法研究其治疗PD的可能性及机制。经鼻通路"致病"和"治病"对于PD的理论和实践意义均十分重大,其研究有望诠释临床上大多数PD患者发病的可能病因、并为PD的有效干预提供新的策略。

90%帕金森病（PD）患者早期即存在嗅觉减退这一重要临床现象，经鼻通路则可能是环境毒物入脑且解释嗅觉减退的路径。本研究通过空气中广泛存在的神经毒物脂多糖(LPS）滴鼻，观察小鼠的行为学、病理学以及神经生物化学等改变，深入探讨LPS经鼻暴露致PD的可能致病性和作用机制。同时，经鼻通路避开了血脑屏障和体循环，是干细胞入脑治疗 PD 的“捷径”。而Rho激酶抑制剂法舒地尔具有辅助干细胞潜能。我们通过经鼻给予小鼠PD模型以法舒地尔预处理的骨髓基质干细（BMSCs），研究其治疗 PD 的可能性及机制。本次试验中我们成功构建了LPS滴鼻小鼠PD模型。发现LPS致小鼠PD的机制可能是通过诱导脑内的小胶质细胞向M1型转化，诱发脑内的炎症反应以及激活Rho激酶实现。另外研究显示，法舒地尔预处理的BMSCs经鼻移植可以明显改善PD小鼠的运动能力，减少黑质多巴胺能神经元的丢失，主要通过抑制脑内的炎症反应，促进M1型小胶质细胞向M2型小胶质细胞转化，增加神经营养因子BDNF的分泌，以及促进内源性干细胞动员等实现。总之，经鼻通路“致病”和“治病”对于 PD 的理论和实践意义均十分重大，其研究有望诠释临床上大多数 PD 患者发病的可能病因、并为PD 的有效干预提供新的策略。