老年性痴呆脑微环境对海马移植NSCs的作用及针刺干预研究

Neural stem cells (NSCs) transplantation has brought about a huge prospect for the nerve regeneration treatment. However, there is no large number of newborn neurons formations in the brain of host after NSCs transplantation, and ultimately affect the efficacy of transplantation for the treatment of dementia. The ultimate fate of implanted NSCs largely depended on the surrounding microenvironment, and the microenvironment closely regulated the process of NSCs transplantation. In this topic, basing on the exact clinical efficacy of acupuncture treatment for senile dementia and the experimental result of acupuncture regulation of endogenous NSCs, we apply "acupuncture regulating Sanjiao and improving brain insufficient" Chinese medicine theory, and combine acupuncture with NSCs transplantation. We are going to use SAMP8 from the view of acupuncture regulating NSCs transplantation microenvironment. We will apply the western blot and real-time quantitative PCR technique to observe the effects of acupuncture on NSCs microenvironment related indicators. And we will apply HE staining, immunofluorescence, immunohistochemistry and flow cytometry techniques to observe the effect of acupuncture on the survival, proliferation, differentiation and the formation of the synaptic network of transplanted NSCs from both NSCs transplantation in vivo and hippocampal slices with NSCs co-cultured in vitro. After that, the inherent connections between the improvement of the NSCs transplantation microenvironment and the improvement of the cognitive impairment of dementia mouse by acupuncture will be analyzed. This will provide new ideas and experimental evidence for the anti-dementia mechanism of acupuncture regulating NSCs transplantation and for the correlation of acupuncture Sanjiao and cerebral diseases.

神经干细胞（NSCs）移植为神经再生修复治疗带来巨大前景，但只有少量细胞能长期存活并发挥功能，最终影响痴呆治疗的效果。植入NSCs的命运很大程度取决于微环境，微环境密切调节着NSCs移植的进程。本课题在针刺治疗老年性痴呆的确切临床疗效和针刺调节内源性NSCs的实验基础上，运用"针刺调理三焦改善脑髓不充"的中医理论，将针刺与NSCs移植相结合，从针刺影响NSCs移植微环境角度，以SAMP8为研究对象，应用免疫印迹和实时荧光定量PCR技术观察针刺对微环境指标的影响，通过体内移植NSCs和建立海马脑片与NSCs共培养体外模型两方面，应用HE染色、免疫荧光、免疫组化、流式等技术观察针刺对移植NSCs存活、增殖、分化和形成突触网络的影响，分析针刺改善NSCs移植微环境和改善痴呆鼠认知障碍的内在联系，从微环境角度为探讨针刺调节NSCs移植的抗痴呆机制及针刺三焦与脑病的相关性等提供新思路和实验依据。

老年痴呆症（Alzheimer's disease，AD）是一种伴衰老出现的以进行性痴呆为主要症状的神经变性疾病，AD患者脑内普遍存在神经元丢失、神经再生修复和神经变性紊乱。神经干细胞（neural stem cells，NSCs）为AD的治疗开辟了美好的前景。本课题从SAMR1小鼠脑海马分离、培养NSCs，并进行扩增、鉴定。将NSCs移植入SAMP8小鼠海马并进行“益气调血，扶本培元”针法治疗后，结果显示针刺能够上调小鼠海马中与影响NSCs存活、增殖、分化、趋化等方面相关的微环境指标bFGF、EGF、BDNF蛋白和基因的表达水平，而对BMP、SDF-1、VEGF蛋白和基因表达无明显影响。体外结果显示经针刺调节的NSCs植入海马微环境对NSCs的生长、增殖和分化具有良好的调节作用。提示针刺能够通过调节NSCs在宿主的存活、增殖、分化、趋化、迁移、血管和突触网络，改善海马NSCs微环境，进而增强NSCs移植防治AD的疗效。本课题同时进行非穴组干预对比，发现本针法具有腧穴特异性。NSCs移植微环境与痴呆密切相关，“益气调血，扶本培元”针法对于调节NSCs移植微环境，改善痴呆小鼠的学习记忆障碍具有重要的作用。针刺在治疗痴呆过程中发挥了整体的调节作用，可能与改善小鼠海马内异常的脑微环境有关，可能通过调整三焦之气，改善三焦失司所致的脑髓不充为基础的脑微环境和脑功能异常，调节NSCs在宿主的存活、增殖、分化、趋化、迁移、血管和突触网络，增强NSCs移植防治AD的疗效。 这种针刺改善痴呆症状与改善NSCs微环境异常的内在联系，为外源性NSCs移植治疗AD提供了针刺增效的实验基础，为临床细胞移植治疗神经退行性疾病奠定了理论依据，为针刺的抗痴呆机制及针刺调节三焦与脑病的相关性研究提供新思路。