化瘀通络灸调控"神经血管小生境"促VD大鼠神经新生胞外信号蛋白cross-talk机制

In the last Fund (NO:30973801) the research group found moxibustion in treatment of vascular dementia (VD) existing the mechanism of promoting angiogenesis in the brain, it is highly consistent with Neurovascular Niche theory which was reported recently. However reciprocal extracellular proteins mechanism of the neuro-vascular alternate promoting is unknown,and become a research target in the next step. Neurovascular Niche was reported recently as a vascular bed microenvironment of the adult brain which the neurogenesis dependent on highly and become a focus of nerve injury reparation researching. in this study,the VD model will be established . the NSCs (neural stem cells) marked with EGFP (enhanced green fluorescent protein)will be applied, co-cultured with the EPCs (endothelial progenitor cells) in vitro to Construct co-implant of Neurovascular niche, and transplanted in VD model rat brain via lateral ventricle.treating of moxibustion on head acupoints and setting control groups. immunofluorescence double labeling and confocal laser microscope will be utilized to track specific protein expression in differentiation, maturation of the NSCs process.the extracellular signaling protein secrete from NSCs or EPCs will be detected synchronously and to be analyzed of cooperative relationship . on the above to ascertain the mechanism of moxibustion regulation of neurogenesis. all of these to research the pathways and mechanisms of moxibustion.the purpose is to provid basis date for the disease treatment by moxibustion. neurovascular niche theory and EGFP tracking technology in acupuncture study field has not been reported. regularity and mechanism .

课题组在上一个基金(NO:30973801)研究中发现,艾灸治疗血管性痴呆(VD)存在着促脑内血管新生机制,这与新近研究Neurovascular Niche(神经血管小生境)理论高度吻合,然艾灸的神经-血管互生促长的胞外蛋白机制不明,成为课题组下一步研究的目标。本研究用增强绿色荧光蛋白(EGFP)标记神经干细胞(NSCs),并与内皮祖细胞(EPCs)共培养,构建"小生境"共植体,移植于VD模型大鼠脑内.艾灸治疗,免疫荧光双标激光共聚焦追踪NSCs的分化、成熟过程特异蛋白表达及其与新生微血管的关系；围绕EGFP阳性神经元,在激光共聚焦显微镜下计数和定位NSCs和EPCs分泌可相互作用的胞外信号蛋白,分析协同关系,探明艾灸调控促神经新生胞外信号蛋白cross-talk机制,研究艾灸治疗VD的作用途径和规律,为艾灸治该病提供依据。"神经血管小生境"理论和EGFP追踪技术在针灸研究领域未见报道。

研究背景：课题组在上一个国家自然科学基金(NO:30973801)研究中发现，艾灸治疗血管性痴呆(VD)存在着促脑内血管新生机制，但促神经新生机制尚缺乏理论的阐述；“Neurovascular Niche”是神经与血管相互促进生长的微环境，如何创造理想神经血管小生境，是研究神经代偿修复的新靶标，为课题组的主要研究目标。研究内容：本研究用增强绿色荧光蛋白(EGFP)标记神经干细胞(NSCs)，并与内皮祖细胞(EPCs)共培养，构建“小生境”共植体，移植于VD模型大鼠脑内。化瘀通络灸治疗，免疫荧光双标激光共聚焦追踪NSCs的分化、成熟过程特异蛋白表达及其与新生微血管的关系；围绕EGFP阳性神经元,在激光共聚焦显微镜下计数和定位NSCs和EPCs分泌可相互作用的胞外信号蛋白，分析彼此协同关系，探明艾灸调控促神经新生的胞外信号蛋白cross-talk机制，研究艾灸的作用途径和规律，为灸治该病提供依据。研究结果：经3个疗程治疗，各组治疗前后水迷宫实验逃避潜伏期及错误次数比较均有显著差异，治疗后组间比较共植体艾灸组与共植体西药组无明显差异，与其它组两两比较有差异，表明艾灸和西药在减少VD大鼠逃避潜伏期、降低错误次数方面均有显著性疗效，且疗效优于单纯的共植体移植空白组和NSCs移植艾灸组。4组治疗组与模型组海马内Nestin、DCX、MAP、NeuN、CD34的表达均有显著差异，且共值体艾灸与西药组表达多于其他治疗组。共植体艾灸组BDNF/TrkB、SDF-1/CXCR4、VEGF/Flt-1表达始终优于其他各组。结论：化瘀通络灸确能改善VD大鼠学习记忆成绩，通过调控脑内微环境促进神经新生，并与血管新生相互促进，其作用机制可能存在着调控神经血管小生境，使其发挥细胞间偶联、协同作用。