GIT1调控ERK1/2的活性促进BMSCs体外移植模型中突触可塑性的机制研究

To evaluate the nonspecificity of green fluorescent protein (GFP), we constructed a model to investigate the interaction between the host and the GFP labeled BMSCs cracking by Sterile hypotonic distilled water in vivo after transplanted via tail vein. In order to trace the functional synapses formation between BMSCs and cortical neurons and explore the G protein-coupled receptor kinase-binding protein 1(GIT1) regulate the synaptic plasticity between BMSCs and cortical neurons and the directional migration of axons via extracellular regulated protein kinase 1/2(ERK1/2). To mimic the intracranial transplantation of BMSCs, we constructed a co-culture system that was combination of BMSCs and cortical neurons. We applied lentiviral vector and RNA interference technology to establish the GIT1 expression and silence model of BMSCs, and analyze the influences of functional synapses formation between BMSCs and cortical neurons and the directional migration of axons after the GIT1 of BMSCs was upregulation and downregulation, to confirm the potential mechanism of synaptic plasticity and directional migration of axons regulated bu GIT1. It is likely that the overexpress of GIT1 in BMSCs induce the phosphorylation of ERK1/2 to promote the functional synapse formation and directional migration of axons. We raise a new target for central nervous system diseases for establishing communication with host cells by BMSCs transplantation.

干细胞移植的功能机制一直备受关注。本研究拟追踪BMSCs与神经元间功能性突触样结构形成,探讨G蛋白偶联受体激酶结合蛋白1(GIT1)介导细胞外调节蛋白激酶(ERK)1/2调控BMSCs与神经元间突触可塑性及神经元轴突定向迁移的关联性。通过低渗蒸馏水裂解GFP标记的BMSCs,收集细胞碎片后模拟BMSCs移植过程死亡的细胞与细胞共培养及体内移植观察宿主细胞表达GFP,评判GFP标记细胞的非特异性;模拟BMSCs颅内移植,建立BMSCs与神经元体外共培养体系。采用慢病毒载体和RNA干扰技术使BMSCs中GIT1过表达和沉默,分析GIT1上下调对BMSCs与神经元间突触可塑性及神经元轴突定向迁移能力的影响,探讨GIT1促进突触可塑性及神经元轴突定向迁移机制：GIT1过表达引起ERK1/2磷酸化促进BMSCs与神经元间功能性突触形成及神经元轴突定向迁移。

在干细胞结构与功能追踪实验研究中，为了区分细胞来源，常常实验前对移植的干细胞使用绿色荧光蛋白（Green Fluorescent Protein，GFP）细胞标记物。本课题通过超声仪裂解GFP标记的BMSCs,收集该细胞碎片后，模拟为BMSCs移植过程中死亡的细胞，通过荧光显微镜、激光共聚焦显微镜检测移植后各组织器官（心、肝、脾、肺、肾、脑）中GFP表达情况，同时使用western blot技术检测移植后各器官组织中的GFP表达情况，并运用PCR技术检测实验组中GFP基因表达，结果GFP标记的BMSCs的碎片经移植入SD大鼠后，各器官中可检测GFP及其基因阳性，故而GFP作为细胞标记物在BMSCs移植治疗中枢神经系统疾病过程中存在非特异性，即宿主细胞可吞噬含GFP的细胞碎片，在实验过程中将追踪到GFP阳性的细胞误判断为移植细胞。. 干细胞移植治疗中枢神经系统疾病的疗效已得到众多学者的证实，但潜在的机制研究一直是当今该领域的热点及难点。该课题组将 BMSCs 与皮层神经元进行共培养，在体外成功建立了 BMSCs 体内移植模型，通过形态学检测两者细胞间的相互作用及潜在机制。结果发现皮层神经元的轴突定向 BMSCs 迁移或沿着 BMSCs 表面生长，并在原子力显微镜、扫描电镜超微结构上发现 BMSCs 与皮层神经元之间突触样结构的形成。因此，共培养体系中两种细胞的生长状态为相互促进，互为共赢。