CB1R对小胶质细胞表型和功能的调控及其机制研究

Cannabinoid 1 receptor (CB1R) is expressed on both neurons and microglial cells in the center nervous system (CNS). Multiple sclerosis (MS) is an inflammatory demyelinating and neurodegenerative disease of the CNS. To date, few studies have focused on the role of CB1R on the phenotype and function of microglial cells, as well as the development of MS. Our previous experiments demonstrate that CB1R plays crucial immunological regulation on the development of experimental autoimmune encephalomyelitis (EAE) in C57B/L6 mice. Nevertheless, the mechanisms of action should be further investigated. Therefore, we hypothesis that CB1R could induce the change of phenotype and function of microglial cells at different time point, which could regulate immune response, and change the microenvironment during the course of development of EAE in C57B/L6 mice. In order to proof our hypothesis, we will culture the BV2 microglial cell line and primary microglial cells in vitro and utilize EAE animal model in vivo to explore the effect of CB1R in immunomodulation and EAE development by using plasmid transfection, RNA interference technique and etc. Taken together, we try to explore the mechanisms of CB1R on immunological regulation and signal transduction of microglial cells in several fields of the molecular, cellular and animal model. This study could improve the theoretical basis of the pathogenesis of MS from the new perspective of CB1R, and find new therapeutic target for prevention and treatment of MS.

大麻素1型受体(CB1R)在神经元和小胶质细胞上均有表达。多发性硬化(MS)是典型的中枢神经系统(CNS)炎性脱髓鞘自身免疫性疾病。迄今，尚未查见CB1R对小胶质细胞表型与功能变化的调控机制及其与MS关系的相关报道。我们的预实验结果提示CB1R通过免疫调节作用参与实验性自身免疫性脑脊髓炎(EAE)的发生发展，但其作用机制尚有待进一步探讨。为此，我们提出假说：CB1R可能通过诱导小胶质细胞表型和功能变化影响CNS免疫调控，参与MS/EAE的发生发展。为验证这一假说，我们将通过细胞培养和动物模型，采用质粒转染、RNA干扰等手段，从分子、细胞和动物整体水平等多方面探讨CB1R在EAE免疫调控中的重要作用，明确小胶质细胞上CB1R免疫调控的分子机制和信号通路。本研究将从CB1R这个新视点完善MS发病机制的理论基础，为MS的防治提供新的思路、开拓新的途径。

目的：从大麻素1型受体（CB1R）这个新视点，通过转染质粒沉默或是上调EAE小鼠脊髓组织CB1R的表达，探讨CB1R在EAE发生发展中的可能机制。.方法： 1.制备RNA干扰靶点序列慢病毒载体颗粒，筛选抑制CB1R表达效果最佳的优势载体(GR603-pLKD.CMV.G&PR.U6.shRNA)；制备基因过表达靶点序列慢病毒载体颗粒，筛选促进CB1R高表达效果最佳的优势载体(CN550-pLOV.CMV.eGFP.2A.EF1a.PuroR）。2.选取正常的C57B/L6小鼠鞘内注射(intrathecal injection,ith) 慢病毒载体颗粒，每只C57B/L6小鼠转染慢病毒数量为1*10e7tus,注射容积为15ul。鞘内注射后观察3天，选取行走正常，尾部翘起的正常C57B/L6小鼠作为EAE模型制备小鼠。C57B/L6小鼠EAE的构建参照文献常规构建。3. 留取起病高峰期和缓解期不同组别的EAE小鼠组织标本，通过免疫组化法和Western Blot法检测靶点蛋白的表达，ELISA法检测细胞因子与神经营养因子的浓度，流式细胞技术分选M1/M2型免疫细胞。.结果：1.沉默CB1R表达的EAE组小鼠在体重、发病时间，病情严重程度等方面与空病毒对照的EAE小鼠均无显著差异。比较于空病毒对照的EAE小鼠，CB1R高表达的EAE组小鼠，EAE起病时间显著推后，病情严重程度显著降低。2. CB1R的过表达可以显著抑制EAE小鼠脊髓组织中NF-kB/p65、TLR-4、IL-1b、IL-6、IL-17和TNF-a的表达，上调IL-10的表达。轻度降低脾脏组织中Il-1b和IL-17的浓度。提升脑组织中IL-10的浓度。3. CB1R过表达的EAE小鼠，脾脏组织单个核细胞CD11b+CD16/32+ 的M1型免疫细胞百分比显著下降， CD11b+CD206+和 CD11b+IL-10+ 双阳性免疫细胞的百分比显著上升。4. CB1R的过表达显著提升脊髓组织中NT-3，BDNF和GDNF等保护性因子的分泌。.结论： CB1R通过多种机制参与免疫调节和神经元变性。EAE小鼠脊髓组织CB1R的过表达可通过抑制微环境炎症反应和提升微环境神经病保护因子发挥神经保护作用。