特异性T细胞对多发性硬化症神经元和轴索损伤及其分子机制的研究

Multiple sclerosis (MS) has Neuronal apoptosis and axonal degeneration in the early stage. The accumulation of axonal injury and neuronal degeneration is the pathological basis of MS patients with the progressive loss of function, so the MS is a kind of neurodegenerative disease which has great significance for treatment. There is a lot of research about the mechanism of neuronal and axonal damage, however, no clear results have been obtained yet. We obvered that specific lymphocytes induced by MBP can act directly on neuronal cell bodies and axon, which might be one of the reasons for the degeneration of nerve cell and axonal damage. This research adopts the common culture and micro island culture with specific CD4+, CD8+T cells induced by MBP or EAE peripheral blood CD4+ CD8+T cells and normal rat neuronal cells, and also the methods of electron microscopy, neuronal apoptosis TUNEL, laser confocal microscopy, Western Blot and related cytokine detection. Through this study, we aim to clarify the variation pattern of specific T cell's damage on neuronal apoptosis and axon, and also to illustrate T cell subsets that directly damage nerve cells and axon and its killing mechanism, with the expectations to expand the pathogenesis of MS, and lay a theoretical foundation for the guide to clinical treatment of patients.

多发性硬化症（MS）早期就有神经元凋亡和轴索变性，且不断积累的轴索损伤和神经元变性是MS患者进行性功能丧失的病理基础, 将MS 看成一种神经变性疾病进行治疗有重要意义。神经元和轴索损害的机制研究很多，但仍不明确。我们以往观察到髓鞘碱性蛋白(MBP)诱导的特异性淋巴细胞能直接作用于神经元和轴索，可能是使神经细胞变性和轴索损害的原因之一。本研究拟采用髓鞘蛋白诱导的特异性CD4+、CD8+T细胞或MS动物模型鼠外周血CD4+、CD8+T细胞与正常鼠神经元细胞共同培养及微岛培养，进行电镜、神经元凋亡末端标记、激光共聚焦显微镜、Western Blot及相关细胞因子检测等方法研究，从而明确特异性T细胞对神经元凋亡和轴索损害的变化规律，阐明对神经细胞和轴索发生直接损害的T细胞亚群及其杀伤机制，期望为拓展MS的发病机制，指导患者的临床治疗奠定理论基础。

神经元凋亡和轴索变性是多发性硬化（MS）的重要病理特征之一，但其形成原因尚不明确。我们前期研究发现髓鞘碱性蛋白（MBP）特异性T淋巴细胞能直接作用于神经元和轴索，这可能是造成神经元损伤的原因机制之一。本研究通过用MBP免疫Lewis大鼠构建EAE模型，将分离培养髓鞘特异性T细胞和神经元进行共培养，发现CD8+T细胞诱导神经元凋亡的能力强于CD4+T细胞，ELISA检测显示活化后的CD8+T细胞分泌更多的穿孔素和颗粒酶，而CD4+T细胞主要分泌IL-17和IFN-γ；Western Blotting检测发现与CD8+T细胞诱导神经元表达凋亡相关蛋白Bax的能力明显高于CD4+T；向培养的神经元中分别加入穿孔素、颗粒酶、IL-17和IFN-γ，发现穿孔素诱导神经元表达Bax能力最强。以上研究结果表明，髓鞘特异性CD8+T细胞是造成神经元坏死的主要T细胞亚群，其主要通过分泌穿孔素诱导神经元凋亡。