抗B7-1单克隆抗体免疫干预促进间充质干细胞修复脊髓损伤及其机制的研究

Mesenchymal stem cell (MSC) has fascinating prospect in treatment of spinal cord injury (SCI) for its special properties of low immunogenicity and multilineage differentiation. However, inflammations immediately followed SCI, cause to damage of specific tissue and disrupt the ability of neuroprotection and regeneration. Additionally, it also inhibits cell survival and neuron differentiation of MSC. Therefore, it has been a crucial problem to control inflammation after SCI and improve MSC clinical efficiency by application of immunomodulators. Besides, the mechanism needs to be clarified. We have successfully developed B7-1 monoclone antibody (B7-1mAb) that could inhibit T cell-mediated immune response of human, rat and mouse and alleviate the production of antoantibodies in Pristane-induced lupus in mouse. Meanwhile, we promoted SCI function recovery in rats with collagen scaffold loaded with bFGF. Based on the preliminary work, in this study, we will firstly crosslink B7-1mAb with collagen and load it with MSC from different human tissues, and then transplant B7-1mAb-Collagen-MSC to injury site during acutephase of SCI in SD rats. After that, we will explore the effect of B7-1mAb on local microenvironment and the impact on SCI outcomes with MSC administration. We will also elucidate the relevant mechanism that B7-1mAb facilitates MSC to repair SCI at cellular and molecular level. This would provide new insights to the relevant of immune intervention with SCI repair and weighted evidence to choose optimal methods for SCI treatment.

间充质干细胞（MSC）在脊髓损伤（SCI）救治中显示了良好的应用前景，但SCI 继发炎症反应不仅破坏自身神经保护和再生，也抑制MSC存活和神经分化。因此，如何及时、有效调控SCI炎症反应，提高MSC疗效，促进自身神经修复是SCI救治的一个关键问题。我们研制的B7-1抗体（B7-1mAb）可抑制T细胞免疫应答及自身抗体的形成；同时利用胶原支架结合碱性成纤维生长因子促进了大鼠脊髓半横断损伤修复。在此基础上，本课题旨在利用大鼠脊髓半横断损伤模型，将B7-1mAb固定于胶原材料并复合人MSC在SCI急性期移植至损伤局部，从细胞、分子等水平探讨B7-1mAb对SCI炎症微环境的免疫干预作用，评价其协同MSC对SCI修复和功能重建的效果，阐明B7-1mAb免疫调控促进MSC修复SCI的作用机制，为深入了解免疫干预与SCI修复内在联系、优化SCI治疗方案提供理论依据。

脊髓损伤后（SCI）形成不利于神经再生的微环境，主要因素包括继发性的炎症环境抑制神经细胞的生长、分化及诱导细胞凋亡；成纤维细胞形成的瘢痕阻碍神经再生；微环境中的细胞因子促进神经干细胞向胶质细胞分化，加剧了神经细胞的蜕变。因此如何构建促进脊髓中枢神经再生的微环境是治疗脊髓损伤的关键问题。本项目以生物材料为载体联合抗B7-1抗体（B7-1mAb）、间充质干细胞（MSCs）构建脊髓损伤微环境，从免疫干预角度探讨其对神经再生和功能重建的生物学作用。研究显示：采用微溶胶方法复合B7-1mAb的静电纺丝支架移植至大鼠半横断脊髓缺损中，局部炎症因子IL-2、IFN-γ等下降，生存率提高，功能BBB评分增加，组织学分析显示有新生神经纤维形成，提示B7-1mAb抑制了局部免疫反应，促进了神经再生；将胶原支架复合大鼠骨髓MSCs，同样移植至大鼠半横断脊髓缺损中，可调节巨噬细胞亚群分化，促进M2型巨噬细胞的极化，抑制细胞凋亡，促进了组织的再生和功能改善；在此基础上，将胶原支架复合人胎盘来源的MSCs移植至大动物比格犬全横断脊髓缺损中，结果表明：尽管2个月后在比格犬脊髓损伤部位未能检查到人来源的MSCs，但在9个月观察期中功能评分（olby评分）移植MSCs 组显著高于对照组，最终的的免疫组化结果证实了该实验组有贯穿损伤区的神经再生，感觉和运动神经元以及神经突触和髓鞘的形成，推测外源性MSCs发挥免疫调节功能促进了内源性MSCs修复脊髓损伤。此外，将胶原支架复合基因工程化含胶原结合域的血管内皮生长因子（CBD-VEGF），移植至大鼠全横断脊髓缺损中，增加了损伤区微小血管形成、改善了损伤局部供血情况，继而促进神经再生和功能改善。我们的研究显示中枢神经再生的微环境是解决脊髓再生的重要策略，以生物材料为基础的组织工程将在脊髓损伤治疗中具有巨大的应有前景。本项目也为临床上优化SCI治疗方案提供了理论依据。