B7-H4在间充质干细胞移植治疗多发性硬化中的作用及机制研究

Bone marrow mesenchymal stem cell (BMSC) is promising in tissue regeneration and autoimmune disease treatment. Besides self renew ability and multy differentiation potentials, immuno-modulating effect is another important characteristic of BMSC, which is paid close attention to by many investigators. B7-H4 is a kind of new negetive co-stimulator. It was indicated in our experiments that B7-H4 was constitutively expressed on BMSC and mediated its immunomodulaing effect on T cells, though the role and the mechanism remain to be clarified. To investigate the role and mechanism of the immuno-modulating effect of mesenchymal stem cells (MSC) mediated by B7-H4, a typical autoimmune disease, multiple sclerosis (MS) is studied. Both MS patients and experimental allergic encephalitis (EAE) mice (the model of MS) are investigated. To explore the treatment effect of MSC mediated by B7-H4 on EAE, the mouse mesenchymal stem cell (MSC) cell line C3H10T1/2, homemade anti-B7-H4 monoclonal antibody and B7-H4 fusion protein are used to interfere the signal of B7-H4.This project will focus on the effect and pathway of B7-H4 expressed on MSC for T cell polarizing, regulartory T cells (Treg) induction and proliferation, as well as the role of denritic cells, which are very important in T cell activation. Among DCs, tolerogenic dentritic cells (tDC) are crucial for Treg induction and proliferation. Nitric oxide (NO), indoleamine-dioxigenase (IDO), and other related molecules are also studied. This study will give a theoretical and experimental evidence for MS and other autoimmun disease treatment by MSC transplantation, also, it may supply a new target molecule for MS treatment.

骨髓间充质干细胞（BMSC)不仅有自我更新、多向分化潜能，还有免疫调节作用，在组织修复、自身免疫病治疗中具潜在应用价值。BMSC的免疫调节机制研究备受关注。B7-H4是新近发现的负性协同刺激分子，实验组前期研究表明B7-H4组成性表达于BMSC并介导其对T细胞的免疫调节作用，但具体机制尚待进一步探讨。本研究拟以神经系统自身免疫病多发性硬化（MS）为切入点，结合临床和MS的动物模型实验性变态反应性脑脊髓炎（EAE），利用组成性表达B7-H4的小鼠间充质干细胞（MSC）株C3H10T1/2，自主研发的抗B7-H4单克隆抗体和B7-H4 Ig融合蛋白等干预B7-H4信号，明确MSC表达的B7-H4在治疗EAE中的体内效应及途径，探讨B7-H4在MSC免疫调节中的作用机制，尤其对T细胞极化、调节性T细胞和树突状细胞等免疫细胞和分子的作用及效应途径，为MS等自身免疫病的治疗开拓新的靶分子和途径。

本项目旨在明确B7-H4 在C3H10 细胞移植于EAE小鼠后的初步效果,探讨B7-H4在MSC治疗MS中的作用及机制。已有结果显示：（1）MSC细胞株C3H10移植治疗MS模型EAE安全、有效,可减轻发作程度，减少组织损伤，病灶周围存在极少量GFP标记的C3H10细胞；B7-H4靶向干扰的C3H10(C3H10-ShRNA-B7-H4)移植组较阴性对照组和C3H10移植组EAE神经功能缺损评分高，组织损伤重，炎性细胞浸润明显；各移植组均未见肿瘤发生。（2）表达B7-H4的C3-H10能抑制PHA激发的小鼠脾淋巴细胞的增殖和活化，阻滞细胞周期于G0/G1期，保护其免受活化诱导的细胞凋亡（AICD）；而下调B7-H4后C3H10的上述作用被逆转。（3）下调B7-H4的C3H10细胞凋亡比例增加；且表面趋化因子受体CXCR4表达明显下调；这与人骨髓MSC（hBMSCs）的研究结果一致。（4) 研究期间，Podojil JR等的论文发表，证明了B7-H4Ig在EAE中通过IL-10/Treg机制发挥免疫抑制作用，这与本实验组预期设想一致。（5）另外，实验组对不同协同刺激分子在hBMSCs和胎盘源MSC（PDMSCs）免疫调节中的作用的研究结果显示：hBMSCs组成性表达B7-H4，而PD-L1为诱导表达，而PDMSCs不组成性表达B7-H4，但组成性表达PD-L1和FasL,PD-L1对T细胞细胞周期、FasL对T细胞凋亡在不同时相发挥免疫抑制作用；另外，hBMSCs和hPMSCs对B细胞也发挥了免疫调节作用。(6）已有结果提示：C3H10移植在EAE中主要通过免疫调节起治疗作用，而非替代损伤细胞；敲除B7-H4的C3H10除了免疫调节作用受损，其低免疫原性也受损，易凋亡，更早被机体的免疫系统清除，在体内维持活性的时间短暂。鉴于MSC移植较B7-H4 Ig治疗EAE既有促修复、又有免疫调节、减少组织损伤的作用，维持时间较长，代价低，且安全，更有优势。本项目初步结论是：表达于C3H10的B7-H4在其免疫调节中，在其移植治疗EAE中起了重要的作用；B7-H4可作为EAE治疗的新靶点。基本证明实验预期结果。本项目已发表SCI 论文1 篇，部分实验结果已投稿，并受邀在国际神经修复协会第六届年会、中华医学会第十六届全国神经病学年会等上口头发言，培养研究生5名，一名已毕业。