血小板αIIb启动子控制卵白蛋白（OVA）靶向诱导抗原特异性免疫耐受及机制的研究

Induction of autoantigen or alloantigen-specific immune tolerance is a desirable therapy for autoimmune diseases or organ transplantation rejection. Our research group have demonstrated that targeting coagulant protein expression to platelets under control of the platelet-specific αIIb promoter can not only results in coagulant protein storage in platelet α-granules, but also induce coagulant protein-specific immune tolerance in hemophilia mice. However, most of autoantigens or alloantigens in autoimmune diseases or organ transplantation rejection are non-coagulant proteins, whether targeting non-coagulant proteins expression to platelets under control of the platelet-specific αIIb promoter can induce immune tolerance to non-coagulant proteins is still unknown, and the mechanism of this induced immune tolerance is also unexplored. In our preliminary experiment we used ovalbumin(OVA) that is a non-coagulant protein to replace autoantigen or alloantigen，data showed that targeting OVA expression to platelets under control of the platelet-specific αIIb promoter can also result in OVA storage in mouse platelet α-granules. In this study, based on the data and mouse model, we are going to explore whether targeting OVA expression to platelets under control of the platelet-specific αIIb promoter can induce OVA-specific immune tolerance, and study the interaction mechanisms between immune cells (OVA specific CD4+ T cell, Treg cell, etc) and platelets in this induced immune tolerance. This research will provide the theories for treatment of autoimmune diseases or organ transplantation rejection through the induction of autoantigen or alloantigen-specific immune tolerance by targeting autoantigen or alloantigen expression to platelets under control of the platelet-specific αIIb promote.

诱导自身或异体靶抗原的特异性免疫耐受可以治疗自身免疫性疾病或器官移植排斥反应。我们课题组报道了血小板αIIb启动子控制凝血因子可使凝血因子储存于小鼠血小板的α颗粒内，并诱导了小鼠对凝血因子产生特异性免疫耐受。但是自身或异体靶抗原多数是非凝血蛋白，而目前尚不明确该启动子控制非凝血蛋白是否也可诱导对非凝血蛋白产生特异性免疫耐受，也未阐明这种诱导免疫耐受的机制。我们前期研究用卵白蛋白（OVA）这种非凝血蛋白来代替靶抗原，发现αIIb启动子控制OVA也可使OVA蛋白储存于小鼠血小板的α颗粒内。本项目将在此基础上，通过研究血小板αIIb启动子控制OVA是否可诱导小鼠对OVA产生特异性免疫耐受，以及OVA特异性CD4+T细胞、Treg细胞等免疫细胞与血小板在这种诱导免疫耐受中的相互作用机制，为今后利用αIIb启动子控制靶抗原诱导靶抗原特异性免疫耐受，治疗自身免疫性疾病或器官移植排斥反应提供理论依据。

诱导对自身或异体靶抗原的特异性免疫耐受是治疗自身免疫性疾病或器官移植排斥反应的最好策略之一。本项目研究血小板αIIb启动子控制OVA诱导小鼠对OVA蛋白产生特异性免疫耐受及其机制。具体如下：（1）包装2bOVA、2bVWFppOVA(2bVpOVA)及2bGFP慢病毒，转染B6/CD45.2供者小鼠的造血干细胞后移植到B6/CD45.1受者小鼠，构建2bOVA、2bVpOVA及2bGFP小鼠模型。PCR检测到2bOVA及2bVpOVA组均表达OVA，而2bGFP及无转染组不表达OVA。（2）电镜检测发现2bOVA组的血小板α-颗粒内检出OVA蛋白。ELISA检测发现：2bOVA及2bVpOVA组分别有95%及98%的OVA蛋白被储存在血小板，且分别有85%及98%的OVA蛋白在血小板激活后可被释放。（3）ELISA检测在OVA蛋白免疫后小鼠的抗OVA抗体水平发现：2bOVA及2bVpOVA组的抗体水平显著低于2bGFP组。CAG-OVA小鼠皮片移植实验发现：2bOVA与2bVpOVA组的移植皮片不会被排斥而存活，而2bGFP及无转染组的移植皮片被排斥脱落。（4）利用OT-II/CD45.2小鼠作为供者小鼠构建了2bOVA、2bVpOVA及2bGFP的三组OT-II转基因小鼠模型。流式细胞术检测发现：2bOVA及2bVpOVA组外周血、脾脏及淋巴结的OVA特异性CD4+T细胞的比例与数量显著低于2bGFP组，Treg细胞的比例显著高于2bGFP组；且脾脏及淋巴结的OVA特异性CD4+T细胞的凋亡比例显著高于2bGFP组。但胸腺中的OVA特异性细胞未受影响。（5）体外T细胞增殖实验发现：在外源性OVA蛋白刺激后，2bOVA及2bVpOVA组的脾脏Treg细胞被诱导并抑制效应性T细胞的增殖。综上所述，本项目研究显示血小板αIIb启动子控制OVA可诱导小鼠对OVA蛋白产生特异性免疫耐受，其机制是通过清除外周的抗原特异性CD4+T细胞以及诱导抗原特异性Treg细胞来诱导免疫耐受的，为今后利用αIIb启动子控制靶抗原诱导对靶抗原特异性免疫耐受，治疗自身免疫性疾病或器官移植排斥反应提供理论依据。现已按计划完成既定目标并已发表2篇SCI论文。