可溶性糖蛋白sgp130通过阻断IL-6/sIL-6R跨信号通路诱导移植免疫耐受的实验研究

The proinflammatory cytokine IL-6 plays an important role in transplant rejection and tolerance induction. IL-6 combined with IL-6R and membrane gp130 activates classical signaling pathway,which can inhibit inflammation and promote cell regeneration.By contrast, IL-6 combined with soluble IL-6R and membrane gp130 activates trans-signaling pathway which can promote inflammation and activate the immune system.Previous studies have shown that completely blocking IL-6 can induce allograft tolerance in a mice cardiac transplantation model. IL-6 antibodies and IL-6R antibodies can block both proinflammatory and anti-inflammatory pathways. Soluble gp130 (sgp130) is a naturally occurring glycoprotein which can be found in plasma. Sgp130 acts as a antagonist of membrane gp130, which can bind IL-6 and soluble IL-6R. Sgp130 can specifically block the trans-signaling pathway, thereby making it a ideal target for IL-6 blocking during the tolerance induction. This project is to investigate the role of sgp130 in allo-immune responses both in vitro and in vivo. The first part is to determine the in role of sgp130 in vitro in T cell proliferation and differentiation as well as cytokine profile during the allo-antigen stimulate.The second part is to determine the in role of sgp130 in vivo in immune response and the underlying mechanism.

炎性细胞因子IL-6在移植排斥和耐受中发挥重要的作用。IL-6和细胞膜表面的IL-6R以及糖蛋白gp130结合激活经典信号通路，发挥抗炎及促进细胞再生的作用。而IL-6与可溶性IL-6受体（sIL-6R）及gp130结合激活跨信号通路，促进炎症反应，激活免疫系统，影响T细胞分化。前期研究结果证实完全阻断IL-6的作用能够诱导免疫耐受。抗IL-6抗体及IL-6R抗体不仅阻断了促炎的跨信号通路，同时也阻断了抗炎的经典信号通路。可溶性糖蛋白sgp130能够与IL-6/sIL-6复合物相结合，拮抗膜表面gp130，特异性地阻断跨信号通路，理论上是诱导免疫耐受更为理想的靶点。本课题拟从体外和体内两个方面研究sgp130对移植免疫的作用：（1）体外研究sgp130对同种异体抗原刺激的淋巴细胞增殖、分化以及细胞因子表达的影响；（2）利用小鼠心脏移植模型，在体内研究sgp130对移植免疫反应的影响及机理。

炎性细胞因子IL-6在移植免疫和缺血再灌注损伤中均发挥了重要的作用。IL-6和细胞膜表面的IL-6R以及糖蛋白gp130结合激活经典信号通路，发挥抗炎及促进细胞再生的作用。而IL-6与可溶性IL-6受体（sIL-6R）及gp130结合激活跨信号通路，促进炎症反应。抗IL-6抗体及IL-6R抗体不仅阻断了促炎的跨信号通路，同时也阻断了抗炎的经典信号通路。可溶性糖蛋白sgp130能够与IL-6/sIL-6复合物相结合，拮抗膜表面gp130，特异性地阻断跨信号通路，理论上是诱导移植免疫耐受以及减轻肾脏缺血再灌注损伤更为理想的靶点。本课题分别研究sgp130特异性阻断IL-6跨信号通路对心脏移植耐受的诱导作用和肾脏缺血再灌注损伤的抑制作用。首先课题组设计并构建了融合蛋白质粒Fc-gp130-pcDNA，在HEK293哺乳动物表达系统中高效分泌表达Fc-sgp130，纯化后获得的蛋白经细胞实验证实能有效抑制IL-6下游信号通路STAT3磷酸化。在心脏移植免疫耐受实验中，课题组证实了sgp130在体外能够显著的抑制淋巴细胞的活化，能够有效抑制炎症细胞因子的表达，而在小鼠同种异体心脏移植模型中，sgp130能够有效延长心脏移植物的存活时间，但IL-6 -/-小鼠受体心脏移植物存活时间更长，否定了先前的假设，特异性阻断sgp130并未表现出显著的生存优势。而sgp130在肾脏缺血再灌注损伤的体外实验中能够显著减轻肾小管上皮细胞的氧化应激损伤，同时在小鼠肾缺血再灌注损伤模型中，sgp130和IL-6R抗体均能有效的减轻肾缺血再灌注损伤，且sgp130在血清学和组织学中均证实对肾脏有更好的保护作用。本课题组证实了sgp130选择性的阻断较完全阻断IL-6信号通路有明显的优势，提示sgp130有潜力成为临床修复急性肾损伤以及围手术期保护肾功能的药物。