PI3K-AKT-mTOR信号通路及其上游信号分子在抗供者HLA特异性抗体介导的移植排斥中的机制研究

Little is known about the molecular mechanism of Anti HLA Specific Antibody (HLA DSA) mediated chronic transplant rejection. The pathological manifestation of chronic rejection including the proliferation of endothelial cell, intimal proliferation and stenosis, cause to graft ischemia and failure. The Mammalian Target of Rapamycin (mTOR) plays an important role in endothelial proliferation and survival. Our previous results showed that the survival of HLA DSA positive kidney transplant patients is better in Rapamycin group than non-Rapamycin group,indicated that Rapamycin has protective effect in HLA DSA positive Solid Organ Transplant rejection. Our pre experiment showed that when HLA DSA incubated with endothelial cells, the important molecules of mTOR pathway- the phosphorylated level of AKT and mTOR were increased. So we speculated that HLA DSA mediated chronic transplant rejection through mTOR pathway. Because HLA molecules lack signal motifs, so they must coupling of other signal molecules to mediated downstream pathway. This study intends to use co-immunoprecipitation, protein mass spectrometry and Western blot techniques, investigating mTOR related molecular mechanism of HLA DSA mediated chronic transplant rejection through vitro endothelial cell line and mouse heart transplantation model, elucidating the mechanism of HLA DSA in graft rejection, and providing theoretical evidence for the prevention and treatment of humoral rejection of rapamycin.

抗供者特异性HLA抗体(HLA DSA)介导的慢性移植排斥在病理上表现为内皮细胞增殖、血管内膜增生和管腔狭窄致移植物缺血失功,但其分子机制仍然不明。哺乳动物雷帕霉素靶蛋白(mTOR)在调控内皮细胞增殖、生长等过程中发挥重要作用。本课题组随访发现HLA DSA阳性患者中,用雷帕霉素治疗的病人预后好于非雷帕霉素组,体外预实验用HLA DSA作用于内皮细胞,Western Blot显示mTOR途径的重要分子-AKT,mTOR磷酸化水平增加,推测HLA DSA通过mTOR信号途径介导移植排斥,HLA分子在HLA DSA的作用下通过偶联其他信号分子介导下游mTOR信号通路。本课题拟通过免疫共沉淀、蛋白质谱和Western blot等技术,从细胞和动物两个层面研究HLA DSA在介导内皮细胞增生和排斥反应中涉及的PI3K-AKT-mTOR途径相关的信号分子, 并为雷帕霉素用于体液排斥提供理论依据。

HLA II类抗体是介导实体器官移植体液性排异反应的主要原因，长期以来对HLA DSA介导的内皮细胞增殖、血管内膜增生分子机制不明。本研究在成功构建人主动脉内皮细胞（HAEC）HLA II类抗原表达的基础上，探讨抗供者特异性HLA II抗体(HLA II DSA)介导的移植排斥作用机制。抗HLA II 抗体(HLA II DSA）刺激表达HLA II类抗原的HAEC后，刺激并活化PI3K/AKT/mTOR信号途径，同时mTOR及其下游信号分子磷酸化水平增加, 荧光示踪 CFSE 标记技术及Transwell 小室迁移技术提示在HLA II DSA刺激下，内皮细胞增殖迁移能力增加，雷帕霉素可以很好地抑制上述信号通路活化。用抗HLA II 抗体免疫共沉淀细胞裂解液，Western Blot检测显示HLA II DSA刺激组 HLA II抗原与Rab8α有相互作用,提示在激活 HLA II 类抗原后，可能通过连接Rab8a信号分子从而激活下游mTOR信号通路。本研究成功构建了Rab8a基因敲除HAEC细胞系,为进一步实验奠定基础。