B和T淋巴细胞衰减子(BTLA)在肾移植术后急性排斥反应中的作用及机制研究

Acute rejection(AR) after kidney transplantation is the main factor affecting the prognosis of allograft; however, the accurate mechanisms are still under investigation. As a new co-stimulator found recently, B and T lymphocyte attenuator(BTLA) could transfer the negative signals and result in the inhibition of the T lymphocytes activation. Our previous studies showed that the expression of BTLA was significantly reduced in the allograft tissue and peripheral blood monocytes of AR recipients; Meanwhile, Belatacept, a novel immunosuppressant, could induce the remarkable decrease of BTLA on the surface of T lymphocytes in vitro. As a result, we hypothesized that BTLA could suppress the occurrence of AR after renal transplant; Moreover, Belatacept could cooperate with BTLA to reduce the occur of AR after kidney transplantation. Based on the established AR model of rat kidney transplantation, the relationship between BTLA and the occurrence of AR would be investigated; Afterwards, the role and related mechanisms of BTLA involved in the development of AR after renal transplant would be studied on the basis of primary T lymphocytes from AR model of rat kidney transplantation by performing various techniques such as the lentiviral vector, PCR and western blot assay; On the other hand, AR model of rat kidney transplantation and its primary T lymphocytes would be both used to validate the role and related mechanisms of Belatacept, which works in coordination with BTLA, in the development of AR post-transplant. The predicted outcomes will provide with a novel target and new clue for further studies of the prevention and treatment of AR after kidney transplantation.

肾移植术后急性排斥反应（AR）是影响移植肾长期预后的关键因素。B和T淋巴细胞衰减子（BTLA）是一种新型协同刺激分子，传递负性调节信号参与抑制T淋巴细胞活化。我们前期发现AR患者移植肾组织及外周血中BTLA表达显著降低；新型免疫抑制剂贝拉西普可诱导T淋巴细胞表面BTLA表达增高。据此，我们提出假说：BTLA可参与抑制AR中T淋巴细胞活化，减少AR发生；而贝拉西普可通过诱导T淋巴细胞表面BTLA高表达，进而抑制肾移植术后AR发生。为了验证上述假说，本课题拟通过构建大鼠肾移植AR模型，于体内探讨BTLA与AR发生的关系，并提取大鼠肾移植AR模型的原代T淋巴细胞，采用慢病毒载体，免疫组织化学及免疫蛋白印迹等技术，体外探究BTLA在AR发生中的作用及相关机制；并在此基础上，进一步探讨贝拉西普协同BTLA抑制AR发生的机制。本研究预期成果将为进一步研究肾移植术后AR的防治措施提供新靶点和线索。

肾移植术后急性排斥反应（AR）是影响移植肾长期预后的关键因素。B和T淋巴细胞衰减子（BTLA）是一种新型协同刺激分子，传递负性调节信号参与抑制T淋巴细胞活化。本研究中，我们首先探究BTLA在肾移植术后AR发生中的作用；构建并鉴定同种异体大鼠肾移植AR模型，探究BTLA对T淋巴细胞活化及肾移植术后AR发生的影响及机制。接下来，我们比较了特异性抑制细胞毒性T淋巴细胞相关蛋白-4（CTLA-4）与过表达BTLA对T细胞分化的影响，并探讨联合使用贝拉西普（CTLA-4特异性抑制剂）与过表达BTLA载体对肾移植术后AR发生的作用。结果表明，BTLA在肾移植术后AR发生的极早期显著升高，而后迅速下降，表明BTLA可能与肾移植术后AR发生密切相关。我们成功构建并鉴定了同种异体大鼠肾移植AR模型，并在此基础上，发现BTLA可通过抑制经典信号通路NF-κB, MAPK, NFAT等关键调控介质的表达以抑制T淋巴细胞活化，减弱肾移植术后T细胞为主的AR发生发展。此外，特异性抑制CTLA-4及过表达BTLA在T细胞分化及肾移植术后急性排斥反应发生过程中的作用及机制各异，即BTLA在肾移植术后AR的早期起到抑制作用，通过减少Th1细胞，抑制T细胞介导的排斥反应；而CTLA-4促进Th0细胞向Th2细胞转化，减少浆细胞及抗体的产生，进而显著抑制抗体介导的排斥反应。由此在细胞及动物模型中，贝拉西普（CTLA-4特异性阻断剂）协同过表达BTLA可显著减缓肾移植术后急性排斥反应。本研究的实验结果不仅发现了BTLA在肾移植术后AR中的重要作用，且在肾移植中发现共刺激分子的协同作用，为肾移植术后AR的靶向治疗提供了新的思路。