PD-1/PD-L1/miR-21通路在创伤后Th17/Treg失衡和免疫抑制形成中作用及机制研究

Surgical trauma contributes to the immune suppression, and its formation mechanism has not been fully elucidated. PD-1/PD-L1, as the negative costimulatory signals, is involved in regulating immune response. Our previous experiment showed that PD-1/PD-L1 in the peripheral blood were abnormal after trauma. Recent reports in the literature, targeted regulation of micro-RNA-21 can damage the PD-1/PD-L1 pathway, then can cause the imbalance of Th17/Treg, and this imbalance can cause immune suppression, so we speculated that PD-1/PD-L1 can participate in the immune suppression after trauma by regulating the balance of Th17/Treg. Aims of this study were to investigate: (1) the expression of PD-1/PD-L1 and Th17/Treg cells in the peripheral blood of traumatic patients, and (2) in vitro interferent the expression of PD-1/PD-L1 can regulate the imbalance of Th17/Treg cells and improve the immune function, and (3) to explore the molecular mechanism of Th17/Treg imbalance, and (4) in the animal experimental to study “silence of PD-1/PD-L1 can reverse the imbalance of Th17/Treg cells and improve immune function after trauma, so as to provide a new strategy for treating the immune dysfunction after trauma.

创伤后机体出现免疫抑制，其发病机制未完全阐明。PD-1/PD-L1作为新发现免疫细胞表面负性共刺激通路参与机体免疫调控。我们前期研究发现，创伤后淋巴细胞表面PD-1/PD-L1表达异常，且与Th17/Treg比例呈负相关；新近又发现miR-21参与PD-1/PD-L1介导Th/Treg失衡，而这一失衡是免疫抑制重要原因，因此我们推测PD-1/PD-L1通过调控miR-21介导Th17/Treg失衡，从而导致创伤后免疫抑制。为此本课题拟进行以下研究：（1）明确创伤后存在PD-1/PD-L1表达异常及Th17/Treg失衡；（2）体外观察过表达/沉默PD-L1对Th17/Treg平衡和免疫功能影响；（3）探讨miR-21参与PD-1/PD-L1调控Th17/Treg平衡分子机制；（4）在体观察阻断PD-1/PD-L1能否纠正创伤后Th17/Treg失衡和免疫抑制，为创伤免疫失衡防治提供新策略。

手术创伤后机体出现免疫抑制是影响临床预后及转归的重要因素和病理生理基础。辅助性T淋巴细胞亚群（Th）在免疫应答的全面调节中起关键性作用。Th细胞在某些信号诱导下可转化为Th17和抑制性Th细胞——调节性T细胞（Treg），Treg通过多种途径抑制免疫效应。程序性死亡因子-1/程序性死亡因子配体-1（PD-1/PD-L1）能够提供负性信号参与免疫抑制。MicroRNA (miR) 分析PD-1基因敲除型小鼠CD4+ T细胞表达miR-21较野生型小鼠明显增加，PD-1是miR-21表达的负性调节因子。本研究检测了手术创伤后Th17/Treg细胞数目，PD-1/PD-L1和miRNA-21的表达，此外，用搭载有Ad-sh-PD1的腺病毒转染从手术创伤患者术后三天的外周血单个核细胞（PBMCs）中分离的CD4+ T细胞，敲降PD-1的表达，再用FCM检测Th17和Treg亚群百分比，用RT-PCR检测RORγt和Foxp3以及miRNA-21的表达，用Western blot检测 PD-1和PD-L1蛋白的表达。用搭载有pre-miRNA-21的腺病毒转染从手术创伤患者术后三天的PBMCs中分离的CD4+ T细胞，过表达miRNA-21后，同样进行上述的检测。探究了PD-1/PD-L1是否通过miRNA-21介导Th17/Treg分化。结果发现手术创伤后Th17细胞百分比减少而Treg细胞百分比增加（P < 0.01），并伴有PD-1（P < 0.05）和PD-L1的表达增加（P < 0.01）。此外，手术创伤后IL-17（P < 0.05）表达降低，而TGF-β1（P < 0.01）表达增加。体外实验表明沉默PD-1促进CD4+ T细胞中miR-21表达（P < 0.05），同时也促进Th17细胞分化（P < 0.01），抑制Treg细胞分化（P < 0.01）。通过pre-miR-21过表达miR-21促进Th17细胞分化（P < 0.01），阻碍Treg细胞分化（P < 0.05）。综上，创伤后存在PD-1/PD-L1表达上调和Th17/Treg失衡，PD-1/PD-L1通路介导Th17/Treg失衡部分依赖于miR-21。