Cripto-1调节巨噬细胞极化对改善异体输血后免疫抑制的机制

Macrophage polarization affects immune function, which is closely related to immune suppression after allogeneic blood transfusion.Our previous study indicated that transfusion of allogeneic blood could down-regulate the expression of mir-449α and the phosphorylation level of PI3K、Akt protein. It was reported that Cripto-1 can lead to macrophage polarization,microRNA-449a bind to Cripto-1 ,respectively. Based on the relationship of miR-449α and Cripto-1, we speculated that miR-449α may regulate Cripto-1 via PI3k/Akt/NF-κB signal pathway to regulate the body's immune response. In this study, we plan to over-express and knock out Cripto-1 gene in the liver of mice using adeno-associated virus,to analyze the fuction of Cripto-1 in immunosuppression after blood transfusion, to observe both in vitro and in vivo how it regulates Macrophage polarization after ransfusion of allogeneic blood via PI3k/Akt/NF-κB signal pathway. Then, the blocking or over-expressing genes in the pathway, using related molecular biology methods will also be applied to study the molecular regulating mechanism of Cripto-1 via the PI3k/Akt/NF-κB signal pathway . This study will elucidate the role and the molecular regulating mechanism of Cripto-1 in immunosuppression after allogeneic blood transfusion , a novel targeting site may therefore be provided for clinical treatment of the disease.

巨噬细胞极化影响机体免疫功能，异体血输注后的免疫抑制与之密切相关。项目组前期研究发现，输注异体血后miR-449α表达降低，同时下调了PI3K、Akt蛋白的磷酸化。根据生物信息学及文献发现，Cripto-1可导致巨噬细胞极化，且miR-449α可与Cripto-1特异性结合，我们推测miR-449α可能调控Cripto-1介导PI3k/Akt/NF-κB信号通路来影响机体的免疫应答。因此本项目拟通过构建Cripto-1相关病毒过表达和基因敲除小鼠模型，分析Cripto-1在输异体血后免疫抑制中的作用；体内外实验研究该基因在输注异体血后对PI3k/Akt/NF-κB信号通路的影响及其对巨噬细胞极性的调控；利用阻断或激活基因表达等相关分子生物学方法分析Cripto-1调节该信号通路的分子机制。由此阐明Cripto-1在异体输血后免疫抑制的作用及机制，为输异体血后免疫抑制治疗提供可能

异体输血作为临床中重要的治疗手段，潜在风险不能忽视，尤其是输血相关的免疫抑制问题是妨碍患者康复的重要因素。巨噬细胞执行固有免疫功能，且在不同微环境下的不同极化表型与机体免疫功能密切相关，目前关于巨噬细胞极化与输血后免疫抑制相关性问题尚未见报道。Cripto-1在免疫应答中对于巨噬细胞的分泌、吞噬等功能具有调控作用。为此，本研究首先通过体外实验，采用了ELISA、RT-PCR、Western blot方法观察到Cripto-1对相关炎症因子的分泌和巨噬细胞极化为M1型的作用，采用PI3K/AKT/NF-κB信号通路抑制剂进行对比观察，证实了此信号通路参与其中，进一步深入地研究发现miR-449a 对Cripto-1促进巨噬细胞极化有负向调控作用。最后通过构建异体输血小鼠模型，在体内证实了体外研究结果。综上所述，Cripto-1可能是未来防治输血后免疫抑制的新策略。