【作者】 吴迪；

【导师】 曹聪；

【作者基本信息】 苏州大学 ， 神经生物学， 2020， 硕士

【摘要】 研究背景和目的:人体免疫系统是一个非常复杂的装置,它不仅保护我们身体免受微生物等外部侵害,而且还保护自身受损或功能失常的组织,其中第一道保护屏障之一是炎症。炎症是先天免疫的主要机制,并在许多过程中受到调节。这种调节步骤之一是细胞因子网络,其中肿瘤坏死因子α(TNF-α)是复杂细胞因子网络的核心角色,发挥着关键的作用。TNF-α生物活性的改变参与调控自身免疫和癌症等多种疾病的发展进程,调节细胞的生长、分化、死亡等。G蛋白是膜内能与鸟苷二磷酸结合,具有GTP水解酶活性的一类信号传导蛋白,在细胞内信号传导途径中发挥重要的作用。近年来研究发现Gαi蛋白通过激活相关信号通路参与介导多种炎症的发生发展,提示Gαi蛋白可能是炎症调控的潜在靶点。在本论文中我们拟在MEFs细胞中探究Gαi蛋白参与介导TNF-α信号转导的相关分子机制,并且通过THP-1和BMDM细胞模型进一步明确Gαi1/3在炎症中的作用,为某些疾病的炎症反应治疗寻找新的靶点。研究方法:通过培养 WT,Gαi1/3 DKO,Gαi1 KO,Gαi3 KO,Gαi2 KO 的 MEFs细胞以及Gab1 KO的MEFs细胞,培育并在MEFs利用shRNA质粒载体靶向敲减Gαi1和/或Gαi3建立稳转细胞株;再转染CRISPR/Case9-KO病毒敲除Gαi1和Gαi3;再在此细胞株中借助于脂质体转染导入外源性Gαi1和Gαi3,获得OE-Gαi1/3 MEFs稳转细胞株;在DKO MEFs中通过脂质体转染重新导入外源性Gαi1和Gαi3,创建不同表达的稳转细胞株,获得Ad-Gαi1 MEFs和Ad-Gαi3 MEFs。对上述细胞株施用TNF-α处理,采用Western blotting(WB)法检测细胞内相关蛋白表达量(Akt-mTORC1、MAPK和NF-κB信号通路)以及下游接头蛋白Gab1的表达情况;运用shRNA干扰法在人单核巨噬细胞(THP-1)中靶向敲减Gαi1和Gαi3,检测Gαi1/3-shRNA THP-1细胞中炎症因子的表达;运用shRNA干扰法在小鼠原代骨髓巨噬细胞(BMDM)中靶向敲减Gαi1和Gαi3,检测Gαi1/3-shRNA BMDM细胞中炎症因子的表达和Akt-mTORC1、MAPK信号通路的活化水平。研究结果:通过WB检测我们发现,在MEFs中,Gαi1/3双敲(DKO)阻断了TNF-α诱导的AK,T-mTOR和MAPK信号通路的活化;此外,在TNF-α处理下,Gαi1/3-shRNA、Gαi1-KO、Gαi3-KO MEFs 下游信号水平均显著降低,而 Gαi2-KO MEFs中无明显变化。同时,TNF-α处理后,Gαi1/3-DKO MEFs中重新表达Gαi1(Ad-Gαi1)和Gαi3(Ad-Gαi3)部分拯救了 TNF-α的下游信号转导,并且OE-Gαi1/3 MEFs中TNF-α下游信号活化显著增强。此外,Gab1 KO MEFs在药物处理后Akt-mTORC1和MAPK通路相关蛋白活化水平显著下降。进一步研究表明,在人单核巨噬细胞THP-1中,Gαi1/3敲减显著降低了炎症因子的表达;并且在小鼠原代骨髓巨噬细胞中,shRNA敲减Gαi1/3降低了 Akt-mTORC1/MAPK信号通路的活化和炎症因子的表达。结论:上述研究结果表明Gαi1/3参与介导TNF-α诱导的下游Akt-mTOR/MAPK信号转导。同时,Gαi1/3参与调控TNF-α诱导的巨噬细胞的炎症反应。本研究丰富了对TNF-α介导的炎症反应活性调控及机制的新认识,由于TNF-α在炎症中发挥重要作用,Gαi1/3可能成为炎症反应临床诊断和治疗的新型靶点。更多还原

【Abstract】 Objective:Human immune system is an extreme complex network,which not only protects our bodies from external attacks by microbes,but also protects internal damaged or dysfunctional tissues.In the network,the inflammatory response is one of the first protective barriers.Inflammation is the primary mechanism of innate immunity and is modulated in many processes,such as cytokine networks.Importantly,tumor necrosis factor α(TNF-α)plays a key role in the cytokine networks.TNF-α can regulate the progression of various diseases such as cancer and autoimmune diseases,as well as the growth,differentiation and death of cells.G protein is a kind of signal transduction protein that has the activity of GTP hydrolase and can bind with GDP,which plays a vital role in the intracellular signal transduction pathway.Recently,it has been found that Gai proteins are required for mediating the initiation and progression of various inflammation by activating related signaling pathways,suggesting that Gαi proteins may be a potential target for inflammation modulation.In this study,we explored the relevant molecular mechanisms of Gαi proteins involved in mediating TNF-α signaling in MEFs cells,and further clarify the role of Gαil/3 in inflammation through the model of THP-1 and BMDM cells,in order to find new targets for the treatment of inflammation in some diseases.Methods:We cultured WT,Gail/3 DKO,Gαil KO,Gαi3 KO,Gai2 KO MEFs and Gab1 KO MEFs.The shRNA strategy was used to knockdown Gail and/or Gαi3 in WT MEFs and stable cell lines are established.CRISPR/Cas9 strategy was used to knockout Gail and Gai3 in WT MEFs.Gail/3 adenovirus constructs were used to re-express the Gail and/or Gai3 in DKO MEFs and increase the expression of Gαil/3 in WT MEFs.Western blotting analysis detected the expression of major proteins(Akt-mTORC1、MAPK and NF-κB pathways)and Gab1 protein in MEFs.The shRNA strategy was used to knockdown Gail and Gαi3 in THP-1 and BMDM cells,in order to establish the stable Gαil/3-shRNA THP-1 and Gαil/3-shRNA BMDM cells.We detected the expression of inflammatory factors in Gαil/3-shRNA THP-1 and Gαil/3-shRNA BMDM cells.The activation levels of Akt-mTORC1 and MAPK pathways were detected in Gαi1/3-shRNA BMDM cells.Results:Western Blot analysis showed that in MEFs,Gαil/3 double knock(DKO)blocked TNF-α-induced activation of AKT-mTOR and MAPK signaling pathways.In addition,the activation levels of AKT-mTOR and MAPK signaling pathways were largely decreased in Gαil/3-shRNA、Gαil-KO、Gαi3-KO MEFs,but not significantly in Gαi2-KO MEFs.However,re-expression of Gail or Gai3 partially rescued TNF-α-induced Akt-mTORC1 and MAPK activation in DKO MEFs;and overexpression of Gail/3 significantly increased the activation levels of AKT-mTOR and MAPK pathways.The activation levels of TNF-α-induced AKT-mTOR and MAPK signaling pathways were also significantly decreased in Gab1-KO MEFs.Further studies showed that knockdown of Gαil/3 significantly suppressed the expression of inflammatory factors in THP-1 and BMDM cells.The activation levels of AKT-mTOR and MAPK pathways were largely decreased in Gail/3-shRNA BMDM cells.Conclusion:Gail/3 is critical for the activation of AKT-mTORC1 and MAPK pathways induced by TNF-α.Meanwhile,Gail/3 is required for the regulation of TNF-α-induced macrophage inflammation.This study enriches the understanding of the mechanism of TNF-α-mediated inflammatory response.In view of the important role of TNF-α in inflammation,Gail/3 may become a new target for the treatment of inflammatory diseases.更多还原