Mer受体酪氨酸激酶调控脂磷壁酸诱导的炎症反应及在金葡菌吞噬中的作用研究

发布时间：2018-07-03 15:01

本文选题：Toll样受体2 + 脂磷壁酸　；参考：《安徽医科大学》2016年博士论文

【摘要】：TAM受体-Tyro3、Axl和Mer,组成受体酪氨酸激酶(RTK)一个独特的亚家族,为单次跨膜蛋白。TAM受体通过二大基本生物学功能(凋亡细胞的吞噬和先天性炎症免疫反应的抑制)来维护体内免疫内环境稳定。我们在本论文中主要针对Mer受体酪氨酸激酶(Mer TK)在脂磷壁酸(LTA)诱导炎症反应调控和金葡菌吞噬中的作用进行研究。一.Mer TK调控脂磷壁酸(LTA)诱导炎症反应的分子机制研究研究背景LTA和LPS分别代表嵌入在革兰氏阳性菌和革兰氏阴性菌细胞壁的二个主要的PAMP分子。目前,有研究证实Mer TK负性调控LPS诱导的炎症反应。然而,在LTA诱导的炎症反应中,Mer TK是否具有类似的作用及其潜在机制仍有待于阐明。研究目的1.LTA刺激RAW264.7巨噬细胞,研究TLR2促炎信号和Mer TK抗炎信号相关蛋白的表达。2.探讨Mer TK信号在LTA刺激RAW264.7巨噬细胞所诱导炎症反应中的作用及其潜在机制。研究方法1.Western Blot检测LTA刺激巨噬细胞中相关蛋白的表达用LTA在不同时间点(4h、8h、12h、24h)刺激小鼠单核巨噬细胞RAW264.7。采用Western Blot方法检测TLR2信号和Mer TK信号相关蛋白分子的表达水平。2.Gas6中和抗体对LTA诱导Mer TK活化的影响首先,使用特异性Mer抑制抗体预处理RAW264.7巨噬细胞1h后,分别用200 ng/ml LTA刺激RAW264.7细胞12h及400 ng/ml Gas6刺激RAW264.7细胞10 min。其次,使用Gas6中和抗体预处理RAW264.7巨噬细胞1h后,用200 ng/ml LTA刺激RAW264.7细胞12h。最后都通过Western Blot方法检测Mer TK活化情况。3.采用免疫细胞化学和Western Blot方法检测靶向干预Mer TK后Mer TK and P-Mer TK的表达使用特异性Mer抑制抗体或Ig G预处理RAW264.7巨噬细胞1h,再用200 ng/ml LTA刺激RAW264.7细胞12h,然后分别采用免疫细胞化学和Western Blot方法检测Mer TK and P-Mer TK的表达水平。4.靶向干预Mer TK对LTA诱导炎症反应及TLR2和Mer TK信号下游相关蛋白表达的影响首先,使用特异性Mer抑制抗体或Ig G预处理RAW264.7巨噬细胞1h,再用200ng/ml LTA刺激RAW264.7细胞48h,然后采用ELISA方法检测培养上清中促炎症因子TNF-αand IL-6的表达水平。其次,使用特异性Mer抑制抗体预处理RAW264.7巨噬细胞1h,再用200 ng/ml LTA刺激RAW264.7细胞12h,然后采用Western Blot方法检测TLR2信号和Mer TK信号下游相关蛋白分子的表达水平变化。5.靶向抑制PI3K对LTA诱导炎症反应及TLR2信号下游相关蛋白表达的影响首先,使用PI3K特异性抑制剂LY294002预处理RAW264.7巨噬细胞1h,再用200ng/ml LTA刺激RAW264.7细胞48h,然后采用ELISA方法检测培养上清中促炎症因子TNF-αand IL-6的表达水平。其次,使用PI3K特异性抑制剂LY294002预处理RAW264.7巨噬细胞1h,再用200 ng/ml LTA刺激RAW264.7细胞12h,然后采用Western Blot方法检测TLR2信号下游相关蛋白分子的表达水平变化。研究结果1.LTA刺激巨噬细胞能够活化TLR2信号通路和Mer TK信号通路RAW264.7细胞在LTA刺激作用下,TLR2信号通路下游相关蛋白分子My D88、IκB-α、p65及ERK被活化;此外,Mer TK信号通路下游相关蛋白分子Akt和SOCS3也被活化。而且,除了ERK之外它们的活化是呈现时间依赖性方式。2.LTA诱导Mer TK活化是Gas6依赖性的首先,使用特异性Mer抑制抗体预处理能显著降低LTA以及Gas6刺激RAW264.7细胞诱导的Mer TK磷酸化水平。其次,使用Gas6中和抗体预处理能明显抑制LTA诱导的Mer TK的活化。3.Mer TK负性调控LTA诱导的炎症反应使用特异性Mer抑制抗体预处理能明显增加LTA刺激RAW264.7细胞诱导促炎症因子TNF-α和IL-6的水平。而且,使用特异性Mer抑制抗体预处理能明显增强NF-κB的活化(P-IκB-α、P-p65表达水平明显升高)。4.Mer TK通过PI3K/Akt信号通路和SOCS3蛋白参与LTA诱导炎症反应的负调控RAW264.7细胞在LTA刺激作用下,Akt和SOCS3活化是Mer TK依赖性的。同时,使用特异性Mer抑制抗体预处理能明显阻止LTA诱导的Akt和SOCS3活化;而且PI3K/Akt通路和SOCS3蛋白抑制TLR2介导的炎症反应。研究结论1.LTA刺激巨噬细胞诱导了TLR2促炎信号和Mer TK抗炎信号通路的活化。2.Mer TK通过PI3K/Akt通路和SOCS3蛋白来负性调控LTA所诱导的炎症反应。二.Mer TK在巨噬细胞吞噬金葡菌中的作用研究研究背景目前,研究证明表达于巨噬细胞表面的许多受体同时参与凋亡细胞和细菌的识别与吞噬,包括SR-A、CD14、整合素、补体受体和Fc受体等。此外,表达于巨噬细胞表面的Mer TK对于凋亡细胞的迅速吞噬是必不可少的。因此,本研究我们探讨Mer TK受体是否也参与对细菌的吞噬。研究目的1.金葡菌刺激RAW264.7巨噬细胞,检测吞噬信号相关蛋白的表达。2.研究Mer TK在RAW264.7巨噬细胞吞噬金葡菌中的作用。研究方法1.Western Blot检测金葡菌刺激巨噬细胞中相关蛋白的表达用金葡菌在不同时间(20min、40min、60min、120min)刺激RAW264.7巨噬细胞。然后,采用Western Blot方法检测在金葡菌刺激RAW264.7巨噬细胞过程中相关蛋白分子的表达水平。2.分别从蛋白分子水平和形态学方面研究Mer TK对巨噬细胞吞噬金葡菌的影响我们从蛋白分子水平检测Mer TK对RAW264.7巨噬细胞吞噬相关蛋白分子的影响。使用特异性Mer抑制抗体预处理RAW264.7巨噬细胞1h,再用金葡菌刺激RAW264.7细胞1h后。首先,通过Western Blot方法检测吞噬相关蛋白P-FAK及GTP-Rac1的表达水平。其次,通过吞噬试验对RAW264.7细胞的吞噬金葡菌的能力进行评估。研究结果1.金葡菌刺激RAW264.7细胞能够引起其表面多种受体分子的活化金葡菌刺激RAW264.7细胞能够引起其表面多种受体分子的活化,包括TLR2信号通路(My D88、p65、JNK、ERK、p38)、SR-A及Mer TK,并且它们的活化呈时间依赖性方式。2.金葡菌刺激RAW264.7细胞能够诱导吞噬相关蛋白FAK及Rac1的活化同时,金葡菌刺激RAW264.7细胞能够引起FAK磷酸化水平明显的增加以及体现Rac1活化的GTP-Rac1表达水平的增高,并且也呈时间依赖性方式。3.靶向干预Mer TK对金葡菌诱导FAK及Rac1的活化无明显影响使用特异性Mer抑制抗体预处理RAW264.7巨噬细胞1h,再用金葡菌刺激RAW264.7细胞1h,之后我们从蛋白分子水平检测了这种特异性Mer抑制抗体对吞噬信号通路相关蛋白P-FAK及GTP-Rac1的影响。我们发现,这种抗体对金葡菌刺激RAW264.7细胞活化的P-FAK和GTP-Rac1的表达水平无明显影响。4.Mer TK对巨噬细胞吞噬金葡菌的能力无显著影响为了进一步证实Mer TK在巨噬细胞吞噬金葡菌中的作用,我们通过吞噬试验对RAW264.7巨噬细胞的吞噬金葡菌的能力进行评估。我们发现,给予特异性Mer抑制抗体预处理后,RAW264.7细胞吞噬金葡菌的能力无显著性变化。研究结论1.金葡菌刺激巨噬细胞诱导吞噬信号及Mer TK的活化。2.然而,Mer TK对巨噬细胞吞噬金葡菌不是重要的。
[Abstract]:TAM receptor -Tyro3, Axl and Mer, which constitute a unique subfamily of receptor tyrosine kinase (RTK), maintain the internal immune stability in the body by two basic biological functions (the phagocytosis of apoptotic cells and innate inflammatory immune response) for the single transmembrane protein.TAM receptor. In this paper, we mainly focus on the Mer receptor tyrosine. The role of kinase (Mer TK) in the regulation of inflammatory response induced by lipophosphoric acid (LTA) and the role of Staphylococcus aureus phagocytosis. A molecular mechanism for the regulation of lipophosphoric acid (LTA) induced inflammatory response by.Mer TK; background LTA and LPS represent two major PAMP molecules embedded in gram positive bacteria and gram negative bacteria cell walls respectively. Studies have confirmed that Mer TK negatively regulates the inflammatory response induced by LPS. However, in the case of LTA induced inflammatory reactions, whether Mer TK has a similar role and its potential mechanism remains to be clarified. The purpose of the study is to stimulate RAW264.7 macrophages and to study the expression of the TLR2 proinflammatory signal and Mer TK anti-inflammatory signal related proteins. The role of TA to stimulate the inflammatory response induced by RAW264.7 macrophages. Methods 1.Western Blot was used to detect the expression of related proteins in macrophages stimulated by LTA and LTA at different time points (4h, 8h, 12h, 24h) stimulated the mononuclear macrophages in mice. The effect of.2.Gas6 neutralization antibody on the activation of Mer TK induced by LTA first. After using specific Mer to pretreat RAW264.7 macrophage 1H, 200 ng/ml LTA stimulates RAW264.7 cell 12h and 400 ng/ml to stimulate 10. Then, the RAW264.7 cell 12h. was stimulated by 200 ng/ml LTA and the activation of Mer TK was detected by Western Blot method. AW264.7 cells 12h, and then using immunocytochemistry and Western Blot method to detect the expression level of Mer TK and P-Mer TK.4. target intervention Mer TK to induce inflammatory response and the expression of downstream related proteins. Ng/ml LTA stimulates RAW264.7 cell 48h, and then uses ELISA method to detect the expression level of TNF- alpha and IL-6 in culture supernatant. Secondly, using specific Mer inhibition antibody preconditioning RAW264.7 macrophage 1H, then 200 ng/ml stimuli are used to stimulate the cells. Changes in the expression level of the associated protein molecules.5. targeting inhibition of PI3K on LTA induced inflammatory response and the expression of downstream related proteins in TLR2 signals first, PI3K specific inhibitor LY294002 pretreated RAW264.7 macrophage 1H, and then 200ng/ml LTA stimulated RAW264.7 cell 48h, and then used to detect proinflammatory in culture supernatant. The expression level of TNF- alpha and IL-6. Second, RAW264.7 macrophage 1H was pretreated with PI3K specific inhibitor LY294002, and RAW264.7 cell 12h was stimulated with 200 ng/ml LTA. 2 signal pathway and Mer TK signaling pathway RAW264.7 cells are activated by LTA, the downstream related protein molecules of the TLR2 signaling pathway, My D88, I kappa B- alpha, p65 and ERK are activated. Besides, the downstream related protein molecules of the Mer signaling pathway are also activated. Besides, the activation of the downstream related proteins in the Mer signaling pathway is also activated in a time dependent manner. TK activation is the first of Gas6 dependence. The use of specific Mer inhibition antibody preconditioning can significantly reduce the level of Mer TK phosphorylation induced by LTA and Gas6 stimulation of RAW264.7 cells. Secondly, the use of Gas6 neutralization antibody preconditioning can significantly inhibit the activation of Mer TK induced by LTA. Antibody preconditioning can significantly increase the level of LTA stimulated RAW264.7 cells to induce TNF- alpha and IL-6. Moreover, the activation of NF- kappa B can be significantly enhanced by specific Mer inhibition antibody preconditioning (P-I kappa B- alpha, P-p65 expression level is obviously elevated).4.Mer TK through the signaling pathway and the negative protein to induce the negative inflammatory reaction Under the action of LTA stimulation, the activation of Akt and SOCS3 is Mer TK dependent. At the same time, the activation of Akt and SOCS3 induced by LTA can be prevented by the use of specific Mer inhibition antibody preconditioning. Moreover, PI3K/Akt pathway and SOCS3 protein inhibit the inflammatory response mediated by LTA. The activation.2.Mer TK of the anti inflammatory signaling pathway of Mer TK through the PI3K/Akt pathway and SOCS3 protein to negatively regulate the inflammatory response induced by LTA. Two.Mer TK in macrophages phagocytic Staphylococcus aureus? Research background is present. Studies have shown that many receptors expressed on the surface of macrophages are involved in the identification of apoptotic cells and bacteria. Phagocytosis, including SR-A, CD14, integrin, complement receptor and Fc receptor. In addition, the rapid phagocytosis of Mer TK expressed on the surface of macrophages is essential. Therefore, we investigate whether the Mer TK receptor is also involved in the phagocytosis of the bacteria. Objective 1. Staphylococcus aureus to stimulate RAW264.7 macrophages and to detect the phagocytic signal The expression of related protein.2. in the study of the role of Mer TK in phagocytosis of Staphylococcus aureus in RAW264.7 macrophages. Methods 1.Western Blot was used to detect the expression of associated proteins in macrophages by Staphylococcus aureus using Staphylococcus aureus at different time (20min, 40min, 60min, 120min) to stimulate RAW264.7 giant macrophages. Then, the Staphylococcus aureus was detected by Western methodology. The expression level of related protein molecules in the process of stimulating RAW264.7 macrophages.2. respectively study the effect of Mer TK on macrophage phagocytic Staphylococcus from protein molecular level and morphology. We detected the effect of Mer TK on the phagocytic protein molecules of RAW264.7 macrophages from the protein molecular level. The specific Mer inhibition antibody was used. RAW264.7 macrophage 1H, then Staphylococcus aureus was used to stimulate RAW264.7 cell 1H. First, the Western Blot method was used to detect the expression level of the phagocytic protein P-FAK and GTP-Rac1. Secondly, the phagocytic ability of RAW264.7 cells phagocytic Staphylococcus was evaluated by phagocytic test. Results 1. Staphylococcus aureus stimulates RAW264.7 cells can cause its table. The activation of RAW264.7 cells by activated Staphylococcus aureus from various receptor molecules can cause activation of a variety of receptor molecules on the surface, including the TLR2 signaling pathway (My D88, p65, JNK, ERK, p38), SR-A and Mer TK, and their activation in a time-dependent manner can induce the activation and activation of the phagocytic associated protein and the activation of the cells. At the time, Staphylococcus aureus stimulated RAW264.7 cells to increase the level of FAK phosphorylation and increase the level of GTP-Rac1 expression of Rac1 activation, and also in a time dependent manner,.3. targeting Mer TK had no significant effect on the activation of FAK and Rac1 induced by Staphylococcus aureus by Mer TK, using specific Mer inhibition antibody preprocessing RAW264.7 macrophage 1 H, then using Staphylococcus aureus to stimulate the RAW264.7 cell 1H, then we detected the effect of this specific Mer inhibitory antibody on the phagocytic signaling pathway related protein P-FAK and GTP-Rac1 from the protein molecular level. We found that this antibody has no significant effect on the P-FAK and GTP-Rac1 expression level of the Staphylococcus aureus stimulated RAW264.7 cell activation of.4.Mer TK to the giant. The ability of phagocytic phagocytosis of Staphylococcus aureus has no significant effect to further confirm the role of Mer TK in macrophage phagocytosis of Staphylococcus aureus. We assessed the ability of RAW264.7 macrophages to phagocytate Staphylococcus aureus by phagocytosis test. We found that RAW264.7 cells phagocytosis of Staphylococcus aureus after specific Mer suppression antibody preconditioning No significant changes. Conclusion 1. Staphylococcus aureus stimulated macrophages to induce phagocytic signal and Mer TK activation.2., however, Mer TK was not important for macrophage phagocytosis of Staphylococcus aureus.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392

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