【作者】 钟河江；

【导师】 杨天德；

【作者基本信息】 第三军医大学 ， 麻醉学， 2005， 硕士

【摘要】 糖皮质激素(Glucocorticoids, GC)是临床上常用抗炎、抗免疫反应药物,普遍认为GC 的抗炎效应是通过糖皮质激素受体(Glucocorticoid receptor, GR)调控多种炎性因子的表达。以往的研究表明严重创伤后多种器官组织GR 表达减少,尤其是GR 核转位减少,提示GR 的转录激活能力减弱,但其具体的分子机制尚不清楚。激活的GR也可直接与其它转录因子相互作用,如核因子-?B(nuclear factor ?B, NF-?B)和活化蛋白-1(activator protein-1,AP-1),AP-1 和NF-?B 是调控炎性基因表达的重要转录因子,GR 与转录因子之间的相互作用可能通过它们共同的辅活化因子来介导。c-Jun激活区结合蛋白1(c-Jun activation domain-binding protein l,JAB1)是一种多功能蛋白质,能与多种蛋白质发生相互作用,调控多种细胞内信号转导通路。JAB1 也能增强多种转录因子的活性如核受体、AP-1 和NF-?B。以往的研究表明JAB1 能与GR-LBD在COS-7 细胞中特异性地发生相互作用,因此,JAB1 均能与GR、AP-1 和NF-?B 发生相互作用,提示JAB1 可能在炎症反应的发生发展中起重要作用,但JAB1 对GR 的具体作用目前仍不清楚。本研究的目的是为了进一步明确JAB1 对GR 转录活性及功能的调控作用及JAB1和GR 在创伤后应激反应紊乱中的作用,阐明安定-氯胺酮对严重烧伤早期应激和炎症反应的调控作用及分子机制,为调控严重创伤后早期应激反应紊乱提供新的途径。本研究首先应用JAB1、hGRa 表达质粒与GRE-CAT 报告质粒共转染COS-7 细胞,通过CAT ELISA方法观察JAB1 及LPS 对GR 转录激活活性的影响。其次通过电穿孔方法将JAB1 表达质粒转染小鼠巨噬细胞系RAW264.7细胞,观察JAB1 高表达对GR 表达水平及功能的影响,并同时观察对RAW264.7 细胞分泌致炎细胞因子TNF-a 及IL-6的影响。最后以严重烧伤小鼠为创伤动物实验模型,旨在观察烧伤后早期肝脏、肺及腹腔巨噬细胞中JAB1、GR 的变化特点及相互关系以及安定-氯胺酮对其影响,进一步明确JAB1、GR 在严重创伤早期的相互关系。主要实验结果: 1. 采用脂质体法将pCDNA3.1-JAB1、pCMV-hGRa 和pMAMneo-CAT GRE报告更多还原

【Abstract】 Glucocorticoids (GC) are used in the treatment of inflammatory and autoimmune diseases. It is widely accepted that anti-inflammatory effects of GC are mediated by the intracellular glucocorticoid receptor (GR) that modulate inflammatory gene expression. Previous works have suggested that the expression and function of GR decreased, especially the translocation of GR from cytoplasm to nucleus reduced after severe trauma. These results indicated that the transcriptional activation of GR is decreased, but the molecular mechanism is not clear. The activated GR may also interact directly with other transcription factor, such as activator protein-1 (AP-1) and nuclear factor-?B (NF-?B), which may be important role in the induction of proinflammatory gene expression. Some of the interactions between GR and transcription factors are mediated by interaction with common coactivator molecule. JAB1 is a multi-functional protein, which interacts with and controls multiple intracellar signaling and potentiates the transactivation properties of most receptors. JAB1 potentiates the activity of a variety of transcription factors, such as nuclear receptors, AP-1 and NF-?B. Previous study showed that JAB1 interacts specifically with GR-LBD in COS-7 cells. JAB1 is common coactivator of GR, AP-1 and NF-?B, so it may play an important role in inflammatory development. But the specific effects of JAB1 on GR still remain obscure. The present study was undertaken to explore the effects of JAB1 on transcriptional activation and function of the GR and the role of JAB1 and GR in post-traumatic stress disorder, and investigate the function and molecular mechanism of diazepam-ketamine in stress and inflammatory, and eventually pave a new way for improve the disorder of stress at early stages after severe trauma. In the present study, we firstly used COS-7 cells transiently cotransfected with JAB1 and hGR expression plasmids and GRE-CAT reporter plasmids by DOTAP liposome, using CAT-ELISA technique observed the expression of the CAT reporter gene to examine the effects of JAB1 and LPS on the transcriptional activation of GR. Secondly, we explored the expression level and function of GR when the JAB1 level increased using transient transfection by electroporation technology in the murine macrophage cell line RAW 264.7 cells. The proinflammatory cytokine levels of TNF-a and IL-6 were detected in the supernatants derived from RAW264.7 cells culture by ELISA. Finally, we used burned mouse as the experimental traumatic animal model and investigated the expression change of JAB1 and GR in the liver, lung and peritoneal macrophages of mice at early stages after severe burn or treatment with diazepam (0.4mg/kg) and ketamine (10mg/kg), in order to clarify the relationship between JAB1 and GR during the traumatic stress. Main results are summarized as follows: 1. COS-7 cells were cultured in vitro, different doses of pCDNA3.1-JAB1 containing the full-length human cDNA encoding JAB1 were cotransfected with eukaryotic vector of pCMV-hGRa and pMAMneo-CAT CAT reporter plasmids containing the GRE by using the transfection reagent DOTAP, The expression of CAT was observed by CAT ELISA after the cotransfected COS-7 cells were cultured with dexamethasone for 36 hours. The concentration of CAT was enhenced with the increasing doses of pcDNA3.1-JAB1. Overexpression of JAB1 promoted the transcriptional activation of GR. This result indicated that JAB1 can enhance the transcriptional activation of GR in a dose-dependent manner. 2. The production of CAT in cotransfected COS-7 cells was significantly decreased after stimulation with LPS in the presence of dexamethasone, but partially reversed cotransfected with pcDNA3.1-JAB1. Thus, LPS could inhibit the transcriptional activation of GR. However, JAB1 overexpression could partially alleviate the repressive effects of LPS on transcriptional activation of GR. 3. RAW264.7 macrophages were incubated with LPS (100ng/ml), dexamethasone (100nM) or both agents for 24 hours and the expression of JAB1 and GR measured by RT-PCR and Western bolt assay. Cytokine levels of TNF-a and IL-6 in the supernatant of cultured RAW264.7 cells were determined by ELISA. We found that LPS-stimulated RAW264.7 macrophages were markedly down-regulated the mRNA and protein expression of JAB1. The protein level of JAB1 was significantly reduced by treatment withdexamethasone for 24h, but JAB1 mRNA did not change significantly. Treatment of RAW264.7 cells with 100nM dexamethasone for 24h led to a significant decrease in the expresson of GR protein. Compared to the control group, GR protein level was significantly increased by LPS treatment of RAW264.7 cells for 24h. Furthermore, the expression of GR protein in RAW264.7 cells costimulated with LPS and dexamethasone was significantly higher than that in the control group. The mean production of proinflammatory cytokines TNF-a and IL-6 in the supernatant of cultured RAW264.7 cells stimulated with LPS was significantly increased compared to that in control cells, and remained heightened through 24h. LPS is a potent inducer of macrophage-derived proinflammatory cytokines TNF-a and IL-6. 4. The expression of JAB1 in RAW264.7 cells was obviously increased after the cells were transfected with pFLAG-CMV2-JAB1 by electroporation procedure. Overexpression of JAB1 in transfected RAW264.7 cells significantly increased the expression of GR protein compared to the control group. ELISA analysis revealed that the prinflammatory cytokine levels of TNF-a and IL-6 were significantly decreased in the supernatant of transfected RAW264.7 cells costimulated with LPS and dexamethasone compared to the non-transfected RAW264.7 cells treated with LPS or LPS plus dexamethason. This analysis indicated that overexpression of JAB1 led to repression of LPS-induced proinflammatory cytokines production in RAW264.7 cells. These results demonstrated that JAB1 could enhance the expression and function of GR in the RAW264.7 cells, and plays a critical role in the suppression of LPS-induced proinflammatory cytokines TNF-a and IL-6 production, and the mechanism may be involved in up-regulation of the expression and function of GR protein by JAB1 overexpression. 5. RT-PCR and Western blot analysis revealed the expression levels of JAB1 and GR in the liver, lung and peritoneal macrophages of mice were markedly decreased after severe burn, and then increased at 12h, but were still lower than normal control group at 24h. The change tendency of protein expression of GR was consistent with the protein and mRNA expression change of JAB1 in mice after severe burn. This phenomenon implied the down-regulation of GR associated with low expression of JAB1 during traumatic stress and eventually resulted in the increase of the proinflammatory cytokine production in mice aftersevere burn. Treatment with diazepam-ketamine could inhibit the down-regulation of expression of JAB1 and GR in the liver, lung and peritoneal macrophages of mice at early stage after severe burn. Taken together, we concluded that: 1. In the presence of dexamethasone, JAB1 enhanced the transcriptional activation of GR in a dose-dependent manner. 2. LPS inhibited the transcriptional activation of GR In the presence of dexamethasone. However, JAB1 overexpression could partially relieve the inhibition of GR transcriptional activation by LPS. 3. The mRNA and protein expression levels of JAB1 were markedly down-regulated in LPS-stimulated RAW264.7 macrophages. The protein level of JAB1 was significantly reduced in RAW264.7 macrophages treated with dexamethasone, but JAB1 mRNA did not change significantly. 4. Dexamethasone led to a significant decrease in the expresson of GR protein in RAW264.7 macrophages. However, GR protein level was significantly increased by LPS. 5. The expression and function of GR in RAW264.7 macrophages was markedly increased by JAB1 overexpression. In murine RAW264.7 macrophages, LPS-induced proinflammatory cytokines production was inhibited by Overexpression of JAB1, this data may be indicated the function of GR was improved by overexpression of JAB1 in RAW264.7 macrophages stimulated with LPS. It was also concluded that overexpression of JAB1 played an important role in the anti-inflammatory response. 6. The expressin of mRNA and protein levels of JAB1 decreased in the liver, lung and peritoneal macrophages of mice at early stage after severe burn. Meanwhile, the expression level of GR also was down-regulated. Taked together above results, this phenomenon may be implied that the expression and function changes of GR were at least partially associated with the down-regulation of JAB1 expression after severe burn. 7. After severe burn injury, the expression levels of the JAB1 and GR in the liver, lung and peritoneal macrophages of mice were significantly elevated by diazepam-ketamine. Diazepam-ketamine partially improved the post-traumatic stress disorder induced by severe burn. This mechanism may be involved in the expression up-regulation of JAB1 and GR.更多还原