【作者】 李军；

【导师】 粟永萍；

【作者基本信息】 第三军医大学 ， 军事预防医学， 2007， 博士

【摘要】 研究证实,严重创伤或感染后发生明显的GCR（GR表达或功能下调）、炎症介质（NF-κB、AP-1）功能增强和炎性细胞因子（TNF-α、IL-1β、IL-6等）大量分泌,三者相互影响,互为因果,形成级联式放大效应,引发SIRS。SIRS是创伤或感染后天然免疫反应过度激活所致,是导致继发性全身性损害的主要原因。伴随SIRS的发生发展,由于内源性抗炎症介质的过量释放,可引起机体发生以细胞免疫为主的免疫功能抑制。因此,深入探讨SIRS发生发展及随后免疫抑制的分子机制,具有重要的临床意义。SRC家族作为核受体和其它转录因子的转录辅活化子,由SRC-1、SRC-2和SRC-3组成,以配体依赖模式与核受体相互作用,通过组蛋白乙酰化/甲基化和募集另外的辅因子,显著增强核受体依赖的转录。尽管目前关于SRC蛋白在调节机体生长发育、能量代谢以及部分肿瘤形成等方面的研究已积累了很多资料,但关于它在炎症/免疫反应中的作用,目前知之甚少。我们通过海外合作的方式从美国休斯顿贝勒医学院成功引进SRC-3基因敲除小鼠的亲代小鼠,并进行了繁殖。在成功繁殖并鉴定的基础上,我们分两个部分探讨了SRC-3在炎症和免疫反应中的作用。首先分别从体内、体外两方面探讨了SRC-3在SIRS发生发展中的作用:1)以5mg/kg体重LPS腹腔注射为炎症反应动物模型,观察SRC-3基因敲除对炎症反应早期炎性细胞因子分泌及肝、脾组织中GR、NF-κB、AP-1表达及核转位的影响;2)在腹腔巨噬细胞原代培养的基础上,在10μg/ml培养体系LPS诱导下,从炎性细胞因子的基因转录、炎症介质的活性等方面观察了SRC-3蛋白缺失对腹腔巨噬细胞激活的影响。其次通过细菌负荷实验观察了SRC-3在天然免疫反应中的作用,并在此基础上,进一步通过烧伤和LPS诱导炎症反应模型,探讨SRC-3与机体免疫功能抑制的关系。主要结果:1、在成功繁殖逐渐扩大种群的基础上,通过PCR和Western blot方法分别对子代小鼠进行基因表型和蛋白表型鉴定,成功获得一批SRC-3基因敲除小鼠,为下步实验打下了良好的基础;2、SRC-3^+/+小鼠肝、脾组织均表达SRC-3蛋白,且脾组织表达水平显著高于肝组织,超出约55.8%;3、5mg/kg LPS腹腔注射后,SRC-3^-/-组小鼠一般情况较SRC-3^+/+组小鼠轻,两组肝、脾、肾、心肌及胸腺病理变化未见明显差别;4、LPS腹腔注射后1h、4h两组血清TNF-α、IL-1β、IL-6、IL-10的水平均显著升高,但SRC-3^+/+组、SRC-3^-/-组小鼠相比,血清TNF-α、IL-1β、IL-6水平SRC-3^-/-组显著低于SRC-3^+/+组,IL-10水平SRC-3^-/-组却显著高于SRC-3^+/+组;5、正常情况下,SRC-3^+/+组腹腔巨噬细胞TNF-α、IL-1β、IL-6、IL-10 mRNA与SRC-3^-/-组相比,表达差异无统计学意义;体外LPS刺激后两组表达水平均显著升高,其中SRC-3^-/-组TNF-α、IL-1β、IL-6 mRNA显著低于SRC-3^+/+组,IL-10 mRNA显著高于SRC-3^+/+组;6、在肝组织,无论是SRC-3^+/+组小鼠还是SRC-3^-/-组LPS刺激引起GR蛋白表达及核转位均显著降低,但SRC-3^-/-组下降幅度显著低于SRC-3^+/+组;同样,LPS刺激后SRC-3^-/-组脾组织GR表达水平下降幅度显著低于SRC-3^+/+组,SRC-3^+/+组小鼠GR核转位明显降低,而SRC-3^-/-组无显著变化;7、LPS刺激早期两组小鼠肝、脾组织IκB-α蛋白水平显著降低,以1h降低最明显,其中SRC-3^-/-组小鼠下降幅度显著低于SRC-3^+/+组;两组小鼠肝、脾组织NF-κB p65/p50蛋白表达水平及核转位程度显著增加,SRC-3^+/+组、SRC-3^-/-组相比,SRC-3^-/-组小鼠NF-κB p65/p50蛋白表达上升幅度显著大于SRC-3^+/+组,而核转位程度显著低于SRC-3^+/+组;无论是SRC-3^+/+组还是SRC-3^-/-组小鼠,LPS刺激引起肝、脾组织IRF-1蛋白表达水平显著增加,并且在相应时相点SRC-3^-/-组小鼠均显著低于SRC-3^+/+组;8、SRC-3^+/+组、SRC-3^-/-组小鼠肝、脾组织在LPS刺激后c-Jun/c-Fos蛋白表达及核转位程度均显著增加,1h为峰值,在相应时相点SRC-3^-/-组小鼠均显著低于SRC-3^+/+组;9、LPS刺激后SRC-3^+/+组、SRC-3^-/-组小鼠肝组织SRC-1蛋白表达均显著降低,SRC-3^-/-组小鼠变化程度显著小于SRC-3^+/+组;肝组织SRC-1蛋白核内水平在SRC-3^+/+组小鼠明显降低,而SRC-3^-/-组小鼠却显著增加;两组脾组织均未明确检测到SRC-1蛋白表达;10、LPS刺激后SRC-3^-/-组小鼠SRC-2蛋白表达及核内水平在相应时相点均显著高于SRC-3^+/+组,其中在肝组织,SRC-3^+/+组、SRC-3^-/-组小鼠均显著增加,而在脾组织,SRC-3^+/+组降低,SRC-3^-/-组升高;11、正常情况下,SRC-3^+/+组腹腔巨噬细胞GR蛋白表达水平与SRC-3^-/-组相比无显著差异,LPS刺激4h后两组均显著降低,但SRC-3^-/-组降低程度显著低于SRC-3^+/+组;12、正常情况下,SRC-3^+/+组腹腔巨噬细胞NF-κB p65显著高于SRC-3^-/-组,LPS刺激4h后两组表达水平均显著升高,但SRC-3^-/-组升高幅度远远高于SRC-3^+/+组;13、正常情况下,SRC-3^+/+组腹腔巨噬细胞c-Jun/c-Fos蛋白表达水平与SRC-3^-/-组相比无显著性差异,LPS刺激4h后两组表达水平均显著升高,但SRC-3^-/-组升高幅度远远低于SRC-3^+/+组;14、通过细菌清除实验发现,SRC-3^-/-小鼠血液中活菌量显著多于SRC-3^+/+小鼠;注射后24h、48h SRC-3^-/-小鼠肝、脾及胸腺组织内细菌含量显著多于SRC-3^+/+小鼠,而肾组织中细菌含量均较少,两者相差不显著;15、正常情况下,两组小鼠血浆IL-2、sIL-2R水平没有显著差别;15%-20%Ⅲ°烧伤后24h,无论是SRC-3^+/+组还是SRC-3^-/-组,血浆IL-2、sIL-2R水平均显著升高,其中SRC-3^-/-组小鼠血浆IL-2显著低于SRC-3^+/+组,血浆sIL-2R含量升高幅度却显著大于SRC-3^+/+组;5mg/kg体重LPS刺激24h后,无论是SRC-3^-/-组小鼠还是SRC-3^+/+组,血浆IL-2水平均未见显著变化,而血浆sIL-2R含量却均显著升高,且SRC-3^-/-组小鼠升高幅度显著高于SRC-3^+/+组;16、正常情况下,SRC-3^-/-组小鼠外周血、脾脏和胸腺CD3⁺、CD4⁺及CD4⁺/CD8⁺比值不同程度降低,CD8⁺升高;烧伤或LPS刺激后24h两组小鼠外周血和脾脏CD3⁺、CD4⁺及CD4⁺/CD8⁺比值均不同程度下降,且SRC-3^-/-组CD3⁺、CD4⁺显著低于SRC-3^+/+组,CD4⁺/CD8⁺比值与SRC-3^+/+组相比无显著差别。结论:1、SRC-3与炎性细胞因子的合成释放有关,其蛋白缺失可抑制LPS诱导的TNF-α、IL-1β、IL-6的基因转录及释放,改善炎症反应中机体的整体效应,缓解腹腔巨噬细胞的激活程度;2、SRC-3对GR蛋白表达及核转位有抑制作用,LPS刺激后GR蛋白表达及核转位显著降低,产生明显的GCR,SRC-3蛋白缺失可缓解其程度;3、SRC-3对NF-κB炎症信号转导通路有正性调控作用。SRC-3蛋白缺失可通过抑制炎症反应早期IκB-α水平下调,减少NF-κB核转位,从而导致其基因转录调控功能下降;4、SRC-3参与AP-1炎症信号转导通路的激活,SRC-3蛋白缺失可抑制LPS诱导的AP-1蛋白表达水平及活性,这可能是由于TNF-α、IL-1β等致炎细胞因子合成释放相对不足所致;5、SRC家族成员间存在相互补偿,SRC-1、SRC-2对SRC-3蛋白缺失具有不同程度的补偿效应,但这种补偿效应是有限的;6、SRC-3在维持机体正常的免疫反应中起重要作用。SRC-3缺失可抑制炎性细胞因子的转录和释放,引起对细菌及产物的吞噬及清除功能下降,导致天然免疫功能减弱,同时引起CD3⁺、CD4⁺及CD4⁺/CD8⁺比值显著下降,使细胞免疫功能低下;7、SRC-3可能通过“双向性”作用精细地调节着细胞免疫,其缺失一方面可促进IL-10表达,导致机体免疫功能抑制加重,另一方面可减轻CD3⁺、CD4⁺及CD4⁺/CD8⁺比值的下降程度,也表明SRC-3可能部分参与了SIRS后免疫抑制的发生发展。更多还原

【Abstract】 Previous study have confirmed that severe trauma or infection can cause glucocorticoid resistance （down-regulation of the expression or the function of glucocorticoid receptor）, hyperactivated mediators of inflammation （nuclear factor-kappa B, activator protein-1） as well as hypersecretion of the pro-inflammatory cytokines （TNF-α, IL-1β, IL-6）. This kind of situation will continue and the interaction of the three factors will lead to the amplification of inflammation reaction and the initiation of systemic inflammatory response syndrome （SIRS）. SIRS is the result of hyperactivity of innate immunity and the main cause of subsequent whole body damage, too. The excessive release of endogenous mediators of anti-inflammatory in concomitant with the development of SIRS is an important causative factor of subsequent immunosuppression, mainly of the cell immune. Therefore, investigating the molecular mechanism of the development of SIRS and the subsequent immunosuppression is of theoretical and clinical significance.The family of steroid receptor coactivator （SRC） is composed of SRC-1, SRC-2 and SRC-3. These cofactors interact with nuclear receptors in a ligand-dependent manner and enhance transcriptional activation of the receptor via histone acetylation/methylation and recruitment of additional cofactors. A great deal of information about SRCs has already been accumulated in regulating growth and development, energy metabolism as well as formation of some tumors. But the regulatory effects of SRCs on inflammatory/immune response still remain unclear.The SRC-3 knockout mice were introduced from Baylor College of Medicine （Huston, USA） to our laboratory and colonized. On the bases of successful reproduction and identification, we carried out two parts of experiments to explore the regulatory effects of SRCs on inflammatory/immune response. In the first part of experiments, the function of SRC-3 during the development of SIRS was studied in vivo and in vitro: 1) on the model of an intraperitoneal injection of LPS （5mg/kg body weight）, the effects of SRC-3 knock-out on the secretion of pro-inflammatory cytokines as well as the expression and nuclear translocation of glucocorticoid receptor, nuclear factor-kappa B, activator protein-1 in liver and spleen were observed; 2) after 10μg/ml LPS challenging, the effects of SRC-3 protein depletion on the activation of primary cultured peritoneal macrophages were studied, including the transcription and bioactivity of pro-inflammatory cytokines. In another part of experiment, the role of SRC-3 in innate immunity was studied on bacteria loading model. The relation of SRC-3 and immunosuppression was studied on animal models of burn- and LPS injection-induced inflammatory response.Main results:1. Based on successful reproduction and colonial expanding, we used PCR and Western blot to characterize the off-springs on both gene and protein level. A number of SRC-3 knock-out mice were identified, which provided a good condition for the following experiments.2. SRC-3 was expressed in the hepatic and splenic tissue from SRC-3^+/+ mice, with much higher expression level （net excess 55.8%） in spleen than in liver tissue.3. After an intraperitoneal injection of LPS with a dose of 5mg/kg body weight, general physiological status in SRC-3^-/- group was much easier than that of SRC-3^+/+ group, but no obvious pathological changes were found in liver, spleen, kidney, myocardium and thymus in mice of the two groups.4. After an intraperitoneal injection of LPS, the two groups exhibited a higher expression of serum TNF-α, IL-1β, IL-6 and IL-10 than the normal level. But in contrast to the SRC-3^+/+ group, the serum level of TNF-α, IL-1βand IL-6 in SRC-3^-/- group was significantly lower, however, the IL-10 significantly higher than that of wildtype mice.5. Under normal condition, no statistic differences in the level of TNF-α, IL-1β, IL-6, IL-10 mRNA of peritoneal macrophages were observed between the SRC-3^+/+ group and SRC-3^-/- group. After LPS stimulation in vitro, the levels of above cytokines in both groups were markedly elevated at 4h, while the increased extent of TNF-α, IL-1β, IL-6 mRNA were obviously less but IL-10 mRNA more in SRC-3^-/- group than that of SRC-3^+/+ group.6. The levels of GR expression and its nuclear translocation in liver were significantly reduced in both SRC-3^+/+ group and SRC-3^-/- group after LPS challenging. However, the reduction extent of SRC-3^-/- group is significant less than that of SRC-3^+/+ group. The reduction extent of GR expression in spleen from SRC-3^-/- group is significant less than that from SRC-3^+/+ group. The nuclear translocation of GR in SRC-3^+/+ mice was significantly reduced, but no significant changes were observed in SRC-3^-/- group.7. After LPS stimulation, the IκB-αin liver and spleen was markedly reduced and reached the nadir at 1h in both groups, and the reduction extent of SRC-3^-/- group was obviously lower than that of SRC-3^+/+ group. The levels of NF-κB p65/p50 expression and its nuclear translocation in liver and spleen in both groups was markedly elevated, and the elevated extent of its expression in SRC-3^-/- group was obviously larger compared with that of SRC-3^+/+ group, but the level of its nuclear translocation in SRC-3^-/- group was obviously lower than that of SRC-3^+/+ group. The interferon regulatory factor-1 （IRF-1） expression in liver and spleen in both groups was markedly elevated, and it was obviously less elevation in SRC-3^-/- group than that of SRC-3^+/+ group at matched time points.8. After LPS stimulation, the levels of c-Jun/c-Fos expression and its nuclear translocation in liver and spleen in both groups were markedly elevated and reached the peak at 1h, but the increased levels in SRC-3^-/- group were significantly lower than those in SRC-3^+/+ group.9. After LPS stimulation, the hepatic SRC-1 in both groups were significantly reduced, but the changes in SRC-3^-/- group were less than those in SRC-3^+/+ group. And the level of intranuclear in the liver was significantly reduced in SRC-3^+/+ group, but increased in SRC-3^-/- group. No SRC-1 in spleen of two groups was detectable10. At matched time points, the expression and intranuclear levels of SRC-2 were obviously higher in SRC-3^-/- group than those of SRC-3^+/+ group. They were both significantly increased in the liver of two groups, but in the spleen, they were reduced in SRC-3^+/+ group and increased in SRC-3^-/- group.11. Under normal condition, the expression level of GR on peritoneal macrophage in SRC-3^+/+ group did not differ from that of SRC-3^-/- group. The GR expression on peritoneal macrophage in both groups at 4h after LPS stimulation were markedly reduced, but the reduction extent of SRC-3^-/- group was obviously lower than that of SRC-3^+/+ group.12. Under normal condition, the expression level of peritoneal macrophage NF-κB p65 in SRC-3^+/+ group was obviously higher than that of SRC-3^-/- group. The expression level of peritoneal macrophage NF-κB p65 in both groups at 4h after LPS stimulation were markedly elevated, but the increased extent of SRC-3^-/- group was obviously larger than that of SRC-3^+/+ group.13. Under normal condition, the expression level of peritoneal macrophage c-Jun/c-Fos in SRC-3^+/+ group was not different from that of SRC-3^-/- group. After LPS stimulation, the expression level of peritoneal macrophage c-Jun/c-Fos in both groups were markedly elevated at 4h, but the increased extent of SRC-3^-/- group was obviously lower than that of SRC-3^+/+ group.14. The results acquired through bacteria loading experiment showed that the viable organism content in peripheral blood in SRC-3^-/- group was obviously more than that of SRC-3^+/+ group. And at 24h and 48h after bacteria injection, the bacterial content in liver, spleen as well as thymus in SRC-3^-/- group was markedly more than those of SRC-3^+/+ group, but the bacterial content in nephridial tissue was low and no difference between two groups.15. Under normal condition, no significant differences in the serum level of IL-2 and sIL-2R were observed between two groups. After TBSA 15%-20%Ⅲ°burn injury, the level of serum IL-2 and sIL-2R significantly reduced at 24h in both SRC-3^+/+ group and SRC-3^-/- group, but serum IL-2 was was markedly lower, serum sIL-2R was obviously higher in SRC-3^-/- group than those of SRC-3^+/+ group, respectively. The levels of serum IL-2 after 5mg/kg body weight of LPS intraperitoneal injection in both groups were no significant difference from normal levels, but sIL-2R was markedly elevated. And the increased extent of serum sIL-2R in SRC-3^-/- group was obviously larger than that of SRC-3^+/+ group.16. Under normal condition, the fraction of CD3⁺, CD4⁺ cell of T cell subgroup decreased with distinct extent in peripheral blood, spleen and thymus in SRC-3^-/- group, in concomitant with the increase of the ratio of CD8⁺ cells and the decrease of the ratio of CD4⁺/CD8⁺. After burn injury or LPS injection, the proportion of CD3⁺, CD4⁺ as well as the ratio of CD4⁺/CD8⁺ in peripheral blood, spleen and thymus were significantly reduced at 24h in both SRC-3^+/+ group and SRC-3^-/- group. However, the reduction of CD3⁺, CD4⁺ in SRC-3^-/- group was significant less than that of SRC-3^+/+ group, and the ratio of CD4⁺/CD8⁺ of two groups were no difference.Conclusion:1. SRC-3 is related with the synthesis and release of pro-inflammatory cytokines. The depletion of SRC-3 protein could suppress the gene transcription and release of TNF-α、 IL-1β、IL-6 induced by LPS, improve system effect during inflammatory reaction, relieve the activation of peritoneal macrophage.2. SRC-3 is relavent to the expression and nuclear translocation of GR. The depletion of SRC-3 protein could relieve the LPS-induced response of down-regulation of the expression and nuclear translocation of GR as well as glucocorticoid resistance.3. The expression and activity of NF-κB may be concerned with the SRC-3 protein which involves in regulation of NF-κB signal transduction pathway. The absence of SRC-3 protein could result in suppression of gene transcription activity through reversing the down-regulation of IκB-αand subsequently inhibiting nuclear translocation of NF-κB during the early stage of inflammatory response.4. SRC-3 is concerned with the activation of AP-1 signal transduction pathway. The absence of SRC-3 protein could result in partial inhibition of LPS-induced the expression and activity of AP-1 owing to relative deficient of the synthesis and release of pro-inflammatory cytokines, such as TNF-α、IL-1β.5. There is compensation effect among the members of SRC family. The SRC-1 and SRC-2 could compensate consequence, to certain extent, induced by the absence of SRC-3 protein, but it is limited.6. SRC-3 plays an important role in mantaining normal immune response. The absence of SRC-3 protein could result in the attenuation of innate immunity function including suppressing the LPS-induced transcription activity and release of TNF-α、IL-1β、IL-6 and cutting down the function of phagocytotic and eliminating bacterium as well as its production and the suppression of cellular immune function through anomaly depression of CD3⁺ and CD4⁺ T cells as well as the CD4⁺/CD8⁺ ratio.7. SRC-3 may work as a fine tuner to adjust the cell immune in a“bi-direction”manner. The absence of SRC-3 protein could aggravate immunosuppression through promoting the expression of IL-10 on one side, and on the other side extenuate the degression of CD3⁺ and CD4⁺ T cells as well as the CD4⁺/CD8⁺ ratio, which indicate SRC-3 involving in the occurance of immunosuppression accompanied with SIRS.更多还原