【作者】 董丽华；

【导师】 王育珊；

【作者基本信息】 吉林大学 ， 内科学， 2016， 博士

【摘要】 目的:脓毒症是由感染引起的全身炎症反应综合,是严重危害人类健康的主要疾病之一。其发病率以每年8.7%的速度逐年增高,但死亡率却没有下降。严重脓毒症和脓毒性休克死亡人数高于急性心肌梗死的死亡人数。脓毒症的发病机制为早期过度的炎症反应及晚期淋巴细胞的过度凋亡。脓毒症时淋巴细胞凋亡的主要机制为激活诱导的细胞死亡(activation induced cell death,AICD)。糖皮质激素早在20世纪40年代就被用于严重性全身感染的治疗,但由于其除了抗炎作用外,尚有免疫抑制作用,至今对其能否提高脓毒性休克患者的生存率、改善预后尚存在争议。糖皮质激素治疗脓毒症引发争议的原因在于糖皮质激素作用的多样性,尤其对于淋巴细胞的活性有双向调节作用:一方面糖皮质激素通过内源性线粒体凋亡途径诱导未成熟的胸腺细胞、成熟的脾T淋巴细胞、前B淋巴瘤细胞及白血病细胞凋亡;另一方面可以通过糖皮质激素受体(Glucocorticoid Receptor,GR),以DNA结合的方式下调Fas L基因的表达,从而抑制AICD。由于糖皮质激素对淋巴细胞的双重作用,使其无法成为脓毒症治疗的理想药物。与GR密切相关的盐皮质激素受体(mineralocorticoid receptors,MR)也在T细胞上高度表达,而在其他具有吞噬作用的白细胞上表达很低,这提示MR可能调节T细胞的功能,而不影响免疫系统的清除细菌的功能。目前临床应用糖皮质激素治疗脓毒性休克的作用机制普遍认为是通过GR介导的。由于GR的过于广泛的基因调节作用,使糖皮质激素治疗有相当多的副作用。MR激动剂与GR激动剂相似,通过糖皮质激素反应元件(glucocorticoid response element,GRE)和负性糖皮质激素反应元件(negative glucocorticoid response element,n GRE)启动子结合位点来发挥其作用,但目前发现的MR作用的靶基因较少。MR对淋巴细胞凋亡相关基因的作用尚无报道。本研究中拟检测两种肾上腺皮质激素受体对于激活诱导原代培养的人外周血T细胞凋亡的调节作用。选择地塞米松(dexamethasone,DEX),一种选择性的GR受体激动剂,和去氧皮质酮(deoxycorticosterone,DOC),一种选择性的MR受体激动剂作为干预药物。(1)探索GR和MR受体激动剂对于有丝分裂原-植物血凝素(phytohemagglutinin,PHA)或者金黄色葡萄球菌超抗原(Staphylococcus aureus enterotoxin B,SEB)诱导的人原代培养的淋巴细胞凋亡的作用;(2)探索DEX和DOC对于外源性凋亡途径的激活(caspase-8)和相关的信号分子IL-2和Fas L的作用;(3)探索GR和MR在基因的诱导、转录抑制和直接DNA结合方面的作用;(4)最后超表达野生型和突变型MR(转录抑制功能逆转为转录激活功能)来验证转录抑制是一种可能的机制,以及DOC的作用的MR依赖性。旨在为盐皮质激素治疗脓毒症提供理论依据,为提高脓毒症的治疗提供有益的线索。方法:从健康志愿者外周血分离淋巴细胞,体外培养2周后用PHA或SEB诱导其凋亡。采用流式记录法检测细胞凋亡及荧光检测法测试细胞caspase-8,caspase-9/6,caspase-3活性,进一步检测DEX和DOC作用下细胞凋亡及上述caspases活性的变化。在PHA与12-豆蔻酸-13-乙酸佛波醇(phorbol 12-myristate 13-acetate,PMA)刺激的Jurkat细胞检测IL-2及IL-2 m RNA和Fas L m RNA水平及DEX与DOC作用下IL-2及IL-2 m RNA和Fas L m RNA水平。为检测DEX和DOC的作用是否为转录水平,引入蛋白合成抑制剂环己酰亚胺(cytoheximide,CHX),检测CHX存在下,DEX和DOC对IL-2 m RNA和Fas m RNA的作用。将Fas L报告基因质粒、NF-κB报告基因质粒、及NF-κB位点突变的Fas L报告基因质粒分别转入Jurkat细胞,检测荧光素酶活性及DEX和DOC作用后对其活性的影响。为进一步探索GR和MR对Fas L基因的作用及其作用机制,采用凝胶电泳迁移实验(EMSA)检测DEX和DOC对于含有NF-κB/AP-1序列的IL-2启动子探针及含有NF-κB/GRE序列的Fas L启动子探针与核蛋白复合体的结合作用的影响。最后为检测DOC的作用的MR依赖性,将NF-κB或Fas L报告基因质粒及野生型MR质粒或显性负突变的MR基因质粒共转染Jurkat细胞,检测Fas L的表达及DEX及DOC对Fas L转录和表达的影响。结果:PHA或SEB均可以诱导原代培养的人外周血T细胞凋亡及caspase-8、caspase-9/6、caspase-3活性升高。DEX或DOC均可减低细胞凋亡百分率及caspase-8、caspase-9/6、caspase-3活性。PHA可以诱导人外周血T细胞产生大量的IL-2及IL-2、Fas L基因的表达,PHA与PMA可以诱导Jurkat细胞产生大量的IL-2,及IL-2、Fas L基因的表达,而DEX及DOC均抑制上述PHA或PHA与PMA诱导的IL-2的升高,及IL-2 m RNA和Fas L m RNA的水平的升高。DEX和DOC均可抑制野生型Fas L及NFκB报告基因表达,但对于NF-κB位点突变的Fas L报告基因表达无作用。无论是DEX,还是DOC都没有改变IL-2启动子探针与NF-κB核蛋白复合体的结合。DEX和DOC也均没有改变Fas L启动子探针与NF-κB核蛋白复合体的结合。在转染了显性负突变MR基因的Jurkat细胞中,DOC对NF-κ报告基因的活性无作用,对Fas L报告基因的活性的抑制作用也消失。而DEX对Fas L报告基因及Fas L m RNA水平的影响也部分被抑制。结论:PHA和SEB均可以诱导的人原代培养的淋巴细胞激活诱导细胞凋亡作用。DEX和DOC通过抑制IL-2与Fas L的表达及IL-2的生成阻断外源性凋亡路径,保护激活诱导的T细胞凋亡。GR与MR均可通过抑制NF-κB而下调Fas L转录,但对抗炎基因的作用不同,提示有着不同的作用机制。DOC抑制NF-κB信号路径和Fas L的产生是经典的核受体作用途径,通过与MR形成配体-受体复合物发挥功能的。DOC的作用是MR依赖性的。MR配体可能成为阻断脓毒症和其他免疫异常疾病的副作用较小的理想治疗药物。更多还原

【Abstract】 Objective: Sepsis is a major public health concern with high morbidity and mortality in the U.S. More recently, sepsis has been viewed as a state of immune dysregulation with excessive activation early followed later by immune-suppression, characterized by secondary infections. This critical late phase has been associated with increased effector T-cell apoptosis. Studies in mice have demonstrated extensive T-cell apoptosis during septic shock and improved outcome with adoptive T-cell transfer or the administration of caspase inhibitors, a numbers of studies have shown an inverse correlation between lymphocyte count and survival. It was clear that activation-induced T cell apoptosis is caused by the upregulation of Fas ligand(Fas L) expression and its subsequent interaction with Fas(CD95). The binding of Fas ligand(Fas L) to its receptor Fas(CD95) activates the caspase cascade and triggers extrinsic T-cell apoptosis. Caspases including caspases-8, caspases-6 or caspases-9 and downstream caspase- 3 cleave target proteins that lead to DNA fragmentation and programmed cell death.Corticosteroids(GCs) therapy has been use in varied doses for sepsis for more than half a century, with no clear benefit on mortality and serious unwanted effects. Stress-dose corticosteroid regimens that target both glucocorticoid receptor(GR) and mineralocorticoid receptors(MR) reverse hypotension in septic shock, but have a variable impact on survival. Lack of a survival benefit has been attributed to secondary infections and other adverse effects mediated through GR. Conversely, the role of mineralocorticoids(MCs) activity in the risks/benefits of corticosteroid therapy in septic shock is largely unknown. The effects of glucocorticoids on lymphocytes are dramatic. Corticosteroids induce apoptosis on immature thymocytes(positive selection and negative selection), mature splenic T cells, pre-B lymphoma cells, and several leukemic cell lines through caspase-dependent manner. Conversely, Glucocorticoids protect activation-induced cell death via down-regulating CD95 ligand gene expression through GR(in a DNA binding dependent manner) in T cells. A negative GR element found at position-990 in the Fas L promoter binds GR in vitro as as in the chromatin context. Fas L promoter contains bingding sites for several transcription factors including NF- κB. Negative glucorticoid response element(n GRE) overlaps with a known NF-κB binding site.GR down-regulates Fas L promoter by competing with NF- κB for binding to the common response element.Glucocorticoids(GCs) may address the hyper-inflammatory early phase of sepsis by trans-repressing inflammatory gene transcription and the late immune suppression phase by inhibiting activation-induced T-cell apoptosis via the down-regulation of Fas L. Closely related to GR, MR also expressed on T cells, but less so in phagocytic leukocytes and therefore may regulate T-cell function while preserving antibacterial mechanisms. In this study we set out to explore the effects Dexamethasone(DEX), a GR agonist, and deoxycorticosterone(DOC), a MR agonist on activation-induced Fas L expression and apoptosis in human peripheral blood-T cells and Jurkat cells.Methods: Human primary T cells were incubated with PHA or SEB in the absence or presence of DEX or DOC, apoptosis were analyzed by flow cytometry using Annexin V/PI staining. The caspase-8, caspase-9/6 and caspase-3 activities were measure by fluorescence method, IL-2 production was measured by ELISA, and IL-2 and Fas L expression level were measured via q RT-PCR. Fas L-driven reporter assays and NF-κB-driven reporter assays in PHA plus PMA-stimulated Jurkat cells were used to explore the effect of DEX and DOC on Fas L transpcription. Then dominant negative mutant MR were transfected into Jurkat cells, DEX and DOC effect on NF-κB and Fas L reporter activity and Fas L gene expression were measured. Further, using the proximal NF-κB/AP-1 composite sequence from the human IL-2 promoter and the putative NF-κB/GRE Fas L composite sequence from the Fas L promoter, EMSAs were performed with Jurkat cell nuclear extracts. Next, to further confirm that both DEX and DOC suppression of IL-2 and Fas L was primarily mediated via a transrepression mechanism, cycloheximide, a protein synthesis inhibitor was used to block any induction of anti-inflammatory genes. Finally human wild type MR and mutant MR(K624A) were over expressed in Jurkat cells simultaneously transfectted with the wild type Fas L reporter construct to test the MR specificity of DOC.Results: Either hytohemagglutinin(PHA) or Staphylococcus aureus enterotoxin B(SEB) can trigger apoptosis in human primary T cell, which were blocked by both DEX and DOC. PHA activated caspase-3,8,9/6 activity in human primary T cells, while both DEX or DOC inhibited those responses. These inhibitory effects of DEX and DOC on PHAinduced caspase-3 activation were dose-dependent. PHA induced IL-2 production in human primary lymphocytes. Both DEX and DOC significantly blocked this response. PHA plus PMA similarly stimulated IL-2 production in Jurkat cells, which was also suppressed by DEX and DOC. PHA plus PMA inhanced Fas L and IL-2 m RNA in Jurkat cells. Both DEX and DOC significantly blocked these responses. Dual specificity phosphatase 1(DUSP1) and glucocorticoid-induced leucine zipper(GILZ) were strongly induced by DEX in primary human lymphocytes, but either unaffected or weakly induced by DOC, respectively. DEX and DOC significantly inhibited NF-κB driven reporter gene activity, which was also were dose-dependent. DEX and DOC inhibited wild type Fas L promoter activity, but had no significant effect on the mutant Fas L promoter reporter construct containing a disrupted mutant NF-κB binding site.Conclusions: DEX, a selective glucocorticoid agonist, and DOC, a selective mineralocorticoid agonist, both block apoptosis in human primary lymphocytes. DEX and DOC inhibit PHA-induced caspase activity in human primary lymphocytes. DEX and DOC block IL-2 and Fas L expression in human primary lymphocytes and Jurkat cells. DEX and DOC differently affect anti-inflammatory genes, but similarily block NF-κB and Fas L promoter driven gene transcription. DEX and DOC inhibition of IL-2 and Fas L expression is consistent with transrepression. DOC suppression of NF-κB signaling and Fas L induction are MR dependent. Selected MR ligands may be useful in blocking the immune dysregulation and possibly have a better risk/bnefit profile than GR agonists in the treatment of sepsis and other immune disorders.更多还原