本文選題：調(diào)節(jié)性T細(xì)胞 + 白細(xì)胞介素-3　； 參考：《天津醫(yī)科大學(xué)》2017年博士論文

【摘要】：近來(lái)研究發(fā)現(xiàn)白介素-3(IL-3)是參與膿毒癥免疫紊亂進(jìn)展,放大炎癥效應(yīng)的重要驅(qū)動(dòng)因子,阻斷IL-3的活性能夠明顯改善膿毒癥預(yù)后,臨床研究亦顯示IL-3水平升高可作為膿毒癥患者預(yù)后的重要預(yù)測(cè)因素。而,調(diào)節(jié)性T細(xì)胞(Treg)對(duì)于維持自身免疫穩(wěn)態(tài)非常關(guān)鍵,在感染免疫中,是影響機(jī)體有效細(xì)菌清除與避免過(guò)度炎癥反應(yīng)病理?yè)p害的重要調(diào)節(jié)因素,其在膿毒癥病生理過(guò)程中的核心作用已得到共識(shí)。但,在膿毒癥免疫炎癥反應(yīng)過(guò)程中,IL-3與Treg之間是否存在內(nèi)在的功能聯(lián)系等,目前尚未見(jiàn)研究報(bào)道。對(duì)兩者間內(nèi)在聯(lián)系與作用機(jī)制的深入研究將無(wú)疑有助于進(jìn)一步闡釋膿毒癥的發(fā)病機(jī)制,并可能提示新的潛在治療靶點(diǎn)與治療策略。為此,本課題首先證實(shí)在Treg細(xì)胞膜表達(dá)有IL-3受體,顯示Treg細(xì)胞可能受到IL-3的功能調(diào)控;進(jìn)而,我們發(fā)現(xiàn)Treg細(xì)胞自身亦能夠生成IL-3;并且,通過(guò)改變Treg細(xì)胞內(nèi)IL-3的生成,我們分析了Treg細(xì)胞功能的變化,結(jié)果顯示下調(diào)IL-3表達(dá),Treg細(xì)胞中Foxp3和CTLA-4表達(dá)上升,IL-10與TGF-β生成增加,對(duì)效應(yīng)T細(xì)胞的增殖抑制活性明顯增強(qiáng),而上調(diào)IL-3表達(dá),Treg細(xì)胞功能呈現(xiàn)相反變化;因此,我們的研究結(jié)果表明:作為一種自分泌機(jī)制,IL-3對(duì)Treg細(xì)胞功能活性的發(fā)揮具有重要的負(fù)反饋調(diào)節(jié)效應(yīng)。在此基礎(chǔ)上,為了探討膿毒癥中IL-3與Treg細(xì)胞間的功能聯(lián)系,我們使用體外LPS刺激模擬膿毒癥細(xì)胞模型,首先分析了外源添加與抗體阻斷IL-3對(duì)LPS活化Treg細(xì)胞功能活性的影響,結(jié)果顯示,IL-3作用會(huì)抑制Treg細(xì)胞中Foxp3和CTLA-4表達(dá)、IL-10與TGF-β生成和對(duì)效應(yīng)T細(xì)胞的增殖效應(yīng),而,應(yīng)用IL-3Ab能夠阻斷上述效應(yīng);進(jìn)一步,我們應(yīng)用慢病毒載體轉(zhuǎn)染,上調(diào)或下調(diào)Treg細(xì)胞內(nèi)IL-3表達(dá),證實(shí)上調(diào)Treg細(xì)胞內(nèi)IL-3的水平會(huì)明顯抑制Treg細(xì)胞Foxp3和CTLA-4表達(dá)、IL-10與TGF-β生成和對(duì)效應(yīng)T細(xì)胞的增殖效應(yīng),反之亦然;因此,以上研究結(jié)果顯示,膿毒癥病生理過(guò)程中,IL-3可能作為一種重要的負(fù)反饋調(diào)節(jié)機(jī)制,負(fù)向調(diào)控LPS對(duì)Treg細(xì)胞的活化效應(yīng),這可能是機(jī)體保持免疫平衡,避免免疫紊亂的一種保護(hù)性機(jī)制。進(jìn)而,通過(guò)采用盲腸結(jié)扎穿孔膿毒癥(CLP)模型,應(yīng)用IL-3Ab,我們首先證實(shí),阻斷IL-3的效應(yīng),能夠有效降低TNF等炎癥因子水平,減輕肝、腎、肺等臟器損害,改善膿毒癥預(yù)后;同時(shí),我們發(fā)現(xiàn)阻斷IL-3,也能夠增加Treg細(xì)胞中Foxp3和CTLA-4的表達(dá),促進(jìn)IL-10與TGF-β的生成,并使其對(duì)效應(yīng)T細(xì)胞的增殖抑制活性明顯增強(qiáng);因此,我們的研究結(jié)果在證實(shí)IL-3Ab的有效性和阻斷炎癥反應(yīng)效應(yīng)的同時(shí),揭示了IL-3參與膿毒癥病生理過(guò)程的新機(jī)制,即,IL-3在放大天然免疫反應(yīng)的同時(shí),也會(huì)下調(diào)Treg細(xì)胞的免疫抑制活性,參與膿毒癥免疫紊亂的發(fā)生發(fā)展,這一研究發(fā)現(xiàn)不僅闡釋了IL-3Ab治療效應(yīng)的作用機(jī)制,也為我們提示了一個(gè)新的免疫調(diào)控治療膿毒癥的策略途徑。綜上所述,本研究首先闡釋了IL-3與Treg細(xì)胞間內(nèi)在聯(lián)系的生物學(xué)基礎(chǔ),即Treg細(xì)胞表達(dá)IL-3R,且自身能生成IL-3,并證實(shí)自分泌的IL-3對(duì)Treg細(xì)胞具有負(fù)反饋調(diào)節(jié)效應(yīng);在此基礎(chǔ)上,通過(guò)體內(nèi)外膿毒癥模型,證實(shí)膿毒癥病生理過(guò)程中,IL-3不僅可通過(guò)促進(jìn)中性粒細(xì)胞等的生成放大炎癥反應(yīng),同時(shí),也會(huì)下調(diào)Treg細(xì)胞的活性,共同參與膿毒癥免疫紊亂的發(fā)生發(fā)展。本項(xiàng)目研究揭示了膿毒癥中IL-3與Treg之間的內(nèi)在聯(lián)系和作用機(jī)制,不僅有助于深化對(duì)膿毒癥病生理機(jī)制的認(rèn)識(shí),也為探索有效防控膿毒癥提供潛在靶點(diǎn)和治療策略的新啟示。
[Abstract]:Recent studies have found that interleukins -3 (IL-3) is an important driving factor involved in the progression of sepsis immune disorder and amplifying the inflammatory effect. Blocking IL-3 activity can significantly improve the prognosis of sepsis. Clinical studies also show that the elevated level of IL-3 can be an important pretest factor for the prognosis of patients with sepsis. And regulatory T cells (Treg) can maintain themselves. Immune homeostasis is very important. In infection immunity, it is an important regulatory factor affecting the pathological damage of effective bacteria clearance and avoidance of excessive inflammation. Its core role in the physiological process of sepsis has been recognized. However, there is an inherent functional relationship between IL-3 and Treg during the immune inflammatory response of sepsis. An in-depth study of the intrinsic links and mechanisms of action between the two will undoubtedly help further explain the pathogenesis of sepsis, and may suggest new potential therapeutic targets and therapeutic strategies. For this reason, this topic first confirms that the expression of IL-3 receptors in the Treg cell membrane, indicating that Treg cells may be subject to the work of IL-3 In addition, we found that Treg cells could also produce IL-3, and by changing the formation of IL-3 in Treg cells, we analyzed the changes in the function of Treg cells. The results showed that the expression of IL-3, the expression of Foxp3 and CTLA-4 in Treg cells increased, IL-10 and TGF- beta was increased, and the proliferation inhibition activity of the effect T cells was significantly enhanced. In addition, the Treg cell function is opposite to IL-3 expression; therefore, our results show that as a kind of autocrine mechanism, IL-3 plays an important negative feedback regulation effect on the function of Treg cells. On this basis, in order to explore the functional relationship between IL-3 and Treg cells in sepsis, we use an in vitro LPS stimulation model. In the Pseudomonas cell model, the effects of exogenous and antibody blocking IL-3 on the functional activity of LPS activated Treg cells were first analyzed. The results showed that the effect of IL-3 could inhibit the expression of Foxp3 and CTLA-4 in Treg cells, the production of IL-10 and TGF- beta and the effect on the proliferation of effect T cells, while IL-3Ab could be used to block the above effects. Further, we applied it. Lentivirus vector transfection, up or down regulation of the expression of IL-3 in Treg cells, confirmed that the level of IL-3 in Treg cells can obviously inhibit the expression of Foxp3 and CTLA-4 in Treg cells, IL-10 and TGF- beta generation and the effect on the proliferation of effect T cells, and vice versa; therefore, the above results show that IL-3 may be a heavy weight in the physiological process of sepsis. The negative feedback regulation mechanism, which negatively regulates the activation effect of LPS on Treg cells, may be a protective mechanism for the body to maintain the immune balance and avoid immune disorders. Then, by using the cecum ligation of the perforated sepsis (CLP) model, the application of IL-3Ab, first of all, to block the effect of IL-3, can effectively reduce the TNF and other inflammatory factors. Level, reduce the liver, kidney, lung and other visceral damage, improve the prognosis of sepsis. At the same time, we found that blocking IL-3, also can increase the expression of Foxp3 and CTLA-4 in Treg cells, promote the formation of IL-10 and TGF- beta, and make it significantly increase the inhibitory activity of the effect T cells; therefore, our research results confirm the effectiveness and blocking of IL-3Ab. At the same time, the effect of inflammatory reaction reveals a new mechanism of IL-3's participation in the physiological process of sepsis. That is, IL-3 also reduces the immunosuppressive activity of Treg cells and participates in the development of the immune disorder of sepsis, while amplifying the natural immune response. This study not only explained the mechanism of the effect of IL-3Ab, but also provided us with the mechanism of the therapeutic effect of the sepsis. A new strategy for the treatment of sepsis with a new immunoregulation is shown. To sum up, this study first explains the biological basis of the intrinsic link between IL-3 and Treg cells, that is, the expression of IL-3R by Treg cells and the self generation of IL-3, and that the autocrine IL-3 has a negative feedback regulation effect on Treg cells; on this basis, it passes through the abscess of the body and the body. In the physiological process of sepsis, it is proved that IL-3 can not only enlarge the inflammatory response by promoting the production of neutrophils, but also reduce the activity of Treg cells, and participate in the development of the immune disorder of sepsis. This project reveals the internal relationship and mechanism of IL-3 and Treg in sepsis, not only It helps deepen our understanding of the physiological mechanism of sepsis and provides new insights into the potential targets and therapeutic strategies for effective prevention and control of sepsis.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R459.7

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