發(fā)布時(shí)間：2018-05-08 18:42

  本文選題：白細(xì)胞介素-27 + MDSCs��； 參考：《浙江大學(xué)》2017年博士論文

【摘要】：本論文分為兩個(gè)部分,分別研究了白細(xì)胞介素-27對(duì)腸炎相關(guān)腸癌的保護(hù)作用和白細(xì)胞介素-27對(duì)小鼠肥胖模型的調(diào)控作用及其機(jī)制。第一部分白細(xì)胞介素-27通過抑制腸上皮細(xì)胞分泌的促炎因子對(duì)腸炎相關(guān)腸癌的保護(hù)作用炎癥和癌癥的相關(guān)性一直是腫瘤免疫的研究重點(diǎn)之一,以往的結(jié)果表明,長(zhǎng)期的炎性環(huán)境會(huì)加速腫瘤的形成。腸炎相關(guān)腸癌是一種典型的炎癥相關(guān)腫瘤,長(zhǎng)期的腸道炎癥導(dǎo)致腸上皮的不斷破壞和修復(fù),長(zhǎng)此以往加速了相關(guān)癌癥相關(guān)基因的突變頻率和腫瘤細(xì)胞的增殖。白細(xì)胞介素-27(Interleukin-27,IL-27)由p28和EBI3兩個(gè)亞基組成,主要由活化的抗原遞呈細(xì)胞分泌,其受體由gp130和WSX-1兩個(gè)亞基組成,表達(dá)于多種免疫細(xì)胞、上皮細(xì)胞及內(nèi)皮細(xì)胞表面。已有文獻(xiàn)報(bào)道,IL-27在各種炎癥相關(guān)的疾病中發(fā)揮調(diào)控功能,并且在不同疾病模型中針對(duì)不同靶細(xì)胞兼有抑炎和促炎兩方面的作用。在腸炎的研究中,IL-27的功能也尚有爭(zhēng)議,兼有其促進(jìn)腸炎和抑制腸炎的報(bào)道,并且目前尚未有關(guān)于IL-27在腸炎相關(guān)腸癌中功能的報(bào)道。因此,本文通過建立由AOM/DSS誘導(dǎo)的腸炎相關(guān)腸癌(colitis-associated cancer)小鼠模型,研究IL-27對(duì)腸癌發(fā)生發(fā)展的作用,以期為炎癥與癌癥的關(guān)系研究提供相關(guān)的理論依據(jù),為腫瘤微環(huán)境中的細(xì)胞因子的作用增添新的認(rèn)識(shí)。我們首先用野生C57BL/6小鼠成功構(gòu)建了 AOM/DSS模型,取小鼠結(jié)直腸腫瘤和癌旁組織,及健康小鼠結(jié)直腸組織,通過熒光定量PCR(Q-PCR)、Western Blot以及免疫熒光等手段檢測(cè)到IL-27的兩個(gè)亞基及其特異性受體亞基WSX-1在腫瘤組織局部表達(dá)升高,并且IL-27主要在腸道固有層細(xì)胞中表達(dá)。隨后,我們使用IL-27R受體亞基WSX-1敲除的(WSX-1 KO)小鼠和野生(WT)小鼠同時(shí)構(gòu)建AOM/DSS模型,結(jié)果發(fā)現(xiàn)與野生小鼠相比,WSX-1KO小鼠腸道腫瘤載量和數(shù)目明顯增多,局部的腫瘤細(xì)胞增殖加強(qiáng),并且伴隨著促炎因子升高、腸道長(zhǎng)度縮短等更嚴(yán)重的腸道炎癥指標(biāo)。為研究上述結(jié)果的機(jī)制,基于我們?cè)赪T和WSX-1 KO小鼠組織中觀察到了不同的炎癥程度,我們進(jìn)一步使用流式細(xì)胞術(shù)檢測(cè)了腸癌模型小鼠的腸道固有層細(xì)胞中的免疫細(xì)胞亞群的分布和變化,結(jié)果顯示促腫瘤生長(zhǎng)的髓系來(lái)源抑制性細(xì)胞(myeloid-derived suppressor cells,MDSCs)在 WSX-1 KO 小鼠的腸道中明顯聚集增加,但其表面關(guān)鍵趨化因子受體CXCR2的表達(dá)并沒有明顯變化。我們進(jìn)一步檢測(cè)了腸上皮細(xì)胞中的趨化因子和促炎因子的表達(dá),發(fā)現(xiàn)在WSX-1 KO腸癌模型小鼠的腸上皮中,除CXCR2的配體CXCL1的表達(dá)明顯升高以外,促炎因子IL-6,TNF-α,GM-CSF的水平也有了明顯升高,提示了 IL-27對(duì)腸上皮細(xì)胞可能發(fā)揮的抑炎作用。同時(shí),免疫熒光實(shí)驗(yàn)也證明腫瘤局部CXCL1的下降與MDSCs數(shù)量的減少是一致的,提示腸上皮細(xì)胞來(lái)源的CXCL1增加可能是MDSCs數(shù)量增加的原因。隨后我們?cè)隗w外證明,使用中和抗體封閉了上皮細(xì)胞來(lái)源的CXCL1后,MDSCs的遷移減少;同時(shí)將IL-6和GM-CSF的中和抗體加入小鼠腸上皮來(lái)源的腫瘤細(xì)胞系CT26和MDSCs的共培養(yǎng)體系后,MDSCs的數(shù)量減少,證明腸上皮細(xì)胞來(lái)源的IL-6和GM-CSF促進(jìn)MDSCs的存活。隨后我們用熒光定量PCR和免疫熒光檢測(cè)了 WT和WSX-1 KO腫瘤建模小鼠的腸道緊密連接蛋白的表達(dá),發(fā)現(xiàn)沒有明顯統(tǒng)計(jì)學(xué)差異,并且血清中LPS也沒有明顯變化,證明促炎因子的變化不是由于局部來(lái)源于腸道菌群的病原相關(guān)模式分子(Pathogen-associated Molecular Pattern,PAMPs)的數(shù)量變化導(dǎo)致的,而可能直接由腸上皮細(xì)胞的反應(yīng)性變化引起。因此我們進(jìn)一步用LPS和IL-27在體外刺激了原代腸上皮細(xì)胞,發(fā)現(xiàn)IL-27可抑制LPS誘導(dǎo)的腸上皮細(xì)胞中促炎因子和趨化因子的分泌。最后,我們用抗生素組合喂食小鼠,清除腸道內(nèi)微生物來(lái)源的PAMPs后,再用AOM/DSS誘導(dǎo)腫瘤模型,我們發(fā)現(xiàn)在清除了微生物來(lái)源的PAMPs后,WT和WSX-1KO模型小鼠腸道腫瘤、腸道固有層MDSCs數(shù)量、腸上皮細(xì)胞促炎因子和趨化因子的表達(dá)均無(wú)統(tǒng)計(jì)學(xué)差異,證明IL-27對(duì)腸炎相關(guān)腸癌的保護(hù)作用是依賴PAMPs的。綜上,本部分研究證明在慢性腸炎相關(guān)的腸癌模型發(fā)病過程中,IL-27可以抑制PAMPs觸發(fā)的腸上皮細(xì)胞中促炎因子和趨化因子的表達(dá),從而降低了促腫瘤的髓系來(lái)源抑制細(xì)胞(MDSCs)的聚集,因此對(duì)腫瘤的發(fā)生發(fā)展起到抑制作用。本研究為闡明腫瘤微環(huán)境中細(xì)胞因子的調(diào)控作用增添了新的理論依據(jù),也可能為腸癌的免疫治療提供新的思路。第二部分白細(xì)胞介素-27受體敲除促進(jìn)小鼠肥胖及其機(jī)制的初步研究肥胖是現(xiàn)代社會(huì)危害人類健康的隱形殺手之一,它所引發(fā)的代謝病癥長(zhǎng)久以來(lái)困擾著人們的身體健康。我們對(duì)脂肪功能的認(rèn)識(shí),也逐漸從簡(jiǎn)單存儲(chǔ)能量,到作為內(nèi)分泌系統(tǒng)的一部分調(diào)節(jié)人體代謝功能。內(nèi)臟脂肪的含量和狀態(tài),對(duì)健康有著非常重要的影響。隨著研究的深入,肥胖被認(rèn)為是一種慢性炎癥狀態(tài),脂肪組織,特別是內(nèi)臟脂肪組織(visceral adipose tissue,VAT)分泌的各種促炎因子如IL-6、TNF-a和C-反應(yīng)蛋白(C-reactiveprotein,CRP)等在肥胖狀態(tài)下均表達(dá)上升,導(dǎo)致胰島素抵抗等相關(guān)表現(xiàn)。近年來(lái)發(fā)現(xiàn),內(nèi)臟脂肪組織中存在一種特殊的調(diào)節(jié)性T細(xì)胞(Treg),脂肪Treg通過分泌IL-10和TGF-β對(duì)脂肪代謝的穩(wěn)態(tài)起到重要調(diào)節(jié)作用。正常狀態(tài)Treg細(xì)胞在脂肪中聚集,VAT Treg占CD4+ T細(xì)胞的比例高于其他淋巴器官和非淋巴器官,而在肥胖狀態(tài)下,Treg細(xì)胞比例在脂肪組織中急劇下降。在進(jìn)行第一部分實(shí)驗(yàn)的時(shí)候,我們發(fā)現(xiàn)在較大年齡(24周以上)的小鼠中,WSX-1 KO小鼠體重略高于WT小鼠,同時(shí)查閱文獻(xiàn)發(fā)現(xiàn)以往并沒有IL-27在肥胖中的作用研究,因此進(jìn)行了第二部分實(shí)驗(yàn)。WSX-1 KO和WT小鼠給予正常飲食飼養(yǎng)在同樣環(huán)境下24周后,我們首先檢測(cè)其體重變化,發(fā)現(xiàn)WSX-1KO小鼠體重相較于WT小鼠有明顯增加,并且內(nèi)臟脂肪的重量也有明顯上升,流式檢測(cè)結(jié)果表明WSX-1 KO小鼠中VATTreg數(shù)量下降。這些結(jié)果提示了 IL-27受體敲除可促進(jìn)小鼠肥胖。隨后,我們進(jìn)一步用高脂飼料誘導(dǎo)肥胖模型,得到同樣結(jié)果。在喂養(yǎng)過程中,雖然兩組進(jìn)食量并沒有差距,但是WSX-1 KO小鼠的體重高于WT小鼠,核磁共振結(jié)果顯示其體脂含量也高于WT小鼠,并且WSX-1 KO小鼠中VAT Treg數(shù)量也顯著降低,VAT Treg的相關(guān)轉(zhuǎn)錄因子Foxp3和PPAR-γ mRNA水平在內(nèi)臟脂肪中的含量也顯著下降,這些結(jié)果進(jìn)一步證明,在高脂飲食誘導(dǎo)的肥胖模型中,IL-27/WSX-1信號(hào)也可以對(duì)肥胖發(fā)揮抑制作用。為了解這種現(xiàn)象是否具有臨床相關(guān)性,我們進(jìn)一步用ELISA檢測(cè)了肥胖患兒血漿中的IL-27含量,但有趣的是,發(fā)現(xiàn)跟對(duì)照組相比,肥胖兒童血漿中IL-27含量升高,我們推測(cè)IL-27可能是以一種代償效應(yīng)發(fā)揮作用,在肥胖狀態(tài)下應(yīng)激分泌。具體機(jī)制還有待于進(jìn)一步深入研究。綜上所述,本部分研究我們初步發(fā)現(xiàn)了 IL-27/WSX-1信號(hào)缺失后可促進(jìn)小鼠肥胖的表型,首次在小鼠肥胖模型中闡述了 IL-27可能發(fā)揮的抑炎作用,希望在日后的研究中,可以為肥胖及相關(guān)并發(fā)癥的機(jī)制研究及其治療提供理論依據(jù)和潛在靶點(diǎn)。
[Abstract]:This thesis is divided into two parts. The protective effect of interleukin -27 on colonic cancer and the regulation of interleukin -27 on the model of obesity in mice and its mechanism. The first part is the protective action of interleukin -27 on the protection of intestinal inflammation and cancer by inhibiting the proinflammatory factors secreted by intestinal epithelial cells Correlation has been one of the key points in the study of tumor immunity. Previous results showed that a long-term inflammatory environment accelerated the formation of tumors. Enteritis related colon cancer is a typical inflammation related tumor. Long term intestinal inflammation leads to the continuous destruction and repair of intestinal epithelium, which has accelerated the mutation frequency of related cancer related genes. The proliferation of tumor cells. Interleukin -27 (Interleukin-27, IL-27) is composed of two subunits of p28 and EBI3, mainly secreted by activated antigen presenting cells. Their receptors are composed of two subunits of gp130 and WSX-1, expressed in various immune cells, epithelial cells and endothelial cells. It has been reported that IL-27 is in various inflammatory related diseases. The role of IL-27 is also controversial in the study of enteritis, and there are also reports on the promotion of enteritis and inhibition of enteritis in the study of enteritis, and there is no report on the function of IL-27 in colitis related colon cancer. To establish a AOM/DSS induced colitis-associated cancer mouse model to study the effect of IL-27 on the development of colon cancer in order to provide a theoretical basis for the study of the relationship between inflammation and cancer, and to add a new understanding to the role of cytokine in the tumor microenvironment. We first succeeded in using wild C57BL/6 mice. The AOM/DSS model was constructed to take the colorectal tumor and para cancerous tissue in mice and the colorectal tissue in healthy mice. The expression of two subunits and specific receptor subunits WSX-1 in the tumor tissues was increased by fluorescence quantitative PCR (Q-PCR), Western Blot and immunofluorescence, and IL-27 was mainly in the intestinal lamina propria cells. Then, we used the IL-27R receptor subunit WSX-1 knockout (WSX-1 KO) mice and the wild (WT) mice to construct the AOM/DSS model simultaneously. The results showed that compared with the wild mice, the intestinal tumor loading and number of WSX-1KO mice increased significantly, the local tumor cell proliferation was stronger, and the proinflammatory factors were increased and the length of the intestine was shortened. A more serious indicator of intestinal inflammation. For the study of the above results, based on the different levels of inflammation observed in WT and WSX-1 KO mice, we further used flow cytometry to detect the distribution and changes in the immune cell subsets in the intestinal lamina propria cells of the colon cancer model mice. The results showed that the tumor growth was promoted. Myeloid-derived suppressor cells (MDSCs) was significantly increased in the intestinal tract of WSX-1 KO mice, but the expression of the key chemokine receptor CXCR2 on the surface was not significantly changed. We further detected the expression of chemokines and proinflammatory factors in the intestinal epithelial cells, and found that the WSX-1 KO colon cancer model was found. In the intestinal epithelium of the mice, the levels of the proinflammatory factor IL-6, TNF- a, and GM-CSF were also significantly elevated in addition to the increase in the expression of the CXCR2 ligand CXCL1, suggesting the possible anti-inflammatory effect of IL-27 on intestinal epithelial cells. At the same time, the immunofluorescence test also showed that the decrease of local CXCL1 in the tumor was consistent with the decrease in the number of MDSCs. The increase of CXCL1 in the intestinal epithelial cells may be the cause of the increase in the number of MDSCs. Subsequently, we demonstrated in vitro that the use of neutralizing antibodies closed the CXCL1 of epithelial cells and reduced the migration of MDSCs; meanwhile, IL-6 and GM-CSF neutralizing antibodies were added to the co culture system of the murine epithelial cells of the intestinal epithelium, CT26 and MDSCs, MDSCs. The number of IL-6 and GM-CSF derived from intestinal epithelial cells promoted the survival of MDSCs. Then we detected the expression of close connexin in the intestinal tract of WT and WSX-1 KO tumor modeling mice by fluorescence quantitative PCR and immunofluorescence, and found no significant difference in statistics, and there was no significant change in serum LPS. The change is not due to the changes in the number of Pathogen-associated Molecular Pattern (PAMPs) derived from the intestinal flora, which may be caused by the reactive changes in the intestinal epithelial cells. Therefore, we further stimulated the primary intestinal epithelial cells with LPS and IL-27 in vitro, and found that IL-27 inhibits LPS. The secretion of pro-inflammatory factors and chemokines in the intestinal epithelial cells was induced. Finally, we fed the mice with antibiotic combination, removed the PAMPs in the intestinal tract and induced the tumor model with AOM/DSS. We found that after the removal of PAMPs from the microbial sources, we found the intestinal tumor in the WT and WSX-1KO model mice, the amount of the intestinal lamina propria MDSCs. There is no statistical difference in the expression of proinflammatory and chemokines in intestinal epithelial cells. It is proved that the protective effect of IL-27 on enterocolitis is dependent on PAMPs. In this part, this part of this study proved that IL-27 could inhibit the proinflammatory factors and chemokines in the intestinal epithelial cells triggered by PAMPs during the pathogenesis of chronic enteritis. Expression, thus reducing the accumulation of myeloid source inhibition cells (MDSCs), can inhibit the development of tumor. This study provides a new theoretical basis for clarifying the regulation of cytokines in tumor microenvironment, and may also provide new ideas for the immunotherapy of colon cancer. The second part of interleukin -27 Receptor knockout promotes obesity and its mechanism in mice. Obesity is one of the invisible killer of human health in modern society. Its metabolic disorder has been perplexing people's health for a long time. Our understanding of fat function is gradually from storing energy simply to regulating human body as part of the endocrine system. Metabolic function. The content and state of visceral fat have a very important impact on health. As the study goes deep, obesity is considered a chronic inflammatory state, and the various proinflammatory agents secreted by visceral adipose tissue (VAT), especially the visceral adipose tissue, such as IL-6, TNF-a, and C- reactive protein (C-reactiveprotein, CRP), are secreted. In recent years, a special regulatory T cell (Treg) has been found in visceral adipose tissue. Fat Treg plays an important role in the homeostasis of fat metabolism by secreting IL-10 and TGF- beta. Treg cells in normal state are aggregated in fat and VAT Treg accounts for CD4+ T. In the first part of the experiment, we found that WSX-1 KO mice were slightly higher than the WT mice at a large age (more than 24 weeks), and the literature found no IL-27 in the past. The role of obesity was studied, so second parts of the experiment.WSX-1 KO and WT mice were given normal diet in the same environment for 24 weeks. We first detected the body weight changes. It was found that the weight of WSX-1KO mice increased significantly compared to the WT mice, and the weight of visceral fat increased significantly, and the flow test results showed WSX-1 KO. The number of VATTreg in mice decreased. These results suggest that IL-27 receptor knockout can promote obesity in mice. Then, we further use high fat diet to induce obesity model and get the same results. In the feeding process, although there is no difference in the intake of two groups, the weight of the WSX-1 KO mice is higher than that of the WT mice, and the MRI results show the body fat. The content was also higher than that of WT mice, and the number of VAT Treg in the WSX-1 KO mice decreased significantly, and the level of VAT Treg related transcription factors Foxp3 and PPAR- gamma mRNA decreased significantly in visceral fat. These results further demonstrated that the IL-27/WSX-1 signal could also inhibit obesity in the high fat diet induced obesity model. In order to understand the clinical relevance of this phenomenon, we further detected the IL-27 content in the plasma of obese children with ELISA, but it is interesting to find that the plasma levels of IL-27 in obese children are higher than those in the control group. We speculate that IL-27 may play a role in a compensatory effect and stress secretion in the obese state. For the first time, we have discovered the phenotype that can promote the obesity in mice after the deletion of IL-27/WSX-1 signal. We first described the possible anti inflammatory effect of IL-27 in the model of obesity in mice. We hope that in the future study, we can study the mechanism of obesity and related complications and the mechanism in the future study. The treatment provides theoretical basis and potential targets.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.3

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9 潘建玲;關(guān)秀茹;馬學(xué)華;;白細(xì)胞介素-18在特發(fā)性血小板減少性紫癜中作用的研究[J];哈爾濱醫(yī)科大學(xué)學(xué)報(bào);2007年01期

10 鄧學(xué)軍;覃數(shù);;白細(xì)胞介素-10、白細(xì)胞介素-6在急性冠狀動(dòng)脈綜合征中的臨床意義[J];四川醫(yī)學(xué);2008年10期

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7 李素，

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