發(fā)布時(shí)間：2018-03-09 04:13

  本文選題：胃癌　切入點(diǎn)：CD40　出處：《蘇州大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：胃癌是我國最常見的惡性腫瘤之一,占消化道腫瘤的50%~60%,病死率居各類惡性腫瘤之首,5年生存率只有30%。近年來發(fā)病率明顯上升,而且越來越年輕化,但因早期癥狀不明顯,缺乏特異性的初篩指標(biāo),多數(shù)患者在就診時(shí)已經(jīng)處于進(jìn)展期,傳統(tǒng)根治手術(shù)及放化療對(duì)進(jìn)展期胃癌的療效不理想。因此,迫切需要新的治療方法。免疫治療是目前的研究熱點(diǎn),啟動(dòng)和建立有效免疫應(yīng)答正成為腫瘤治療的新希望。目前眾多研究發(fā)現(xiàn)一些協(xié)同刺激分子在多種腫瘤中異常表達(dá)。CD40/CD40L是介導(dǎo)特異性免疫應(yīng)答的一對(duì)極其重要的共刺激分子。CD40表達(dá)于淋巴系統(tǒng)的惡性細(xì)胞,包括幾乎所有的B細(xì)胞惡性腫瘤,以及許多實(shí)體瘤,例如膀胱癌,腎癌,胰腺癌,胃癌,乳腺癌,大腸癌,肺癌等。臨床研究表明CD40的表達(dá)與腫瘤的進(jìn)展和轉(zhuǎn)移有密切關(guān)聯(lián)。腫瘤細(xì)胞協(xié)同刺激分子的表達(dá)改變,包括正性協(xié)同刺激分子表達(dá)的下降或者突變,以及負(fù)性協(xié)同刺激分子的異常高表達(dá),導(dǎo)致T細(xì)胞的凋亡及功能抑制,參與了腫瘤免疫逃逸。而腫瘤免疫逃逸通常與髓源性抑制細(xì)胞(myeloid derived suppressor cell,MDSC)的聚集和調(diào)節(jié)性T細(xì)胞(Regulatory T cell,Treg)的增多相關(guān)。Treg在維持免疫耐受和破壞抗腫瘤反應(yīng)中扮演重要角色。MDSC通過誘導(dǎo)活化的T細(xì)胞產(chǎn)生凋亡、釋放抑制性細(xì)胞因子、誘導(dǎo)Treg的產(chǎn)生以及與NK細(xì)胞和Treg細(xì)胞等形成復(fù)雜的網(wǎng)絡(luò)而產(chǎn)生負(fù)向調(diào)控功能。其抑制作用主要與精氨酸酶1(arginase-1,ARG1)和誘導(dǎo)型一氧化氮合酶(inducible nitrieoxidesyntnase,i Nos)有關(guān)。已有研究表明MDSC可通過抑制T細(xì)胞免疫反應(yīng)以及擴(kuò)增Treg發(fā)揮其免疫抑制功能的。本研究以CD40為切入點(diǎn),探討其對(duì)MDSC生物學(xué)功能的調(diào)控作用及相關(guān)機(jī)制,為認(rèn)識(shí)MDSC在免疫逃逸中的作用提供有價(jià)值的線索。第一部分:小鼠胃癌模型中MDSC上CD40的表達(dá)及其與腫瘤進(jìn)展的關(guān)系目的:檢測CD40分別在荷瘤小鼠脾臟來源和腫瘤浸潤的MDSC上的表達(dá)水平,分析其表達(dá)在胃癌進(jìn)展過程中可能的作用。方法:利用小鼠胃癌細(xì)胞系MFC皮下注射C57BL/6j構(gòu)建小鼠胃癌模型。隨著腫瘤進(jìn)展,每隔5天脫頸處死3只模型鼠,剝離腫瘤和脾臟,通過流式細(xì)胞術(shù)檢測腫瘤不同進(jìn)展階段脾臟及腫瘤浸潤組織中MDSC比例及其表面CD40表達(dá)情況,統(tǒng)計(jì)學(xué)分析其表達(dá)的意義。結(jié)果:與正常小鼠脾臟MDSC相比,荷瘤小鼠脾臟來源的MDSC表面CD40的表達(dá)明顯上升(P=0.0024);脾臟MDSC以及腫瘤組織浸潤MDSC比例隨著腫瘤生長逐漸增高;MDSC上CD40表達(dá)水平隨腫瘤進(jìn)展逐漸降低后穩(wěn)定至一定水平。此外,我們尚發(fā)現(xiàn)Wilde Type(WT)小鼠大約在5~6天成瘤,而CD40-/-小鼠約在7天后成瘤,腫瘤生長速度和大小明顯小于WT小鼠。隨著腫瘤進(jìn)展,WT小鼠腫瘤浸潤性MDSC的累積水平明顯高于CD40-/-小鼠。上述結(jié)果提示,在小鼠胃癌模型中,腫瘤相關(guān)MDSC表面共刺激分子CD40高表達(dá),且與腫瘤進(jìn)展相關(guān)。第二部分:探討CD40對(duì)MDSC生物學(xué)功能的調(diào)控作用目的:以CD40分子為切入點(diǎn),探討其在調(diào)節(jié)MDSC凋亡、對(duì)T細(xì)胞增殖抑制以及誘導(dǎo)Treg方面的調(diào)控作用及相關(guān)機(jī)制。方法:自脾臟分離純化的Gr-1+CD11b+細(xì)胞調(diào)整細(xì)胞濃度鋪于24孔板。每孔加入2 l GM-CSF后,分別用IL-4、IL-6、IL-10、TNF-α、GM-CSF、IFN-γ、LPS、PEG2和MFC刺激24 h、48h、72h后,收集細(xì)胞,用流式細(xì)胞術(shù)分別檢測CD40在MDSC上的表達(dá)。取經(jīng)磁珠分選獲得的小鼠脾臟MDSC懸液,鋪入96孔板中,對(duì)照組加入PBS,實(shí)驗(yàn)組加入激發(fā)型CD40抗體,5%CO2孵箱培養(yǎng)24 h后,分別離心收集各孔細(xì)胞,流式細(xì)胞術(shù)分析MDSC細(xì)胞凋亡率。將分選獲得的小鼠脾臟MDSC與CFSE染色T細(xì)胞按照1:3的比例,用anti-CD3和anti-CD28進(jìn)行刺激,72小時(shí)后檢測CFSE熒光衰減,分析T細(xì)胞增殖情況。將WT小鼠和CD40KO小鼠的脾臟細(xì)胞分離出來后,流式細(xì)胞術(shù)檢測IL-4,IL-17,IFN-γ,Foxp3的表達(dá)。結(jié)果:LPS體外刺激MDSC后能夠上調(diào)表面分子CD40的表達(dá),且在48小時(shí)后,CD40呈進(jìn)一步顯著性上調(diào)。胃癌微環(huán)境下,CD40能夠顯著抑制MDSC的凋亡;而且CD40促進(jìn)MDSC介導(dǎo)的抑制T細(xì)胞增殖功能;CD40參與了MDSC介導(dǎo)的Treg誘導(dǎo)-分化,促進(jìn)Foxp3表達(dá),增強(qiáng)MDSC免疫抑制功能,在胃癌免疫逃逸中發(fā)揮關(guān)鍵作用。第三部分:Genechip分析CD40-/-與CD40low胃癌荷瘤小鼠脾臟細(xì)胞差異基因表達(dá)目的:通過基因芯片技術(shù)分析CD40-/-與CD40low胃癌荷瘤小鼠脾臟細(xì)胞基因表達(dá)概況,尋找差異表達(dá)基因,分析CD40對(duì)MDSC生物功能作用的可能分子機(jī)制。方法:參照第一部分構(gòu)建胃癌荷瘤小鼠模型,當(dāng)腫瘤直徑達(dá)1cm后,頸椎脫臼法處死WT荷瘤小鼠與CD40-/-荷瘤小鼠,手術(shù)取出完整的脾臟。經(jīng)150目鋼網(wǎng)研磨,裂紅后,加入1ml Trizol備用。Genechip委托北京博奧生物有限公司進(jìn)行檢測。結(jié)果:通過基因芯片技術(shù)對(duì)CD40-/-和WT小鼠脾臟細(xì)胞進(jìn)行全基因組檢測,尋找差異表達(dá)基因,分析CD40對(duì)MDSC生物功能作用的可能分子機(jī)制。結(jié)果顯示兩組細(xì)胞基因表達(dá)譜有差異。篩選兩組間差異倍數(shù)(經(jīng)log2轉(zhuǎn)換)2倍以上的基因?yàn)椴町惐磉_(dá)基因,篩選出2890個(gè)差異表達(dá)基因,其中表達(dá)上調(diào)593個(gè),表達(dá)下調(diào)1419個(gè)。Pathway分析結(jié)果顯示差異基因主要涉及信號(hào)通路、細(xì)胞代謝、免疫應(yīng)答、炎癥反應(yīng)、細(xì)胞凋亡等。結(jié)論 本研究表明CD40是腫瘤相關(guān)MDSC的重要標(biāo)志,能夠調(diào)控MDSC介導(dǎo)的免疫抑制功能,從而影響腫瘤的發(fā)生和發(fā)展。這對(duì)分析腫瘤免疫耐受的產(chǎn)生機(jī)制并探索有效的免疫治療方法具有重要意義。為胃癌的有效干預(yù)治療提供了新的思路。
[Abstract]:Gastric cancer is one of the most common malignant tumors in China, accounting for 50%~60% and gastrointestinal tumors, the mortality rate among all malignant tumors, 5 year survival rate is only 30%. in recent years the incidence rate increased significantly, and more and more young, but because the early symptoms are not obvious, the lack of specific screening index, most patients have in the advanced stage at the time of treatment, the curative effect of traditional radical surgery and chemotherapy for advanced gastric cancer is not ideal. Therefore, an urgent need for new treatments. Immunotherapy is the research hotspot at present, start and establish an effective immune response is becoming the new hope for the treatment of tumor. At present, many studies have found that some costimulatory molecules in a variety of the abnormal expression of.CD40/CD40L in tumor specific immune response is mediated by a pair of important costimulatory molecules.CD40 expression in the lymphatic system malignant cells, including almost all of the B cell malignant tumor, to And many solid tumors, such as bladder cancer, kidney cancer, pancreatic cancer, gastric cancer, breast cancer, colorectal cancer, lung cancer. Clinical studies have shown that closely related to progression and metastasis of tumors. The expression of CD40 and expression of costimulatory molecules in tumor cells, including positive expression of costimulatory molecules decreased or mutation, abnormal high the expression of negative costimulatory molecules, induce apoptosis and function of T cells involved in tumor suppression, immune escape and tumor immune escape cells usually associated with myeloid derived suppressor (myeloid derived suppressor cell, MDSC) and aggregation of regulatory T cells (Regulatory T cell, Treg) increased in immune related.Treg damage tolerance and plays an important role in.MDSC induced apoptosis through activation of T cells in antitumor response, inhibit the release of cytokines, induce the expression of Treg and NK cells and Treg cells form a complex The network has a negative regulatory function. Its inhibitory effect and arginase 1 (arginase-1, ARG1) and inducible nitric oxide synthase (inducible nitrieoxidesyntnase, I Nos). Studies have shown that MDSC can inhibit T cell immune response and amplification of Treg exerts its immunosuppressive function. In this study, CD40 as the starting point, to explore its role in the regulation of MDSC biological function and mechanism, provide valuable clues for understanding the role of MDSC in immune escape. The first part: the objective relationship between the expression of MDSC CD40 on the mouse model of gastric carcinoma and advances in tumor: the detection of CD40 in spleen derived tumor bearing mice and tumor infiltration of MDSC on the expression level of its expression may be in progress in the process of gastric cancer. Methods: the construction of mouse gastric cancer mouse model of gastric cancer cell line MFC by subcutaneous injection of C57BL/6j with tumor. In every 5 days, 3 rats were sacrificed, tumor and spleen, spleen and tumor MDSC expression by flow cytometry in different stages of tumor invasive tissue proportion and the surface of CD40, the expression of the meaning of statistical analysis. Results: compared with normal mice spleen MDSC surface expression of MDSC CD40 in spleen the source of tumor bearing mice was significantly increased (P=0.0024); spleen MDSC and tumor infiltrating MDSC with tumor growth ratio gradually increased; the MDSC expression of CD40 with tumor progression decreased gradually after stable to a certain level. In addition, we have found that Wilde Type (WT) mice at about 5~6 and CD40-/- about tumor Tiancheng, in mice 7 days after tumor formation, tumor growth rate and size was less than WT mice. With the progression of cancer, the cumulative levels of tumor infiltrating MDSC WT mice was significantly higher than that of CD40-/- mice. The results suggest that in the mouse stomach In the model of cancer, tumor associated MDSC costimulatory molecules CD40 expression and associated with tumor progression. The second part: To investigate the role of CD40 in regulation of the biological functions of MDSC Objective: to CD40 molecules as the starting point, to investigate the regulation of MDSC apoptosis and related mechanism of regulation of T cell proliferation inhibition and induced by Treg since the separation of spleen. Methods: adjust the cell concentration of purified Gr-1+CD11b+ cells plated on 24 well plates. Each hole with 2 L GM-CSF, respectively IL-4, IL-6, IL-10, TNF- alpha, GM-CSF, LPS, PEG2 and IFN- gamma, MFC stimulated for 24 h, 72h, 48h, cells were collected and detected the expression CD40 on MDSC by flow cytometry. From multisort obtained mice spleen MDSC suspension, spread in 96 well plates, the control group joined PBS agonist CD40 antibody was added in experimental group, 5%CO2 incubator after 24 h culture were collected by centrifugation of the hole cells, flow cytometry analysis of MDSC cells 鑳?yōu)鍑嬩骸鐜?灝嗗垎閫夎幏寰楃殑灝忛紶鑴捐剰MDSC涓嶤FSE鏌撹壊T緇嗚優(yōu)鎸夌収1:3鐨勬瘮渚，

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