【摘要】： NK細(xì)胞不僅直接殺傷腫瘤細(xì)胞和病毒感染的細(xì)胞,還分泌細(xì)胞因子調(diào)節(jié)機(jī)體的獲得性免疫功能。樹突狀細(xì)胞(DC)是激活初始T細(xì)胞、啟動(dòng)特異性免疫應(yīng)答的重要抗原遞呈細(xì)胞。前期研究表明:NK細(xì)胞與樹突狀細(xì)胞(DC)之間的對(duì)話(crosstalk)不僅影響天然免疫功能,還對(duì)獲得性免疫的形成及發(fā)展有深遠(yuǎn)的影響。DC激活NK細(xì)胞,并通過上調(diào)NK細(xì)胞的活性而增強(qiáng)其抗腫瘤作用。反之,活化的NK細(xì)胞促進(jìn)DC成熟,并誘導(dǎo)Th0細(xì)胞向Th1方向發(fā)展,從而增強(qiáng)機(jī)體的免疫防御和免疫監(jiān)視功能。 MHC I類相關(guān)分子A(Major histocompatibility complex class I chain-related protein A,MICA),是NK細(xì)胞活化性受體NKG2D的配體,MICA與NKG2D結(jié)合后激活NK細(xì)胞,發(fā)揮其細(xì)胞毒活性和分泌IFN-γ等細(xì)胞因子。盡管單核細(xì)胞表面無MICA的表達(dá),胞漿內(nèi)卻存在MICA分子。因此,本文試圖研究MICA如何介導(dǎo)DC與NK細(xì)胞的相互作用,以及MICA參與DC與NK細(xì)胞之間的對(duì)話是否與結(jié)腸癌的免疫逃逸有關(guān),從而深入探討MICA參與腫瘤發(fā)病的機(jī)制。本研究分為三部分: 第一部分:DC表達(dá)MICA對(duì)NK細(xì)胞活性的影響 目的:觀察單核細(xì)胞分化為DC時(shí)是否表達(dá)MICA,以及DC表達(dá)MICA后對(duì)NK細(xì)胞活性的影響。 方法:首先分離正常個(gè)體和結(jié)腸癌患者外周血單個(gè)核細(xì)胞(PBMC),經(jīng)重組GM-CSF、IL-4誘導(dǎo)為未成熟DC(iDC)。然后分別用LPS、TNF-α、CD40L、IL-15和IFN-α刺激成熟,流式細(xì)胞儀(FCM)檢測(cè)MICA在單核細(xì)胞、iDC以及成熟DC(mDC)細(xì)胞表面的表達(dá)。并觀察DC表達(dá)MICA后對(duì)NK細(xì)胞表達(dá)CD69、細(xì)胞毒活性和IFN-γ分泌的影響。最后分別利用重組NKG2D-Ig蛋白和IL-12抗體阻斷實(shí)驗(yàn)觀察對(duì)DC激活NK細(xì)胞的影響。 結(jié)果:健康個(gè)體和結(jié)腸癌患者單核細(xì)胞均微量表達(dá)MICA,iDC表面MICA表達(dá)量均提高。與iDC相比,LPS、TNF-α和CD40L刺激的mDC表面MICA表達(dá)無明顯變化,但I(xiàn)FN-α、IL-15可刺激健康個(gè)體的成熟DC上調(diào)表達(dá)MICA,卻不能促進(jìn)結(jié)腸癌患者的成熟DC上調(diào)MICA的表達(dá)。受IFN-α刺激成熟的DC促進(jìn)NK細(xì)胞表達(dá)CD69、分泌IFN-γ、殺傷K562細(xì)胞。在DC激活NK細(xì)胞的培養(yǎng)體系中加入NKG2D-Ig蛋白可下調(diào)NK細(xì)胞毒活性和分泌IFN-γ,而加入IL-12抗體僅下調(diào)NK細(xì)胞分泌IFN-γ,對(duì)細(xì)胞毒功能無影響。 結(jié)論:正常個(gè)體iDC和mDC細(xì)胞表面均表達(dá)MICA,IFN-α、IL-15刺激mDC上調(diào)MICA表達(dá)。DC表達(dá)MICA增強(qiáng)NK細(xì)胞活性,且其細(xì)胞毒活性的提高依賴于DC表面相關(guān)配體與NK細(xì)胞表面NKG2D的相互作用。結(jié)腸癌患者體內(nèi)mDC低表達(dá)MICA可能是其免疫逃逸的機(jī)制之一。 第二部分:受MICA刺激的NK細(xì)胞對(duì)DC活性的影響 目的:觀察固相MICA(iMICA)是否協(xié)同NK細(xì)胞對(duì)iDC的作用,以及受NK細(xì)胞攻擊的MICA陽(yáng)性靶細(xì)胞是否促進(jìn)DC吞噬。 方法:首先分別取異體新鮮分離的NK細(xì)胞、或受固相MICA刺激的異體NK細(xì)胞,以及IL-2、或IL-2聯(lián)合iMICA刺激的自體NK細(xì)胞與iDC按5:1孵育,24小時(shí)后FCM分析CD86或HLA-DR陽(yáng)性細(xì)胞頻率。其次取自體NK細(xì)胞以IL-2、或IL-2聯(lián)合iMICA刺激后按1:5比例與iDC孵育,24小時(shí)后FCM檢測(cè)HLA-DR分子的表達(dá)變化,并通過IFN-γ抗體阻斷實(shí)驗(yàn)觀察iMICA刺激的NK細(xì)胞是否通過分泌IFN-γ促進(jìn)DC成熟。最后分別將受LAK細(xì)胞攻擊的、或絲裂霉素C處理的CFSE標(biāo)記的K562、K562-MICA細(xì)胞,與iDC作用24小時(shí)后FCM檢測(cè)HLA-DR與CFSE雙陽(yáng)性細(xì)胞頻率。 結(jié)果:當(dāng)NK與iDC孵育比例為5:1時(shí),同種異體新鮮NK細(xì)胞及自體活化NK細(xì)胞均能殺傷iDC,而固相MICA無協(xié)同作用。當(dāng)NK與iDC孵育比例為1:5時(shí),固相MICA協(xié)同自體NK細(xì)胞誘導(dǎo)iDC成熟,而加入IFN-γ抗體可以下調(diào)DC表達(dá)HLA-DR。當(dāng)K562、K562-MICA細(xì)胞受LAK細(xì)胞攻擊或絲裂霉素C處理后,K562-MICA細(xì)胞易被DC吞噬。 結(jié)論:當(dāng)IL-2活化的NK細(xì)胞殺傷iDC時(shí),無需iMICA刺激。但固相MICA可以刺激NK細(xì)胞分泌IFN-γ促進(jìn)DC成熟。MICA陽(yáng)性的腫瘤細(xì)胞被NK細(xì)胞攻擊后促進(jìn)iDC吞噬。 第三部分:結(jié)腸癌患者外周血單核細(xì)胞MICA表達(dá)與NK細(xì)胞相關(guān)表型的關(guān)聯(lián)分析 目的:比較結(jié)腸癌患者與健康個(gè)體外周血MICA表達(dá)的單核細(xì)胞頻率,以及NKG2D、CD16、CD69陽(yáng)性NK細(xì)胞頻率的差異,并探討體內(nèi)單核細(xì)胞MICA表達(dá)與NK活性相關(guān)受體的表達(dá)是否存在關(guān)聯(lián)。 方法:取結(jié)腸癌患者以及健康個(gè)體外周血,常規(guī)分離PBMC,用不同顏色熒光單克隆抗體分別標(biāo)記CD14/MICA,CD56/NKG2D,CD56/CD69,CD56/CD16,流式細(xì)胞儀分析雙陽(yáng)性細(xì)胞頻率。利用直線相關(guān)回歸分析單核細(xì)胞MICA表達(dá)頻率與NK表達(dá)NKG2D及CD69頻率的關(guān)聯(lián)性。 結(jié)果:結(jié)腸癌患者與健康個(gè)體外周血CD14+/MICA+單核細(xì)胞頻率無明顯差異。結(jié)腸癌患者CD56+/NKG2D+細(xì)胞,CD56+/CD69+細(xì)胞頻率與健康個(gè)體相比明顯下調(diào),但CD56+/CD16+細(xì)胞頻率無差異。另外,單核細(xì)胞MICA的表達(dá)與NK細(xì)胞NKG2D及CD69的表達(dá)無明顯關(guān)聯(lián)。 結(jié)論:結(jié)腸癌患者與健康個(gè)體外周血MICA陽(yáng)性的單核細(xì)胞頻率均很低,結(jié)腸癌患者體內(nèi)由NKG2D受體介導(dǎo)的NK細(xì)胞活性較健康個(gè)體明顯降低,但外周血單核細(xì)胞MICA的表達(dá)與NK細(xì)胞功能無明顯關(guān)聯(lián)。 綜上所述,MICA分子直接不僅介導(dǎo)DC與NK細(xì)胞之間的對(duì)話,并在MICA陽(yáng)性腫瘤患者體內(nèi)發(fā)揮著激活NK細(xì)胞,誘導(dǎo)DC吞噬凋亡腫瘤碎片并交叉遞呈抗原、啟動(dòng)特異性免疫應(yīng)答的作用。因此,基于增強(qiáng)DC與NK細(xì)胞對(duì)話的生物治療措施將具有顯著的抗腫瘤效果。
[Abstract]:NK cells not only directly kill tumor cells and virus infected cells, but also secrete cytokines to regulate the acquired immune function of the body. Dendritic cells (DC) are the important antigen presenting cells that activate the initial T cells and initiate specific immune responses. Previous studies showed that the dialogue between NK cells and dendritic cells (DC) is not only a crosstalk. Affecting the natural immune function, it also has a profound influence on the formation and development of the acquired immune system, which has a profound effect on the activation of NK cells by.DC, and enhances its anti-tumor effect by up regulating the activity of NK cells. On the contrary, the activated NK cells promote the maturation of DC and induce the Th0 cells to develop into Th1 direction, thus strengthening the immune defense and immune monitoring function of the body.
The MHC I related molecule A (Major histocompatibility complex class I chain-related protein A, MICA) is the ligand to activate the cell activation receptor, and activates its cytotoxic activity and secretes the cytokines. This article tries to investigate how MICA mediates the interaction between DC and NK cells, and whether the dialogue between MICA and DC and NK cells is related to the immune escape of colon cancer, thus exploring the mechanism of MICA involvement in the pathogenesis of cancer. This study is divided into three parts:
Part one: the effect of DC expressing MICA on the activity of NK cells.
Objective: To observe whether MICA is expressed in DC when monocytes differentiate into DC, and the effect of DC expression on NK cell activity.
Methods: first, peripheral blood mononuclear cells (PBMC) were isolated from normal individuals and colon cancer patients. After recombinant GM-CSF, IL-4 was induced to be immature DC (iDC). Then, LPS, TNF- a, CD40L, IL-15 and IFN- alpha were used to stimulate maturation. The effect of NK cells on the expression of CD69, cytotoxic activity and IFN- gamma secretion. Finally, the effects of recombinant NKG2D-Ig protein and IL-12 antibody on DC activation of NK cells were blocked respectively.
Results: the mononuclear cells of the healthy individuals and colon cancer patients both expressed MICA, and the expression of MICA on the surface of iDC increased. Compared with iDC, the MICA expression on the mDC surface stimulated by LPS, TNF- A and CD40L had no significant changes, but IFN- a, IL-15 could stimulate the expression of mature DC. DC stimulated by IFN- alpha stimulated NK cells to express CD69, secrete IFN- gamma and kill K562 cells. The addition of NKG2D-Ig protein in the culture system of DC activated NK cells could down regulate the cytotoxic activity of NK and secrete IFN- gamma.
Conclusion: the surface of iDC and mDC cells in normal individuals all express MICA, IFN- alpha, and IL-15 stimulates mDC to increase MICA expression to express MICA enhanced NK cell activity, and its cytotoxic activity is dependent on the interaction between DC surface related ligands and NK cell surfaces.
The second part: the effect of MICA stimulated NK cells on DC activity.
Objective: To observe whether solid MICA (iMICA) synergies with NK cells in iDC and whether MICA positive target cells attacked by NK cells promote DC phagocytosis.
Methods: first, the fresh isolated NK cells, or the allogenic NK cells stimulated by the solid phase MICA, and the IL-2, or IL-2 combined iMICA stimulated autologous NK cells were incubated at 5:1, and the FCM CD86 or HLA-DR positive cells were analyzed 24 hours later. 4 hours later, FCM detected the changes in the expression of HLA-DR molecules, and through the IFN- gamma antibody blocking experiment to observe whether the NK cells stimulated by iMICA stimulated the maturation of DC by secreting IFN- gamma. Finally, LAK cells were attacked and CFSE markers of mitomycin C treatment were K562, K562-MICA cells were tested after 24 hours of action. Cell frequency.
Results: when the proportion of NK and iDC incubation was 5:1, both the allogenic fresh NK cells and the autologous NK cells could kill iDC, while the solid-phase MICA had no synergistic effect. When NK and iDC incubated in 1:5, the solid-phase MICA coordinated autologous NK cells to induce maturation. After being treated with mitomycin C or K562-MICA, the DC cells were easily swallowed.
Conclusion: when IL-2 activated NK cells kill iDC, no iMICA stimulation is needed, but solid phase MICA stimulates the secretion of IFN- gamma by NK cells to promote DC mature.MICA positive tumor cells to promote iDC phagocytosis after NK cells attack.
The third part: correlation analysis of MICA expression in peripheral blood mononuclear cells and NK cell related phenotype in patients with colon cancer
Objective: To compare the frequency of mononuclear cells expressed by MICA in patients with colon cancer and healthy peripheral blood, and the difference in the frequency of NKG2D, CD16, CD69 positive NK cells, and to explore whether there is a correlation between the expression of MICA and the expression of NK activity related receptors in the monocytes in the body.
Methods: PBMC was isolated from patients with colorectal cancer and healthy peripheral blood. CD14/MICA, CD56/NKG2D, CD56/CD69, CD56/CD16, and flow cytometry were used to analyze the frequency of double positive cells with different color fluorescent monoclonal antibodies. The relationship between the expression of MICA expression of mononuclear cell MICA and the expression of NKG2D and CD69 in NK was analyzed by linear correlation regression. Sex.
Results: there was no significant difference in the frequency of CD14+/MICA+ mononuclear cells from patients with colon cancer and healthy individuals. The frequency of CD56+/CD69+ cells in CD56+/NKG2D+ cells of colon cancer patients was significantly lower than that of healthy individuals, but there was no difference in the frequency of CD56+/CD16+ cells. In addition, the expression of MICA in monocyte was not significantly associated with the expression of NKG2D and CD69 in NK cells.
Conclusion: the frequency of MICA positive mononuclear cells in patients with colon cancer and healthy peripheral blood is very low, and the activity of NK cells mediated by NKG2D receptor in colon cancer patients is significantly lower than that of healthy individuals, but the expression of MICA in peripheral blood mononuclear cells is not significantly related to the function of NK cells.
To sum up, MICA molecules directly not only mediate the dialogue between DC and NK cells, but also play a role in activating NK cells in the MICA positive tumor patients, inducing DC to phagocytic apoptotic tumor fragments, cross presenting antigen, and initiate specific immune responses. Therefore, biological therapy based on enhanced DC and NK cells will have significant resistance. Tumor effect.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

【共引文獻(xiàn)】

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1 潘建平;γ-干擾素對(duì)樹突狀細(xì)胞分化和功能成熟的調(diào)控及其基因修飾的樹突狀細(xì)胞抗腫瘤免疫機(jī)制研究[D];浙江大學(xué);2002年

2 郭軍;熱休克腫瘤細(xì)胞上清及EXOSOME中趨化因子的發(fā)現(xiàn)及一種非細(xì)胞結(jié)構(gòu)性新型高效腫瘤疫苗的研究[D];第二軍醫(yī)大學(xué);2002年

3 蔣宇光;血IP-10、Mig、BLC水平和CXCR3基因多態(tài)性對(duì)腎移植預(yù)后的影響及尿IP-10、Mig、MIP-3α水平對(duì)術(shù)后早期移植腎功能異常診斷價(jià)值的研究[D];浙江大學(xué);2007年

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1 翁躍頌;血管活性腸肽對(duì)樹突狀細(xì)胞趨化活性及細(xì)胞因子分泌的調(diào)節(jié)作用[D];浙江大學(xué);2006年

2 何宋兵;趨化因子MIP-1α動(dòng)員的DC經(jīng)基因修飾后抗胃癌效應(yīng)的研究[D];蘇州大學(xué);2006年

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本文編號(hào)：2135064