MICA介导DC与NK细胞相互作用的初步研究
发布时间：2018-07-21 09:20
【摘要】： NK细胞不仅直接杀伤肿瘤细胞和病毒感染的细胞,还分泌细胞因子调节机体的获得性免疫功能。树突状细胞(DC)是激活初始T细胞、启动特异性免疫应答的重要抗原递呈细胞。前期研究表明:NK细胞与树突状细胞(DC)之间的对话(crosstalk)不仅影响天然免疫功能,还对获得性免疫的形成及发展有深远的影响。DC激活NK细胞,并通过上调NK细胞的活性而增强其抗肿瘤作用。反之,活化的NK细胞促进DC成熟,并诱导Th0细胞向Th1方向发展,从而增强机体的免疫防御和免疫监视功能。 MHC I类相关分子A(Major histocompatibility complex class I chain-related protein A,MICA),是NK细胞活化性受体NKG2D的配体,MICA与NKG2D结合后激活NK细胞,发挥其细胞毒活性和分泌IFN-γ等细胞因子。尽管单核细胞表面无MICA的表达,胞浆内却存在MICA分子。因此,本文试图研究MICA如何介导DC与NK细胞的相互作用,以及MICA参与DC与NK细胞之间的对话是否与结肠癌的免疫逃逸有关,从而深入探讨MICA参与肿瘤发病的机制。本研究分为三部分: 第一部分:DC表达MICA对NK细胞活性的影响 目的:观察单核细胞分化为DC时是否表达MICA,以及DC表达MICA后对NK细胞活性的影响。 方法:首先分离正常个体和结肠癌患者外周血单个核细胞(PBMC),经重组GM-CSF、IL-4诱导为未成熟DC(iDC)。然后分别用LPS、TNF-α、CD40L、IL-15和IFN-α刺激成熟,流式细胞仪(FCM)检测MICA在单核细胞、iDC以及成熟DC(mDC)细胞表面的表达。并观察DC表达MICA后对NK细胞表达CD69、细胞毒活性和IFN-γ分泌的影响。最后分别利用重组NKG2D-Ig蛋白和IL-12抗体阻断实验观察对DC激活NK细胞的影响。 结果:健康个体和结肠癌患者单核细胞均微量表达MICA,iDC表面MICA表达量均提高。与iDC相比,LPS、TNF-α和CD40L刺激的mDC表面MICA表达无明显变化,但IFN-α、IL-15可刺激健康个体的成熟DC上调表达MICA,却不能促进结肠癌患者的成熟DC上调MICA的表达。受IFN-α刺激成熟的DC促进NK细胞表达CD69、分泌IFN-γ、杀伤K562细胞。在DC激活NK细胞的培养体系中加入NKG2D-Ig蛋白可下调NK细胞毒活性和分泌IFN-γ,而加入IL-12抗体仅下调NK细胞分泌IFN-γ,对细胞毒功能无影响。 结论:正常个体iDC和mDC细胞表面均表达MICA,IFN-α、IL-15刺激mDC上调MICA表达。DC表达MICA增强NK细胞活性,且其细胞毒活性的提高依赖于DC表面相关配体与NK细胞表面NKG2D的相互作用。结肠癌患者体内mDC低表达MICA可能是其免疫逃逸的机制之一。 第二部分:受MICA刺激的NK细胞对DC活性的影响 目的:观察固相MICA(iMICA)是否协同NK细胞对iDC的作用,以及受NK细胞攻击的MICA阳性靶细胞是否促进DC吞噬。 方法:首先分别取异体新鲜分离的NK细胞、或受固相MICA刺激的异体NK细胞,以及IL-2、或IL-2联合iMICA刺激的自体NK细胞与iDC按5:1孵育,24小时后FCM分析CD86或HLA-DR阳性细胞频率。其次取自体NK细胞以IL-2、或IL-2联合iMICA刺激后按1:5比例与iDC孵育,24小时后FCM检测HLA-DR分子的表达变化,并通过IFN-γ抗体阻断实验观察iMICA刺激的NK细胞是否通过分泌IFN-γ促进DC成熟。最后分别将受LAK细胞攻击的、或丝裂霉素C处理的CFSE标记的K562、K562-MICA细胞,与iDC作用24小时后FCM检测HLA-DR与CFSE双阳性细胞频率。 结果:当NK与iDC孵育比例为5:1时,同种异体新鲜NK细胞及自体活化NK细胞均能杀伤iDC,而固相MICA无协同作用。当NK与iDC孵育比例为1:5时,固相MICA协同自体NK细胞诱导iDC成熟,而加入IFN-γ抗体可以下调DC表达HLA-DR。当K562、K562-MICA细胞受LAK细胞攻击或丝裂霉素C处理后,K562-MICA细胞易被DC吞噬。 结论:当IL-2活化的NK细胞杀伤iDC时,无需iMICA刺激。但固相MICA可以刺激NK细胞分泌IFN-γ促进DC成熟。MICA阳性的肿瘤细胞被NK细胞攻击后促进iDC吞噬。 第三部分:结肠癌患者外周血单核细胞MICA表达与NK细胞相关表型的关联分析 目的:比较结肠癌患者与健康个体外周血MICA表达的单核细胞频率,以及NKG2D、CD16、CD69阳性NK细胞频率的差异,并探讨体内单核细胞MICA表达与NK活性相关受体的表达是否存在关联。 方法:取结肠癌患者以及健康个体外周血,常规分离PBMC,用不同颜色荧光单克隆抗体分别标记CD14/MICA,CD56/NKG2D,CD56/CD69,CD56/CD16,流式细胞仪分析双阳性细胞频率。利用直线相关回归分析单核细胞MICA表达频率与NK表达NKG2D及CD69频率的关联性。 结果:结肠癌患者与健康个体外周血CD14+/MICA+单核细胞频率无明显差异。结肠癌患者CD56+/NKG2D+细胞,CD56+/CD69+细胞频率与健康个体相比明显下调,但CD56+/CD16+细胞频率无差异。另外,单核细胞MICA的表达与NK细胞NKG2D及CD69的表达无明显关联。 结论:结肠癌患者与健康个体外周血MICA阳性的单核细胞频率均很低,结肠癌患者体内由NKG2D受体介导的NK细胞活性较健康个体明显降低,但外周血单核细胞MICA的表达与NK细胞功能无明显关联。 综上所述,MICA分子直接不仅介导DC与NK细胞之间的对话,并在MICA阳性肿瘤患者体内发挥着激活NK细胞,诱导DC吞噬凋亡肿瘤碎片并交叉递呈抗原、启动特异性免疫应答的作用。因此,基于增强DC与NK细胞对话的生物治疗措施将具有显著的抗肿瘤效果。
[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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2135064
[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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
【共引文献】
相关博士学位论文 前3条
1 潘建平;γ-干扰素对树突状细胞分化和功能成熟的调控及其基因修饰的树突状细胞抗肿瘤免疫机制研究[D];浙江大学;2002年
2 郭军;热休克肿瘤细胞上清及EXOSOME中趋化因子的发现及一种非细胞结构性新型高效肿瘤疫苗的研究[D];第二军医大学;2002年
3 蒋宇光;血IP-10、Mig、BLC水平和CXCR3基因多态性对肾移植预后的影响及尿IP-10、Mig、MIP-3α水平对术后早期移植肾功能异常诊断价值的研究[D];浙江大学;2007年
相关硕士学位论文 前2条
1 翁跃颂;血管活性肠肽对树突状细胞趋化活性及细胞因子分泌的调节作用[D];浙江大学;2006年
2 何宋兵;趋化因子MIP-1α动员的DC经基因修饰后抗胃癌效应的研究[D];苏州大学;2006年
，本文编号：2135064
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2135064.html
最近更新
教材专著
