组蛋白修饰酶调控NK细胞成熟与功能

发布时间：2018-05-29 06:23

本文选题：NK + 细胞　；参考：《天津医科大学》2017年博士论文

【摘要】：目的第一部分:当前NK细胞毒性的研究一直专注于激活性受体和抑制性受体的平衡、信号转导、钙离子内流、免疫突触的形成和细胞脱颗粒等机制。然而,在NK细胞被激活或识别靶细胞的过程中,其细胞核内染色质的结构和状态是否发生改变我们仍然不清楚。在这一部分中,我们通过综合分析一套表达谱芯片数据和两套ChIP-seq数据,研究NK细胞在被激活的过程中其组蛋白甲基化修饰状态的变化。用一系列特异于催化H3K4和H3K27修饰酶的小分子抑制剂处理NK细胞以改变NK细胞甲基化修饰状态,研究NK细胞的脱颗粒水平,以及IFN-γ和TNF-α的表达。以期进一步分析组蛋白甲基化修饰状态对NK细胞激活的影响。第二部分:尽管NK细胞激活的机制研究还不够清晰,但很多研究显示正常的钙离子信号是NK细胞完成脱颗粒过程的必要因素。当钙离子信号被破坏后,NK细胞毒性颗粒外泌能力受阻,杀伤能力降低。然而,钙离子信号升高对NK细胞脱颗粒和杀伤的影响却鲜有研究。我们课题组研究发现小分子抑制剂UNC1999(特异于EZH2)可以上调NK细胞的钙流以及脱颗粒水平。因此,本课题的主要目的是研究UNC1999上调NK细胞钙流的机制,以及进一步研究钙离子信号与NK细胞脱颗粒和杀伤能力之间的关系。第三部分:我们前期研究发现,在小鼠造血干细胞中敲除mEzh2(mouse Ezh2),可以观察到小鼠骨髓、肝和脾中成熟NK细胞的绝对数和百分比被显著上调。而且基因芯片结果发现,mEzh2敲除的NKp细胞中,促进NK细胞后期成熟的转录因子Tox的表达被显著上调。因此,我们怀疑组蛋白甲基化酶mEzh2可以调控小鼠NK细胞的终端成熟。基于以上理由,我们用mEzh2fl/fl小鼠与Ncr1i Cre小鼠杂交,繁殖特异于NK细胞的mEzh2条件敲除小鼠,旨在研究mEzh2与NK细胞晚期发育和抗肿瘤能力之间的关系。方法第一部分:1.通过综合分析一套表达谱芯片数据和两套ChIP-seq数据,研究NK细胞在被激活的过程中,组蛋白甲基化修饰酶的表达变化和相关基因启动子区组蛋白甲基化修饰状态的变化。2.qPCR和Western blot研究NK细胞被激活前后,组蛋白甲基转移酶和去甲基化酶的表达水平。3.ChIP-qPCR研究NK活性相关基因的启动子区H3K4me3和H3K27me3的修饰在NK细胞被激活前后的变化。4.用一系列特异于催化H3K4和H3K27修饰酶的小分子抑制剂处理NK细胞以改变NK细胞甲基化修饰状态,用流式细胞术检测其脱颗粒水平和细胞中IFN-γ与TNF-α的表达水平。第二部分1.小分子抑制剂UNC1999和GSK343分别处理NK细胞,抑制EZH2的酶活性。用慢病毒法感染NK细胞,抑制NK细胞中EZH2的表达。2.流式细胞术检测NK细胞在处理前后的脱颗粒水平、杀伤能力和钙离子流。3.激光共聚焦显微镜观察NK细胞在处理前后与靶细胞结合、免疫突触形成和细胞毒颗粒极化的能力。4.利用高通量RNA测序技术筛选调控钙离子上调表型的因子。5.qPCR、Western blot和ChIP-qPCR研究UNC1999上调钙离子信号的分子机制。第三部分1.Western blot检测mEzh2条件敲除小鼠NK细胞中mEzh2的表达,以及H3K27me3修饰水平。2.流式细胞术检测NK细胞表面分化标志物以及功能蛋白IFN-γ的表达。3.小鼠尾静脉注射肺转移模型检测mEzh2条件敲除小鼠的抗肿瘤能力。结果第一部分:1.表达谱芯片中发现八个组蛋白甲基转移酶和组蛋白去甲基化酶的表达在NK细胞被激活后发生变化。2.qPCR和Western blot验证组蛋白甲基转移酶JARID2和组蛋白去甲基化酶UTY与KDM6B在NK细胞被激活后表达上调。3.ChIP-seq分析发现27个与NK功能相关的基因启动子区被H3K4me3和H3K27me3同时修饰,并且这些基因中的77.8%在NK细胞被激活后表达上调。4.ChIP-qPCR验证在NK细胞被激活后,PI3KCA、NFATC1和TNFSF9基因启动子区H3K4me3和H3K27me3的修饰发生显著变化。5.小分子抑制剂UNC1999可以上调NK细胞的脱颗粒水平,而OG-L002和MM102可以提高NK细胞中IFN-γ与TNF-α的表达。第二部分:1.小分子抑制剂UNC1999和GSK343抑制NK细胞中EZH2酶活性,或用sh RNA抑制NK细胞中EZH2表达,可以上调NK细胞的脱颗粒水平和钙离子信号,下调NK细胞短时间内的杀伤效率。2.抑制EZH2酶活性不影响NK细胞与靶细胞结合、免疫突触形成和毒性颗粒极化。3.抑制EZH2酶活性导致钙离子信号异常升高,进而导致异常毒性颗粒释放,最终导致很少的靶细胞可以获得毒性颗粒。4.抑制EZH2酶活性,导致钙离子通道PKD2启动子区H3K27me3修饰被抑制,并最终上调钙离子通道PKD2的表达。第三部分:1.mEzh2条件敲除小鼠的NKp46+CD3-NK细胞中mEzh2表达被显著下调;与此同时,H3K27me3的修饰水平也显著降低。2.mEzh2条件敲除小鼠的脾、骨髓和淋巴结中NKp46+CD3-NK细胞的百分比和绝对数同时上调;而且mEzh2条件敲除小鼠的NK细胞更加成熟。3.mEzh2条件敲除小鼠的NKp46+CD3-NK细胞被激活后,IFN-γ的表达水平显著高于野生型小鼠的NK细胞;而且小鼠尾静脉注射肺转移模型中观察到mEzh2条件敲除小鼠的抗肿瘤能力更强。结论第一部分:1.NK细胞在静息状态下,很多功能相关基因的启动子区被H3K4me3和H3K27me3同时修饰,处于“poised”状态。2.NK细胞在激活过程中,部分组蛋白甲基转移酶和组蛋白去甲基化酶表达发生变化,而与NK功能相关基因的启动子区甲基化修饰状态也发生变化,说明组蛋白甲基化修饰在NK细胞激活过程中起重要的作用。3.针对不同组蛋白甲基转移酶和去甲基化酶的小分子抑制剂UNC1999、OG-L002和MM102可能提高NK细胞毒性,为调控NK细胞毒性和免疫调节能力提供了新的思路。第二部分:1.抑制EZH2的表达或者酶活性可以上调NK细胞内质网中钙离子的储存,从而上调NK细胞内的钙离子信号,进而导致NK细胞在短时间内释放大量毒性颗粒到很少的靶细胞,下调NK细胞短时间内的杀伤效率。2.静息状态下的NK细胞中钙离子通道PKD2的启动子区被H3K4me3和H3K27me3同时修饰,抑制EZH2的表达或酶活性,可以改变PKD2启动子区的甲基化修饰状态,从而上调PKD2的表达水平。第三部分:1.在小鼠NK细胞的发育过程中,mEzh2不仅参与NK细胞的早期分化,而且调控NK细胞的终端成熟。2.mEzh2条件敲除小鼠的NK细胞在被激活后表达更高的IFN-γ,能更好的控制体内肿瘤的增殖和转移。
[Abstract]:Objective Part 1: the current study of NK cytotoxicity has focused on the balance of active and inhibitory receptors, signal transduction, calcium ion inflow, the formation of immune synapses and cell degranulation. However, whether the structure and state of chromatin in the nucleus changes in the process of activation or identification of target cells in NK cells. In this part, we study the changes in the methylation modification of NK cells in the process of activation by a comprehensive analysis of a set of expressed spectral chip data and two sets of ChIP-seq data. A series of small molecular inhibitors that catalyze H3K4 and H3K27 modified enzymes are used to process NK cells to change NK fines. The demylation status, the degranulation level of NK cells, and the expression of IFN- gamma and TNF- alpha, in order to further analyze the effect of the histone methylation modification on the activation of NK cells. Second part: Although the mechanism of NK cell activation is not clear enough, a lot of studies show that normal calcium signals are NK cells complete defragmentation. When the calcium signal was destroyed, the extracellular secretion of NK cytotoxic particles was hindered and the killing ability decreased. However, the effect of the increase of calcium ion signal on the degranulation and killing of NK cells was rarely studied. Our research group found that the small molecule inhibitor UNC1999 (specific to EZH2) could increase the calcium flow in NK cells. The main purpose of this project is to study the mechanism of UNC1999 to increase the calcium flow in NK cells and to further study the relationship between calcium ion signal and the degranulation and killing ability of NK cells. The third part: we found that the mouse bone marrow can be observed by knocking out the mEzh2 (mouse Ezh2) in the mouse hematopoietic stem cells. The absolute number and percentage of mature NK cells in the liver and spleen were significantly up-regulated. Moreover, the gene chip results showed that the expression of Tox, the transcription factor promoting the late maturation of NK cells, was significantly up-regulated in the mEzh2 knockout NKp cells. Therefore, we suspect that the histone methylase mEzh2 can regulate the terminal maturation of the mouse NK cells. Based on the above reasons, We hybridized mEzh2fl/fl mice with Ncr1i Cre mice and propagated mEzh2 conditional knockout mice specific to NK cells. The purpose of this study was to study the relationship between the late development of mEzh2 and NK cells and the anti-tumor ability of NK cells. Method first part: 1. the process of activation of NK cells was studied through a comprehensive analysis of a set of expression chip data and two sets of ChIP-seq data. Changes in the expression of histone methylation modified enzymes and changes in the methylation modification status of related genes in promoter region.2.qPCR and Western blot study the expression level of histone methyltransferase and demethylation enzyme.3.ChIP-qPCR before and after the activation of NK cells to study the repair of H3K4me3 and H3K27me3 in the promoter region of the NK active phase gene. Changes in NK cells before and after activation of.4. cells use a series of small molecular inhibitors that catalyze H3K4 and H3K27 modifier to treat NK cells to change the methylation status of NK cells. Flow cytometry is used to detect the level of degranulation and the expression level of IFN- gamma and TNF- a in cells. Second part 1. small molecule inhibitors, UNC1999 and GSK343 points Do not deal with NK cells and inhibit the activity of EZH2. NK cells were infected with lentivirus, and the expression of EZH2 in NK cells was suppressed by.2. flow cytometry. The degranulation level of NK cells before and after treatment was detected. The killing ability and calcium ion flow.3. laser confocal microscope observed the combination of NK cells with the target cells before treatment and the formation of immune synapses and cytotoxicity. Particle polarization ability.4. uses high throughput RNA sequencing technology to screen factor.5.qPCR regulating the up-regulated calcium ion, Western blot and ChIP-qPCR to study the molecular mechanism of UNC1999 up regulation of calcium ion signals. Third part 1.Western blot detection of mEzh2 in NK cells in mEzh2 conditional knockout mice and fine flow refinement Detection of the surface differentiation markers of NK cells and the expression of functional protein IFN- gamma by cytosolic technique,.3. mice were injected with lung transfer model in the tail vein to detect the anti-tumor ability of mEzh2 conditional knockout mice. Results the first part: the 1. expression spectrum chip found that eight histone methyltransferases and histone demethylation enzymes were expressed after the activation of NK cells. .2.qPCR and Western blot verifying histone methyltransferase JARID2 and histone demethylation enzyme UTY and KDM6B up regulated.3.ChIP-seq analysis after NK cells were activated, and found that 27 gene promoter regions associated with NK function were modified by H3K4me3 and H3K27me3, and 77.8% of these genes were expressed after the NK cells were activated. After activation of NK cells, the modification of H3K4me3 and H3K27me3 in the promoter region of PI3KCA, NFATC1 and TNFSF9 genes changes significantly after the activation of the PI3KCA, NFATC1 and TNFSF9 genes. The.5. small molecule inhibitor UNC1999 can increase the degranulation level of NK cells, while OG-L002 and MM102 can increase the expression of.5. and alpha in the cells. Second part: 1. small molecule inhibitors UNC1999 and GSK343 inhibit the activity of EZH2 enzyme in NK cells, or the inhibition of EZH2 expression in NK cells by SH RNA, it can up regulate the degranulation level and calcium ion signal of NK cells. Down regulation of the killing efficiency of NK cells in short time inhibits EZH2 enzyme activity and does not affect the binding of the cells to the target cells, the formation of immunization synapses and the inhibition of the toxic granule polarization. The activity leads to the abnormal increase of calcium ion signal, which leads to the release of abnormal toxic particles, which eventually leads to the inhibition of EZH2 enzyme activity by a few target cells.4., resulting in the inhibition of H3K27me3 modification in the promoter region of the calcium channel PKD2 and the expression of PKD2 in the calcium channel. The third part: 1.mEzh2 conditional knockout mice The expression of mEzh2 in the NKp46+CD3-NK cells was significantly downregulated; at the same time, the modification level of H3K27me3 also significantly reduced the spleen of the.2.mEzh2 knockout mice, the percentage and the absolute number of NKp46+CD3-NK cells in the bone marrow and lymph nodes, and the NK cells in the mEzh2 conditional knockout mice were more mature in the.3.mEzh2 conditional knockout mice. After the activation of D3-NK cells, the expression level of IFN- gamma was significantly higher than that of NK cells in the wild type mice; moreover, the mouse tail vein injection model of the lung was used to observe the anti-tumor ability of the mEzh2 conditional knockout mice. Conclusion the first part: the 1.NK cells in the resting state, the promoter region of many functional related genes is the same as H3K4me3 and H3K27me3. Time modification, the expression of partial histone methyltransferase and histone demethylation in the activation process of "poised" state.2.NK cells changes, and the methylation modification of the promoter region of NK function related genes also changes, indicating that the histone methylation repair plays an important role in the activation process of NK cells,.3. needle. Small molecular inhibitors, UNC1999, OG-L002 and MM102, of different histone methyltransferases and demethylation enzymes, may improve the toxicity of NK cells and provide new ideas for the regulation of NK cytotoxicity and immunoregulation. Second: 1. inhibition of the expression of EZH2 or enzyme activity can increase the storage of calcium ions in the endoplasmic reticulum of NK cells. Modulation of calcium ion signals in NK cells, resulting in NK cells releasing a large number of toxic particles in a short period of time to a few target cells, down the killing efficiency of NK cells in a short time,.2. in the resting state of NK cells, the promoter region of the calcium channel PKD2 is trimming simultaneously by H3K4me3 and H3K27me3, inhibiting the expression of EZH2 or enzyme activity, which can be changed. The methylation status of PKD2 promoter and up regulation of PKD2 expression. Third part: 1. in the development of NK cells in mice, mEzh2 not only participates in the early differentiation of NK cells, but also regulates the higher IFN- gamma expression in the NK cells of the terminal mature.2.mEzh2 knockout mice of NK cells after being stimulated, which can better control the body. The proliferation and metastasis of the tumor.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R3416

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