本文選題：NK + 細胞�。� 參考：《天津醫(yī)科大學》2017年博士論文

【摘要】：目的第一部分:當前NK細胞毒性的研究一直專注于激活性受體和抑制性受體的平衡、信號轉(zhuǎn)導、鈣離子內(nèi)流、免疫突觸的形成和細胞脫顆粒等機制。然而,在NK細胞被激活或識別靶細胞的過程中,其細胞核內(nèi)染色質(zhì)的結(jié)構(gòu)和狀態(tài)是否發(fā)生改變我們?nèi)匀徊磺宄Ｔ谶@一部分中,我們通過綜合分析一套表達譜芯片數(shù)據(jù)和兩套ChIP-seq數(shù)據(jù),研究NK細胞在被激活的過程中其組蛋白甲基化修飾狀態(tài)的變化。用一系列特異于催化H3K4和H3K27修飾酶的小分子抑制劑處理NK細胞以改變NK細胞甲基化修飾狀態(tài),研究NK細胞的脫顆粒水平,以及IFN-γ和TNF-α的表達。以期進一步分析組蛋白甲基化修飾狀態(tài)對NK細胞激活的影響。第二部分:盡管NK細胞激活的機制研究還不夠清晰,但很多研究顯示正常的鈣離子信號是NK細胞完成脫顆粒過程的必要因素。當鈣離子信號被破壞后,NK細胞毒性顆粒外泌能力受阻,殺傷能力降低。然而,鈣離子信號升高對NK細胞脫顆粒和殺傷的影響卻鮮有研究。我們課題組研究發(fā)現(xiàn)小分子抑制劑UNC1999(特異于EZH2)可以上調(diào)NK細胞的鈣流以及脫顆粒水平。因此,本課題的主要目的是研究UNC1999上調(diào)NK細胞鈣流的機制,以及進一步研究鈣離子信號與NK細胞脫顆粒和殺傷能力之間的關(guān)系。第三部分:我們前期研究發(fā)現(xiàn),在小鼠造血干細胞中敲除mEzh2(mouse Ezh2),可以觀察到小鼠骨髓、肝和脾中成熟NK細胞的絕對數(shù)和百分比被顯著上調(diào)。而且基因芯片結(jié)果發(fā)現(xiàn),mEzh2敲除的NKp細胞中,促進NK細胞后期成熟的轉(zhuǎn)錄因子Tox的表達被顯著上調(diào)。因此,我們懷疑組蛋白甲基化酶mEzh2可以調(diào)控小鼠NK細胞的終端成熟。基于以上理由,我們用mEzh2fl/fl小鼠與Ncr1i Cre小鼠雜交,繁殖特異于NK細胞的mEzh2條件敲除小鼠,旨在研究mEzh2與NK細胞晚期發(fā)育和抗腫瘤能力之間的關(guān)系。方法第一部分:1.通過綜合分析一套表達譜芯片數(shù)據(jù)和兩套ChIP-seq數(shù)據(jù),研究NK細胞在被激活的過程中,組蛋白甲基化修飾酶的表達變化和相關(guān)基因啟動子區(qū)組蛋白甲基化修飾狀態(tài)的變化。2.qPCR和Western blot研究NK細胞被激活前后,組蛋白甲基轉(zhuǎn)移酶和去甲基化酶的表達水平。3.ChIP-qPCR研究NK活性相關(guān)基因的啟動子區(qū)H3K4me3和H3K27me3的修飾在NK細胞被激活前后的變化。4.用一系列特異于催化H3K4和H3K27修飾酶的小分子抑制劑處理NK細胞以改變NK細胞甲基化修飾狀態(tài),用流式細胞術(shù)檢測其脫顆粒水平和細胞中IFN-γ與TNF-α的表達水平。第二部分1.小分子抑制劑UNC1999和GSK343分別處理NK細胞,抑制EZH2的酶活性。用慢病毒法感染NK細胞,抑制NK細胞中EZH2的表達。2.流式細胞術(shù)檢測NK細胞在處理前后的脫顆粒水平、殺傷能力和鈣離子流。3.激光共聚焦顯微鏡觀察NK細胞在處理前后與靶細胞結(jié)合、免疫突觸形成和細胞毒顆粒極化的能力。4.利用高通量RNA測序技術(shù)篩選調(diào)控鈣離子上調(diào)表型的因子。5.qPCR、Western blot和ChIP-qPCR研究UNC1999上調(diào)鈣離子信號的分子機制。第三部分1.Western blot檢測mEzh2條件敲除小鼠NK細胞中mEzh2的表達,以及H3K27me3修飾水平。2.流式細胞術(shù)檢測NK細胞表面分化標志物以及功能蛋白IFN-γ的表達。3.小鼠尾靜脈注射肺轉(zhuǎn)移模型檢測mEzh2條件敲除小鼠的抗腫瘤能力。結(jié)果第一部分:1.表達譜芯片中發(fā)現(xiàn)八個組蛋白甲基轉(zhuǎn)移酶和組蛋白去甲基化酶的表達在NK細胞被激活后發(fā)生變化。2.qPCR和Western blot驗證組蛋白甲基轉(zhuǎn)移酶JARID2和組蛋白去甲基化酶UTY與KDM6B在NK細胞被激活后表達上調(diào)。3.ChIP-seq分析發(fā)現(xiàn)27個與NK功能相關(guān)的基因啟動子區(qū)被H3K4me3和H3K27me3同時修飾,并且這些基因中的77.8%在NK細胞被激活后表達上調(diào)。4.ChIP-qPCR驗證在NK細胞被激活后,PI3KCA、NFATC1和TNFSF9基因啟動子區(qū)H3K4me3和H3K27me3的修飾發(fā)生顯著變化。5.小分子抑制劑UNC1999可以上調(diào)NK細胞的脫顆粒水平,而OG-L002和MM102可以提高NK細胞中IFN-γ與TNF-α的表達。第二部分:1.小分子抑制劑UNC1999和GSK343抑制NK細胞中EZH2酶活性,或用sh RNA抑制NK細胞中EZH2表達,可以上調(diào)NK細胞的脫顆粒水平和鈣離子信號,下調(diào)NK細胞短時間內(nèi)的殺傷效率。2.抑制EZH2酶活性不影響NK細胞與靶細胞結(jié)合、免疫突觸形成和毒性顆粒極化。3.抑制EZH2酶活性導致鈣離子信號異常升高,進而導致異常毒性顆粒釋放,最終導致很少的靶細胞可以獲得毒性顆粒。4.抑制EZH2酶活性,導致鈣離子通道PKD2啟動子區(qū)H3K27me3修飾被抑制,并最終上調(diào)鈣離子通道PKD2的表達。第三部分:1.mEzh2條件敲除小鼠的NKp46+CD3-NK細胞中mEzh2表達被顯著下調(diào);與此同時,H3K27me3的修飾水平也顯著降低。2.mEzh2條件敲除小鼠的脾、骨髓和淋巴結(jié)中NKp46+CD3-NK細胞的百分比和絕對數(shù)同時上調(diào);而且mEzh2條件敲除小鼠的NK細胞更加成熟。3.mEzh2條件敲除小鼠的NKp46+CD3-NK細胞被激活后,IFN-γ的表達水平顯著高于野生型小鼠的NK細胞;而且小鼠尾靜脈注射肺轉(zhuǎn)移模型中觀察到mEzh2條件敲除小鼠的抗腫瘤能力更強。結(jié)論第一部分:1.NK細胞在靜息狀態(tài)下,很多功能相關(guān)基因的啟動子區(qū)被H3K4me3和H3K27me3同時修飾,處于“poised”狀態(tài)。2.NK細胞在激活過程中,部分組蛋白甲基轉(zhuǎn)移酶和組蛋白去甲基化酶表達發(fā)生變化,而與NK功能相關(guān)基因的啟動子區(qū)甲基化修飾狀態(tài)也發(fā)生變化,說明組蛋白甲基化修飾在NK細胞激活過程中起重要的作用。3.針對不同組蛋白甲基轉(zhuǎn)移酶和去甲基化酶的小分子抑制劑UNC1999、OG-L002和MM102可能提高NK細胞毒性,為調(diào)控NK細胞毒性和免疫調(diào)節(jié)能力提供了新的思路。第二部分:1.抑制EZH2的表達或者酶活性可以上調(diào)NK細胞內(nèi)質(zhì)網(wǎng)中鈣離子的儲存,從而上調(diào)NK細胞內(nèi)的鈣離子信號,進而導致NK細胞在短時間內(nèi)釋放大量毒性顆粒到很少的靶細胞,下調(diào)NK細胞短時間內(nèi)的殺傷效率。2.靜息狀態(tài)下的NK細胞中鈣離子通道PKD2的啟動子區(qū)被H3K4me3和H3K27me3同時修飾,抑制EZH2的表達或酶活性,可以改變PKD2啟動子區(qū)的甲基化修飾狀態(tài),從而上調(diào)PKD2的表達水平。第三部分:1.在小鼠NK細胞的發(fā)育過程中,mEzh2不僅參與NK細胞的早期分化,而且調(diào)控NK細胞的終端成熟。2.mEzh2條件敲除小鼠的NK細胞在被激活后表達更高的IFN-γ,能更好的控制體內(nèi)腫瘤的增殖和轉(zhuǎn)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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R3416

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