本文選題：NK + 細(xì)胞��； 參考：《天津醫(yī)科大學(xué)》2017年博士論文

【摘要】：目的第一部分:當(dāng)前NK細(xì)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(hào)轉(zhuǎn)導(dǎo)、鈣離子內(nèi)流、免疫突觸的形成和細(xì)胞脫顆粒等機(jī)制。然而,在NK細(xì)胞被激活或識(shí)別靶細(xì)胞的過(guò)程中,其細(xì)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�。在這一部分中,我們通過(guò)綜合分析一套表達(dá)譜芯片數(shù)據(jù)和兩套ChIP-seq數(shù)據(jù),研究NK細(xì)胞在被激活的過(guò)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(xì)胞以改變NK細(xì)胞甲基化修飾狀態(tài),研究NK細(xì)胞的脫顆粒水平,以及IFN-γ和TNF-α的表達(dá)。以期進(jìn)一步分析組蛋白甲基化修飾狀態(tài)對(duì)NK細(xì)胞激活的影響。第二部分:盡管NK細(xì)胞激活的機(jī)制研究還不夠清晰,但很多研究顯示正常的鈣離子信號(hào)是NK細(xì)胞完成脫顆粒過(guò)程的必要因素。當(dāng)鈣離子信號(hào)被破壞后,NK細(xì)胞毒性顆粒外泌能力受阻,殺傷能力降低。然而,鈣離子信號(hào)升高對(duì)NK細(xì)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(xì)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(xì)胞鈣流的機(jī)制,以及進(jìn)一步研究鈣離子信號(hào)與NK細(xì)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(xì)胞中敲除mEzh2(mouse Ezh2),可以觀察到小鼠骨髓、肝和脾中成熟NK細(xì)胞的絕對(duì)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(xì)胞中,促進(jìn)NK細(xì)胞后期成熟的轉(zhuǎn)錄因子Tox的表達(dá)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(xì)胞的終端成熟�；谝陨侠碛�,我們用mEzh2fl/fl小鼠與Ncr1i Cre小鼠雜交,繁殖特異于NK細(xì)胞的mEzh2條件敲除小鼠,旨在研究mEzh2與NK細(xì)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(guò)綜合分析一套表達(dá)譜芯片數(shù)據(jù)和兩套ChIP-seq數(shù)據(jù),研究NK細(xì)胞在被激活的過(guò)程中,組蛋白甲基化修飾酶的表達(dá)變化和相關(guān)基因啟動(dòng)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(xì)胞被激活前后,組蛋白甲基轉(zhuǎn)移酶和去甲基化酶的表達(dá)水平。3.ChIP-qPCR研究NK活性相關(guān)基因的啟動(dòng)子區(qū)H3K4me3和H3K27me3的修飾在NK細(xì)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(xì)胞以改變NK細(xì)胞甲基化修飾狀態(tài),用流式細(xì)胞術(shù)檢測(cè)其脫顆粒水平和細(xì)胞中IFN-γ與TNF-α的表達(dá)水平。第二部分1.小分子抑制劑UNC1999和GSK343分別處理NK細(xì)胞,抑制EZH2的酶活性。用慢病毒法感染NK細(xì)胞,抑制NK細(xì)胞中EZH2的表達(dá)。2.流式細(xì)胞術(shù)檢測(cè)NK細(xì)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(xì)胞在處理前后與靶細(xì)胞結(jié)合、免疫突觸形成和細(xì)胞毒顆粒極化的能力。4.利用高通量RNA測(cè)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(hào)的分子機(jī)制。第三部分1.Western blot檢測(cè)mEzh2條件敲除小鼠NK細(xì)胞中mEzh2的表達(dá),以及H3K27me3修飾水平。2.流式細(xì)胞術(shù)檢測(cè)NK細(xì)胞表面分化標(biāo)志物以及功能蛋白IFN-γ的表達(dá)。3.小鼠尾靜脈注射肺轉(zhuǎn)移模型檢測(cè)mEzh2條件敲除小鼠的抗腫瘤能力。結(jié)果第一部分:1.表達(dá)譜芯片中發(fā)現(xiàn)八個(gè)組蛋白甲基轉(zhuǎn)移酶和組蛋白去甲基化酶的表達(dá)在NK細(xì)胞被激活后發(fā)生變化。2.qPCR和Western blot驗(yàn)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(xì)胞被激活后表達(dá)上調(diào)。3.ChIP-seq分析發(fā)現(xiàn)27個(gè)與NK功能相關(guān)的基因啟動(dòng)子區(qū)被H3K4me3和H3K27me3同時(shí)修飾,并且這些基因中的77.8%在NK細(xì)胞被激活后表達(dá)上調(diào)。4.ChIP-qPCR驗(yàn)證在NK細(xì)胞被激活后,PI3KCA、NFATC1和TNFSF9基因啟動(dòng)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(xì)胞的脫顆粒水平,而OG-L002和MM102可以提高NK細(xì)胞中IFN-γ與TNF-α的表達(dá)。第二部分:1.小分子抑制劑UNC1999和GSK343抑制NK細(xì)胞中EZH2酶活性,或用sh RNA抑制NK細(xì)胞中EZH2表達(dá),可以上調(diào)NK細(xì)胞的脫顆粒水平和鈣離子信號(hào),下調(diào)NK細(xì)胞短時(shí)間內(nèi)的殺傷效率。2.抑制EZH2酶活性不影響NK細(xì)胞與靶細(xì)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(dǎo)致鈣離子信號(hào)異常升高,進(jìn)而導(dǎo)致異常毒性顆粒釋放,最終導(dǎo)致很少的靶細(xì)胞可以獲得毒性顆粒。4.抑制EZH2酶活性,導(dǎo)致鈣離子通道PKD2啟動(dòng)子區(qū)H3K27me3修飾被抑制,并最終上調(diào)鈣離子通道PKD2的表達(dá)。第三部分:1.mEzh2條件敲除小鼠的NKp46+CD3-NK細(xì)胞中mEzh2表達(dá)被顯著下調(diào);與此同時(shí),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(xì)胞的百分比和絕對(duì)數(shù)同時(shí)上調(diào);而且mEzh2條件敲除小鼠的NK細(xì)胞更加成熟。3.mEzh2條件敲除小鼠的NKp46+CD3-NK細(xì)胞被激活后,IFN-γ的表達(dá)水平顯著高于野生型小鼠的NK細(xì)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(qiáng)。結(jié)論第一部分:1.NK細(xì)胞在靜息狀態(tài)下,很多功能相關(guān)基因的啟動(dòng)子區(qū)被H3K4me3和H3K27me3同時(shí)修飾,處于“poised”狀態(tài)。2.NK細(xì)胞在激活過(guò)程中,部分組蛋白甲基轉(zhuǎn)移酶和組蛋白去甲基化酶表達(dá)發(fā)生變化,而與NK功能相關(guān)基因的啟動(dòng)子區(qū)甲基化修飾狀態(tài)也發(fā)生變化,說(shuō)明組蛋白甲基化修飾在NK細(xì)胞激活過(guò)程中起重要的作用。3.針對(duì)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(xì)胞毒性,為調(diào)控NK細(xì)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(dá)或者酶活性可以上調(diào)NK細(xì)胞內(nèi)質(zhì)網(wǎng)中鈣離子的儲(chǔ)存,從而上調(diào)NK細(xì)胞內(nèi)的鈣離子信號(hào),進(jìn)而導(dǎo)致NK細(xì)胞在短時(shí)間內(nèi)釋放大量毒性顆粒到很少的靶細(xì)胞,下調(diào)NK細(xì)胞短時(shí)間內(nèi)的殺傷效率。2.靜息狀態(tài)下的NK細(xì)胞中鈣離子通道PKD2的啟動(dòng)子區(qū)被H3K4me3和H3K27me3同時(shí)修飾,抑制EZH2的表達(dá)或酶活性,可以改變PKD2啟動(dòng)子區(qū)的甲基化修飾狀態(tài),從而上調(diào)PKD2的表達(dá)水平。第三部分:1.在小鼠NK細(xì)胞的發(fā)育過(guò)程中,mEzh2不僅參與NK細(xì)胞的早期分化,而且調(diào)控NK細(xì)胞的終端成熟。2.mEzh2條件敲除小鼠的NK細(xì)胞在被激活后表達(dá)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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R3416

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