本文關(guān)鍵詞： NK細(xì)胞 丙戊酸 表觀遺傳學(xué) 腫瘤　出處：《吉林大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景:NK細(xì)胞約占外周血淋巴細(xì)胞的10%~15%,是固有免疫的重要組成部分,可以直接殺傷病毒感染的細(xì)胞和惡性腫瘤細(xì)胞而不需要預(yù)先致敏,故在控制病毒感染和腫瘤免疫監(jiān)測方面發(fā)揮重要作用。惡性腫瘤因其高發(fā)病率和高死亡率,嚴(yán)重威脅人類健康,成為關(guān)系到我國社會和經(jīng)濟發(fā)展的重大問題。隨著腫瘤免疫學(xué)的發(fā)展、生物技術(shù)的日益完善,腫瘤的免疫治療得到廣泛關(guān)注,通過過繼性細(xì)胞免疫治療來恢復(fù)腫瘤患者的免疫能力,從而達到治療腫瘤的目的,已經(jīng)越來越受到人們的重視。但同時也面臨一些問題,例如,腫瘤患者外周血的NK細(xì)胞細(xì)胞毒性明顯下降,這可能是因為膜表面KIR受體與自身主要組織相容性復(fù)合物(major histocompatibility complex,MHC)抗原相互作用的干擾,同時,從腫瘤患者中新鮮分離出來的腫瘤浸潤NK細(xì)胞對自體腫瘤細(xì)胞并沒有殺傷作用。結(jié)果,輸注患者自體NK細(xì)胞并沒有顯示出明顯的臨床獲益以消滅腫瘤。很多藥物可以提升NK細(xì)胞的功能,包括細(xì)胞因子和靶向PD1、KIR和腫瘤抗原的抗體類藥物,特別是用細(xì)胞因子活化和擴增后,NK細(xì)胞對表達自身MHC分子的腫瘤細(xì)胞的殺傷能力明顯增強。目前已經(jīng)報道,T細(xì)胞可以被設(shè)計表達能識別腫瘤細(xì)胞上特異性抗原分子的嵌合性抗原受體。該方法已經(jīng)顯示出了顯著了臨床療效,可以誘導(dǎo)進展期的白血病患者緩解,然而,應(yīng)用CAR-T細(xì)胞治療其他腫瘤仍具有挑戰(zhàn)性,主要是因為致命的細(xì)胞因子釋放綜合征、對正常組織的抗原非特異性毒性、以及病毒載體對初始T細(xì)胞轉(zhuǎn)染效率低下。越來越多的證據(jù)表明,表觀遺傳學(xué)機制可以調(diào)控腫瘤免疫。例如,腫瘤細(xì)胞通過表觀遺傳學(xué)抑制免疫基因而逃避免疫監(jiān)測,包括MHC class II、CD40、MHC class I、I類肽遞呈通路中的成分、B7-1/2、NKG2D配體和一些腫瘤抗原。類似的,表觀遺傳學(xué)方法,包括DNA甲基化和組蛋白修飾,已經(jīng)被用來調(diào)控重要的免疫系統(tǒng)相關(guān)基因的表達,近而影響免疫反應(yīng)的發(fā)展。然而,目前尚不清楚,是否這些表觀遺傳學(xué)方法可以被用來提高NK細(xì)胞介導(dǎo)的抗腫瘤治療。研究目的:在本研究中,我們檢測了很多已知的可以促進體細(xì)胞重編程為多潛能干細(xì)胞的小分子物質(zhì),觀察其是否可以通過表觀遺傳學(xué)機制活化NK細(xì)胞,進一步提高NK細(xì)胞的抗腫瘤能力,最終提高NK細(xì)胞過繼性免疫治療的療效。研究方法:(1)利用鈣黃綠素釋放法,檢測不同效靶比時,不同藥物處理后的NK細(xì)胞對不同靶細(xì)胞(K562,Jurkat,Hep G2)的細(xì)胞毒性作用。(2)用流式細(xì)胞術(shù)檢測不同濃度VPA處理后NK細(xì)胞的凋亡情況、CD107a和NKG2D表達情況,并用ELISA試劑盒檢測IFN-γ分泌。(3)Western blot檢測NK細(xì)胞NKG2D表達,以及STAT5干擾素通路活性。(4)應(yīng)用實時熒光定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)q RT-PCR檢測VPA處理組和未處理組之間PD-1及其配體PD-L1 m RNA表達水平,以及VPA處理后NKG2D m RNA表達水平的差異。(5)利用亞硫酸鹽測序法(BSP)分析NKG2D啟動子區(qū)域DNA甲基化狀態(tài),利用Ch IP法分析NKG2D啟動子區(qū)域組蛋白甲基化狀態(tài),探討NKG2D的表觀遺傳學(xué)調(diào)控機制。研究結(jié)果:(1)甲狀腺激素T3可以輕度增加NK細(xì)胞對K562細(xì)胞的毒性作用,但是沒有統(tǒng)計學(xué)意義,Vit-C和5-azacytidine對NK細(xì)胞的細(xì)胞毒性作用沒有影響,而組蛋白去乙�；敢种苿￢PA,在不同的效靶比時,均可明顯抑制NK細(xì)胞對K562、Jurkat、Hep G2的細(xì)胞毒作用(P0.05)。另外,在培養(yǎng)液中去除細(xì)胞因子IL-2和抗CD3單克隆抗體,并未明顯影響這些小分子藥物對NK細(xì)胞毒性的作用。VPA對NK細(xì)胞細(xì)胞毒性的抑制作用是劑量依賴性的,即VPA濃度越大,其對NK細(xì)胞的抑制作用越強,NK細(xì)胞對K562細(xì)胞的殺傷作用越弱。并且,在去除VPA后繼續(xù)培養(yǎng)NK細(xì)胞48 h,其毒性是部分可逆的。(2)不同濃度VPA處理NK細(xì)胞24h后,與PBS對照組相比較(5.15%),PI+/Annexin-V+細(xì)胞的比例,隨VPA濃度的增加而輕度增加(7.35%,8.38%,10.6%,13.1%),這說明,誘導(dǎo)細(xì)胞凋亡可能是NK細(xì)胞毒性下降的部分原因,而不是全部原因。(3)VPA處理后,NK細(xì)胞表面CD107a表達隨VPA濃度升高而下降,說明其脫顆粒效應(yīng)表達下降。這表明,VPA處理不僅誘導(dǎo)NK細(xì)胞凋亡,也抑制其他影響NK細(xì)胞細(xì)胞毒性的分子的生成。(4)VPA處理后,NK細(xì)胞IFN-γ分泌下降,并且隨著VPA濃度上升,IFN-γ分泌逐漸下降,呈現(xiàn)劑量依賴性,表明組蛋白乙�；梢燥@著降低細(xì)胞因子產(chǎn)生,這與NK細(xì)胞細(xì)胞毒性下降有關(guān)。同樣,VPA處理可以使NK細(xì)胞內(nèi)STAT5干擾素通路活性下降。(5)實時定量PCR結(jié)果顯示,應(yīng)用2m M的VPA處理后,NK細(xì)胞PD-1及其配體PD-L1表達均明顯上調(diào),表明在VPA引起的NK細(xì)胞細(xì)胞毒性下降中,PD-1抑制性通路也參與其中。(6)細(xì)胞表面NKG2D表達隨著VPA劑量的增加而逐漸下降,表現(xiàn)為NKG2D陽性細(xì)胞比例和平均熒光強度的下降,并呈現(xiàn)明顯的劑量依賴性。q RT-PCR結(jié)果顯示NKG2D m RNA表達的下降呈現(xiàn)VPA濃度依賴性,利用Western blot也檢測到了相似的結(jié)果,即NK細(xì)胞NKG2D的表達隨著VPA劑量的增加而逐漸下降。這些結(jié)果顯示VPA引起的NKG2D表達下調(diào),可能是NK細(xì)胞細(xì)胞毒性下降的一個重要的機制。(7)VPA處理后的NK細(xì)胞NKG2D啟動子區(qū)域DNA甲基化有輕度的增加,表明DNA甲基化在NKG2D基因表達下調(diào)中只起到了一小部分作用,而NKG2D基因啟動子區(qū)域H3K9me2的增加,可能與NK細(xì)胞表面NKG2D受體表達下降有關(guān)。研究結(jié)論:(1)組蛋白去乙�；敢种苿￢PA抑制NK細(xì)胞對白血病細(xì)胞系的殺傷作用,并呈劑量依賴性。(2)用VPA預(yù)處理NK細(xì)胞后,可以下調(diào)IFN-γ分泌,降低CD107a脫顆粒,并且通過激活PD-1/PD-L1通路而誘導(dǎo)凋亡。(3)VPA通過誘導(dǎo)啟動子區(qū)域組蛋白K9過度甲基化和DNA甲基化,下調(diào)活化性受體NKG2D的表達。
[Abstract]:Background: NK cells in peripheral blood lymphocytes accounted for about 10%~15%, is an important part of innate immunity, can directly kill virus infected cells and tumor cells without prior sensitization, it plays an important role in the control of viral infection and tumor immune monitoring of malignant tumor. Because of its high incidence and high the mortality rate, a serious threat to human health, has become a major problem related to social and economic development in China. With the development of tumor immunology, biological technology is increasingly perfect, tumor immunotherapy has attracted considerable attention by adoptive cellular immunotherapy to restore tumor immunity, so as to achieve the purpose of treatment of cancer, has been more and more the attention of people. But it also faces some problems, for example, the NK cell cytotoxicity in peripheral blood of tumor patients was significantly decreased, which may be due to the membrane surface by KIR The body and its major histocompatibility complex (major histocompatibility, complex, MHC) interference, antigen interaction at the same time, from patients with tumor of freshly isolated tumor infiltrating NK cells and no cytotoxicity to autologous tumor cells. The results showed that the patients with infusion of autologous NK cells did not show significant clinical benefit to eradicate tumors many drugs can enhance the function of NK cells, including cytokines and antibody drugs targeting PD1, KIR and tumor antigen, especially activated by cytokines and amplification, the killing effect of NK cells on the expression of MHC molecules in tumor cells significantly enhanced. At present already reported that T cells can be engineered to express to identify the tumor cell specific antigen chimeric antigen receptor. This method has shown significant clinical efficacy, remission can induce progression of leukemia patients, however However, application of CAR-T cells in the treatment of other tumors is still challenging, mainly because of lethal cytokine release syndrome, antigen nonspecific toxicity to normal tissues, and viral vectors for the initial T cell transfection efficiency. Increasing evidence suggests that epigenetics mechanism can regulate tumor immunity. For example, the escape immune surveillance of tumor cells by epigenetic suppression of immune genes, including MHC class II, CD40 MHC, class I, I peptide presenting pathway components, B7-1/2, NKG2D ligand and tumor antigen. Similarly, epigenetics, including DNA methylation and histone modifications have been used to express the important genes related to regulation of the immune system, development and influence the immune response. However, it is unclear whether these epigenetic methods can be used to improve the anti tumor therapy mediated by NK cells. Objective: in this study, we examined many known to promote the reprogramming of somatic cells into stem cells of multiple small molecules to observe its potential, whether through epigenetic mechanisms of activation of NK cells, NK cells and further improve the ability of anti tumor, finally to improve the efficacy of high NK cells for adoptive immunotherapy. Research methods: (1) using the calcein release assay to detect different effector target ratio, on different target cells after different treatment of NK cells (K562, Jurkat, Hep, G2) cytotoxicity. (2) the apoptosis of NK cells were detected by flow cytometry with VPA concentration after treatment the expression of NKG2D, CD107a and IFN-, and gamma ELISA kit to detect secretion. (3) the expression of Western blot and STAT5 NKG2D to detect NK cells, interferon pathway activity. (4) using real-time quantitative reverse transcriptase polymerase chain reaction Q RT-PCR VPA treatment group and no detection PD-1 PD-L1 m and its ligand RNA expression level between treatment group, and after treatment with VPA NKG2D m RNA expression. (5) by bisulfite sequencing (BSP) analysis of the NKG2D promoter region methylation status of DNA, using the Ch IP method for analysis of the promoter region of NKG2D histone methylation state of NKG2D apparent the genetic regulation mechanism. Results: (1) thyroid hormone T3 can slightly increased toxicity of NK cells to K562 cells, but not statistically significant, did not affect the cytotoxicity of Vit-C and 5-azacytidine on NK cells, and the histone deacetylase inhibitor, VPA, in different ratio of effector and target, could be significantly inhibited Jurkat NK of K562 Hep, G2 cells, cytotoxicity (P0.05). In addition, in the culture medium for removal of cytokines IL-2 and CD3 monoclonal antibody,.V did not significantly influence these small molecule drugs on NK cell toxicity The inhibition effect of PA on NK cell cytotoxicity was dose dependent, i.e. VPA higher concentration, the stronger inhibitory effect on NK cells, cytotoxicity of NK cells to K562 cells is weak. And continue to culture NK cells at 48 h after VPA removal, the toxicity is partially reversible. (2) not the same concentration of VPA in NK cells treated with 24h, and PBS compared to the control group (5.15%), the proportion of PI+/Annexin-V+ cells, with the increase of VPA concentration and increased slightly (7.35%, 8.38%, 10.6%, 13.1%), which shows that the induction of apoptosis may be due in part to decreased cytotoxicity in NK cells, but not all of the reasons. (3) after VPA treatment, NK cell surface expression of CD107a decreased with the increase of VPA concentration, indicating a decline in the degranulation effect expression. This indicates that VPA not only induced apoptosis in NK cells, generating molecules also inhibit other cytotoxic effects of NK cells. (4) after VPA treatment, NK cells secreting IFN- drop, And with the increase of VPA concentration, IFN- secretion decreased in a dose-dependent manner, suggesting that histone acetylation can significantly reduce cytokine production, and the NK cell cytotoxicity decreased. Similarly, VPA can make NK cells within the STAT5 interferon pathway activity decreased. (5) real time quantitative PCR results showed that the application of 2m M after VPA treatment, the expression of NK cell PD-1 and its ligand PD-L1 were significantly up-regulated, showed decreased NK cell cytotoxicity in VPA induced, PD-1 inhibitory pathway is also involved. (6) the cell surface expression of NKG2D was increased with the dose of VPA decreased gradually, decreased the proportion of NKG2D positive cells and the mean fluorescence intensity, and was dose-dependent..q RT-PCR results showed that the expression of NKG2D decreased m RNA showed VPA concentration dependent, the use of Western blot has detected a similar result, the expression of NK cells with NKG2D With the increase of the dose of VPA decreased. These results suggest that VPA induced down-regulation of NKG2D expression may be an important mechanism of decreased NK cell cytotoxicity. (7) NK NKG2D cells after treatment with VPA promoter DNA methylation slightly increase, indicating that DNA methylation plays down only a small part in the expression of NKG2D gene and NKG2D gene increased with the promoter region of H3K9me2, and NK cell surface expression of NKG2D receptor decreased. Conclusions: (1) the histone deacetylase inhibitor VPA inhibits NK cell killing effect on leukemia cells, in a dose-dependent manner. (2) pretreatment with VPA NK cells, can down regulate IFN- secretion, reduce CD107a degranulation, and activate the PD-1/PD-L1 pathway to induce apoptosis. (3) VPA induced by promoter region hypermethylation and histone K9 methylation of DNA, decreased the activation of receptor NK The expression of G2D.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R96

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