丙戊酸对NK细胞抗肿瘤作用的影响及机制研究

发布时间：2018-01-31 10:06

本文关键词： NK细胞丙戊酸表观遗传学肿瘤　出处：《吉林大学》2016年博士论文　论文类型：学位论文

【摘要】：研究背景:NK细胞约占外周血淋巴细胞的10%~15%,是固有免疫的重要组成部分,可以直接杀伤病毒感染的细胞和恶性肿瘤细胞而不需要预先致敏,故在控制病毒感染和肿瘤免疫监测方面发挥重要作用。恶性肿瘤因其高发病率和高死亡率,严重威胁人类健康,成为关系到我国社会和经济发展的重大问题。随着肿瘤免疫学的发展、生物技术的日益完善,肿瘤的免疫治疗得到广泛关注,通过过继性细胞免疫治疗来恢复肿瘤患者的免疫能力,从而达到治疗肿瘤的目的,已经越来越受到人们的重视。但同时也面临一些问题,例如,肿瘤患者外周血的NK细胞细胞毒性明显下降,这可能是因为膜表面KIR受体与自身主要组织相容性复合物(major histocompatibility complex,MHC)抗原相互作用的干扰,同时,从肿瘤患者中新鲜分离出来的肿瘤浸润NK细胞对自体肿瘤细胞并没有杀伤作用。结果,输注患者自体NK细胞并没有显示出明显的临床获益以消灭肿瘤。很多药物可以提升NK细胞的功能,包括细胞因子和靶向PD1、KIR和肿瘤抗原的抗体类药物,特别是用细胞因子活化和扩增后,NK细胞对表达自身MHC分子的肿瘤细胞的杀伤能力明显增强。目前已经报道,T细胞可以被设计表达能识别肿瘤细胞上特异性抗原分子的嵌合性抗原受体。该方法已经显示出了显著了临床疗效,可以诱导进展期的白血病患者缓解,然而,应用CAR-T细胞治疗其他肿瘤仍具有挑战性,主要是因为致命的细胞因子释放综合征、对正常组织的抗原非特异性毒性、以及病毒载体对初始T细胞转染效率低下。越来越多的证据表明,表观遗传学机制可以调控肿瘤免疫。例如,肿瘤细胞通过表观遗传学抑制免疫基因而逃避免疫监测,包括MHC class II、CD40、MHC class I、I类肽递呈通路中的成分、B7-1/2、NKG2D配体和一些肿瘤抗原。类似的,表观遗传学方法,包括DNA甲基化和组蛋白修饰,已经被用来调控重要的免疫系统相关基因的表达,近而影响免疫反应的发展。然而,目前尚不清楚,是否这些表观遗传学方法可以被用来提高NK细胞介导的抗肿瘤治疗。研究目的:在本研究中,我们检测了很多已知的可以促进体细胞重编程为多潜能干细胞的小分子物质,观察其是否可以通过表观遗传学机制活化NK细胞,进一步提高NK细胞的抗肿瘤能力,最终提高NK细胞过继性免疫治疗的疗效。研究方法:(1)利用钙黄绿素释放法,检测不同效靶比时,不同药物处理后的NK细胞对不同靶细胞(K562,Jurkat,Hep G2)的细胞毒性作用。(2)用流式细胞术检测不同浓度VPA处理后NK细胞的凋亡情况、CD107a和NKG2D表达情况,并用ELISA试剂盒检测IFN-γ分泌。(3)Western blot检测NK细胞NKG2D表达,以及STAT5干扰素通路活性。(4)应用实时荧光定量逆转录聚合酶链反应q RT-PCR检测VPA处理组和未处理组之间PD-1及其配体PD-L1 m RNA表达水平,以及VPA处理后NKG2D m RNA表达水平的差异。(5)利用亚硫酸盐测序法(BSP)分析NKG2D启动子区域DNA甲基化状态,利用Ch IP法分析NKG2D启动子区域组蛋白甲基化状态,探讨NKG2D的表观遗传学调控机制。研究结果:(1)甲状腺激素T3可以轻度增加NK细胞对K562细胞的毒性作用,但是没有统计学意义,Vit-C和5-azacytidine对NK细胞的细胞毒性作用没有影响,而组蛋白去乙酰化酶抑制剂VPA,在不同的效靶比时,均可明显抑制NK细胞对K562、Jurkat、Hep G2的细胞毒作用(P0.05)。另外,在培养液中去除细胞因子IL-2和抗CD3单克隆抗体,并未明显影响这些小分子药物对NK细胞毒性的作用。VPA对NK细胞细胞毒性的抑制作用是剂量依赖性的,即VPA浓度越大,其对NK细胞的抑制作用越强,NK细胞对K562细胞的杀伤作用越弱。并且,在去除VPA后继续培养NK细胞48 h,其毒性是部分可逆的。(2)不同浓度VPA处理NK细胞24h后,与PBS对照组相比较(5.15%),PI+/Annexin-V+细胞的比例,随VPA浓度的增加而轻度增加(7.35%,8.38%,10.6%,13.1%),这说明,诱导细胞凋亡可能是NK细胞毒性下降的部分原因,而不是全部原因。(3)VPA处理后,NK细胞表面CD107a表达随VPA浓度升高而下降,说明其脱颗粒效应表达下降。这表明,VPA处理不仅诱导NK细胞凋亡,也抑制其他影响NK细胞细胞毒性的分子的生成。(4)VPA处理后,NK细胞IFN-γ分泌下降,并且随着VPA浓度上升,IFN-γ分泌逐渐下降,呈现剂量依赖性,表明组蛋白乙酰化可以显著降低细胞因子产生,这与NK细胞细胞毒性下降有关。同样,VPA处理可以使NK细胞内STAT5干扰素通路活性下降。(5)实时定量PCR结果显示,应用2m M的VPA处理后,NK细胞PD-1及其配体PD-L1表达均明显上调,表明在VPA引起的NK细胞细胞毒性下降中,PD-1抑制性通路也参与其中。(6)细胞表面NKG2D表达随着VPA剂量的增加而逐渐下降,表现为NKG2D阳性细胞比例和平均荧光强度的下降,并呈现明显的剂量依赖性。q RT-PCR结果显示NKG2D m RNA表达的下降呈现VPA浓度依赖性,利用Western blot也检测到了相似的结果,即NK细胞NKG2D的表达随着VPA剂量的增加而逐渐下降。这些结果显示VPA引起的NKG2D表达下调,可能是NK细胞细胞毒性下降的一个重要的机制。(7)VPA处理后的NK细胞NKG2D启动子区域DNA甲基化有轻度的增加,表明DNA甲基化在NKG2D基因表达下调中只起到了一小部分作用,而NKG2D基因启动子区域H3K9me2的增加,可能与NK细胞表面NKG2D受体表达下降有关。研究结论:(1)组蛋白去乙酰化酶抑制剂VPA抑制NK细胞对白血病细胞系的杀伤作用,并呈剂量依赖性。(2)用VPA预处理NK细胞后,可以下调IFN-γ分泌,降低CD107a脱颗粒,并且通过激活PD-1/PD-L1通路而诱导凋亡。(3)VPA通过诱导启动子区域组蛋白K9过度甲基化和DNA甲基化,下调活化性受体NKG2D的表达。
[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.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

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