γ干扰素诱导肿瘤再生细胞休眠的机制及相关应用的研究

发布时间：2018-04-27 01:30

  本文选题：干扰素 + 诱导　； 参考：《北京协和医学院》2017年硕士论文

【摘要】：肿瘤细胞与免疫系统的相互作用被归纳为3E理论(Dunn et al.,2004;Schreiber et al.,2011),包括 Eradication(消灭),Equilibrium(平衡),Evasion(逃逸)。在机体中,肿瘤细胞在免疫因素的作用下会出现以上三种生物现象:肿瘤细胞被免疫系统消灭清除;在免疫系统的监视下肿瘤细胞与机体达到均势;肿瘤细胞逃脱免疫系统的控制进而侵害全身(Romero et al.,2014)。免疫诱导的肿瘤休眠(tumordormancy),是指肿瘤细胞在机体免疫系统作用下,出现细胞周期阻滞、增殖相关基因表达下调、代谢减慢等现象。此种现象是肿瘤与机体免疫系统相互作用的过程中进入平衡稳定状态的集中体现(Sosa et al.,2014),与免疫监视这一假说相呼应。目前的科学研究由于缺少有效公认的肿瘤休眠的体内外模型,免疫诱导的肿瘤休眠缺乏有效的手段研究其深入的机制。在对于肿瘤细胞休眠这一领域的科学研究中,目前的研究成果仅限于通过运用中和抗体和转基因动物探究该现象的存在,及确定与之相关的免疫分子如Interferonγ(伽马干扰素,IFNγ)。Interferonγ作为免疫效应分子,在肿瘤免疫中,IFNγ主要由肿瘤微环境中的杀伤性T细胞(CTLs)所分泌,对控制肿瘤的生长具有重要的作用(Akira etal.,2006;Dunn etal.,2006),可能会引起肿瘤细胞的休眠。但IFNγ如何作用于肿瘤细胞休眠,其内在的作用机制尚未阐明。肿瘤细胞具有异质性,不同的亚群具有不同的特性。其中,肿瘤干细胞即肿瘤再生细胞(tumorigenic cells,TRCs)是一群具有很强成瘤能力和增殖分裂能力的细胞,且表现为较强的耐药特性,与肿瘤的复发转移具有紧密关系(Enver et al.,2009;Visvader and Lindeman,2008)。目前尚未有研究证明肿瘤干细胞是否存在肿瘤休眠这一现象,且其内在的机制也不清楚。本课题研究旨在利用本课题组已经建立起来的3D(软三维胶体外扩增培养肿瘤再生细胞)技术,来探究以下三个问题:IFNγ能否诱导肿瘤的休眠;IFNγ可具体诱导哪群肿瘤细胞休眠;IFNγ诱导肿瘤细胞休眠的机制。本论文分为三部分:1.IFNγ体外诱导肿瘤干细胞的休眠的探究;2.IFNγ诱导肿瘤干细胞休眠的机制探究;3.运用相关分子的抑制剂逆转肿瘤休眠对肿瘤细胞影响的探究第一部分:IFNγ体外诱导肿瘤干细胞的休眠的研究目的:研究IFNγ是否可诱导肿瘤干细胞休眠,并区别于传统2D培养肿瘤细胞;且探究IFNγ诱导的肿瘤干细胞是否存在对化疗药物甲氨蝶呤(Methotrexate)和紫杉醇(Paclitaxel)的耐药性。方法:该研究选用本实验组之前使用过的小鼠黑色素瘤细胞系B16及三文鱼来源的纤维蛋白原建立的3D肿瘤干细胞培养方法(Liu et al.,2012),来建立体外模型。1.IFNγ处理小鼠来源的B16黑色素瘤细胞、H22肝癌细胞系及CT26结肠癌细胞系:1)1OOng/ml IFNγ分别作用于传统2D培养的B16细胞、H22细胞及CT26细胞72h;2)100ng/ml IFNγ作用于3D培养的B16细胞、H22细胞及CT26细胞72h,检测其对两种不同培养模式的三种肿瘤细胞增殖及凋亡代谢情况。2.化疗药物甲氨蝶呤(MTX)和紫杉醇(Paclitaxel)分别处理3D培养的B16细胞:1)细胞对照组:2D培养B16细胞+PBS;2D培养B16细胞+IFNγ;2D培养B16细胞+MTX;2D培养B16细胞+IFNγ+MTX;2)实验细胞组:3D培养B16细胞+ MTX;3D培养B16细胞+IFNγ+MTX;IFNγ预处理72h的3D培养B16细胞+MTX+ IFNγ;IFNγ预处理72h后3D培养B16细胞+MTX。紫杉醇分组同甲氨蝶呤。结果:1.用不同浓度的IFNγ处理传统2D培养皿和3D培养的B16细胞、H22细胞和CT26细胞96h后,2D肿瘤细胞的凋亡明显,3D细胞未见明显凋亡。3D细胞主要表现为克隆变小,细胞周期变慢,葡萄糖消耗量下降,及增殖相关基因下调。2.本研究选用常规化疗药物甲氨蝶呤和紫杉醇处理IFNγ诱导的休眠的肿瘤细胞和正常培养的2D细胞和3D细胞,发现休眠的肿瘤细胞有更强的耐药性。第二部分:IFNγ在诱导肿瘤细胞休眠的机制目的:在已经建立的IFNγ体外诱导肿瘤休眠模型的条件下探究其内在机制方法:在已经建立IFNγ体外诱导肿瘤休眠的模型中,我们运用PCR,Western-blot,质粒构建,病毒包装,CRISPR-Cas9,免疫荧光,流式分析、分选等分子生物学、细胞生物学的手段来鉴定并验证相关信号通路及机制。结果:1.根据文献报道及PCR验证,我们确定了吲哚胺2,3双加氧酶(indoleamine 2,3-dioxygenase,IDO)在被IFNγ处理过的B16肿瘤细胞中发生了很大的改变(Takikawa et al.,1990),并且TRCs被IFNγ处理后IDO上调相较于普通2D细胞更为明显,说明IDO在TRCs的休眠的过程中具有重要作用;2.我们将过表达IDO的TRCs进行分析,发现其细胞克隆大小及细胞周期均发生较明显的变化,出现了克隆变小及周期变慢的现象;3.IDO是一种催化酶,主要催化色氨酸(Tryptophan,Trp)的代谢,使其代谢成为犬尿氨酸(kynurenine,Kyn),我们用200uM到500uM浓度的Kyn处理TRCs也得到了克隆变小及周期减慢的结果,说明Kyn在TRCs的休眠中扮演着重要的角色;4.我们从文献中得知,Kyn为多环芳香烃受体(Aryl hydrocarbon receptor,Ahr)的配体,可以导致Ahr诱导下游基因的表达(Opitz et al.,2011),其中可能包括细胞周期抑制相关的蛋白,我们通过免疫荧光、western-blot及荧光素酶实验确定了 Ahr在TRCs中得到激活;5.同时我们用western-blot筛选相关周期蛋白,发现在IFNγ处理后,Cdknlb(cyclin-dependent kinase inhibitor 1B,P27kip)出现了 很明显的变化,而其他周期蛋白没有较为明显的变化,说明P27可能在休眠中扮演重要的角色;6.我们用Kyn处理TRCs72小时后用western-blot检测P27的表达水平,发现其出现了明显上调,并同时调取P27基因的启动子,构建荧光素报告质粒,并同时联合AHR基因行瞬时转染后,用Kyn处理后检测,发现荧光素信号在AHR、Kyn及IFNγ处理后有明显增强,说明IFNγ对P27的调控主要是通过IDO-Kyn-AHR这一信号通路;7.经典的 IFNγ 信号主要通过 STAT1(signal transducer and activator of transcription 1)发挥作用,并且主要对肿瘤细胞引起凋亡的作用,我们通过western-blot及免疫荧光及流式证实了这一信号通路在普通培养的B16中存在,但在休眠的细胞中,这一通路被明显抑制,而且我们通过Ip-western发现IFNy-Stat1这一信号通路与IFNγ-IDO-AHR-P27这一信号通路存在交互作用,即P27结合p-STAT1蛋白阻止其入核;8.我们也在后来建立的IDO、AHR、P27敲低敲除细胞系中进一步验证了这一结论。第三部分:在体外实验中运用相关分子的抑制剂逆转肿瘤休眠目的:通过运用相关分子的抑制剂联合IFNy看能否打破肿瘤细胞的休眠,并对肿瘤治疗提出新的想法方法:运用3D培养、western-blot及流式检测等相关手段检测相关分子抑制剂联合IFNγ处理后的肿瘤细胞的增殖凋亡情况。结果:1.我们联合 IDO 抑制剂 1-MT(1-Methyl-L-tryptophan)及 IFNγ 处理3D培养的B16细胞后,其出现克隆数减少,克隆大小变小的表现,并在整体细胞水平及分子水平出现了凋亡的增加,说明IFNγ联合IDO抑制剂可以有效的打破休眠,杀伤肿瘤;2.我们联合AHR抑制剂DMF(3',4'-Dimethoxyflavone)及IFNγ处理3D培养的B16细胞后,得到了与IDO抑制剂相同的结果,即休眠打破凋亡增加。说明这两个抑制剂在今后的免疫治疗中可能有较好的前景。总结:肿瘤休眠这一现象在肿瘤的发生、发展、转移及治疗预后中均有重要的意义,免疫所引起的休眠,更是这一现象的很好的体现,我们通过运用3D培养肿瘤再生细胞这一方法,很好的在体外模拟了肿瘤休眠这一过程,即用IFNγ在体外处理3D培养的细胞72小时,就会诱导其进入休眠,并在进一步的研究中我们发现,在诱导肿瘤体外休眠的过程中,吲哚胺2,3双加氧酶(indoleamine 2,3-dioxygenase,IDO),犬尿氨酸(kynurenine,kyn),芳香烃受体(aryl hydrocarbon receptor,AhR)即IDO-kyn-AhR这一代谢调控的转录因子信号通路发挥着重要的作用。IFNγ作用于肿瘤再生细胞后,可诱导其进入休眠,具体表现为IFNγ诱导肿瘤再生细胞内IDO与AHR的表达上调,IDO的表达上调继而产生大量的内源性的Kyn,Kyn与AhR结合并激活AhR,AhR进入细胞核并结合到相应基因的启动子上,导致相关周期阻滞基因的表达,从而产生细胞周期的阻滞。我们通过进一步的筛选,发现在这一现象休眠之中,细胞周期阻滞蛋白P27出现了很明显的变化,并且受到AhR基因的转录调控。IFNγ-IDO-AhR-P27这一信号通路对肿瘤再生细胞的休眠具有巨大的作用,我们同时通过运用相关抑制剂联合IFNγ,发现肿瘤的休眠可以被打破,并取得了较好的治疗效果,为今后临床上对肿瘤的治疗提出了新的思路。
[Abstract]:The interaction of tumor cells with the immune system is summed up as 3E theory (Dunn et al., 2004; Schreiber et al., 2011), including Eradication (elimination), Equilibrium (balance), Evasion (escape). In the body, the tumor cells will present more than three biological phenomena under the action of immune factors: tumor cells are eliminated by the immune system; in exemption Under the surveillance of the epidemic system, the tumor cells and the body reach the balance; the tumor cells escape the immune system and then infringe the whole body (Romero et al., 2014). The immune induced tumor dormancy (tumordormancy) refers to the cell cycle arrest, the downregulation of the related gene expression and the metabolic slowing down of the tumor cells under the action of the immune system of the body. This phenomenon is the concentration of equilibrium and stability in the process of interaction between the tumor and the immune system of the body (Sosa et al., 2014), which corresponds to the hypothesis of immune surveillance. The current scientific research is lacking effective means of immune induced dormancy due to the lack of an effectively recognized model of tumor dormancy in vivo and in vivo. In a scientific study of the field of tumor cell dormancy, the current research results are limited to the use of neutralizing antibodies and genetically modified animals to explore the presence of the phenomenon and to determine the immune molecules such as Interferon gamma (gamma interferon, IFN gamma).Interferon gamma as an immune response molecule. In the epidemic, IFN gamma is secreted mainly by the killer T cells (CTLs) in the tumor microenvironment. It plays an important role in controlling the growth of tumor (Akira etal., 2006; Dunn etal., 2006), which may cause the dormancy of tumor cells. However, the internal mechanism of the tumor cells has not been elucidated. The tumor cells have heterogeneity, Different subgroups have different characteristics. Among them, tumor stem cells (tumorigenic cells, TRCs) are a group of cells with strong tumor forming ability and proliferation and division, and exhibit strong resistance characteristics, closely related to tumor recurrence and metastasis (Enver et al., 2009; Visvader and Lindeman, 2008). There have been no studies to prove whether tumor stem cells exist in tumor dormancy, and its intrinsic mechanism is not clear. This study aims to explore the following three questions: whether IFN gamma can induce the dormancy of tumor by using the 3D (soft three-dimensional colloid amplification and proliferation of tumor regenerative cells) established by our group: IFN gamma can induce the tumor's dormancy; IFN gamma can be used Which group of tumor cells to induce dormancy and IFN gamma induced dormancy of tumor cells. This paper is divided into three parts: 1.IFN gamma induced dormancy of tumor stem cells in vitro; mechanism of 2.IFN gamma induced dormancy of tumor stem cells; 3. the first part of the exploration of the effect of tumor dormancy on tumor cells using inhibitors of related molecules: the first part: IFN gamma The purpose of in vitro induction of tumor stem cell dormancy is to investigate whether IFN gamma can induce tumor stem cells dormancy and differentiate between traditional 2D and tumor cells; and explore whether IFN gamma induced cancer stem cells have resistance to chemotherapy drugs methotrexate (Methotrexate) and paclitaxel (Paclitaxel). The mouse melanoma cell line B16 used before and the 3D tumor stem cell culture method (Liu et al., 2012) derived from salmon derived fibrinogen to establish B16 melanoma cells derived from.1.IFN gamma treatment mice in vitro model, H22 hepatoma cell line and CT26 colon cancer cell line: 1) 1OOng/ml IFN gamma action on traditional 2D The cultured B16 cells, H22 cells and CT26 cells 72h; 2) 100ng/ml IFN gamma acts on B16 cells cultured in 3D, H22 cells and CT26 cells 72h. The proliferation and apoptosis metabolism of three tumor cells in two different culture modes were detected by.2. chemotherapeutic drugs methotrexate and taxol, respectively, 1) cells The control group: 2D culture B16 cells +PBS; 2D culture B16 cells +IFN gamma; 2D culture B16 cells +MTX; 2D culture B16 cells +IFN gamma; 2) experimental cell group. Results: 1. the apoptosis of 2D tumor cells was obvious after the treatment of traditional 2D culture dish and 3D cultured B16 cells, H22 cells and CT26 cells 96h, and the.3D cells of 3D cells did not have obvious apoptosis, and the main manifestations of the.3D cells were the small clone, the slow cell cycle, the decrease of glucose consumption, and the.2. of proliferation related genes. The treatment of methotrexate and paclitaxel treated IFN gamma induced dormant tumor cells and normal 2D and 3D cells, and found that the dormant tumor cells have stronger resistance. The second part: the mechanism of IFN gamma in inducing tumor cells dormancy: explore the inside of the tumor dormancy model under the established IFN gamma body. In the mechanism method: in the model of IFN gamma induced tumor dormancy in vitro, we use PCR, Western-blot, plasmid construction, virus packaging, CRISPR-Cas9, immunofluorescence, flow analysis, sorting and other molecular biology, cell biology methods to identify and verify the related signaling pathways and mechanisms. Results: 1. according to the literature and PCR test It was confirmed that the indolamine 2,3 dioxygenase (indoleamine 2,3-dioxygenase, IDO) had a great change in the B16 tumor cells treated by IFN gamma (Takikawa et al., 1990), and TRCs was more obvious after TRCs was treated by IFN gamma than that of ordinary B16 cells, indicating that it plays an important role in the dormancy process; 2. We analyzed the TRCs of the expression of IDO, and found that the cell clone size and cell cycle were obviously changed, the clone became smaller and the cycle slowed down; 3.IDO was a kind of catalytic enzyme, which catalyzed the metabolism of Tryptophan, Trp, so that the metabolism became kynurenine, Kyn, and we used 200uM to 500uM concentration. The degree of Kyn treatment TRCs also obtained the result of cloning and periodic slowing down, indicating that Kyn plays an important role in the dormancy of TRCs; 4. we have learned from the literature that Kyn is a ligand of polycyclic aromatic hydrocarbon receptor (Aryl hydrocarbon receptor, Ahr), which can lead to the expression of the Ahr induced gene (Opitz et, 2011), which may include finer The proteins associated with cell cycle inhibition, we determined that Ahr was activated in TRCs by immunofluorescence, Western-blot and luciferase experiment; 5. at the same time, we screened the related cyclin with Western-blot, and found that after IFN gamma treatment, Cdknlb (cyclin-dependent kinase inhibitor 1B, P27kip) had obvious changes, and other cycles. There is no obvious change in protein, indicating that P27 may play an important role in dormancy; 6. we detected the expression level of P27 with Western-blot after TRCs72 hours of Kyn treatment, and found that it was obviously up-regulated, and the promoter of P27 gene was transferred simultaneously, the fluorescein plasmid was constructed, and the AHR gene was simultaneously transfected with the AHR gene. After Kyn treatment, it was found that the fluorescein signal was obviously enhanced after AHR, Kyn and IFN gamma treatment, indicating that the regulation of IFN gamma to P27 was mainly through the IDO-Kyn-AHR signal pathway; 7. the classic IFN gamma signal was mainly played by STAT1 (signal transducer and), and it was mainly caused by the tumor cells. The role of apoptosis was confirmed by Western-blot and immunofluorescence and flow cytometry in the common culture of B16, but in dormant cells, this pathway was obviously suppressed, and we found that the signal pathway of IFNy-Stat1 was interacted with IFN gamma -IDO-AHR-P27 signaling pathway through Ip-western, that is, P2 7 combined with p-STAT1 protein to prevent its nucleation; 8. we also further verified this conclusion in the later IDO, AHR, and P27 knockout cell lines. The third part: using inhibitors of relevant molecules to reverse the tumor dormancy in vitro experiments: the possibility of breaking the dormancy of the tumor cells by using the inhibitors of the relevant molecules combined with IFNy And a new way of thinking for tumor therapy is put forward: using 3D culture, Western-blot and flow detection to detect the proliferation and apoptosis of tumor cells associated with IFN gamma treatment. Results: 1. we combine the IDO inhibitor 1-MT (1-Methyl-L-tryptophan) and IFN gamma treatment of 3D cultured B16 cells, and they appear. The number of clones decreased, the size of clones became smaller, and the apoptosis increased at the whole cell level and molecular level. It showed that IFN gamma combined with IDO inhibitor could effectively break the dormancy and kill the tumor; 2. we are the same as IDO inhibitors with the AHR inhibitor DMF (3', 4'-Dimethoxyflavone) and IFN gamma treatment of 3D cultured B16 cells. The results indicate that dormancy breaks the increase of apoptosis. It shows that these two inhibitors may have good prospects in the future immunotherapy. Conclusion: the phenomenon of tumor dormancy is of great significance in the occurrence, development, metastasis and treatment of tumor, and the dormancy caused by immunization is a good manifestation of this phenomenon, and we use 3D The culture of tumor regenerative cells is a good method to simulate the process of tumor dormancy in vitro, that is, IFN gamma is used to treat 3D cells for 72 hours in vitro, and it will induce its entry into dormancy. In further study, we found that in the process of inducing the tumor to sleep in vitro, the indolamine 2,3 dioxygenase (indoleamine 2,3-dioxygena) Se, IDO), kynurenine (KYN), aromatic hydrocarbon receptor (aryl hydrocarbon receptor, AhR), a metabolic transcriptional factor signaling pathway, plays an important role in the action of.IFN gamma on tumor regenerative cells and can induce its entry into dormancy. The expression of IDO is up and then produces a large number of endogenous Kyn. Kyn combined with AhR and activates AhR, AhR enters the nucleus and binds to the promoter of the corresponding gene, resulting in the expression of the related cycle block gene, resulting in cell cycle arrest. The protein P27 changes obviously, and the signaling pathway of the AhR gene transcription regulation.IFN gamma -IDO-AhR-P27 has a great effect on the dormancy of the tumor regenerated cells. We also find that the dormancy of the tumor can be broken through the use of the associated inhibitor combined with IFN gamma, and we have obtained a better therapeutic effect for the future clinical practice. A new way of thinking for the treatment of tumor is put forward.

【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.5
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本文编号：1808596