人类γδT细胞配体分子MutS同源蛋白2—应激性膜异位表达的信号调控机制及在细胞恶变中的膜异位表达

发布时间：2018-01-01 02:19

本文关键词：人类γδT细胞配体分子MutS同源蛋白2—应激性膜异位表达的信号调控机制及在细胞恶变中的膜异位表达　出处：《北京协和医学院》2012年博士论文　论文类型：学位论文

【摘要】：人类γδT细胞具有天然免疫细胞特征,可直接识别某些应激诱导的、细胞表面异常表达的抗原分子并启动γδT细胞的早期活化,在清除病原体和维持机体免疫稳态过程中发挥了重要作用。人MutS同源蛋白2(Human MutS homologue2, hMSH2)是DNA错配修复系统成员之一,主要在胞浆合成,并在细胞核内发挥DNA损伤的错配修复作用。hMSH2缺陷或表达异常多见于各种肿瘤细胞。本课题组的前期研究发现,hMSH2在正常细胞膜上几乎不表达,而在肿瘤细胞膜表面呈广泛的异位表达。这种膜异位表达能被Epstein-barr病毒(Epstein-barr virus, EBV)感染所诱导。异位表达的hMSH2可为γδT细胞受体(78T cell receptor, TCRγδ)和NK细胞受体成员2D (Natural killer group2member D, NKG2D)双重识别,从而促进γδT细胞对病毒感染细胞的清除。然而,目前hMSH2膜异位表达的调控方式尚不明确。澄清γδT细胞应激性配体分子膜表达的分子机制,将有助于进一步揭示γδT细胞在免疫监视中的重要作用。鉴此,本研究主要针对以下三个科学问题展开：一是hMSH2是否为γδT细胞的应激性配体分子?二是hMSH2应激性膜异位表达的信号调控机制是什么?三是细胞恶变过程中,hMSH2的膜异位表达变化及其对γδT细胞杀伤恶变细胞存在什么影响?本文分两部分就上述三个科学问题进行研究,证实了hMSH2是γδT细胞的应激性配体分子,并发现MAPK家族的p38丝裂原活化蛋白激酶(p38mitogen-activated protein kinase, p38-MAPK)通路和应激活化蛋白激酶/c-Jun氨基末端激酶(Stress-activated protein kinase/c-Jun N-terminal kinase, SAPK-JNK)通路是调控hMSH2应激性膜异位表达的重要信号通路,初步揭示了细胞的恶变过程可伴随着hMSH2膜异位表达水平升高的规律性变化。本研究的第一部分包括三项内容。第一项内容旨在证实应激环境中,hMSH2在肿瘤细胞上的膜异位表达是否具有可诱导性。通过建立热应激和氧化应激等细胞应激模型,分析了hMSH2应激时膜异位表达情况。流式细胞术(Flow cytometry, FCM)榆测结果表明,hMSH2在肾癌细胞表面呈低水平的组成性表达；应激环境中,hMSH2膜异位表达呈不同程度上调,其中以氧化应激诱导hMSH2膜异位趋势最明显(从8-10%上调至40-50%),而抗氧化剂N-乙酰半胱氨酸(N-acetyl-L-cysteine, NAC)能抑制应激诱导的hMSH2膜异位表达(从40-50%下调至6-10%),提示hMSH2的膜异位表达可能被应激环境所诱导。随后,在几种对γδT细胞杀伤作用敏感的血液来源肿瘤细胞(黑素瘤、淋巴瘤和白血病肿瘤)中,证实了hMSH2在肿瘤细胞膜上的异位表达具有可诱导性。运用实时荧光定量PCR (Quantitative real-time PCR, qRT-PCR)和免疫印迹技术,发现氧化应激可诱导hMSH2mRNA及总蛋白表达水平升高,提示氧化应激可能在转录水平上调hMSH2的表达。本研究还发现,hMSH2与γδT细胞经典的应激性配体分子MHC-class I类链相关抗原A/B (MHC-class I chain related A and B, MICA/B)在胞膜的应激性表达上调趋势一致,进一步证实了应激环境中,hMSH2在肿瘤细胞上的膜异位表达具有可诱导性,提示该分子可能为危险相关分子模式(Danger associated molecular pattern, DAMP)家族的成员。本研究第一部分的第二项工作,是利用肾癌细胞建立的氧化应激模型,探讨了hMSH2应激性膜异位表达的信号调控机制。Western blot结果显示,氧化应激时MAPK家族的p38-MAPK、SAPK-JNK和丝裂原活化蛋白／胞外信号调节激酶(Mitogen-activated protein/extracellular signal-regulated kinase, MEK-ERK)通路均处于磷酸化活化状态。利用qRT-PCR、FCM和Western blot技术联合三条信号通路特异性抑制剂,对应激前后hMSH2mRNA、膜异位和总蛋白表达进行分析,初步证实了p38-MAPK和SAPK-JNK通路主要参与应激时hMSH2膜异位表达的调控,而未见MEK/ERK通路发挥调控作用。进而,当促进p38-MAPK和SAPK-JNK通路上游共同的结点激酶——凋亡信号激酶1(Apoptosis signal-regulating kinase1, ASK1)活化时,hMSH2在肾癌细胞上的膜异位表达上调,验证了上述两条通路对hMSH2膜异位表达的调节作用。采用qRT-PCR和Wesetern blot方法,发现p38-MAPK和SAPK-JNK通路下游的激活转录因子3(Activating transcription factor3,ATF3)对氧化应激敏感,且hMSH2调控区存在ATF3的结合位点。运用小RNA干扰(Small interference RNA, siRNA)技术靶向敲低肾癌细胞ATF3表达时,氧化应激诱导的hMSH2膜异位表达水平明显降低,而MICA/B的膜表达变化不明显,提示氧化应激时ATF3对hMSH2膜异位表达的调控作用可能具有特异性。本研究第一部分的第三项内容,旨在进一步确定氧化应激诱导hMSH2膜异位表达中关键的影响因素。运用酶联免疫吸附试验(Enzyme linked immunosorbentassay, ELISA),证实了氧化应激能促进肾癌细胞自分泌白细胞介素(Interleukin, IL)-18。FCM结果表明,肾癌细胞组成性表达IL-18受体α(Interleukin18receptor α, IL-18Ra),且其表达呈应激性上调。给予肾癌细胞外源性IL-18作用时,hMSH2的膜异位表达上调。利用siRNA技术敲低肾癌细胞IL-18Ra表达时,发现氧化应激诱导肾癌细胞自分泌的IL-18,对hMSH2膜异位表达发挥了直接的促进作用。运用ELISA和胞内免疫荧光染色技术,证实了氧化应激可刺激γδT细胞分泌干扰素(Interferon, IFN)-γ,且.外源性IFN-γ可进一步增强hMSH2的应激性膜异位表达。同时,氧化应激、外源性IL-18或IFN-γ作用能有效增强γδT细胞对肾癌细胞的杀伤作用。由此,氧化应激中γδT细胞产生的IFN-γ可能上调hMSH2在肿瘤细胞上的膜异位表达,且诱导hMSH2膜异位表达的因素可促进γδT细胞清除肾癌细胞。由于hMSH2的膜异位表达主要存在于上皮及血液来源的肿瘤细胞而非止常细胞表面,因此本研究的第二部分内容,分析了肾正常上皮细胞系HK-2在受到三甲基胆蒽(3-methylcholanthrene,3-MCA)诱变时,hMSH2的膜异位表达变化。通过分析HK-2细胞形态和染色体数目,利用刀豆蛋白A (Concanavalin A, ConA)凝集试验和软琼脂克隆形成试验,证实了3-MCA可诱导HK-2细胞发生一定程度的恶性转变。同时,FCM检测发现hMSH2膜表达出现相应的上调。在诱变后期,hMSH2的膜表达在30-40%,且异位表达的hMSH2促进了γδT细胞对恶变细胞的杀伤。综上,本研究得出以下主要结论：1、应激环境可诱导hMSH2在肿瘤细胞上膜异位表达上调；2、氧化应激时p38-MAPK和SAPK-JNK是调控hMSH2应激性膜异位的重要信号通路；3、氧化应激刺激肾癌细胞自分泌的IL-18和γδT细胞产生的IFN-γ可促进hMSH2在肾癌细胞上膜异位表达,并增强γδT细胞对相应靶细胞的杀伤作用；4、化学诱变引发的细胞恶变过程可伴随着hMSH2膜异位表达上调。本研究的创新点：1、证实γδT细胞配体分子hMSH2具有应激性膜异位表达特征,提示hMSH2可能为DAMP成员,是应激环境中向γδT细胞预警的靶标分子；2、首次报道氧化应激时,p38-MAPK和SAPK-JNK通路为调控hMSH2在肿瘤细胞应激性膜异位表达的重要信号通路,初步揭示了hMSH2膜异位表达的分子调控机制；3、发现氧化应激刺激肾癌细胞自分泌的IL-18及γδT细胞产生的IFN-γ,为促进hMSH2膜异位表达的关键因素,提出ROS联合上述两种细胞因子构成一正反馈回路,维持应激环境中hMSH2在肿瘤细胞上的高水平膜异位表达这一调控模式。本文主要科研成果已在JBC上发表,表明国际学术界对本文的学术结果已有认同。
[Abstract]:Human gamma delta T cells possess the characteristics of natural immune cells. They can directly recognize some stress induced abnormal antigen molecules on the cell surface and initiate the early activation of gamma delta T cells, which play an important role in clearing pathogens and maintaining immune homeostasis.
Human MutS homolog 2 (Human MutS, homologue2, hMSH2) is one of the members of the DNA mismatch repair system, mainly in the cytoplasm of synthesis, and play the DNA damage effect of.HMSH2 mismatch repair defects or abnormal expression in a variety of tumor cells in the nucleus. Ourprevious study found that almost no hMSH2 expression in normal the cell membrane, and is widely expressed in the tumor cell surface. Ectopic expression of this membrane can be ectopic Epstein-barr virus (Epstein-barr virus, EBV) induced by infection. The ectopic expression of hMSH2 for gamma delta T cell receptor (78T cell receptor, TCR gamma delta NK cell receptor) and 2D (Natural killer group2member members D, NKG2D) dual recognition, so as to promote T cells to virus infected cells removed. However, the current regulation of ectopic expression of the hMSH2 film is not clear. To clarify the gamma delta T cells stress-induced ligand molecules expressed on the surface of points The submechanism will help to further reveal the important role of gamma delta T cells in immune surveillance.
In view of this, this study focuses on the following three scientific questions: one is whether hMSH2 is a stress-induced ligand of gamma delta T cells? Two signal regulatory mechanism of hMSH2 expression of stress endometriosis is what? Three is in the process of malignant transformation of cells, ectopic hMSH2 expression and membrane of gamma delta T cells what are the effects of malignant cells? This paper is divided into two parts of the three scientific issues of research, demonstrates that hMSH2 is a stress-induced ligand of gamma delta T cells, and found that the MAPK family of p38 mitogen activated protein kinase (p38mitogen-activated protein, kinase, p38-MAPK) pathway and stress activated protein kinase /c-Jun N-terminal kinase (Stress-activated protein kinase/c-Jun N-terminal kinase, SAPK-JNK) pathway is an important pathway to regulate the expression of hMSH2 membrane stress heterotopic, reveal the process of malignant transformation of cells can be accompanied by hMSH The regular changes in the level of ectopic expression of 2 membranes.
The first part of this study includes three contents. The first is to confirm the contents of stress, the expression of hMSH2 on tumor cells is induced by ectopic membrane. The cell stress model of heat stress and oxidative stress, analyzes the situation of ectopic expression of hMSH2 membrane stress. Flow cytometry (Flow cytometry FCM), test results showed that hMSH2 was constitutively expressed at low levels in renal carcinoma cell surface; stress environment, ectopic expression of hMSH2 membrane showed different degrees of increase, the oxidative stress induced ectopic hMSH2 membrane most obvious trend (up from 8-10% to 40-50%), and the antioxidant of N- acetylcysteine (N-acetyl-L-cysteine, NAC) expression hMSH2 can inhibit the stress induced by endometriosis (down from 40-50% to 6-10%), suggesting that the membrane of ectopic hMSH2 expression may be induced by environmental stress. Then, in the role of several sensitive to gamma delta T cells killing The source of blood tumor cells (melanoma, lymphoma and leukemia), confirmed that the ectopic expression of hMSH2 in tumor cell membrane can be induced. The real-time fluorescence quantitative PCR (Quantitative real-time PCR, qRT-PCR) and Western blot, found that oxidative stress can induce an increase in the expression level of hMSH2mRNA and total protein expression of oxidation stress may be at the transcriptional level by hMSH2. This study also found that stress-induced ligand MHC-class hMSH2 and gamma delta T cells classical class I chain related antigen A/B (MHC-class I chain related A and B, MICA/B) on the cell membrane of the stress-induced upregulation of the expression of the same trend, further confirmed the stress environment, can induce with ectopic expression of hMSH2 membrane on tumor cells, suggesting that the molecule may be danger associated molecular patterns (Danger associated molecular pattern, DAMP) of the family members.
The first part of the research work of second, is the use of oxidative stress in renal carcinoma cell model established, signal regulation mechanism of.Western blot on expression of hMSH2 results of stress endometriosis showed that p38-MAPK of the MAPK family of oxidative stress, SAPK-JNK and mitogen activated protein / extracellular signal regulated kinase (Mitogen-activated protein/extracellular signal-regulated kinase, MEK-ERK) pathway in the state of activation of phosphorylation. Using qRT-PCR, FCM and Western blot technology combined with the three signaling pathway specific inhibitor hMSH2mRNA on stress before and after the analysis of ectopic expression of membrane and total protein, confirmed that the regulation of the expression of hMSH2 p38-MAPK and SAPK-JNK pathway in endometriosis is mainly involved in stress, but not the MEK/ERK pathway play a regulatory role. Furthermore, when promoting p38-MAPK and SAPK-JNK pathway upstream common node kinase - signal induced apoptosis Enzyme 1 (Apoptosis signal-regulating kinase1, ASK1) activation, expression of hMSH2 in renal cell carcinoma on endometriosis, verify the regulatory role of these two pathways on the expression of hMSH2 in endometriosis. Using qRT-PCR and Wesetern blot, found that p38-MAPK and SAPK-JNK pathway downstream of the activating transcription factor 3 (Activating transcription, Factor3, ATF3) sensitive to oxidative stress, and hMSH2 regulatory region have ATF3 binding sites. The use of small interfering RNA (siRNA Small interference RNA) technology targeted knockdown of ATF3 expression in renal cell carcinoma, the expression level of hMSH2 in endometriosis induced oxidative stress significantly decreased, while MICA/B membrane expression did not change significantly and the regulatory effect of ATF3 on the expression of hMSH2 endometriosis suggests that oxidative stress may have the specificity.
Third the content of the first part of this study, in order to determine the expression of influence factors of key hMSH2 endometriosis induced oxidative stress. By using enzyme-linked immunosorbent assay (Enzyme linked, Immunosorbentassay, ELISA), confirmed that oxidative stress can promote cell autocrine secretion of interleukin (Interleukin, IL) -18.FCM results showed that the renal carcinoma cell the constitutive expression of IL-18 receptor alpha (Interleukin18receptor alpha, IL-18Ra), and its expression was up-regulated. Exogenous stress induced renal cell carcinoma cell IL-18, upregulate the expression of ectopic hMSH2 membrane. By using siRNA technology on low expression of renal cell carcinoma IL-18Ra cells, found that oxidative stress induced autocrine renal cell carcinoma cells IL-18 expression plays a direct to promote the role of hMSH2 membrane by ELISA and ectopic. Intracellular immunofluorescence staining confirmed that oxidative stress can stimulate the secretion of interferon gamma delta T cells (Interfero N, IFN) - gamma, gamma and exogenous IFN- can further enhance the stress film ectopic expression of hMSH2. At the same time, oxidative stress, exogenous IL-18 or IFN- gamma function can effectively enhance the killing effect of T cells on renal cells. Thus, IFN- gamma gamma delta T cells produce oxidative stress in May expression of hMSH2 on tumor cells membrane and hMSH2 membrane induced factors of ectopic ectopic expression can promote cell removal of gamma delta T cells.
The expression of ectopic hMSH2 membrane mainly existed in epithelial and blood derived tumor cells instead of normal cell surface, so the second part of this study, analysis of the normal renal epithelial cell line HK-2 in three by 3-methylcholanthrene (3-methylcholanthrene, 3-MCA) mutation, expression of ectopic hMSH2. Through the analysis of film form number and HK-2 cell chromosome, using concanavalin A (Concanavalin A ConA) agglutination test and soft agar colony formation assay confirmed that 3-MCA could induce malignant transformation of HK-2 cells to a certain extent. At the same time, FCM test found that hMSH2 membrane expression of the corresponding increase. In the late hMSH2 mutation, the expression of 30-40% in the film, and ectopic expression of hMSH2 promotes the killing of gamma delta T cells on malignant cells.
In summary, this study draws the following conclusions: 1, stress can induce hMSH2 in tumor cell membrane of ectopic expression; 2, oxidative stress and p38-MAPK SAPK-JNK is an important signal transduction pathway of hMSH2 stress endometriosis; 3, IFN- gamma oxidative stress stimulates the production of IL-18 and T cells in renal cell carcinoma by autocrine can promote hMSH2 in renal cell carcinoma cell membrane on ectopic expression, and enhance the killing effect of gamma delta T cells on the target cells; 4 cell malignant transformation caused by chemical mutagenesis can be accompanied by hMSH2 membrane ectopic expression.
The innovation of this research: 1, confirmed that the gamma delta T cells hMSH2 ligands have stress membrane ectopic expression characteristics, suggesting that hMSH2 may be a member of the DAMP, is the stress to the target molecule of gamma delta T cells early warning; 2 reported for the first time, oxidative stress, p38-MAPK and SAPK-JNK pathways for regulating the expression of an important signaling pathway hMSH2 in tumor cell membrane stress of ectopic, molecular mechanism revealed ectopic expression of the hMSH2 film; 3, IFN- found that gamma oxidative stress stimulates the production of autocrine renal cell carcinoma cells IL-18 and T cells, as the key factor to promote the expression of hMSH2 in ectopic membrane, the ROS combined these two cytokines constitute a a positive feedback loop, to maintain a high level of hMSH2 membrane stress in cancer cells by ectopic expression of this control mode. The main research results have been published in JBC, shows that the international academic circles on the academic results have been Agree.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392.1

【共引文献】