發(fā)布時間：2018-01-01 02:19

  本文關鍵詞：人類γδT細胞配體分子MutS同源蛋白2—應激性膜異位表達的信號調控機制及在細胞惡變中的膜異位表達　出處：《北京協(xié)和醫(yī)學院》2012年博士論文　論文類型：學位論文

  更多相關文章： hMSH2 γδT細胞 氧化應激 p38-MAPK SAPK-JNK IL-18 IFN-γ

【摘要】：人類γδT細胞具有天然免疫細胞特征,可直接識別某些應激誘導的、細胞表面異常表達的抗原分子并啟動γδT細胞的早期活化,在清除病原體和維持機體免疫穩(wěn)態(tài)過程中發(fā)揮了重要作用。 人MutS同源蛋白2(Human MutS homologue2, hMSH2)是DNA錯配修復系統(tǒng)成員之一,主要在胞漿合成,并在細胞核內發(fā)揮DNA損傷的錯配修復作用。hMSH2缺陷或表達異常多見于各種腫瘤細胞。本課題組的前期研究發(fā)現(xiàn),hMSH2在正常細胞膜上幾乎不表達,而在腫瘤細胞膜表面呈廣泛的異位表達。這種膜異位表達能被Epstein-barr病毒(Epstein-barr virus, EBV)感染所誘導。異位表達的hMSH2可為γδT細胞受體(78T cell receptor, TCRγδ)和NK細胞受體成員2D (Natural killer group2member D, NKG2D)雙重識別,從而促進γδT細胞對病毒感染細胞的清除。然而,目前hMSH2膜異位表達的調控方式尚不明確。澄清γδT細胞應激性配體分子膜表達的分子機制,將有助于進一步揭示γδT細胞在免疫監(jiān)視中的重要作用。 鑒此,本研究主要針對以下三個科學問題展開：一是hMSH2是否為γδT細胞的應激性配體分子?二是hMSH2應激性膜異位表達的信號調控機制是什么?三是細胞惡變過程中,hMSH2的膜異位表達變化及其對γδT細胞殺傷惡變細胞存在什么影響?本文分兩部分就上述三個科學問題進行研究,證實了hMSH2是γδT細胞的應激性配體分子,并發(fā)現(xiàn)MAPK家族的p38絲裂原活化蛋白激酶(p38mitogen-activated protein kinase, p38-MAPK)通路和應激活化蛋白激酶/c-Jun氨基末端激酶(Stress-activated protein kinase/c-Jun N-terminal kinase, SAPK-JNK)通路是調控hMSH2應激性膜異位表達的重要信號通路,初步揭示了細胞的惡變過程可伴隨著hMSH2膜異位表達水平升高的規(guī)律性變化。 本研究的第一部分包括三項內容。第一項內容旨在證實應激環(huán)境中,hMSH2在腫瘤細胞上的膜異位表達是否具有可誘導性。通過建立熱應激和氧化應激等細胞應激模型,分析了hMSH2應激時膜異位表達情況。流式細胞術(Flow cytometry, FCM)榆測結果表明,hMSH2在腎癌細胞表面呈低水平的組成性表達；應激環(huán)境中,hMSH2膜異位表達呈不同程度上調,其中以氧化應激誘導hMSH2膜異位趨勢最明顯(從8-10%上調至40-50%),而抗氧化劑N-乙酰半胱氨酸(N-acetyl-L-cysteine, NAC)能抑制應激誘導的hMSH2膜異位表達(從40-50%下調至6-10%),提示hMSH2的膜異位表達可能被應激環(huán)境所誘導。隨后,在幾種對γδT細胞殺傷作用敏感的血液來源腫瘤細胞(黑素瘤、淋巴瘤和白血病腫瘤)中,證實了hMSH2在腫瘤細胞膜上的異位表達具有可誘導性。運用實時熒光定量PCR (Quantitative real-time PCR, qRT-PCR)和免疫印跡技術,發(fā)現(xiàn)氧化應激可誘導hMSH2mRNA及總蛋白表達水平升高,提示氧化應激可能在轉錄水平上調hMSH2的表達。本研究還發(fā)現(xiàn),hMSH2與γδT細胞經典的應激性配體分子MHC-class I類鏈相關抗原A/B (MHC-class I chain related A and B, MICA/B)在胞膜的應激性表達上調趨勢一致,進一步證實了應激環(huán)境中,hMSH2在腫瘤細胞上的膜異位表達具有可誘導性,提示該分子可能為危險相關分子模式(Danger associated molecular pattern, DAMP)家族的成員。 本研究第一部分的第二項工作,是利用腎癌細胞建立的氧化應激模型,探討了hMSH2應激性膜異位表達的信號調控機制。Western blot結果顯示,氧化應激時MAPK家族的p38-MAPK、SAPK-JNK和絲裂原活化蛋白／胞外信號調節(jié)激酶(Mitogen-activated protein/extracellular signal-regulated kinase, MEK-ERK)通路均處于磷酸化活化狀態(tài)。利用qRT-PCR、FCM和Western blot技術聯(lián)合三條信號通路特異性抑制劑,對應激前后hMSH2mRNA、膜異位和總蛋白表達進行分析,初步證實了p38-MAPK和SAPK-JNK通路主要參與應激時hMSH2膜異位表達的調控,而未見MEK/ERK通路發(fā)揮調控作用。進而,當促進p38-MAPK和SAPK-JNK通路上游共同的結點激酶——凋亡信號激酶1(Apoptosis signal-regulating kinase1, ASK1)活化時,hMSH2在腎癌細胞上的膜異位表達上調,驗證了上述兩條通路對hMSH2膜異位表達的調節(jié)作用。采用qRT-PCR和Wesetern blot方法,發(fā)現(xiàn)p38-MAPK和SAPK-JNK通路下游的激活轉錄因子3(Activating transcription factor3,ATF3)對氧化應激敏感,且hMSH2調控區(qū)存在ATF3的結合位點。運用小RNA干擾(Small interference RNA, siRNA)技術靶向敲低腎癌細胞ATF3表達時,氧化應激誘導的hMSH2膜異位表達水平明顯降低,而MICA/B的膜表達變化不明顯,提示氧化應激時ATF3對hMSH2膜異位表達的調控作用可能具有特異性。 本研究第一部分的第三項內容,旨在進一步確定氧化應激誘導hMSH2膜異位表達中關鍵的影響因素。運用酶聯(lián)免疫吸附試驗(Enzyme linked immunosorbentassay, ELISA),證實了氧化應激能促進腎癌細胞自分泌白細胞介素(Interleukin, IL)-18。FCM結果表明,腎癌細胞組成性表達IL-18受體α(Interleukin18receptor α, IL-18Ra),且其表達呈應激性上調。給予腎癌細胞外源性IL-18作用時,hMSH2的膜異位表達上調。利用siRNA技術敲低腎癌細胞IL-18Ra表達時,發(fā)現(xiàn)氧化應激誘導腎癌細胞自分泌的IL-18,對hMSH2膜異位表達發(fā)揮了直接的促進作用。運用ELISA和胞內免疫熒光染色技術,證實了氧化應激可刺激γδT細胞分泌干擾素(Interferon, IFN)-γ,且.外源性IFN-γ可進一步增強hMSH2的應激性膜異位表達。同時,氧化應激、外源性IL-18或IFN-γ作用能有效增強γδT細胞對腎癌細胞的殺傷作用。由此,氧化應激中γδT細胞產生的IFN-γ可能上調hMSH2在腫瘤細胞上的膜異位表達,且誘導hMSH2膜異位表達的因素可促進γδT細胞清除腎癌細胞。 由于hMSH2的膜異位表達主要存在于上皮及血液來源的腫瘤細胞而非止常細胞表面,因此本研究的第二部分內容,分析了腎正常上皮細胞系HK-2在受到三甲基膽蒽(3-methylcholanthrene,3-MCA)誘變時,hMSH2的膜異位表達變化。通過分析HK-2細胞形態(tài)和染色體數(shù)目,利用刀豆蛋白A (Concanavalin A, ConA)凝集試驗和軟瓊脂克隆形成試驗,證實了3-MCA可誘導HK-2細胞發(fā)生一定程度的惡性轉變。同時,FCM檢測發(fā)現(xiàn)hMSH2膜表達出現(xiàn)相應的上調。在誘變后期,hMSH2的膜表達在30-40%,且異位表達的hMSH2促進了γδT細胞對惡變細胞的殺傷。 綜上,本研究得出以下主要結論：1、應激環(huán)境可誘導hMSH2在腫瘤細胞上膜異位表達上調；2、氧化應激時p38-MAPK和SAPK-JNK是調控hMSH2應激性膜異位的重要信號通路；3、氧化應激刺激腎癌細胞自分泌的IL-18和γδT細胞產生的IFN-γ可促進hMSH2在腎癌細胞上膜異位表達,并增強γδT細胞對相應靶細胞的殺傷作用；4、化學誘變引發(fā)的細胞惡變過程可伴隨著hMSH2膜異位表達上調。 本研究的創(chuàng)新點：1、證實γδT細胞配體分子hMSH2具有應激性膜異位表達特征,提示hMSH2可能為DAMP成員,是應激環(huán)境中向γδT細胞預警的靶標分子；2、首次報道氧化應激時,p38-MAPK和SAPK-JNK通路為調控hMSH2在腫瘤細胞應激性膜異位表達的重要信號通路,初步揭示了hMSH2膜異位表達的分子調控機制；3、發(fā)現(xiàn)氧化應激刺激腎癌細胞自分泌的IL-18及γδT細胞產生的IFN-γ,為促進hMSH2膜異位表達的關鍵因素,提出ROS聯(lián)合上述兩種細胞因子構成一正反饋回路,維持應激環(huán)境中hMSH2在腫瘤細胞上的高水平膜異位表達這一調控模式。本文主要科研成果已在JBC上發(fā)表,表明國際學術界對本文的學術結果已有認同。
[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.

【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R392.1

【共引文獻】

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1 王媛媛;鉀通道Kv1.3在動脈粥樣硬化巨噬細胞活化中的作用及信號轉導通路[D];山東大學;2012年

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