本文關(guān)鍵詞： γδT細(xì)胞 組蛋白去乙�；敢种苿� 免疫調(diào)節(jié) 殺傷 Notch信號(hào)通路　出處：《浙江大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：γδT細(xì)胞作為T細(xì)胞中的一個(gè)亞群,其識(shí)別抗原的方式與αβT細(xì)胞不同,γδT細(xì)胞通過(major histocompatibility complex, MHC)限制性方式識(shí)別腫瘤相關(guān)抗原,發(fā)揮免疫調(diào)控作用。目前,不少研究報(bào)道了γδT細(xì)胞及其亞群在感染、自身免疫性疾病以及抗腫瘤免疫監(jiān)視中發(fā)揮重要的作用,因此,這類細(xì)胞應(yīng)用于臨床的腫瘤免疫治療有著光明前景。盡管近年來γδT細(xì)胞在臨床上治療難治耐藥的惡性腫瘤取得一定進(jìn)展,但是大部分惡性腫瘤患者對(duì)這類細(xì)胞無反應(yīng),或者緩解后復(fù)發(fā)。本實(shí)驗(yàn)室前期的體外研究也發(fā)現(xiàn)急性髓系白血病細(xì)胞(acute myeloid leukemia, HDAC的nRNA表達(dá)水平無顯著變化,HDAC6和HDAC10的mRA表達(dá)較未處理的稍降低,有少部分HDAC家族成員,如HDAC9和HDA感。因此,γδT細(xì)胞的臨床應(yīng)用仍然阻礙重重。 γδT細(xì)胞的活化依賴于其表面受體對(duì)抗原的識(shí)別,活化后的γδT細(xì)胞產(chǎn)生生物學(xué)效應(yīng),分泌細(xì)胞因子或發(fā)揮細(xì)胞毒效應(yīng)。但是,對(duì)γδT細(xì)胞殺傷不敏感的腫瘤細(xì)胞大多由于表面抗原無法識(shí)別或發(fā)揮負(fù)調(diào)控機(jī)制,導(dǎo)致丫δT細(xì)胞未能活化。令人興奮的是,研究者發(fā)現(xiàn)了一些藥物,如雙膦酸類化合物可作為類似抗原的刺激物,促進(jìn)γδT細(xì)胞增殖和活化,增強(qiáng)抗腫瘤活性。因此,采用藥物調(diào)控γδT細(xì)胞免疫反應(yīng)并促進(jìn)殺傷功能的方案可能更易于提高惡性腫瘤的免疫治療效果。由于對(duì)γδT細(xì)胞活化和抗腫瘤機(jī)制的認(rèn)識(shí)非常有限,目前可用于調(diào)控γδT細(xì)胞生物學(xué)特性、激活其功能的藥物屬于未知的領(lǐng)域。那么,是否有合適的抗腫瘤藥物可作為促進(jìn)γδT細(xì)胞功能的理想藥物呢?只有探索和尋找合適的藥物并深入研究其對(duì)γδT細(xì)胞的調(diào)控機(jī)制,才能有效提高γδT細(xì)胞在抗腫瘤免疫治療中的效果。 我們實(shí)驗(yàn)室前期研究發(fā)現(xiàn)去甲基化藥物地西他濱可促進(jìn)調(diào)節(jié)性γδT細(xì)胞(regulatory γδT,γδTreg)細(xì)胞增殖,并增強(qiáng)對(duì)移植物抗宿主病(graft-versus-host disease, GVHD)的抑制效應(yīng)。其他研究者發(fā)現(xiàn),組蛋白去乙酰化酶抑制劑可導(dǎo)致T細(xì)胞激活,CD8+T細(xì)胞以及NK細(xì)胞表面激活型受體受組蛋白乙�；{(diào)控。更多的研究表明,T細(xì)胞的功能性基因與表觀遺傳修飾密切相關(guān),而組蛋白去乙�；敢种苿┰谂R床多用于治療T細(xì)胞相關(guān)的血液系統(tǒng)腫瘤。鑒于此,我們推測(cè)組蛋白去乙酰化酶抑制劑可作為調(diào)控γδT細(xì)胞功能的理想藥物。 在第一部分研究中,本課題采用組蛋白去乙�；敢种苿㎜BH589處理人γδT細(xì)胞,研究結(jié)果發(fā)現(xiàn)低濃度的LBH589(5nM)對(duì)γδT細(xì)胞的擴(kuò)增無顯著影響,如克隆形成能力、γδT細(xì)胞占外周血單個(gè)核細(xì)胞比例以及γδT細(xì)胞絕對(duì)數(shù)量均未出現(xiàn)明顯變化；但較高濃度或高濃度的LBH589(5nM)則顯著抑制γδT細(xì)胞擴(kuò)增,γδT細(xì)胞克隆形成數(shù)量減少,絕對(duì)數(shù)量明顯下降,但在外周血單個(gè)核細(xì)胞中的比例無顯著下降。隨LBH589濃度增加,γδT細(xì)胞表面活化分子CD69和CD25表達(dá)水平均未發(fā)生改變,各處理組γδT細(xì)胞免疫表型均大部分為CD45RA-/CD27-的效應(yīng)記憶型細(xì)胞。與殺傷功能相關(guān)的活化性受體NKG2D (natural killer group2, member D)高表達(dá)于γδT細(xì)胞,并且不隨LBH589濃度增加而改變。我們還檢測(cè)γδT細(xì)胞表達(dá)IFN-γ的能力,結(jié)果發(fā)現(xiàn)LBH589并未對(duì)IFN-γ的表達(dá)產(chǎn)生影響。 為進(jìn)一步研究LBH589對(duì)γδT細(xì)胞的細(xì)胞毒性效應(yīng)影響,我們選擇對(duì)γδT細(xì)胞不敏感的AML細(xì)胞株作為靶細(xì)胞。LBH589預(yù)處理γδT細(xì)胞后以不同效靶比對(duì)AML細(xì)胞株進(jìn)行殺傷,發(fā)現(xiàn)在較高或高濃度的LBH589(≥10nM)預(yù)處理后,γδT細(xì)胞對(duì)原先不敏感的AML細(xì)胞株(HL-60)殺傷效應(yīng)增強(qiáng),具有顯著統(tǒng)計(jì)學(xué)差異。但對(duì)另一型的AML細(xì)胞株(KG-1)的殺傷效應(yīng)無增強(qiáng)作用。研究結(jié)果提示LBH589對(duì)γδT細(xì)胞的擴(kuò)增有顯著抑制效應(yīng),但是對(duì)其表面活化分子以及免疫表型、細(xì)胞因子表達(dá)無明顯影響。與抑制增殖相反的是,LBH589可顯著增強(qiáng)γδT細(xì)胞的殺傷功能。因此,本研究首次發(fā)現(xiàn)組蛋白去乙�；敢种苿┐龠M(jìn)γδT細(xì)胞的細(xì)胞毒性效應(yīng),并且不會(huì)影響其活化狀態(tài)及細(xì)胞因子表達(dá),該結(jié)果可為提高血液系統(tǒng)惡性疾病患者采用過繼輸注或激活自體γδT細(xì)胞行免疫治療的療效提供實(shí)驗(yàn)室數(shù)據(jù)支持。 γδT細(xì)胞表面活化型受體與抗原識(shí)別后發(fā)出信號(hào),傳遞至胞內(nèi),激活一系列信號(hào)傳導(dǎo)通路,如絲裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)通路、磷脂酰肌醇3-激酶(phosphatidylinositol3-kinase, PI3K)通路,此外,Notch信號(hào)通路也被發(fā)現(xiàn)參與γδT細(xì)胞的活化和殺傷作用。但是究竟哪條通路與γδT細(xì)胞殺傷AML細(xì)胞的功能相關(guān)?LBH589增強(qiáng)γδT細(xì)胞細(xì)胞毒效應(yīng)與這些信號(hào)通路是否有關(guān),或者有另外的信號(hào)通路參與這一過程?對(duì)此,我們進(jìn)行深入研究。 第二部分研究發(fā)現(xiàn),LBH589并不抑制γδT細(xì)胞的組蛋白去乙�；�(histone deacetylase, HDAC)的mRNA表達(dá)水平,HDAC蛋白表達(dá)也無顯著影響。隨著LBH589處理γδT細(xì)胞時(shí)間延長或處理濃度增加,ERK、JNK、Akt等蛋白均未發(fā)生磷酸化,并且JNK總蛋白表達(dá)水平還隨著時(shí)間增加而降低,但ERK上游蛋白R(shí)af-1隨時(shí)間延長持續(xù)高表達(dá)；我們另外發(fā)現(xiàn)Notch2蛋白表達(dá)顯著升高,但其下游轉(zhuǎn)錄因子RBP Jκ表達(dá)無顯著改變。Notch抑制劑可逆轉(zhuǎn)LBH589對(duì)γδT細(xì)胞殺傷增強(qiáng)的效應(yīng),提示Notch信號(hào)通路與γδT細(xì)胞殺傷功能相關(guān)。此外,我們發(fā)現(xiàn)γδT細(xì)胞凋亡通路的蛋白表達(dá)水平未見明顯改變,提示LBH589對(duì)γδT細(xì)胞擴(kuò)增的抑制不是通過促進(jìn)其凋亡,可能是通過其他生存、增殖相關(guān)信號(hào)通路,調(diào)控γδT細(xì)胞的生存和凋亡過程。因此,本研究表明Notch通路被LBH589激活后,參與調(diào)節(jié)γδT細(xì)胞的殺傷效應(yīng)。Notch通路可作為γδT細(xì)胞發(fā)揮抗腫瘤功能作用的觸發(fā)點(diǎn),提高γδT細(xì)胞的免疫治療效果。 綜上所述,我們的研究首次發(fā)現(xiàn)γδT細(xì)胞殺傷血液系統(tǒng)惡性腫瘤的效應(yīng)可通過組蛋白去乙酰化酶抑制劑獲得提高,并且不會(huì)影響免疫調(diào)節(jié)功能,Notch信號(hào)通路參與組蛋白去乙�；敢种苿⿲�(duì)γδT細(xì)胞的調(diào)控作用,研究其具體機(jī)制可為優(yōu)化血液系統(tǒng)惡性疾病的免疫治療提供新策略。
[Abstract]:Gamma delta T cells as T cells in a subpopulation of different antigen recognition and alpha beta T cells, T cells (major histocompatibility complex MHC, by way of limiting) recognition of tumor associated antigen, immune regulation. At present, many studies have reported the gamma delta T cells and its subsets in infection, autoimmune diseases and anti tumor immune surveillance play an important role, therefore, this kind of cells used in clinical tumor immunotherapy has bright prospects. Although some progress in recent years of gamma delta T cells in the clinical treatment of refractory resistant malignant tumor, but the majority of patients with malignant tumors do not respond to this type of cells or relapse after remission. In our previous study also found that acute myeloid leukemia cells (acute myeloid leukemia, HDAC nRNA expression level did not change significantly, HDAC6 and HDAC10 mRA expression Not treated slightly, with a small number of members of the HDAC family, such as HDAC9 and HDA. Therefore, the clinical application of gamma delta T cells is still obstructing.
The activation of T cells depends on their surface receptors for antigen recognition, activation of gamma delta T cells to produce biological effects, the secretion of cytokines or exert cytotoxic effects. However, the gamma delta T cells is not sensitive to the tumor cell surface antigen mostly due to unrecognized or play a negative regulatory mechanism, leading to ya Delta T cells did not activate. Exciting, researchers found that some drugs, such as bisphosphonates as a stimulant similar antigen, promote the proliferation of T cells and activation, enhanced antitumor activity. Therefore, the drug regulation of gamma delta T cell immune response and promote the cytotoxic function scheme may be more to improve the effect of immunotherapy of malignant tumors. The understanding of gamma delta T cell activation and anti tumor mechanism is very limited, currently available for regulation of gamma delta T cells biological characteristics, drug activation of its function belonging to the unknown Then, is there any suitable antitumor drug as an ideal drug to promote the function of gamma delta T cells? Only by exploring and finding the appropriate drugs and further studying the regulatory mechanism of the gamma delta T cells can we effectively improve the effect of gamma delta T cells in anti-tumor immunotherapy.
Our previous study found that demethylation drug decitabine can promote the regulation of gamma delta T cells (regulatory gamma delta T, gamma delta Treg) cell proliferation, and enhance of graft-versus-host disease (graft-versus-host disease, GVHD). The inhibitory effect of other researchers found that histone deacetylase inhibitors can lead to the activation of T cell, CD8+T cell and NK cell surface receptor activation by histone acetylation. Many studies suggest that functional genes of T cells with epigenetic modifications are closely related, and histone deacetylase inhibitors used in clinical treatment of T cell related hematological malignancies. In view of this we speculate that the ideal drug, histone deacetylase inhibitors can be used as the control function of gamma Delta T cells.
In the first part of the study, this paper uses the histone deacetylase inhibitor LBH589 treated human gamma delta T cells, the results showed that low concentration of LBH589 (5nM) had no significant effect on the amplification of gamma delta T cells, such as clone formation ability of gamma delta T cells accounted for significant changes were not found in peripheral blood nuclear cell proportion and absolute number of gamma delta T cells; but high concentration or high concentration of LBH589 (5nM) inhibited gamma delta T cells, T cells clone formation decreased, the absolute number decreased significantly, but in peripheral blood mononuclear cells in proportion with the concentration of LBH589 decreased significantly. Increased gamma activation molecules CD69 and CD25 surface expression of delta T cells were not changed, each group of gamma delta T cell phenotype were mostly effector memory CD45RA-/CD27- cells. The activation of NKG2D receptor associated with the cytotoxicity of killer (NATURAL group2, memb Er D) is highly expressed in gamma delta T cells, and does not change with increasing LBH589 concentration. We also detect the ability of gamma delta T cells to express IFN- gamma, and it is found that LBH589 has no effect on IFN- gamma expression.
For the cytotoxic effect of LBH589 on the further study of gamma delta T cells, we choose the AML cell line is not sensitive to gamma delta T cells as target cells pretreated with.LBH589 gamma delta T cells after killing by different effector target ratio of AML cells were found in the high or high concentration of LBH589 (10nM) pretreatment after gamma delta T cells on AML cells was not sensitive (HL-60) enhanced the killing effect, the difference was statistically significant. But on another type of AML cell line (KG-1) of the killing effect of enhancing effect. The results suggest that the LBH589 of gamma delta T cells was significantly inhibited, but the the surface of activated molecules and immune phenotype, cytokine expression was not affected. In contrast with the inhibition of proliferation is that LBH589 can significantly enhance the gamma delta T cell killing function. Therefore, this is the first study found that histone deacetylase inhibitors promote cytotoxic gammadelta T cells Sex effect will not affect its activation state and cytokine expression. This result can provide laboratory data support for improving the efficacy of adoptive infusion or activation of autologous gamma delta T cells in patients with hematological malignancies.
The surface of gamma delta T cells activation receptor and antigen recognition after the signal transmitted to the intracellular, activate a series of signal transduction pathways, such as mitogen activated protein kinase (mitogen-activated protein, kinase, MAPK) pathway, phosphatidylinositol 3- kinase (phosphatidylinositol3-kinase, PI3K) pathway, in addition, Notch signal pathway were also found to be involved in gamma delta T cell activation and cytotoxicity. But what pathways and gamma delta T cells killing AML cell function? LBH589 enhanced gamma delta T cells and cytotoxic effect of these signaling pathways is related, or other signaling pathways involved in this process? In this regard, we conduct in-depth research.
The second part of the study found that LBH589 did not inhibit T cells of histone deacetylase (histone deacetylase, HDAC) mRNA expression, HDAC protein expression had no significant impact. With the LBH589 processing of gamma delta T cells prolonged or the increase of the concentration, ERK, JNK, Akt protein were not phosphorylated the expression of JNK, and total protein levels also decreased with time increasing, but upstream of the ERK protein Raf-1 with the prolonging of sustained high expression; we also found that Notch2 protein expression was significantly increased, but the downstream transcription factor RBP expression had no significant change J kappa.Notch inhibitor could reverse LBH589 of gamma delta T cells enhanced effect, suggesting that Notch signal pathway and gamma delta T cell killing function. In addition, we found that gamma delta T cells apoptosis protein expression level did not change significantly, suggesting that inhibition of LBH589 amplification of gamma delta T cells is not through the promotion of Apoptosis may be mediated by other survival, proliferation related signal pathway, survival and apoptosis regulation of gamma delta T cells. Therefore, this study suggests that the Notch pathway is activated by LBH589, is involved in the regulation of gamma delta T cell killing effect of.Notch pathway can be used as gamma delta T cells play the anti-tumor function of the trigger points. To improve the immune therapeutic effect of gamma delta T cells.
In summary, our study is the first to find the effect of gamma delta T cells of hematologic malignancies by histone deacetylase inhibitors have improved, and will not affect the immune function, the Notch signaling pathway is involved in regulation of histone deacetylase inhibitors of gamma delta T cells, to study its specific mechanism may provide a new strategy for the treatment of immune optimization of malignant hematological diseases.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R730.51

【共引文獻(xiàn)】

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3 趙杰;瘦素增強(qiáng)人外周血γδT細(xì)胞對(duì)肺癌細(xì)胞殺傷作用的實(shí)驗(yàn)研究[D];蘇州大學(xué);2011年

4 于松濤;人類腫瘤浸潤γδT淋巴細(xì)胞的體外擴(kuò)增及體內(nèi)外功能研究[D];中國協(xié)和醫(yī)科大學(xué);1998年

5 何小鵑;人類γδT細(xì)胞抗原表位肽的鑒定和健康人γδT細(xì)胞克隆的建立及鑒定[D];中國協(xié)和醫(yī)科大學(xué);2008年

6 張鐵;人γδT細(xì)胞過繼免疫治療的臨床前研究[D];中國協(xié)和醫(yī)科大學(xué);2007年

7 田軍;聯(lián)合重組纖維連接蛋白活化異基因殺傷細(xì)胞過繼治療腎癌的實(shí)驗(yàn)研究[D];中國協(xié)和醫(yī)科大學(xué);2008年

8 郭陽;TCRγ9/δ2 CDR3δ結(jié)合腫瘤抗原的分子基礎(chǔ)腫瘤反應(yīng)性TCRγ9/δ2移植的人源化抗體/抗體片段的抗腫瘤作用研究[D];中國協(xié)和醫(yī)科大學(xué);2009年

9 鄭靜;TCRγδ-Ig融合蛋白抗人卵巢癌活性的研究及功能性抗人TCRγδ抗體及單鏈抗體的制備和鑒定[D];北京協(xié)和醫(yī)學(xué)院;2012年

10 喬志新;吉西他濱協(xié)同西達(dá)本胺或雷公藤甲素誘導(dǎo)胰腺癌細(xì)胞凋亡的研究[D];吉林大學(xué);2013年

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4 李秉擘;具有生物活性的天然產(chǎn)物根皮素的合成與純化的相關(guān)研究[D];浙江大學(xué);2012年

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6 葛y泍，

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