本文選題：腫瘤 + 免疫抑制　； 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：背景腫瘤在其形成中產(chǎn)生了獨特的微環(huán)境,具有異質(zhì)性,結(jié)構(gòu)復(fù)雜,能分泌刺激性生長因子和細胞因子,募集不同類型的細胞,通過多種機制抑制免疫應(yīng)答,逃避免疫監(jiān)視和免疫系統(tǒng)的攻擊。復(fù)雜的炎癥和免疫抑制環(huán)境會大大影響腫瘤反應(yīng)性T細胞活性, 回輸?shù)腡細胞也會被腫瘤微環(huán)境所抑制,降低治療的有效性。針對某一種抑制策略的療法通常不能廣泛和長期有效,我們通過增強回輸?shù)腡細胞活性,使之能夠抵抗腫瘤中已知和未知的免疫抑制因子,可能會是一種有效的方法。對T細胞的信號通路進行分析,發(fā)現(xiàn)絲氨酸/蘇氨酸蛋白酶Akt(蛋白激酶B, PKB)在T細胞發(fā)揮效應(yīng)功能中處于信號中樞節(jié)點,通過調(diào)節(jié)Akt來提高T細胞抗腫瘤活性有顯著的研究意義。方法我們在表達卵清蛋白(OVA)抗原的黑色素瘤(B16-OVA)模型中,檢測腫瘤微環(huán)境里T細胞的Akt磷酸化水平,然后通過逆轉(zhuǎn)錄病毒轉(zhuǎn)導(dǎo)Akt至特異性靶向OVA的OT-1小鼠的T細胞中,檢測轉(zhuǎn)導(dǎo)Akt后OT-1細胞的體內(nèi)外抗腫瘤活性。在人前列腺癌PC3M模型中,我們構(gòu)建了聯(lián)合表達靶向上皮細胞粘附分子(EpCAM)的嵌合抗原受體(CAR)和Akt的逆轉(zhuǎn)錄病毒載體,轉(zhuǎn)導(dǎo)至人外周血細胞,檢測過表達Akt的腫瘤特異性T細胞的體內(nèi)外抗腫瘤活性。結(jié)果腫瘤微環(huán)境中T細胞的Akt磷酸化水平是受抑制的,OT-1過表達Akt后在B16-OVA細胞的刺激下分泌的IFN-γ增多且增殖能力增強�；剌斶^表達Akt的OT-1細胞治療B16-OVA腫瘤,其抑制腫瘤的生長能力更強且延長了小鼠的生存期。PC3M細胞也表現(xiàn)多種免疫抑制表型,相比靶向P3CM表面抗原EpCAM的CAR-T細胞,過表達Akt的CAR-T細胞在PC3M微環(huán)境中增殖受到的抑制最少,凋亡水平降低,體外殺傷能力提高；回輸過表達Akt的CAR-T細胞治療免疫缺陷的NOD/SCID荷瘤小鼠時,對PC3M腫瘤生長的抑制能力更強。結(jié)論腫瘤特異性T細胞中過表達Akt能幫助其抵抗腫瘤微環(huán)境對T細胞活性的抑制,增強腫瘤微環(huán)境中T細胞的增殖和殺傷活性,提高回輸T細胞的體內(nèi)抗腫瘤效果。我們的研究為改善過繼T細胞回輸治療的療效提供了一種研究策略,是未來臨床轉(zhuǎn)化的一種思路。背景腫瘤細胞異于正常細胞的快速增殖特性使其可以被某些化療藥物相對特異性殺傷；化療常分多個周期進行以降低毒性,但在化療的休息階段,機體正常組織恢復(fù)的同時,某些腫瘤組織也在恢復(fù),伴隨耐藥、復(fù)發(fā)和轉(zhuǎn)移的隱患。有些化療藥物有骨髓抑制的副作用,對分裂增殖的骨髓和免疫細胞同樣有毒性。有臨床研究發(fā)現(xiàn)對晚期腫瘤病人進行化療不能提高生活質(zhì)量和延長生存期。另一方面近期研究揭示有些化療藥物可以誘導(dǎo)腫瘤細胞免疫原性死亡,減少免疫抑制性細胞,激發(fā)抗腫瘤免疫。我們對不同化療周期后荷瘤小鼠的免疫狀態(tài)進行研究,探究化療過程中抗腫瘤免疫功能的變化,以期更為精準的聯(lián)合免疫治療,增強療效。方法我們建立了多周期5-FU治療小鼠CT26腫瘤的模型,觀察不同周期化療對腫瘤生長的抑制和小鼠生存期的影響。我們檢測了荷瘤小鼠一到三周期化療后,體外抗腫瘤活性,重點比較一周期和三周期化療后,脾臟中各免疫細胞亞群的細胞數(shù)和腫瘤中免疫細胞的比例,及外周血中細胞因子的變化；在腫瘤抗原刺激下各亞群細胞的增殖和分泌的細胞因子變化,及脾細胞對CT26的殺傷；并探究早期化療聯(lián)合細胞因子誘導(dǎo)的殺傷細胞(CIK)和PD-L1抗體治療的抗腫瘤效果。結(jié)果5-FU多周期化療相比一周期抑瘤生長效果更明顯,但各治療組在總生存率上沒有顯著差異。一周期和三周期化療后,相比同期荷瘤對照組,脾臟中CD8+T細胞和NK細胞絕對數(shù)沒有顯著變化；但三周期化療后,混合淋巴細胞腫瘤細胞培養(yǎng)(MLTC)中增殖的CD8+T細胞減少,分泌的IFN-γ也減少,脾細胞對CT26細胞的殺傷水平降低。而一周期化療后,腫瘤組織中浸潤的CD8+T細胞比例升高；脾細胞體外殺傷CT26增強,MLTC中增殖的CD8+T細胞數(shù)增多,分泌的IFN-γ也增多�；熀竺庖咭种埔蜃尤鏣GF-β、IL-10和腫瘤組織表達的PD-L1上升；一周期化療聯(lián)合CIK細胞和PD-L1抗體治療,相比四周期的化療和單純免疫治療,腫瘤抑制效果和總生存率都提高。結(jié)論一周期5-FU化療促進了CT26腫瘤特異性免疫應(yīng)答,而多周期化療損傷了性抗腫瘤的細胞免疫功能。化療停止后雖然免疫細胞亞群數(shù)量可以恢復(fù),但有效的抗腫瘤免疫應(yīng)答受損,化療早期即聯(lián)合免疫治療可以提高抗腫瘤療效。伴隨腫瘤免疫治療的發(fā)展,我們的研究以期為更合理有效的進行腫瘤化療和聯(lián)合免疫治療提供思路。
[Abstract]:Background tumor produces a unique microenvironment in its formation, which is heterogeneous and complex. It can secrete stimulating growth factors and cytokines, raise different types of cells, inhibit immune response through a variety of mechanisms and escape the attack of immune surveillance and immune system. Complex inflammation and immunosuppression environment will greatly affect tumor reaction. In response to the activity of the T cells, the retransfused T cells can also be suppressed by the tumor microenvironment, reducing the effectiveness of the treatment. A therapy against a certain inhibitory strategy is usually not widely and long-term. We can be effective by enhancing the retransmission of T cell activity to enable it to resist known and unknown immunosuppressive factors in the tumor. Methods. The signal pathway of T cells was analyzed. It was found that serine / threonine protease Akt (protein kinase B, PKB) was in the signal center node of T cells, and it was significant to improve the anti-tumor activity of T cells by regulating Akt. Methods we were expressing the ovalbumin (OVA) antigen of melanoma (B16-OVA). In the model, the Akt phosphorylation level of T cells in the tumor microenvironment was detected and then the antitumor activity of the OT-1 cells after the transduction of Akt was detected by retroviral transduction Akt to the T cells of the specifically targeted OVA OT-1 mice. In the PC3M model of human prostate cancer, we constructed a joint expression target epithelial cell adhesion molecule (EpCAM). The retroviral vector of the chimeric antigen receptor (CAR) and Akt, transduced to the human peripheral blood cells, detected the antitumor activity of the tumor specific T cells expressing Akt. Results the level of Akt phosphorylation of T cells in the tumor microenvironment was inhibited, and the IFN- gamma secreted by B16-OVA cells increased and increased after the OT-1 overexpressed Akt. Increased colonization ability. OT-1 cells transfused with Akt expressed B16-OVA tumor, which inhibited tumor growth and extended the survival time of.PC3M cells to a variety of immunosuppressive phenotypes. Compared to CAR-T cells targeting the P3CM surface antigen EpCAM, the CAR-T cells that overexpressed Akt were inhibited at least in PC3M microenvironment. The level of apoptosis and the ability to kill in vitro increase in vitro, and when the CAR-T cells transfused with Akt expressed in the NOD/SCID bearing mice with immune deficiency, the inhibition of PC3M tumor growth is stronger. Conclusion the overexpression of Akt in the tumor specific T cells can help it resist the inhibition of the activity of T cells in the tumor microenvironment and enhance the T cells in the tumor microenvironment. Our study provides a research strategy to improve the efficacy of adoptive T cell retransmission therapy, and it is a way of thinking in the future clinical transformation. Background tumor cells are different from normal cells in the rapid proliferation of T cells, which can be relatively specific to some chemotherapeutic drugs. Kill; chemotherapy is often divided into several cycles to reduce toxicity, but in the rest stage of the chemotherapy, while the body's normal tissue is restored, some tumor tissues are also recovering, accompanied by resistance, recurrence and metastasis. Some chemotherapeutic drugs have side effects of bone marrow suppression and are also toxic to the split and proliferating bone marrow and immune cells. It is found that chemotherapy on advanced cancer patients can not improve the quality of life and prolong life. On the other hand, recent studies have revealed that some chemotherapeutic drugs can induce immunogenic death of tumor cells, reduce immunosuppressive cells and stimulate anti-tumor immunity. We study the immune status of tumor bearing mice after different chemotherapy weeks. We studied the changes in anti tumor immune function during the chemotherapy, in order to make a more accurate combined immunotherapy and enhance the effect. Methods we established a multi cycle 5-FU model of CT26 tumor in mice, and observed the inhibition of tumor growth and the effect of mice survival on different cycles of chemotherapy. External antitumor activity, the number of cells in the spleen of the spleen and the proportion of immune cells in the tumor, and the changes in the cytokines in the peripheral blood after the one cycle and three cycles of chemotherapy, and the changes in the proliferation and secretion of the subgroups of the subgroups under the stimulation of the tumor antigen, and the killing of the spleen cells to CT26. The effect of chemotherapy combined with cytokine induced cytokine induced killer cells (CIK) and PD-L1 antibody was found. Results the effect of 5-FU multi cycle chemotherapy was more obvious than one cycle of tumor growth, but there was no significant difference in the total survival rate of each treatment group. One cycle and three cycles of chemotherapy were compared with the same period of the tumor bearing control group, the CD8+T cells in the spleen and the NK fine. After three cycles of chemotherapy, the proliferation of CD8+T cells in the mixed lymphocyte tumor cell culture (MLTC) decreased, the secretion of IFN- gamma decreased and the killing level of the spleen cells decreased to CT26 cells. After a period of chemotherapy, the proportion of CD8+T cells infiltrated in the tumor tissues increased and the spleen cells killed CT26 in vitro. The number of CD8+T cells proliferated in MLTC increased and the secretion of IFN- gamma increased. After chemotherapy, the immunosuppressive factors such as TGF- beta, IL-10 and the expression of PD-L1 in tumor tissues increased; one cycle chemotherapy combined with CIK cell and PD-L1 antibody, compared with four cycles of chemotherapy and simple immunotherapy, the effect of swelling and total survival increased. Conclusion a cycle of 5 -FU chemotherapy promotes the specific immune response of CT26 tumor, while multi cycle chemotherapy injure the cellular immune function of sexual antitumor. Although the number of immune cell subgroups can be recovered after chemotherapy, the effective anti tumor immune response is impaired, and early chemotherapy combined with immunotherapy can improve the antitumor effect. Development, our research will provide ideas for more rational and effective chemotherapy and combined immunotherapy.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R730.3
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本文編號：1894211