發(fā)布時(shí)間：2018-06-29 12:52

  本文選題：嵌合抗原受體修飾的T細(xì)胞 + 上皮細(xì)胞粘附分子　； 參考：《成都醫(yī)學(xué)院》2017年碩士論文

【摘要】：嵌合抗原受體(Chimeric Antigen Receptor,CAR)是將識(shí)別腫瘤抗原的抗體分子和T細(xì)胞活化信號(hào)耦合的融合分子。經(jīng)CAR修飾的T細(xì)胞將單克隆抗體的精確靶向特異性與細(xì)胞毒性T細(xì)胞的強(qiáng)毒性和持久性相結(jié)合,能夠特異性識(shí)別腫瘤相關(guān)抗原而不依賴(lài)MHC限制,從而高效持久地殺傷腫瘤細(xì)胞。迄今為止,CAR T細(xì)胞免疫治療已經(jīng)在血液惡性腫瘤的臨床治療中取得了顯著的療效,但對(duì)實(shí)體腫瘤的治療并未取得突破性進(jìn)展。究其原因可能和實(shí)體腫瘤組織表面高度特異性腫瘤抗原的缺失有關(guān)。一個(gè)理想的靶抗原應(yīng)當(dāng)在腫瘤組織表面高表達(dá),在正常組織不表達(dá)或低表達(dá)。上皮細(xì)胞粘附分子(Epithelial cell adhesion molecule,EpCAM)是結(jié)腸癌、肝癌等惡性腫瘤的干細(xì)胞標(biāo)志之一,在腫瘤組織高表達(dá),在正常組織僅在基底膜外側(cè)表達(dá)。本研究中,我們以EpCAM為靶點(diǎn),構(gòu)建靶向EpCAM的CAR T細(xì)胞,探究靶向EpCAM的CAR T細(xì)胞對(duì)EpCAM+結(jié)腸癌細(xì)胞的殺傷能力。目的本課題以EpCAM為研究靶點(diǎn),構(gòu)建穩(wěn)定表達(dá)EpCAM-CAR基因的T細(xì)胞,探究靶向EpCAM的CAR T細(xì)胞對(duì)EpCAM+結(jié)腸癌細(xì)胞的殺傷能力。材料與方法(1)轉(zhuǎn)化、鑒定、擴(kuò)增pCLK-EF-1 kana EpCAM-CAR,psPAX2、pMD2.G這三個(gè)病毒包裝質(zhì)粒;(2)利用磷酸鈣法轉(zhuǎn)染293T細(xì)胞以制備重組慢病毒顆粒,超高速離心以濃縮重組慢病毒顆粒,實(shí)時(shí)熒光定量PCR法檢測(cè)重組慢病毒的滴度;(3)利用免疫蛋白印跡法和流式細(xì)胞術(shù)檢測(cè)EpCAM在五種結(jié)腸癌細(xì)胞株(SW620、SW480、HCT116、LoVo、HT-29)中的表達(dá);(4)人外周血單核細(xì)胞的提取及T細(xì)胞的活化、培養(yǎng);(5)用攜帶EpCAM-CAR基因表達(dá)框的重組慢病毒顆粒轉(zhuǎn)染人T淋巴細(xì)胞;(6)利用免疫蛋白印跡法、定量PCR法及流式細(xì)胞術(shù)檢測(cè)轉(zhuǎn)染后T細(xì)胞中CAR的表達(dá);利用流式細(xì)胞術(shù)檢測(cè)轉(zhuǎn)染后中央記憶型T細(xì)胞的比例;(7)以攜帶epcam-car基因的t細(xì)胞為實(shí)驗(yàn)組效應(yīng)細(xì)胞,以未轉(zhuǎn)染的t細(xì)胞為對(duì)照組效應(yīng)細(xì)胞,與epcam高表達(dá)的結(jié)腸癌細(xì)胞sw620按0.5:1、1:1、2:1、4:1、8:1、16:1的效靶比共培養(yǎng),與結(jié)腸癌細(xì)胞sw480、hct116、lovo、ht-29按16:1的效靶比共培養(yǎng)。通過(guò)乳酸脫氫酶釋放實(shí)驗(yàn)檢測(cè)cart細(xì)胞對(duì)結(jié)腸癌細(xì)胞的殺傷能力;(8)以攜帶epcam-car基因的t細(xì)胞為實(shí)驗(yàn)組效應(yīng)細(xì)胞,以未轉(zhuǎn)染的t細(xì)胞為對(duì)照組效應(yīng)細(xì)胞,與epcam高表達(dá)的結(jié)腸癌細(xì)胞sw620按0.5:1、1:1、2:1、4:1、8:1、16:1的效靶比共培養(yǎng),與結(jié)腸癌細(xì)胞sw480、hct116、lovo、ht-29按16:1的效靶比共培養(yǎng)。利用酶聯(lián)免疫吸附法檢測(cè)cart細(xì)胞釋放炎性細(xì)胞因子的水平。結(jié)果(1)成功轉(zhuǎn)化、擴(kuò)增pclk-ef-1kanaepcam-car、pspax2、pmd2.g這三個(gè)質(zhì)粒;(2)成功包裝攜帶epcam-car基因表達(dá)框的慢病毒;(3)結(jié)腸癌細(xì)胞行免疫蛋白印跡及流式細(xì)胞術(shù)結(jié)果顯示:結(jié)腸癌細(xì)胞株sw620、sw480、hct116、lovo、ht-29表面epcam的陽(yáng)性表達(dá)率分別為97.5%、85.4%、78.3%、75.4%、67.3%;(4)成功分離、激活t淋巴細(xì)胞,并大量增殖;(5)攜帶epcam-car基因表達(dá)框的慢病毒轉(zhuǎn)染t淋巴細(xì)胞,rt-pcr和wb檢測(cè)顯示;轉(zhuǎn)染后t細(xì)胞中存在epcam-car的表達(dá);流式細(xì)胞術(shù)檢測(cè)顯示:轉(zhuǎn)染后t細(xì)胞表面epcam-car的表達(dá)為50.4%;(6)與對(duì)照組相比,轉(zhuǎn)染后的t細(xì)胞組,中央記憶型t細(xì)胞的比例增多;(7)實(shí)驗(yàn)組和對(duì)照組t細(xì)胞與五種結(jié)腸癌細(xì)胞共培養(yǎng)后行乳酸脫氫酶釋放實(shí)驗(yàn)檢測(cè)epcam-cart細(xì)胞對(duì)腫瘤細(xì)胞的殺傷作用,結(jié)果顯示:epcam-cart細(xì)胞對(duì)epcam+結(jié)腸癌細(xì)胞發(fā)揮殺傷作用,殺傷能力隨著效靶比及腫瘤細(xì)胞表面epcam表達(dá)升高而逐漸增強(qiáng);(8)實(shí)驗(yàn)組和對(duì)照組t細(xì)胞與五種結(jié)腸癌細(xì)共培養(yǎng)后行酶聯(lián)免疫吸附實(shí)驗(yàn)檢測(cè)細(xì)胞因子(IL-2、IFN-γ、IL-6)的釋放水平,結(jié)果顯示:與對(duì)照組相比,實(shí)驗(yàn)組T細(xì)胞炎性細(xì)胞因子(IL-2、IFN-γ、IL-6)的分泌水平更高,其分泌水平隨著效靶比及腫瘤細(xì)胞表面EpCAM表達(dá)升高而逐漸升高。結(jié)論1.成功構(gòu)建靶向EpCAM的CAR T細(xì)胞;2.靶向EpCAM的CAR T細(xì)胞可以識(shí)別并殺傷EpCAM+的結(jié)腸癌細(xì)胞;3.CAR T細(xì)胞對(duì)腫瘤細(xì)胞的殺傷能力依賴(lài)于CAR T細(xì)胞的數(shù)量和腫瘤細(xì)胞表面EpCAM的表達(dá);
[Abstract]:Chimeric Antigen Receptor (CAR) is a fusion molecule that identifies the antibody molecules of the tumor antigen and the activation signal of T cells. The CAR modified T cells bind the exact target of the monoclonal antibody to the strong toxicity and persistence of the cytotoxic T cells, and can specifically identify the tumor related antigens without the specific identification of the tumor associated antigens. It is dependent on MHC restriction to kill tumor cells efficiently and persistently. So far, CAR T cell immunotherapy has achieved significant effect in the clinical treatment of hematological malignancies, but the treatment of solid tumors has not made a breakthrough. The reason may be due to the lack of high specific tumor antigen on the surface of the solid tumor tissue. An ideal target antigen should be highly expressed on the surface of the tumor tissue, not in normal tissue or in low expression. Epithelial cell adhesion molecule (EpCAM) is one of the stem cell markers of cancer, such as colon and liver cancer, and is highly expressed in the tumor group, and is expressed only in the lateral basal membrane of the normal tissue. In this study, we use EpCAM as the target to construct CAR T cells targeting EpCAM, and explore the killing ability of CAR T cells targeting EpCAM to EpCAM+ colon cancer cells. Materials and methods (1) transformation, identification, amplification of pCLK-EF-1 kana EpCAM-CAR, psPAX2, pMD2.G as three virus packaging plasmids; (2) transfection of 293T cells with calcium phosphate to prepare recombinant lentivirus particles, hypervelocity centrifugation to concentrate recombinant lentivirus particles and real-time quantitative PCR method to detect the titer of recombinant lentivirus; (3) use immunoblotting. The expression of EpCAM in five kinds of colon cancer cell lines (SW620, SW480, HCT116, LoVo, HT-29) was detected by method and flow cytometry; (4) extraction of mononuclear cells from human peripheral blood and activation of T cells and culture; (5) using recombinant lentivirus particles carrying EpCAM-CAR gene expression frame to dye human T lymphocyte; (6) quantitative PCR and flow using immunoblotting method. The expression of CAR in transfected T cells was detected by cytometry, and the proportion of the central memory T cells after transfection was detected by flow cytometry; (7) the T cells carrying epcam-car gene were used as the experimental group, and the untransfected T cells were used as the control cells, and the colon cancer cell SW620 with the EpCAM high expression was in 0.5:1,1:1,2:1,4:1,8:1,16:1. The effect target ratio co culture was co cultured with colon cancer cells SW480, HCT116, LoVo, HT-29 according to the target ratio of 16:1. Through the lactate dehydrogenase release test, the killing ability of cart cells to colon cancer cells was detected. (8) T cells carrying epcam-car gene were used as experimental group, and the untransfected T cells were used as control cells, and the expression of EpCAM was high. The colon cancer cell SW620 was co cultured according to the target ratio of 0.5:1,1:1,2:1,4:1,8:1,16:1, and the colon cancer cells SW480, HCT116, LoVo, HT-29 were co cultured according to the target ratio of 16:1. The levels of inflammatory cytokines released by cart cells were detected by enzyme linked immunosorbent assay. Results (1) the three substances were successfully transformed and amplified pclk-ef-1kanaepcam-car, pspax2, pmd2.g. (2) successfully packaged the lentivirus carrying epcam-car gene expression frame; (3) the results of immunoblotting and flow cytometry of colon cancer cells showed that the positive rates of EpCAM in colon cancer cell lines, SW620, SW480, HCT116, LoVo, and HT-29 were 97.5%, 85.4%, 78.3%, 75.4%, 67.3%; (4) successfully separated, activated T lymphocytes and proliferated in large numbers; (5) T lymphocyte transfected by lentivirus carrying epcam-car gene expression frame, RT-PCR and WB detection showed that there was epcam-car expression in T cells after transfection, and flow cytometry showed that the expression of epcam-car on the surface of T cells after transfection was 50.4%; (6) the proportion of central memory T cells in the infected T cell group was increased compared with the control group; (7) the proportion of the central memory type T cells increased. After co culture of T cells and five kinds of colon cancer cells in the control group and five kinds of colon cancer cells, lactic dehydrogenase release test was used to detect the killing effect of epcam-cart cells on the tumor cells. The results showed that epcam-cart cells played a killing effect on epcam+ colon cancer cells, and the killing ability gradually increased with the target ratio and the increase of the expression of EpCAM on the surface of the tumor cells; (8 The release levels of cytokines (IL-2, IFN- gamma, IL-6) in the experimental group and the control group of T cells and five kinds of colon cancer were detected by enzyme linked immunosorbent assay. The results showed that compared with the control group, the level of T cell inflammatory cytokines (IL-2, IFN- gamma, IL-6) in the experimental group was higher than that in the control group, and the secretion level was with the target ratio and the surface E on the tumor cell surface. The expression of pCAM increased gradually. Conclusion 1. successfully constructed CAR T cells targeting EpCAM, and CAR T cells targeting EpCAM can identify and kill colon cancer cells of EpCAM+, and the killing ability of 3.CAR T cells to tumor cells depends on the number of CAR T cells and the expression of tumor cells.
【學(xué)位授予單位】：成都醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R730.51

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