靶向EpCAM的嵌合抗原受体修饰的T细胞的构建及其对结肠癌细胞的杀伤研究

发布时间：2018-06-29 12:52

本文选题：嵌合抗原受体修饰的T细胞 + 上皮细胞粘附分子　；参考：《成都医学院》2017年硕士论文

【摘要】：嵌合抗原受体(Chimeric Antigen Receptor,CAR)是将识别肿瘤抗原的抗体分子和T细胞活化信号耦合的融合分子。经CAR修饰的T细胞将单克隆抗体的精确靶向特异性与细胞毒性T细胞的强毒性和持久性相结合,能够特异性识别肿瘤相关抗原而不依赖MHC限制,从而高效持久地杀伤肿瘤细胞。迄今为止,CAR T细胞免疫治疗已经在血液恶性肿瘤的临床治疗中取得了显著的疗效,但对实体肿瘤的治疗并未取得突破性进展。究其原因可能和实体肿瘤组织表面高度特异性肿瘤抗原的缺失有关。一个理想的靶抗原应当在肿瘤组织表面高表达,在正常组织不表达或低表达。上皮细胞粘附分子(Epithelial cell adhesion molecule,EpCAM)是结肠癌、肝癌等恶性肿瘤的干细胞标志之一,在肿瘤组织高表达,在正常组织仅在基底膜外侧表达。本研究中,我们以EpCAM为靶点,构建靶向EpCAM的CAR T细胞,探究靶向EpCAM的CAR T细胞对EpCAM+结肠癌细胞的杀伤能力。目的本课题以EpCAM为研究靶点,构建稳定表达EpCAM-CAR基因的T细胞,探究靶向EpCAM的CAR T细胞对EpCAM+结肠癌细胞的杀伤能力。材料与方法(1)转化、鉴定、扩增pCLK-EF-1 kana EpCAM-CAR,psPAX2、pMD2.G这三个病毒包装质粒;(2)利用磷酸钙法转染293T细胞以制备重组慢病毒颗粒,超高速离心以浓缩重组慢病毒颗粒,实时荧光定量PCR法检测重组慢病毒的滴度;(3)利用免疫蛋白印迹法和流式细胞术检测EpCAM在五种结肠癌细胞株(SW620、SW480、HCT116、LoVo、HT-29)中的表达;(4)人外周血单核细胞的提取及T细胞的活化、培养;(5)用携带EpCAM-CAR基因表达框的重组慢病毒颗粒转染人T淋巴细胞;(6)利用免疫蛋白印迹法、定量PCR法及流式细胞术检测转染后T细胞中CAR的表达;利用流式细胞术检测转染后中央记忆型T细胞的比例;(7)以携带epcam-car基因的t细胞为实验组效应细胞,以未转染的t细胞为对照组效应细胞,与epcam高表达的结肠癌细胞sw620按0.5:1、1:1、2:1、4:1、8:1、16:1的效靶比共培养,与结肠癌细胞sw480、hct116、lovo、ht-29按16:1的效靶比共培养。通过乳酸脱氢酶释放实验检测cart细胞对结肠癌细胞的杀伤能力;(8)以携带epcam-car基因的t细胞为实验组效应细胞,以未转染的t细胞为对照组效应细胞,与epcam高表达的结肠癌细胞sw620按0.5:1、1:1、2:1、4:1、8:1、16:1的效靶比共培养,与结肠癌细胞sw480、hct116、lovo、ht-29按16:1的效靶比共培养。利用酶联免疫吸附法检测cart细胞释放炎性细胞因子的水平。结果(1)成功转化、扩增pclk-ef-1kanaepcam-car、pspax2、pmd2.g这三个质粒;(2)成功包装携带epcam-car基因表达框的慢病毒;(3)结肠癌细胞行免疫蛋白印迹及流式细胞术结果显示:结肠癌细胞株sw620、sw480、hct116、lovo、ht-29表面epcam的阳性表达率分别为97.5%、85.4%、78.3%、75.4%、67.3%;(4)成功分离、激活t淋巴细胞,并大量增殖;(5)携带epcam-car基因表达框的慢病毒转染t淋巴细胞,rt-pcr和wb检测显示;转染后t细胞中存在epcam-car的表达;流式细胞术检测显示:转染后t细胞表面epcam-car的表达为50.4%;(6)与对照组相比,转染后的t细胞组,中央记忆型t细胞的比例增多;(7)实验组和对照组t细胞与五种结肠癌细胞共培养后行乳酸脱氢酶释放实验检测epcam-cart细胞对肿瘤细胞的杀伤作用,结果显示:epcam-cart细胞对epcam+结肠癌细胞发挥杀伤作用,杀伤能力随着效靶比及肿瘤细胞表面epcam表达升高而逐渐增强;(8)实验组和对照组t细胞与五种结肠癌细共培养后行酶联免疫吸附实验检测细胞因子(IL-2、IFN-γ、IL-6)的释放水平,结果显示:与对照组相比,实验组T细胞炎性细胞因子(IL-2、IFN-γ、IL-6)的分泌水平更高,其分泌水平随着效靶比及肿瘤细胞表面EpCAM表达升高而逐渐升高。结论1.成功构建靶向EpCAM的CAR T细胞;2.靶向EpCAM的CAR T细胞可以识别并杀伤EpCAM+的结肠癌细胞;3.CAR T细胞对肿瘤细胞的杀伤能力依赖于CAR T细胞的数量和肿瘤细胞表面EpCAM的表达;
[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.
【学位授予单位】：成都医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.51

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