CAR-T细胞增强抗多发性骨髓瘤的实验研究

发布时间：2018-03-12 13:54

  本文选题：多发性骨髓瘤　切入点：CAR-T细胞疗法　出处：《华东师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：多发性骨髓瘤是最常见的恶性浆细胞病,由于骨髓瘤细胞的高侵袭性、转移性和耐药性,故所有患者最终都将复发或耐药。目前还没有一种有效的方法将其治愈,攻克骨髓瘤是我们不可懈怠的使命。免疫治疗的发展给骨髓瘤的治愈提供了新方向,对于该疾病,首选的治疗手段仍为干细胞移植术,但该方法不仅容易复发,还易发生移植物抗宿主反应,带来极高的发病率和死亡率。骨髓瘤的免疫治疗目前存在的主要问题是:1)骨髓瘤细胞表面缺乏特异性表达的靶标分子;2)患者骨髓的局部微环境易产生免疫抑制和免疫逃脱并且容易产生免疫耐受。CAR-T细胞免疫疗法的出现给骨髓瘤的治疗带来了新的希望。CAR分子具有同时特异性定位到靶标分子和活化T细胞的功能,并且通过与信号域的结合实现T细胞持续的激活。靶向CD19的CART细胞临床试验表现出惊人和持久的结果,随着CART疗法在治疗血液病的成功,人们开始将目光转向其他领域,关于骨髓瘤目前有三个临床试验正在进行,其中有两个靶向BCMA和一个靶向CD138分子。本研究选取在骨髓瘤细胞表面高表达的CD138,BCMA,CD38,CD40分子和B细胞恶性肿瘤表面特异性表达的CD19分子为研究的靶标,通过freepatent online网站查找各研究靶标单抗序列的scFv段,获取其序列。电泳鉴定完后片段与用EcoRI和XbaI酶切好的工具质粒进行重组,电泳结果显示scFv片段、工具质粒、重组质粒的条带位置符合预期结果。重组质粒DNA测序的结果对该结果进一步确认。各靶标重组质粒转染293T细胞24小时,感染293T细胞48小时后均得到了很好的结果,通过对各靶标慢病毒的浓缩与纯化,得到了平均滴度达到10^9 IU/ml的慢病毒。K562稳定细胞株的抗原表达量均高于96%,CAR分子转导T细胞,BCMA-CAR的转导效率达64%,LDH法的杀伤实验结果表明,各CART细胞杀伤效果明显,最高达65%;我们成功的将杀伤实验结果与IFN-γ、IL-2和TNF细胞因子释放量建立相关性,本研究可为CAR-T细胞在应用到临床时方案的选择、细胞剂量的确定和临床治疗效果的预测提供重要依据。
[Abstract]:Multiple myeloma is the most common malignant plasmacytosis. Due to the high invasion, metastasis and drug resistance of myeloma cells, all patients will eventually recur or resistant to drugs. The development of immunotherapy provides a new direction for the cure of myeloma. For this disease, the preferred treatment is stem cell transplantation, but it is not only easy to recur. Also prone to graft-versus-host response, The main problem with immunotherapy for myeloma is that the bone marrow microenvironment of patients with myeloma is prone to immunosuppression, which is a target molecule with no specific expression on the surface of myeloma cells. Immune escape and easy to produce immune tolerance. CAR-T cell immunotherapy brings new hope for the treatment of myeloma. Car molecule has the function of targeting target molecule and activating T cell at the same time. The clinical trials of CART cells targeting CD19 showed surprising and lasting results. With the success of CART therapy in the treatment of hematological diseases, people began to turn their eyes to other fields. With regard to myeloma, there are currently three clinical trials under way. In this study, we selected CD138BCMA-CD38 CD40 molecule and CD19 molecule specifically expressed on the surface of B cell malignant tumor. The scFv fragment of each target McAb sequence was searched by freepatent online website, and its sequence was obtained. After electrophoresis, the fragment was recombined with the tool plasmid digested by EcoRI and XbaI. The result of electrophoresis showed that scFv fragment, tool plasmid, and so on. The band position of the recombinant plasmid was in accordance with the expected results. The results of DNA sequencing of the recombinant plasmids confirmed the results. After transfection of each target recombinant plasmid into 293T cells for 24 hours, good results were obtained after 48 hours of infection with 293T cells. Through the concentration and purification of various target lentivirus, the antigenic expression of lentivirus. K562 stable cell line with an average titer of 10 ^ 9 IU/ml was higher than that of 96 car molecular transduction T cell line BCMA-CAR. The results showed that the transduction efficiency of BCMA-CAR reached 64%. The killing effect of each CART cell was significant, up to 65%. We successfully established a correlation between the killing results and the release of IL-2 and TNF cytokines. This study can be used as a choice for the regimen of CAR-T cells in clinical application. The determination of cell dose and the prediction of clinical therapeutic effect provide important basis.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.3
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本文编号：1601858