不同抗原表位CART-30细胞对经典型霍奇金淋巴瘤细胞株的杀伤作用及优化研究

发布时间：2018-07-04 18:01

本文选题：霍奇金淋巴瘤 + 嵌合抗原受体　；参考：《中国人民解放军医学院》2017年硕士论文

【摘要】：研究背景:经典型霍奇金淋巴瘤(classical Hodgkin's lymphoma,cHL)是霍奇金淋巴瘤的病理类型之一,是起源于淋巴造血系统的恶性肿瘤,主要发生于年轻及老年患者。目前常规化疗疗效较好,但难治/复发型cHL仍缺少有效的治疗方法,因此如何减少并发症、优化、个体化治疗成为目前研究重点。嵌合抗原受体修饰的T细胞(chimeric antigen receptor-modified T cell,CART )是近年来迅速发展的免疫治疗领域的研究热点。目前靶向性针对CD30-CART细胞免疫治疗在我院临床治疗工作中已收获一定成就,但疗效仍待进一步优化。目的:本研究选择不同抗原表位的两种单克隆抗体Ki-4及Ber-H2,优化性构建不同的CAR基因片段的载体,比较两种CART细胞对CD30阳性的cHL细胞系L428的杀伤作用,以及其机制与NF-κB信号通路相关性进行研究。方法:①构建携带不同抗原表位的CD30-CAR基因片段的慢病毒载体;②两种不同抗原表位的CART-30细胞的培养;③两种不同抗原表位CART-30细胞对cHL细胞株的杀伤效率的比较;④CD30-CARs对cHL细胞株NF-κB机制的影响。结果:①成功构建出不同抗原表位的CD30-CART细胞,阳性率平均值约为65.7%左右;②Ki-4组效靶比为1:1时杀伤效率的平均值为35.15%,效靶比为10:1时杀伤效率的平均值为66.99%; Ber-H2组效靶比为1:1时杀伤效率的平均值为31.95%,效靶比为10:1时杀伤效率的平均值为56.11%; CD19组效靶比为1:1时杀伤效率的平均值为21.92%,效靶比为10:1时杀伤效率的平均值为45.49%; CIK组效靶比为1:1时杀伤效率的平均值为16.99%,效靶比为10:1时杀伤效率的平均值为32.22%。P0.05,不同效应细胞间及不同效靶比间的杀伤效应均有差异,且此差异具有统计学意义。③Western蛋白印迹法结果示:TNF-α组及Ki-42h及3h组P-P65呈强阳性,IκBα蛋白呈弱阳性;而空白对照组、Ki-4的Oh组及Ber-H2各组P-P65及IκBα蛋白均呈现弱阳性。结论:①不同抗原表位的CD30-CART细胞对经典型霍奇金淋巴瘤细胞杀伤作用不同;②Ki-4的scFv能有效激活NF-κB信号通路,但Ber-H2-CARs不能激活NF-κB信号通路。
[Abstract]:Background: classical Hodgkin's lymphoma (classical Hodgkin's lymphoma) is one of the pathological types of Hodgkin's lymphoma. It is a malignant tumor originating from the lymphatic hematopoietic system and mainly occurs in young and elderly patients. The efficacy of conventional chemotherapy is good, but there is still a lack of effective treatment for refractory / relapsed cHL. Therefore, how to reduce complications, optimize, individualized treatment has become the focus of current research. Chimeric antigen receptor modified T cell (chimeric antigen receptor-modified T cell cart is a hot topic in the field of immunotherapy. At present, targeted CD30-cart cellular immunotherapy has achieved some achievements in our clinical treatment, but the curative effect still needs to be optimized. Aim: in this study, we selected two monoclonal antibodies Ki-4 and Ber-H2 from different antigenic epitopes to construct the vectors of different car gene fragments and compare the killing effects of two kinds of cart cells on CD30 positive CHL cell line L428. The relationship between NF- 魏 B signaling pathway and its mechanism was studied. Methods the cell culture of CART-30 cells containing different epitopes of CD30-CAR gene containing different antigen epitopes was carried out. The cytotoxicity of two different epitopes of CART-30 cells to cHL cell line was compared. 4 the effect of CD30-CARs on the mechanism of NF- 魏 B in CHL cells. Results the CD30-CART cells with different epitopes were successfully constructed with the average positive rate of 65.7%. 2The average killing efficiency of Ki-4 group was 35.15 when 1:1, 66.99 at 10:1, 31.95 at 1:1 in Ber-H2 group, 56.11 at 1:1 in Ber-H2 group. The average killing efficiency of CIK group is 21.92 at 1:1 and 45.49 at 10:1, the average killing efficiency of CIK is 16.999when 1:1, the average killing efficiency is 32.22.P0.05at 1:1. The cytotoxicity between cells and different target ratios was different. The difference was statistically significant. The results of Western blot showed that P-P65 was weakly positive in the w TNF- 伪 group, Ki-42 h and 3 h group, but weakly positive in the blank control group in the Oh group of Ki-4 and Ber-H2 group, and the protein of P-P65 and I 魏 B 伪 was weakly positive in the control group. Conclusion CD30-CART cells with different antigen epitopes have different killing effects on classical Hodgkin's lymphoma cells. SCFv of 2Ki-4 can effectively activate NF- 魏 B signaling pathway, but Ber-H2-CARs can not activate NF- 魏 B signaling pathway.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.1

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