肿瘤相关抗原P1A的诱导表达及其增强细胞毒T细胞杀伤恶性肿瘤的研究

发布时间：2018-06-23 12:56

本文选题：淋巴瘤 + 癌/睾丸抗原　；参考：《中国人民解放军军医进修学院》2010年博士论文

【摘要】： 当前,恶性肿瘤(包括恶性血液系统疾病)的发生率和死亡率正处于不断上升之中。尽管包括异基因干细胞移植等先进的医疗技术和治疗手段大大改善了恶性血液系统疾病患者的预后,但晚期和多次治疗后复发的患者预后仍然很差。因此探索新的治疗方法,已成为改善此类患者预后的迫切需要。恶性肿瘤的免疫治疗早已成为人们关注的焦点之一。在过去的十年中,免疫治疗在多种肿瘤治疗中都获得了令人注目的成就,这些治疗手段包括细胞因子治疗,局部免疫刺激,肿瘤疫苗和异基因干细胞移植后的全身淋巴细胞输注(DLI)等。然而,这些治疗方法获得部分成功的同时,却未能使大多数肿瘤患者实现真正意义的治愈,甚至连肿瘤部分控制或缩小的目标也未达到。其原因是肿瘤细胞的免疫逃避,或者是机体免疫细胞的无能。进一步的研究表明,这种令人失望的现象是因为肿瘤细胞缺乏有效的免疫源性从而导致机体的免疫细胞无法识别或抗原呈递细胞无法有效地递呈抗原。因此,如何增强肿瘤组织／细胞的抗原性,是肿瘤免疫研究领域一个殛待解决的首要问题。癌／睾丸抗原(Cancer/testis antigens CTAs)是一类在胚胎组织和滋养层细胞优势表达的抗原,并在大约40%的恶性肿瘤患者中存在,而在正常组织中几乎没有表达。表观遗传学研究表明,该类基因在肿瘤中的表达大多数情况下处于低水平或沉默状态,其原因是其该类基因启动子的异常甲基化造成的。由于睾丸等组织的免疫豁免特性和CTA抗原的分布特点,使得该类抗原成为近年来免疫治疗的重要靶点之一。我们的前期研究表明,P1A是一种重要鼠类CT抗原,在多种肿瘤细胞有异常分布。该基因的表达受甲基化调控,5-杂氮胞苷等药物处理可恢复其表达。鉴于上述背景,我们从下述几个方面进行研究,旨在增强肿瘤细胞的免疫源性,并通过有效的抗原呈递,进而有效刺激机体产生有效的抗肿瘤作用。一、用5-杂氮胞苷处理多种肿瘤细胞,并在5-杂氮胞苷处理前后对所处理肿瘤细胞进行P1A的DNA和mRNA检测,进而阐明5-杂氮胞苷对肿瘤细胞P1A表达的影响,筛选受甲基化调控、便于体外、体内免疫研究的肿瘤细胞株(本研究中T细胞淋巴瘤细胞系EL-4符合上述条件)。二、对P1A的抗原表位(多肽片段)进行人工合成,以此多肽片段刺激经细胞因子诱导成熟的树突状细胞(dendritic cells, DCs),而后以一定数量经多肽刺激的DCs多次免疫BALB／c小鼠,使之产生针对P1A多肽的特异性细胞毒T细胞(Cytotoxic T cell, CTL). 三、以免疫磁珠法分离小鼠脾脏内的CTL(CD90+T细胞),以流式细胞仪检测P1A特异性CTL对肿瘤细胞的体外杀伤效果；同时分离CD4+和CD8+,进一步探讨其各自在免疫杀伤中的作用。四、构建荷瘤动物模型,在一定的时机经尾静脉给予荷瘤小鼠注射经特异免疫刺激产生的细胞毒T细胞,并以生理盐水、无关肽刺激的细胞毒T细胞为对照,观察P1A特异性CTL对肿瘤组织生长的抑制作用。五、以CFSE为示踪剂,进一步检测了特异性CTL在荷瘤小鼠体内的归巢运动及分布,进一步探讨克服免疫逃逸的有效方法。六、通过对不同方法处理的肿瘤细胞进行流式检测,探讨CTL杀伤肿瘤细胞机制。结果发现,5-杂氮胞苷诱导癌／睾丸抗原P1A的表达存在明显的量效和时效关系。通过DC细胞的递呈,有效地刺激小鼠产生P1A特异性细胞毒T细胞,体外、体内均证实其对P1A恢复表达的肿瘤细胞或组织均有杀伤作用；我们同时还发现CD4+T细胞在特异性杀伤中起重要的作用,这种杀伤与癌／睾丸抗原的表达和识别密切相关。此外,我们对P1A特异性CTL杀伤的机制进行了初步探讨。
[Abstract]:At present, the incidence and mortality of malignant tumors (including malignant hematological diseases) are on the rise. Although advanced medical techniques and treatments, including allogeneic stem cell transplantation, have greatly improved the prognosis of patients with malignant hematological diseases, the prognosis of patients with advanced and repeated treatment is still poor. This exploration of new treatment has become an urgent need to improve the prognosis of such patients.
Immunotherapy for malignant tumors has long been one of the focus of attention. In the past ten years, immunotherapy has achieved remarkable achievements in various tumor treatments, including cytokine therapy, local immune stimulation, tumor vaccine and allogeneic stem cell transplantation after allogeneic lymphocyte infusion (DLI). However, while these treatments are partially successful, most of the cancer patients fail to achieve a true cure, even the target of the tumor part control or reduction is not reached. The reason is the immune escape of the tumor cells, or the inability of the immune cells in the body. As a result of the lack of effective immunogenicity in tumor cells, the immune cells of the body can not be identified or the antigen presenting cells can not deliver the antigen effectively. Therefore, how to enhance the antigenicity of the tumor tissue / cell is the first problem to be solved in the field of tumor immunology.
Cancer / testicular antigen (Cancer/testis antigens CTAs) is a class of antigen expressed predominantly in embryonic tissue and trophoblast cells, and exists in about 40% of the cancer patients and almost no expression in normal tissues. Epigenetic studies show that the expression of this gene in the swelling is at a low level or sink in most cases. The reason is that the abnormal methylation of the promoter of the gene is caused by the abnormal methylation of the gene promoter. Due to the immunity immunity of the testis and the distribution characteristics of CTA antigen, this kind of antigen has become one of the important targets in the immunotherapy in recent years. Our previous study showed that P1A is an important mouse CT antigen and is different in a variety of tumor cells. Normal distribution. The expression of this gene is regulated by methylation. 5- heterocytidine and other drugs can be treated to restore its expression. In view of the above background, we have studied the following aspects to enhance the immunogenic activity of tumor cells and to effectively stimulate the body to produce effective anti-tumor effects through effective antigen presentation.
First, 5- heterocytidine is used to treat a variety of tumor cells, and the DNA and mRNA detection of P1A in the treated tumor cells before and after the treatment of 5- heterocytidine, and then clarify the effect of 5- heterocytidine on the expression of P1A in the tumor cells, screening the methylation regulation to facilitate the in vitro, in vivo immunological research of the tumor cell line (T cell lymphoma fine in this study) The cell line EL-4 conforms to the above conditions.
Two, the antigen epitopes (polypeptide fragments) of P1A were artificially synthesized to stimulate the mature dendritic cells (dendritic cells, DCs) induced by cytokines and then immunize BALB / c mice several times by a certain number of DCs stimulated DCs to produce specific cytotoxic T cells (Cytotoxic T cell) against P1A peptides.
Three, CTL (CD90+T cells) in the spleen of mice were separated by immunomagnetic beads, and the effect of P1A specific CTL on the tumor cells in vitro was detected by flow cytometry, and CD4+ and CD8+ were isolated at the same time, and their respective roles in immune killing were further explored.
Four, the tumor bearing animal model was constructed. At a certain time, the tumor bearing mice were injected into the tail vein to injecting the cytotoxic T cells produced by the specific immune stimulation, and the control of the cytotoxic T cells with the physiological saline and the unrelated peptide stimulation was used to observe the inhibitory effect of P1A specific CTL on the growth of the tumor tissue.
Five, using CFSE as tracer, we further detected the homing movement and distribution of specific CTL in tumor bearing mice, and further explored the effective way to overcome immune escape.
Six, we investigated the mechanism of CTL killing tumor cells by flow cytometry.
The results showed that the expression of cancer / testicular antigen P1A induced by 5- has obvious dose effect and aging relationship. Through the delivery of DC cells, it effectively stimulates the production of P1A specific cytotoxic T cells in mice. In vitro, in vitro, it has proved that it has a killing effect on the tumor cells or tissues of the P1A restored expression in vitro; and we also found that CD4+T is fine. Cell plays an important role in the specific killing, which is closely related to the expression and recognition of cancer / testicular antigen. In addition, we have preliminarily discussed the mechanism of P1A specific CTL killing.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392.12;R730.5

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