本文選題：淋巴瘤 + 癌/睪丸抗原��； 參考：《中國人民解放軍軍醫(yī)進修學院》2010年博士論文

【摘要】： 當前,惡性腫瘤(包括惡性血液系統(tǒng)疾病)的發(fā)生率和死亡率正處于不斷上升之中。盡管包括異基因干細胞移植等先進的醫(yī)療技術和治療手段大大改善了惡性血液系統(tǒng)疾病患者的預后,但晚期和多次治療后復發(fā)的患者預后仍然很差。因此探索新的治療方法,已成為改善此類患者預后的迫切需要。 惡性腫瘤的免疫治療早已成為人們關注的焦點之一。在過去的十年中,免疫治療在多種腫瘤治療中都獲得了令人注目的成就,這些治療手段包括細胞因子治療,局部免疫刺激,腫瘤疫苗和異基因干細胞移植后的全身淋巴細胞輸注(DLI)等。然而,這些治療方法獲得部分成功的同時,卻未能使大多數(shù)腫瘤患者實現(xiàn)真正意義的治愈,甚至連腫瘤部分控制或縮小的目標也未達到。其原因是腫瘤細胞的免疫逃避,或者是機體免疫細胞的無能。進一步的研究表明,這種令人失望的現(xiàn)象是因為腫瘤細胞缺乏有效的免疫源性從而導致機體的免疫細胞無法識別或抗原呈遞細胞無法有效地遞呈抗原。因此,如何增強腫瘤組織／細胞的抗原性,是腫瘤免疫研究領域一個殛待解決的首要問題。 癌／睪丸抗原(Cancer/testis antigens CTAs)是一類在胚胎組織和滋養(yǎng)層細胞優(yōu)勢表達的抗原,并在大約40%的惡性腫瘤患者中存在,而在正常組織中幾乎沒有表達。表觀遺傳學研究表明,該類基因在腫瘤中的表達大多數(shù)情況下處于低水平或沉默狀態(tài),其原因是其該類基因啟動子的異常甲基化造成的。由于睪丸等組織的免疫豁免特性和CTA抗原的分布特點,使得該類抗原成為近年來免疫治療的重要靶點之一。我們的前期研究表明,P1A是一種重要鼠類CT抗原,在多種腫瘤細胞有異常分布。該基因的表達受甲基化調(diào)控,5-雜氮胞苷等藥物處理可恢復其表達。鑒于上述背景,我們從下述幾個方面進行研究,旨在增強腫瘤細胞的免疫源性,并通過有效的抗原呈遞,進而有效刺激機體產(chǎn)生有效的抗腫瘤作用。 一、用5-雜氮胞苷處理多種腫瘤細胞,并在5-雜氮胞苷處理前后對所處理腫瘤細胞進行P1A的DNA和mRNA檢測,進而闡明5-雜氮胞苷對腫瘤細胞P1A表達的影響,篩選受甲基化調(diào)控、便于體外、體內(nèi)免疫研究的腫瘤細胞株(本研究中T細胞淋巴瘤細胞系EL-4符合上述條件)。 二、對P1A的抗原表位(多肽片段)進行人工合成,以此多肽片段刺激經(jīng)細胞因子誘導成熟的樹突狀細胞(dendritic cells, DCs),而后以一定數(shù)量經(jīng)多肽刺激的DCs多次免疫BALB／c小鼠,使之產(chǎn)生針對P1A多肽的特異性細胞毒T細胞(Cytotoxic T cell, CTL). 三、以免疫磁珠法分離小鼠脾臟內(nèi)的CTL(CD90+T細胞),以流式細胞儀檢測P1A特異性CTL對腫瘤細胞的體外殺傷效果；同時分離CD4+和CD8+,進一步探討其各自在免疫殺傷中的作用。 四、構建荷瘤動物模型,在一定的時機經(jīng)尾靜脈給予荷瘤小鼠注射經(jīng)特異免疫刺激產(chǎn)生的細胞毒T細胞,并以生理鹽水、無關肽刺激的細胞毒T細胞為對照,觀察P1A特異性CTL對腫瘤組織生長的抑制作用。 五、以CFSE為示蹤劑,進一步檢測了特異性CTL在荷瘤小鼠體內(nèi)的歸巢運動及分布,進一步探討克服免疫逃逸的有效方法。 六、通過對不同方法處理的腫瘤細胞進行流式檢測,探討CTL殺傷腫瘤細胞機制。 結果發(fā)現(xiàn),5-雜氮胞苷誘導癌／睪丸抗原P1A的表達存在明顯的量效和時效關系。通過DC細胞的遞呈,有效地刺激小鼠產(chǎn)生P1A特異性細胞毒T細胞,體外、體內(nèi)均證實其對P1A恢復表達的腫瘤細胞或組織均有殺傷作用；我們同時還發(fā)現(xiàn)CD4+T細胞在特異性殺傷中起重要的作用,這種殺傷與癌／睪丸抗原的表達和識別密切相關。此外,我們對P1A特異性CTL殺傷的機制進行了初步探討。
[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.
【學位授予單位】：中國人民解放軍軍醫(yī)進修學院
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R392.12;R730.5

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本文編號：2057186