【作者】 李纪明；

【导师】 祁元明； 王柏生；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2005， 硕士

【摘要】 目的: 免疫识别理论已阐明,肿瘤的免疫机制是以细胞免疫为主,即细胞毒性T淋巴细胞通过识别肿瘤细胞表面与MHC-Ⅰ连接的抗原肽,进而杀死肿瘤细胞。对于肿瘤患者,研究表明其细胞毒性T淋巴细胞同正常人并没有区别,只是肿瘤细胞表面抗原肽表达异常,而使肿瘤细胞逃避了淋巴细胞的监视。所以寻找肿瘤细胞表面的抗原肽成为了肿瘤免疫治疗的核心问题。本研究旨在从食管癌TE-1细胞寻找细胞表面与MHC-Ⅰ相结合的抗原肽,进而为食管癌抗原肽的研究打下较好的基础。 肿瘤肽疫苗在黑色素瘤患者临床治疗上取得的理想效果,以及对肝癌、胃癌、肺癌、白血病和黑素瘤抗原肽的成功鉴定与制备,预示了对食管癌抗原肽结构与功能的研究有着广阔的前景。我国是食管癌发病率最高的国家,但食管癌免疫治疗方面研究甚少。若能加快这方面研究的进展,有望给食管癌的治疗开辟一条新的途径。本研究探讨了用负载食管癌抗原肽树突状细胞刺激的肿瘤特异性细胞毒性T淋巴细胞杀死食管癌细胞的可行性,为食管癌的免疫治疗提供一些实验依据。 方法: 1.TE-1细胞培养:用含体积分数为10%胎牛血清的RPMI 1640培养基在37℃,更多还原

【Abstract】 Objective:The theory of immunity recognition demonstrated that the tumor immunity mechanism is characterized of cell immunity. The cytotoxic T lymphocytes kill tumor cells by recognizing the peptides presented by MHC-1 molecules on the membranes. There is no difference in the cytotoxic T lymphocytes between healthy persons and tumor patients, for tumor patients, peptides on the membranes change so that tumor cells can not be recognized by lymphocytes. Therefor, the study of antigen peptides play a key role on the tumor immunotherapy. The experiment is to seek antigen peptides presented by MHC-1 molecules in human esophageal carcinoma TE-1 cell line and to seek a effective way to obtain peptides, so that it can contribute to improve the study of human esophageal tumor antigen peptides.The study demonstrated that tumor antigen-based vaccine is a effective way to cure melanoma, and antigen peptides were obtained from liver cancer、 gastric cancer、 lung cancer、 leucocythemia and melanoma. These studies indicate the feasibility of investigation about esophageal cancer antigen peptides. In China , though the number of esophageal cancer patients is the largest in the world, the study is not enough. If we can do more work, maybe we can find a new way about esophageal tumor immunotherapy. The experiment is to investigate the killing activity of cytotoxic T lymphocytes activated by dendritic cells loaded with esophageal cancer antigen andmake progress in esophageal tumor immunotherapy. Methods:1. TE-l cell is cultivated in RPMI 1640 culture medium supplemented with 10% fetal calf serum at 37 ℃ and 5 % CO2.2. Mild acid wash method: TE-l cells are cultivated in 250ml culture flasks. TE-1 cells for 3 days were treated as follows: the medium was removed, and each flask was washed three times with phosphate-buffered saline ( PBS ) to remove residual tissue culture medium. Each flask was treated for 50 seconds at room temperature with citrate-phosphate buffer at pH=3.3, to denature MHC-1 molecules and to release MHC-1 -associated peptides into the supernatant.3. The first step of purification: The acid solution, containing peptides, was desalted on Sep-Pak C18 column according to the manufacturer’s instructions, vacuum concentrated, and centrifuged on a Centricon-3. The filtered material was vacuum lyophilized and stored at - 20 ℃.4. The second step of purification with reverse-phase high performance liquid chromatography (RP-HPLC): acid eluted peptides from TE-l cells were fractionated by RP-HPLC. Every peak was collected manually, desalted, vacuum concentrated, lyophilized, and stored at - 20℃.5. The screened volunteer’s dendritic cells were gained in vitro: The volunteer’speripheral blood mononuclear cells were isolated with the method of density gradient centrifugation and were cultivated in RPMI 1640 culture medium supplemented with rhGM-CSF、 rhIL-4 and autologous serum for 8 days. 6. The screened volunteer’s lymphocytes extracted from peripheral blood were cultivated in vitro in RPMI 1640 culture medium supplemented with 10 % fetal calf serum and IL-2 at 37 ℃ and 5 % CO2.7. Induction of specific cytotoxic T lymphocytes in vitro: Dendritic cells pulsed with peptides stimulated peripheral blood lymphocytes for 5 days, then cytotoxic T lymphocytes can be gained.8. 51Cr-release assays: Target cells were labeled with 100 μ Ci 51Cr sodium chromate/4×106 cells. After 1 hour at room temperature, cells washed three times and resuspended in RPMI 1640 culture medium at a concentration of 5×104cells/ml. Cells were added at 100:1 effector to target cell ratio (E:T) in wells of 96-well U-bottom plates(200 μl/well) for 4 hours. Assays were performed in triplicate. Chromium release was assessed in culture supernatants, using a gamma counter. Wells containing lymphocytes, cytotoxic T lymphocytes activated by dendritic cells non-pulsed with peptides and different target cells were used as controls.Results:1. There are some peaks in the chromatogram;2. The killing rate of cytotoxic T lymphocytes activated by dendritic cells loadedwith esophageal cancer antigen to TE-1 was higher significantly than the killing rate of cytotoxic T lymphocytes activated by dendritic cells loaded without esophageal cancer antigen (P < 0.05);3. The killing rate of cytotoxic T lymphocytes activated by dendritic cells loadedwith esophageal cancer antigen to TE-1 cells which were stimulated with γ-IFN was higher significantly than to TE-1 cells which were not stimulated with γ-IFN(P<0.05);4. The killing rate of cytotoxic T lymphocytes activated by dendritic cells loaded with esophageal cancer antigen to TE-1 was higher significantly than to Eca-109,786-0and A-549 (P < 0.05 );5. The killing rate of cytotoxic T lymphocytes activated by dendritic cells loaded with esophageal cancer antigen to T2 cells which were reacted with esophageal cancer was higher significantly than to T2 which were not reacted with esophageal cancer antigen( P < 0.05 ).Conclusions:1. Acid elution can get effective esophageal cancer antigen peptides from TE-1 cellmembranes;2. Cytotoxic T lymphocytes activated by dendritic cells loaded with esophageal cancel antigen can kill efficiently human esophageal cancer cell specifically;3. There were effective antigen peptides which can link with HLA-A2 molecular and generate specific cytotoxic T lymphocytes by activating lymphocytes among esophageal cancer antigen peptide obtained by acid elution.更多还原