【作者】 李艳萍； 徐惠绵； 吕大鹏； 高宏；

【Author】 Li Yan-Ping Xu Hui-Mian Lv Da-Peng Gao Hong Xu Xiao-ning Department of oncosurgery, Beijing Shijitan Hospital ,Beijing 100038,China. Department of Surgical Oncology, First Affiliated Hospital of China Medical University.Shenyang,110001,China,Molecular Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, ,Headington, Oxford Ox3 9DS,Uk)

【机构】 北京市世纪坛医院肿瘤外科； 中国医科大学附属第一医院肿瘤外科；

【摘要】 目的:人癌症中的自身抗原是免疫系统识别的最普遍抗原,但由于自身耐受,难以在体内引出抗癌免疫。组织学和流式细胞仪分析提示FasL表达细胞能引起局部中性粒细胞浸润,产生强烈的炎症。Anna—Katharina Simon等使用B16F10恶性黑色素瘤C57BL／6小鼠,皮下注射转染 FasL的B16F10细胞后,野生型B16F10细胞激发两次,肿瘤细胞不生长,小鼠产生对肿瘤细胞的排斥称保护鼠。保护鼠能识别恶性黑色素瘤自身分化抗原,但肿瘤免疫主要由抗体介导,为诱导小鼠细胞免疫,保护鼠用表达黑色素细胞分化抗原的重组痘苗病毒来加强。CD8+T淋巴细胞又称细胞毒T细胞,在抗肿瘤免疫中起关键作用。MHC—I类分子肽四聚体复合物可以通过荧光激活细胞分类仪(FACS)直接检测抗原特异性CTL,同时进行细胞定量,极大地扩展了我们对肿瘤细胞免疫的认识。我们希望通过诱导、检测小鼠恶性黑色素瘤模型在不同实验条件下CTL产生情况,探索打破恶性肿瘤自身耐受的方法,帮助我们确定新的肿瘤表面标志物。本文应用四聚体(tetramer)技术检测恶性黑色素瘤小鼠B16F10细胞毒T淋巴细胞(CTL)数量,探讨一种刺激产生抗肿瘤免疫应答的新途径。方法:选择在恶性黑色素瘤 B16F10中占免疫优势的抗原表位人糖蛋白100(hgp100),鼠糖蛋白100(mgpl00)按其氨基酸序列合成抗原肽,制作相应四聚体;小鼠分成4组诱导:FasL(Fas配体)处理组,含鼠糖蛋白100(mgp100)蛋白序列的重组痘苗病毒(rVV)处理组,FasL+rVVmgp100处理组,阴性对照和阳性对照组。初始鼠脾细胞与抗原肽孵育加强刺激一次,体内四聚体检测,各组脾细胞体外培养建立细胞系,一组用人糖蛋白100 (hgp100)抗原肽刺激,另一组hgp100,mgp100两种四聚体交叉检测抗原特异CTL。结果:除阳性对照鼠血液、淋巴结及脾中检出抗原特异性CTL,其他各组血液、淋巴结、脾四聚体检测均阴性;细胞系第一组,FasL+rVVmgp100鼠脾细胞四聚体hgp100检测阳性,其他组阴性。第二组, FasL+rVVmgp100鼠脾细胞出现四聚体交叉检测阳性,其他组阴性。结论:FasL与rVV两种因素联合能打破恶性黑色素瘤B16F10的自身耐受状态,产生抗原特异性CTL,还能诱导固有免疫,产生抗原交叉反应。更多还原

【Abstract】 Objective: The purpose of immunotherapy in cancer is to induce an effective anti-tumor specific cy-totoxic T lymphocytes (CTL) response, however, major his-tocompatibility complex (MHC) class I is usually expressed at low levels by tumors and can’ t elicit effective immune responses. Self-antigens are the most common antigens that are recognized in human cancer. Immune responses to self-antigens are difficult to induce because of self-tolerance. Even if the immune system recognizes and makes a response to self antigens, it is often too weak to reject the tumor.This study was designed to evaluate the antigen specific cytotoxic T lymphocytes (CTL) in melanoma B16F10 mice by tetramers . Methods: Mice were divided into 4 groups for priming and boosting : B16F10-FasL, recom-binant vaccinia virus(rVV) mgp100 and NP, B16F10-FasL followed by rVV, naive mice. Mice were also boosted with spleen cells loaded with peptides before tetramer staining. Spleen cells of the different groups are cultured to set up CTL lines. one group was stimulated with hgp100, the other group were stimulated by hgp100 and mgp100 peptides and crisscross staining was performed after finial stimulation. Results: No antigen specific CTLs were detected ex vivo except mouses that received vaccinia-NP and was stained with NP tetramer used as a positive control. Tetramer positive CTLs were observed in cell lines made from mice that received FasL+rVVmgp 100, tested by tetramer hgp 100, all other groups were negative. The cell line made from FasL+rVVmgp 100 mice also stained positive with crisscross tetramers, while the other CTL lines negative. Conclusion: It concludes that tetramer can detect antigen specific CTL accurately. Combination of FasL and rVVmgp100 can break self-tolerance to B16F10 melanoma, induces possibly an innate immune response, Our observation and results will provide a basis to develop new anti-tumor vaccine and immunotherapy.更多还原