角蛋白19致敏的DC/CTL对MCF-7体外抑瘤作用的实验研究

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (10): 1247-1250.

角蛋白19致敏的DC/CTL对MCF-7体外抑瘤作用的实验研究

李鸣芳, 罗荣城, 尤长宣, 涂小玉

南方医科大学南方医院肿瘤中心, 广东, 广州, 510515

出版日期:2005-10-20 发布日期:2005-10-20
基金资助:
收稿日期:2005-6-28。
基金项目:广东省自然科学基金(037050)
作者简介:李鸣芳,女,硕士,主治医师,电话:027-82930525,E-mail:limf161@126.com
通讯作者:罗荣城,教授,主任医师,博士生导师,电话:020-61641651,E-mail:lrc@nfhoc.com

Cytotoxicity of cytotoxic T lymphcytes induced by keratin 19-sensitized dendritic cells against MCF-7 cells in vitro

LI Ming-fang, LUO Rong-cheng, YOU Chang-xuan, TU Xiao-yu

南方医科大学南方医院肿瘤中心, 广东, 广州, 510515

Online:2005-10-20 Published:2005-10-20

摘要/Abstract

摘要： 目的探讨体外经角蛋白19(K19)致敏的树突状细胞(DC)诱导的细胞毒T细胞(CTL)对MCF-7细胞株的抑瘤作用。方法经抗原多肽K19体外致敏外周血来源的DC诱导特异性CTL,流式细胞仪检测DC表面标志及细胞因子分泌,³H-TdR掺入法检测DC诱导CTL增殖,⁵¹Cr释放法测定CTL对MCF-7细胞的杀伤活性。结果外周血单个核细胞经体外扩增培养出可见树突状形状的DC,流式细胞仪检测细胞表面CD83、CD40、CD80及CD86高表达,混合淋巴细胞反应观察经K19致敏的DC刺激的自体淋巴细胞增殖明显较对照组高(P<0.01)。在同一效靶比时,K19致敏的DC诱导的CTL对MCF-7细胞的杀伤率与对照组比较差异有显著性(P<0.01),且随着效靶比增加,杀伤作用增强(P<0.01)。结论 DC在体外经K19抗原致敏后,可诱导产生对MCF-7细胞有特异性杀伤作用的效应细胞杀伤MCF-7细胞,且随效靶比增加,杀伤力增强。

Abstract: Objective To investigate the cytotoxicity of cytotoxic T lymphcytes (CTL) induced by keratin 19 (K19)-sensitized dendritic cells (DC) against MCF-7 cells in vitro. Methods DC isolated from peripheral blood mononuclear cells were cultured in vitro and sensitized by K19 peptide to generate specific CTL. The phenotypes and intracytoplasm cytokine of DC were analyzed by flow cytometry. Mixed leukocyte responses were evaluated by ³H-TdR incorporation assay. The T cells were generated by DC pulsed with K19 antigen and T cell cytotoxicity was measured by ⁵¹Cr release assay. Results The DCs derived from peripheral blood mononuclear cells expressed high levels of CD40, CD86, CD80 and CD83. Mixed leukocyte responses induced by the DCs pulsed with K19 was stronger than that induced by naive DCs (P<0.01). The cytotoxicity rate of CTL induced by the sensitized DCs was higher than that of CTL induced by naive DCs (P<0.01). The cytotoxity activity was enhanced by increasing the effector/target ratio (P<0.01). Conclusion After sensitization with K19 antigen, the DCs can stimulate T cell proliferation and induce cytotoxicity activity against MCF-7 cells. Increased effector/target ratio may enhance the cytotoxity activity. Sensitized DCs possess the potential for amplification in immunotherapy of carcinoma.

中图分类号:

李鸣芳, 罗荣城, 尤长宣, 涂小玉. 角蛋白19致敏的DC/CTL对MCF-7体外抑瘤作用的实验研究[J]. 南方医科大学学报, 2005, 25(10): 1247-1250.

LI Ming-fang, LUO Rong-cheng, YOU Chang-xuan, TU Xiao-yu. Cytotoxicity of cytotoxic T lymphcytes induced by keratin 19-sensitized dendritic cells against MCF-7 cells in vitro[J]. Journal of Southern Medical University, 2005, 25(10): 1247-1250.

参考文献

[1] Rosenblatt J, Kufe D, Avigan D. Dendritic cell fusion vaccines for cancer immunotherapy [J]. Expert Opin Biol Ther, 2005, 5 (5):703-15.
[2] Hayashi T, Nakao K, Nagayama Y, et al. Vaccination with dendritic cells pulsed with apoptotic cells elicits effective antitumor immunity in murine hepatoma models[J]. Int J Oncol, 2005, 26(5): 1313-9.
[3] Okano F, Merad M, Furumoto K, et al. In vivo manipulation of dendritic cells overcomes tolerance to unmodified tumor-associated self antigens and induces potent antitumor immunity [J]. J Immunol, 2005, 174(5): 2645-52.
[4] 尤长宣,罗荣城,苏瑾,等.腺相关病毒介导乳腺癌BA46基因转移的实验研究[J].中华微生物与免疫学杂志,2004,24(9):724-7.You CX, Luo RC, Su J, et al. Adeno-associated virus mediate the breast cancer BA46 gene transfer into dendritic cells [J]. Chin J Microbiol Immunol, 2004, 24(9): 724-7.
[5] Liu Y, You CX, Luo RC, et al. Use and specificity of breast cancer antigen/milk protein BA46 for generating anti-self-cytotoxic T lymphocytes by recombinated virus-based gene loading of dendritic cells[J]. Cancer Gene Ther, 2004, 12(1): 1-9.
[6] Zitvoge IL, Mayordomo J, Tjandrawan T, et al. Therapy of murine tumors with tumor peptide pulsed dendritic cells: dependence on T cells, B7 co-stimulation, and T help cell 1 associated cytokines[J]. J Exp Med, 1996, 183(1): 87-97.
[7] Nakamura I, Kajino K, Bamba H, et al. Phenotypic stability of mature dendritic cells tuned by TLR or CD40 to control the efficiency of cytotoxic T cell priming [J]. Microbiol Immunol, 2004, 48 (3):211-9.
[8] Colombo MP, Trinchieri G. Interleukin-12 in anti-tumor immunity and immunotherapy [J]. Cytokine Growth Factor Rev, 2002, 13(1):155-68.
[9] Heystek HC, Mudde GC, Ohler R, et al. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has opposing effects on the capacity of monocytes versus monocyte-derived dendritic cells to stimulate the antigen-specific proliferation of a human T cell clone [J]. Clin Exp Immunol, 2000, 120(3): 440-7.
[10] Lutz MB, Schnare M, Menges M, et al. Differential functions of IL-4 receptor types Ⅰ and Ⅱ for dendritic cell maturation and IL-12 production and their dependency on GM-CSF [J]. J Immunol, 2002,169(7): 3574-80.
[11] Wan Y, Lu L, Bramson JL, et al. Dendritic cell-derived IL-12 is not required for the generation of cytotoxic, IFN-gamma-secreting, CD8 (+) CTL in vivo[J]. J Immunol, 2001, 167(9): 5027-33.
[12] Yang S, Vervaert CE, Burch J Jr, et al. Murine dendritic cells transfected with human GP100 elicit both antigen-specific CD8 (+) and CD4(+) T-cell responses and are more effective than DNA vaccines at generating anti-tumor immunity [J]. Int J Cancer, 1999, 83(4):532-40.