中华皮肤科杂志 ›› 2011, Vol. 44 ›› Issue (1): 18-22.

• 论著 • 上一篇    下一篇

跨膜型血型A抗原模拟肽疫苗在黑素瘤细胞B16中表达及细胞毒效应检测

岑东芝1,孟辉2,张积仁1   

  1. 1. 广州市南方医科大学珠江医院肿瘤中心
    2.
  • 收稿日期:2010-04-12 修回日期:2010-07-10 出版日期:2011-01-15 发布日期:2011-01-10
  • 通讯作者: 张积仁 E-mail:zjzhangjiren@163.com

Expression and cytotoxic effect of transmembrane form of human blood group A antigen mimotope vaccine in a malignant melanoma cell line B16

  • Received:2010-04-12 Revised:2010-07-10 Online:2011-01-15 Published:2011-01-10

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摘要：

目的　建立稳定表达跨膜型血型A抗原模拟肽疫苗的恶性黑素瘤细胞株B16，并检测体外细胞毒活性。方法 采用脂质体转染法将前期构建的模拟肽疫苗稳定转染B16细胞进行表达，RT-PCR 及Western印迹法检测模拟肽/Fas融合基因及巨噬细胞炎症蛋白3β（Mip3β）的 mRNA及蛋白表达。细胞计数试剂盒-8（CCK-8）法检测疫苗介导的补体依赖性细胞毒效应（CDC）及抗体依赖的细胞介导细胞毒效应（ADCC）。结果 RT-PCR 在预期位置检测到特异性条带。Western印迹表明，表达产物具有特异的结合抗血型A抗体活性。析因分析提示不同分组之间CDC效应总体差异有统计学意义（F = 244.522，P < 0.01），ADCC效应总体差异也有统计学意义（F = 71.593，P < 0.01）。组间比较示M-pIRES组CDC效应与空质粒pIRES组无统计学差异，两组均明显低于P/F-M-pIRES组和P/F-pIRES组；P/F-M-pIRES组、P/F-pIRES组ADCC效应明显强于M-pIRES组与pIRES组，P/F-M-pIRES组ADCC效应明显强于P/F-pIRES组（F = 15.42，P < 0.05）。结论　跨膜型血型A抗原模拟肽疫苗可在B16细胞膜稳定表达，且可通过介导CDC和ADCC发挥对黑素瘤细胞B16的杀伤作用。

关键词: 黑色素瘤

Abstract:

Objective To establish a stable cell line expressing transmembrane form of human blood group A antigen mimotope vaccine by transfecting malignant melanoma cell line B16, and to detect the cytotoxicity of the vaccine against melanoma cells. Methods Cultured B16 cells were classified into 4 groups, i.e., P/F-M-pIRES group ［transfected with the recombinant plasmid mimotope peptide/Fas-macrophage inflammatory protein （Mip）-pIRES］, P/F-pIRES group （transfected with the recombinant plasmid mimotope peptide/Fas-pIRES）, M-pIRES group （transfected with the recombinant plasmid Mip-pIRES）, and pIRES group （transfected with the empty plasmid pIRES）. B16 cells were transfected through Lipofectamine 2000. Subsequently, RT-PCR and Western blotting were performed to detect the mRNA and protein expressions of the mimotope peptide/Fas fusion gene and Mip3β in transfected B16 cells. Cell counting kit-8 （CCK-8） was used to evaluate the vaccine-mediated complement dependent cytotoxicity （CDC） and antibody-dependent cell-mediated cytotoxicity （ADCC） against B16 cells. Results RT-PCR yielded specific DNA fragments with expected size. Western blotting revealed the anti-A antibody-binding activity of the recombinant mimotope peptide/Fas fusion protein. Factor analysis indicated significant differences in CDC （F = 244.522, P < 0.01） and ADCC （F = 71.593, P < 0.01） against B16 cells between the 4 groups. Group comparisons demonstrated more intense CDC and ADCC in P/F-M-pIRES and P/F-pIRES groups compared with M-pIRES and pIRES groups, stronger ADCC in P/F-M-pIRES group in comparison with P/F-pIRES group （F = 15.42, P < 0.05）, but no significant difference in CDC was observed between M-pIRES and pIRES group. Conclusions The transmembrane form of human blood group A antigen mimotope vaccine could be stably expressed in B16 cells, and mediate ADCC and CDC against B16 cells in vitro.

Key words: Malignant melanoma