p53基因修饰树突细胞诱导特异性抗肿瘤免疫应答的实验研究

目的　探讨含p5 3基因的质粒pC5 3 SN3转染的树突细胞 (DC)体外诱导特异性抗肿瘤细胞毒性T淋巴细胞 (CTL)。方法　应用脂质体介导将质粒pC5 3 SN3转染肺癌患者单个核细胞 [HLA A2 + ]衍生的DC ,然后与未经纯化的自体T细胞混合培养以诱导CTL(T pC5 3 SN3) ,并通过乳酸脱氢酶(LDH)释放实验测定其对p5 3基因突变的HLA A2 + 人肺癌细胞系Calu 6的杀伤活性。结果　用质粒pC5 3 SN3转染DC后 ,发现CD1a、CD83显著升高 ,转染前表达率分别为 (5 .4 5± 0 .89) %、(3.2 6± 0 .4 7) % ,转染后分别为 (5 2 .15± 11.5 6 ) %、(2 5 .78± 12 .35 ) %。但CD86 、CD4 0 、HLA DR等DC相关标志与转染前无明显改变。LDH释放实验显示 ,以Calu 6作为靶细胞 ,T pC5 3 SN3诱导的杀伤作用显著高于T IL 2(IL 2 10 0U ml刺激外周血单个核细胞产生的CTL) ,效靶比为 10∶1时其杀伤活性分别为 (5 6 .79±15 .6 7) %和 (39.33± 9.88) %。同时细胞表面标记检测可见T pC5 3 SN3细胞中以CD8+ 细胞为主 ,且CD6 9、CD4 5RO CD8表达均显著升高。结论　p5 3基因修饰的DC对p5 3突变的人肺癌细胞系可有效诱导HLA A2限制的特异性CTL。

Experimental study of the specific immunoresponse induced by p53 modified dendritic cells

OBJECTIVE: To explore the specific cytotoxic T lymphocyte (CTL) induced by dendritic cells (DC), which were transfected by the plasmid pC53-SN3 encoding p53 gene. METHODS: DC derived from HLA-A2(+) mononuclear cells of the 24-lung cancer patients was transfected with the plasmid pC53-SN3 by lipofectamine and then co-cultured with auto-unpurified T cells to induce potent CTL (T-pC53-SN3). The cytolysis of specific CTL against Calu-6, a HLA-A2(+) human lung cancer cell line, was measured by using lactate dehydrogenase (LDH) releasing assay. RESULTS: The expression of CD(1a) and CD(83), the correlative markers of DC, increased apparently after transfected with plasmid pC53-SN3, the expression rate was (5.45 +/- 0.89)% and (3.26 +/- 0.47)% versus (52.15 +/- 11.56)% and (25.78 +/- 12.35)%. CD(14) decreased apparently, but other DC correlative markers of CD(1a), CD(40), CD(86), and HLA-DR remained almost the same as that before transfection. Compared with T-IL-2, the CTL derived from PBMNC stimulated by IL-2 (100 U/ml), the cytolytic activity of T-pC53-SN3 against Calu-6 cell line showed a significant increase, but cytolytic activity was 56.79 +/- 15.67 and 39.33 +/- 9.88, respectively, when effect cells: target cells was 10:1. The expression of the CD(8), CD(69), and CD(45)RO/CD(8) of T-pC53-SN3 cells increased significantly, but that of CD(3), CD(4), CD(86), ect, was not significantly different from those of T-pCMV-neo. CONCLUSIONS: It showed that DC transfected by p53 gene could induce potent HLA-A(2) restrictive CTL to kill tumor cell efficiently.