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血管内皮生长因子受体-2胞外区基因修饰树突状细胞的抗实验性肺转移作用
作者姓名:Pan JP  Weng YS  Wu QQ
作者单位:310031,杭州,浙江大学免疫学研究所
基金项目:浙江省科技攻关计划重点资助项目(2005C23005)
摘    要:目的研究血管内皮生长因子受体-2胞外区基因修饰树突状细胞(DC)的抗肿瘤转移作用及其机制。方法电穿孔法基因转染DC,ELISA检测基因转染DC表达sVEGFR-2水平;经尾静脉给C57BL/6小鼠分别注射PBS、DC、pcDNA3.1修饰的DC(DC-vector)、pcDNA3.1/sVEGFR-2修饰的DC(DC-sVEGFR-2),连续3次,每次间隔1周,最后一次免疫注射后10 d,以51Cr释放法检测小鼠脾细胞VEGFR-2特异性CTL活性,藻酸钠小珠法检测肿瘤细胞诱导的新生血管形成,以B16黑色素瘤肺转移模型观察DC-sVEGFR-2免疫的抗肿瘤转移作用。以抗-CD4单抗或抗-CD8单抗分别剔除体内CD4+T细胞及CD8+T细胞,研究DC-sVEGFR-2免疫抗肿瘤转移作用的免疫学机制。结果DC- sVEGFR-2能有效表达sVEGFR-2,而DC-vector和DC不表达sVEGFR-2。DC-sVEGFR-2免疫小鼠脾细胞能有效杀伤VEGFR-2+的靶细胞H5V和3LL-sVEGFR,与对照组相比,差异有统计学意义(P<0.01),但对VEGFR-2-的同系EL4和3LL细胞无杀伤作用。DC-sVEGFR-2免疫能显著抑制肿瘤诱导的新生血管形成,DC-sVEGFR-2免疫小鼠藻酸钠小珠中FITC-dextran的摄取量仅为对照组的50%,与对照组相比,差异有统计学意义(P<0.01)。DC-sVEGFR-2免疫组小鼠B16黑色素瘤肺转移灶数目显著减少,瘤灶体积显著小于对照组,与DC-vector免疫组相比,肺表面转移灶数目下降81.9%(49.7±12.7:9.0±3.2,P<0.01)。体内T细胞亚群剔除试验显示,DC-sVEGFR-2免疫的抗肿瘤转移作用由CD8+T细胞介导。结论DC-sVEGFR-2免疫能打破自身免疫耐受,诱导针对VEGFR-2的CTL应答,抑制肿瘤诱导的新生血管形成,显著抑制肿瘤的转移,其抗肿瘤转移作用由CD8+T细胞介导。

关 键 词:血管内皮生长因子受体-2  树突状细胞  新生血管形成  黑色素瘤
收稿时间:09 6 2005 12:00AM
修稿时间:2005-09-06

Anti-metastatic effect of vascular endothelial growth factor receptor 2 extracellular domain gene-modified dendritic cell vaccination in murine model with experimental pulmonary metastasis
Pan JP,Weng YS,Wu QQ.Anti-metastatic effect of vascular endothelial growth factor receptor 2 extracellular domain gene-modified dendritic cell vaccination in murine model with experimental pulmonary metastasis[J].Chinese Journal of Oncology,2006,28(9):646-649.
Authors:Pan Jian-ping  Weng Yue-song  Wu Qian-qian
Institution:Institute of Immunology, Zhejiang University, Hangzhou 310031, China. jppan@zju.edu.cn
Abstract:OBJECTIVE: To investigate the anti-metastatic effect of vascular endothelial growth factor receptor 2 extracellular domain gene-modified dendritic cell (DC-sVEGFR-2) vaccination. METHODS: Dendritic cells (DC) were electroporated with pcDNA3. 1/sVEGFR-2 plasmid DNA. Expression of sVEGFR-2 was determined by ELISA. For immunization, C57BL/6 mice were intravenously injected three times with 1 x 10(5) cells per mouse of DC, pcDNA3. 1-transfected DC (DC-vector) , DC-sVEGFR-2, or 100 microl of PBS at 7-day intervals. At 10 days after the last immunization, the immunized mice were subjected to assessment of cytotoxic T lymphocyte ( CTL) response to VEGFR-2, alginate bead analysis of tumor cell-induced angiogenesis, and observation of the anti-metastatic effect in B16 melanoma metastasis model. CTL activity was determined by a standard 4-h 51Cr release assay against VEGFR-2 + vascular endothelial cell line H5V, 3LL cells stably transfected with pcDNA3. 1/sVEGFR-2 (3LL,-sVEGFR-2), and VEGFR-2- cell lines EL-4 and 3LL. Monoclonal antibodies GK1.5 anti-CD4 and 2.43 anti-CD8 were used to deplete in vivo CD4 + T cells and CD8' T cells, respectively. RESULTS: DC-sVEGFR-2 could effectively express sVEGFR-2, whereas DC-vector and DC could not. Immunization of mice with DC-sVEGFR-2 significantly induce CTL activity against VEGFR-2 + cell lines H5V and 3LL-sVEGFR-2, however, no significant CTL activity was observed when VEGFR-2- syngeneic cell lines EL-4 and 3LL. were used as target cells, implying this CTL activity was VEGFR-2 specific. Alginate bead analysis of in vivo neoangiogenesis showed that the inhibition reached 50% in mice vaccinated with DC-sVEGFR-2 compared with mice vaccinated with DC, DC-vector or PBS. Anti-metastatic experiment showed that profound reduction in pulmonary metastases was found in mice immunized with DC-sVEGFR-2, while mice immunized with PBS, DC, DC-vector developed extensive pulmonary metastases. The number of tumor nodules on lung surface decreased by 81.9% in mice immunized with DC-sVEGFR-2 when compared with mice immunized with DC-vector (49.7+/-12.7 vs. 9.0+/-3.2). In vivo T cell subset depletion experiments showed that the anti-metastatic effect of DC-sVEGFR-2 vaccination was abrogated in CD8 + T cell-depleted but not in CD4+ T cell-depleted mice. CONCLUSION: Immunization of mice with DC-sVEGFR-2 could break self-tolerance and induce a significant CTL response to VEGFR-2, leading to profound inhibition of tumor-cell induced angiogenesis and metastasis. This anti-metastatic effect is mainly mediated by CD8+ T cells.
Keywords:Vascular endothelial growth factor receptor 2  Dendritic cell  Angiogenesis  Melanoma
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