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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。
Objective To investigate the anti-tumor metastasis and its mechanism of vascular endothelial growth factor receptor-2 extracellular domain-modified dendritic cells (DCs). Methods DCs were transfected with DCs by electroporation, and sVEGFR-2 was transfected into DCs by ELISA. C57BL / 6 mice were injected with DCs, DCs and pcDNA3.1 modified DCs (DC-vector) and pcDNA3 by tail vein. (DC-sVEGFR-2) modified with 1 / s VEGFR-2 for 3 consecutive times, at intervals of 1 week and 10 days after the last immunization, the specific CTL activity of VEGFR-2 was detected by 51Cr release assay The tumor cell-induced neovascularization was detected by sodium alginate beads method. The anti-tumor effect of DC-sVEGFR-2 immunization was observed by B16 melanoma lung metastasis model. The anti-CD4 monoclonal antibody or anti-CD8 monoclonal antibody were used to excise CD4 + T cells and CD8 + T cells respectively in order to study the immunological mechanism of anti-tumor immunity of DC-sVEGFR-2. Results DC-sVEGFR-2 effectively expressed sVEGFR-2, while DC-vector and DC did not express sVEGFR-2. The splenocyte immunized with DC-sVEGFR-2 could effectively kill H5V and 3LL-sVEGFR of target cells of VEGFR-2 +, compared with the control group, the difference was statistically significant (P <0.01) - The same line of EL4 and 3LL cells without killing effect. DC-sVEGFR-2 immunization can significantly inhibit tumor-induced neovascularization. The uptake of FITC-dextran in DC-sVEGFR-2 immunized mice was only 50% of that in control group. Compared with the control group, The difference was statistically significant (P <0.01). The number of lung metastases of B16 melanoma in DC-sVEGFR-2 immunized group was significantly decreased, the tumor volume was significantly smaller than that in control group, and the number of lung surface metastases decreased by 81.9% (49.7 ± 12.7: 9.0 ± 3.2, P <0.01). In vivo T-cell subset knockout assays showed that the anti-tumor metastatic effect of DC-sVEGFR-2 immunization is mediated by CD8 + T cells. Conclusion Immunization of DC-sVEGFR-2 can abrogate autoimmune tolerance, induce CTL response to VEGFR-2, inhibit tumor-induced neovascularization, significantly inhibit tumor metastasis, and anti-tumor metastasis mediated by CD8 + T cells.