【摘要】 目的探讨抑制MCF-7乳癌细胞血管内皮生长因子(VEGF)的表达对树突细胞(DC)免疫功能的影响。方法针对VEGF基因设计小干扰RNA(siRNA),以脂质体转染法将siRNA导入乳癌细胞MCF-7,RT-PCR检测干扰VEGF基因后mRNA的表达,Western blotting法检测VEGF蛋白的表达,观察VEGF基因沉默效果。以MCF-7乳癌细胞的培养上清和干扰VEGF基因后MCF-7乳癌细胞培养上清液同时添加粒-单细胞集落刺激因子、白细胞介素-4、肿瘤坏死因子α培养正常异基因DC,利用MTT法和Hoechst33258检测DC刺激正常外周血T细胞增殖的能力及DC激发的细胞毒T淋巴细胞(CTL)杀伤活性;ELISA法检测DC培养上清液中白细胞介素12(IL-12)及DC与外周血单个核细胞(PBMC)共同培养液中干扰素γ(IFN-γ)的含量。结果VEGF基因经siRNA处理24 h后,MCF-7乳癌细胞VEGF mRNA和蛋白的表达明显降低,其诱生的DC与MCF-7乳癌细胞培养上清液诱生的DC相比,siRNA处理DC刺激T淋巴细胞增殖能力、CTL杀伤活性以及DC分泌IL-12和协同刺激外周血PBMC分泌INF-γ的能力明显增强。结论siRNA可靶向抑制乳癌MCF-7细胞VEGF的表达,下调VEGF后的MCF-7细胞上清液对DC分化成熟及功能的抑制作用明显降低。更多还原

【Abstract】 Objective To investigate the effect of inhibition of VEGF on immune function of dendritic cell(DC) secreted by MCF-7 breast cancer cells. Methods siRNA-VEGF was constructed and transfected into MCF-7 breast cancer cell by using cationic liposome Lipofectamine 2000TM as transfecting agent,the changes of VEGF gene expression were measured by reverse transcription-polymerase chain reaction(RT-PCR),and Western blotting in vitro,the VEGF silencing result was observed.Mononuclear cells were cultured in the culture supernatants from fresh primary MCF-7 cells(control group) and from siRNA-VEGF treated MCF-7 cells(siRNA-VEGF group) with granulocyte-macrophage colony stimulating factor(GM-CSF),interleukin 4(IL-4) and tumor necrosis factor-α(TNF-α).The proliferative ability of T cells from healthy volunteers stimulated by DCs and the tumoricidal activity of CTL primed by DCs were measured by MTT assay and Hoechst33258.The concentrations of IL-12 in the supernatant of cultured DCs and IFN-γ in the supernatant of cocultivated DCs periphra1 blood mononuclear cells(PBMC) were detected by ELISA. Results siRNA directed against VEGF gene effectively down-regulated the expression of VEGF in the level of mRNA and protein.The tumoricidal activity against MCF-7 breast cancer cells,and the concentration of IL-12 secreted by DCs and IFN-γ produced by PBMC were all significantly higher than that in the control group. Conclusion siRNA can aim at VEGF secreted by MCF-7 cells to inhibit its expression,the inhibition of the supernatant from MCF-7 cells on DCs decreased markedly after VEGF gene was down-regulated by siRNA.更多还原