抑制MCF7乳癌细胞VEGF表达对树突细胞免疫功能影响_医学论文.docVIP

抑制MCF乳癌细胞VEGF表达对树突细胞免疫功能影响_医学论文 抑制MCF乳癌细胞VEGF表达对树突细胞免疫功能影响_医学论文 作者：王海燕 谭丽莉 江彩玉 葛银林 【摘要】 目的 探讨抑制MCF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，RT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乳癌细胞的培养上清和干扰VEGF基因后MCF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、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）及DC与外周血单个核细胞(PBMC)共同培养液中干扰素γ（IFN）的含量。结果 VEGF基因经siRNA处理24 h后，MCF乳癌细胞VEGF mRNA和蛋白的表达明显降低，其诱生的DC与MCF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和协同刺激外周血PBMC分泌INF的能力明显增强。结论 siRNA可靶向抑制乳癌MCF细胞VEGF的表达，下调VEGF后的MCF细胞上清液对DC分化成熟及功能的抑制作用明显降低。 【关键词】 树突细胞；血管内皮生长因子；RNA干扰；MCF细胞 ［ABSTRACT］ Objective To investigate the effect of inhibition of VEGF on immune function of dendritic cell (DC) secreted by MCF 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 it