抑制MCF7乳癌细胞VEGF表达对树突细胞免疫功能影响.docVIP

　　抑制MCF7乳癌细胞VEGF表达对树突细胞免疫功能影响 ：王海燕 谭丽莉 江彩玉 葛银林 【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，CF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。 【关键词】 树突细胞；血管内皮生长因子；RNA干扰；MCF7细胞 ［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 e Lipofectamine 2000TM as transfecting agent, the changes of VEGF gene expression easured by reverse transcriptionpolymerase chain reaction (RTPCR), and CF7 cells (control group) and from siRNAVEGF treated MCF7 cells (siRNAVEGF group) acrophage 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 easured 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) RNA 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 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 RNA水平上关闭VEGF基因的表达，观察VEGF基因沉默前后肿瘤细胞分泌的上清液对DC功能的影响，以进一步探讨VEGF在肿瘤DC免疫抑制机制中的作用，为乳癌的临床治疗寻找新的靶点。 1 材料与方法 1.1 材料 1.1.1 试剂及引物 Lipofectamine 2000TM及TRIzol Reagent购自Invitrogen公司；AMV 逆转录试剂盒、兔抗VEGF多克隆抗体（sc507）、ECL发光