应用siRNA技术探讨MCF-7乳腺癌细胞分泌的VEGF对树突状细胞的影响

为探讨MCF-7乳腺癌细胞分泌的血管内皮生长因子（ vascular endothelial growth factor, VEGF）对树突状细胞（dendritic cell, DC）功能及其分化的影响，针对VEGF基因设计siRNA（small interfering RNA, siRNA),采用脂质体转染法以100 nmol/L最佳转染浓度导入MCF-7乳腺癌细胞（siRNA组），以脂质体Lipofectamine　2000TM转染MCF-7 乳腺癌细胞培养上清培养正常DC作为对照（对照组），采用ELISA法检测经siRNA 干扰VEGF基因后的MCF-7 乳腺癌细胞分泌的VEGF因子含量， Western 印迹检测VEGF蛋白表达,以探讨siRNA的基因沉默效果；以siRNA组和对照组培养上清分别培养外周血单个核细胞，用流式细胞仪检测所诱导DC表型CD1a、CD80、CD83、CD86和HLA-DR的表达，用MTT法检测转染前后两组DC 诱导的细胞毒性T淋巴细胞（cytotoxic T lymphocyte, CTL）对MCF-7细胞的细胞毒作用.结果显示,MCF-7 乳腺癌细胞培养上清能明显抑制正常DC分化成熟及抗原递呈能力，干扰VEGF基因后MCF-7 乳腺癌细胞培养上清对DC的影响明显降低，CD80、CD83、CD86和HLA-DR的表达较对照组显著升高，而CD1a表达下降（P＜0.01）.转染前后DC 诱导的CTL对MCF-7细胞的杀伤活性有明显差异（P＜0.01）.由此可见，siRNA可靶向抑制MCF-7乳腺癌细胞VEGF的表达，下调VEGF后的MCF-7 细胞上清对DC分化成熟及功能的抑制作用明显降低，从而推测VEGF在肿瘤的发生、发展和免疫抑制方面可能起着重要的作用.

Effects of VEGF Secreted by MCF-7 Breast Cancer Cells on Dendritic Cells Using siRNA

To investigate the effects of VEGF secreted by MCF-7 breast cancer cells on differentiation, maturation and function of dendritic cells (DCs) secreted by MCF-7 breast cancer cells, small interfering RNAs (siRNAs) directed against VEGF gene were designed and transfected into MCF-7 breast cancer cells at theoptimal concentration (100 nmol/L) using cationic liposome Lipofectamine　2000 TM, while the control group was transfected with Lipofectamine　2000TMonly. The culture supernatants were tested on normal DCs. The silening of VEGF gene expression in MCF-7 cell treated with siRNA was measured by Western blotting and ELISA assay to detect the VEGF protein expression and VEGF concentration, respectively Mononuclear cells were cultured with the culture supernatants from primary MCF-7 cells (control group) and from siRNAs treated MCF-7 cells (siRNA group). The phenotypes of DCs including CD1a,CD80,CD83,CD86 and HLA-DR were evaluated by flow cytometry. MTT assay was used to detect the cytotoxicity (inhibition rates) of DCsmediated tumor-specific cytotoxic T lymphocyte (CTL) against MCF-7 cells in twodifferent culture supernatants. The results showed that DCs in siRNA group had significantly higher expression of CD86,CD80,CD83 and HLA-DR than the control group while CD1a was much lower in siRNA group (P＜0.01). The killing activities of CTLs mediated by DCs were significantly altered before and after siRNA transfection (P＜0.01). In summary, our designed siRNAs specifically inhibited VEGFexpression in MCF-7 cells, the inhibition of DC maturation and function in culturesupernatants from siRNA transfected cells was significantly decreased. This suggested that VEGF might play an important role in tumor developmemt, progress and immunorepression.