稳定转染小鼠IL-17全长基因的乳腺癌细胞株的建立及基因转染对4T1细胞的影响

目的建立稳定转染小鼠IL-17基因全长的小鼠乳腺癌4T1细胞株，并进行鉴定。方法脂质体法将携带小鼠IL-17基因全长的真核表达载体pcDNA3．1转染小鼠乳腺癌细胞4T1，经G418筛选出稳定表达IL-17的细胞株；镜下观察细胞形态，RT-PCR法、Western印迹和激光共聚焦法检测目的基因和蛋白的表达；收集转染和未转染IL．17的4T1细胞的培养上清，分别与巨噬细胞RAW264．7共培养，ELISA法检测RAw264．7细胞培养上清中IL-6水平；MTS法检测细胞的增殖情况，流式细胞技术检测细胞周期、凋亡以及细胞表面MHCI、MHC11、淋巴细胞功能相关抗原-1（LFA-1）等分子表达。结果获得1株稳定表达IL-17的4T1细胞，而且其培养上清可刺激小鼠巨噬细胞RAW264．7产生IL-6，证明该细胞可表达功能性IL-17。结论成功建立了稳定转染IL-17基因的小鼠乳腺癌细胞株。

Construction of a transfected breast cancer cell line expressing mouse IL-17 and effect of gene transfection on 4T1 cell

Objective To establish a stable mouse full-length IL-17 gene transfecting 4TI cell line and identify its the function. Method We transfected 4T1 mouse breast cancer cell line with IL-17 full length gene （inserted into pcDNA3.1 vector） using lipofectamine 2000 and selected cells with G418. First, we observed cellular morphology and then checked target gene and protein expression through RT-PCR, Western blotting and confocal microscopy. We cultured mouse macro- phage cell line RAW264.7 by adding conditioned medium collected from transfected and untransfected 4T1 cells respective- ly,checked [L-6 secretion by RAW264.7 cells using ELISA kit. The proliferation of cells was observed by MTS assay. Cell cycle, apoptosis, and cell surface expression of MHC I , MHC ]I and LFA-1 were checked by FACS. Results We ac- quired 4T1 cell line which was stablly transfected with mice full length IL-17. The supernatant of this cell line could induce macrophage RAW264.7 into secreting IL-6, suggesting that this cell line could express functional IL-17. Conclusion We have successfully established a mouse breast cancer cell line which is stably transfected with IL-I7 gene.