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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、MHCⅡ、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。
Objective To establish a mouse breast cancer 4T1 cell line stably transfected with the full-length mouse IL-17 gene and identify it. Methods Liposome method was used to transfect mouse breast cancer cell line 4T1, which contains the full-length of mouse IL-17 gene, and the cell line stably expressing IL-17 was screened by G418. The cell morphology was observed under microscope RT-PCR, Western blotting and confocal laser scanning were used to detect the expression of target gene and protein. The supernatant of 4T1 cells transfected and untransfected with IL-17 was collected and co-cultured with RAW264.7 macrophages, The levels of IL-6 in the culture supernatant of RAW264.7 cells were detected by ELISA. The proliferation of RAW264.7 cells was detected by MTS assay. The cell cycle and apoptosis were detected by flow cytometry. The expressions of MHC Ⅰ, MHC Ⅱ and LFA -1) and other molecular expression. Results A strain of 4T1 cells stably expressing IL-17 was obtained, and its culture supernatant stimulated RAW264.7 mouse macrophages to produce IL-6, demonstrating that the cells express functional IL-17. Conclusion The mouse breast cancer cell line stably transfected with IL-17 gene was successfully established.