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目的通过模拟体内乳腺癌微环境,探讨4T1小鼠乳腺癌细胞培养上清液在体外对ANA-1巨噬细胞中精氨酸酶1(Arg-1)含量的影响。方法用添加或不添加4T1培养上清液培养ANA-1巨噬细胞,分别在6、8、10、24 h用光学显微镜观察ANA-1细胞形态变化。实时荧光定量PCR(qRT-PCR)检测诱导型一氧化氮合酶(i NOS)、Arg-1 mRNA水平,免疫荧光染色、Western blot法检测i NOS、Arg-1蛋白水平。结果实时荧光定量PCR结果显示,添加4T1上清液的巨噬细胞i NOS mRNA表达水平较对照组减少,Arg-1 mRNA水平较对照组显著升高,且在8 h最显著。与对照组相比,免疫荧光染色和Western blot实验也发现Arg-1表达增强,但添加或不添加4T1培养上清液的细胞i NOS的表达差异不明显。结论 4T1乳腺癌细胞培养上清液诱导ANA-1细胞Arg-1分泌增加。
Objective To investigate the effect of 4T1 mouse breast cancer cell culture supernatant on the content of arginase 1 (Arg-1) in ANA-1 macrophages by simulating the breast cancer microenvironment in vivo. Methods ANA-1 macrophages were cultured with or without 4T1 culture supernatant. The morphological changes of ANA-1 cells were observed under light microscope at 6, 8, 10 and 24 hours. Real-time quantitative PCR (qRT-PCR) was used to detect the expression of iNOS and Arg-1 mRNA, and the levels of iNOS and Arg-1 were detected by immunofluorescence staining and Western blot. Results The real-time PCR results showed that the iNOS mRNA expression of macrophages with 4T1 supernatant decreased compared with that of the control group, and the level of Arg-1 mRNA increased significantly compared with the control group. Compared with the control group, Arg-1 expression was also found to be enhanced by immunofluorescence staining and Western blotting, but there was no significant difference in iNOS expression between the 4T1 culture supernatants and the control group. Conclusion 4T1 breast cancer cell culture supernatant induces an increase of Arg-1 secretion in ANA-1 cells.