4T1乳腺癌细胞培养上清液促进ANA-1巨噬细胞精氨酸酶1分泌

The supernatant of 4T1 breast cancer cell culture increases arginase 1 content in ANA-1 macrophages

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 explore the effect of the supematant of 4T1 mudne breast cancer cell culture on arginase 1 （Arg-l） in ANA-1 macrophages in vitro by simulating the microenvironment of breast cancer. Methods The experimental ANA-1 macrophages were treated with the supernatant of 4T1 culture, and meanwhile, the control cells were cultured in the absence of the supematant. Morphological changes of the ANA-I macrophages were observed with a light microscope at 6, 8, 10, 24 hours, respectively. Real-time quantitative PCR （qRT-PCR） was performed to detect the levels of inducible nitric oxide synthase （iNOS） and Arg-1 mRNAs. Immunofluorescence and Western blotting were used to determine the levels of iNOS and Arg-I proteins. Results The qRT-PCR indicated that the level of iNOS mRNA decreased in the experiment group compared with the control group, while Arg-1 mRNA level significantly increased compared with the control group and it reached a peak at the 8th hour. The immunofluorescence and Western blotting also demonstrated that Arg-t protein expression was enhanced in the experimental group compared with the control group. However, iNOS protein expression was no significantly different between the experiment group and the control group. Conclusion The supernatant of 4T1 cell culture increases Arg-1 production in ANA-1 macrophages. [