1.Department of Obstetrics and Gynecology, the First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310000, China; 2.Department of Laboratory, the First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310000, China
DOU Xiaoqing,WEN Mingxiao,ZHU Yingping, et al. M2 phenotype in THP-1 macrophages induced by supernatants of cervical cancer cell culture[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2020, 50(5): 371-376.
Abstract：Objective: To study the effect of supernatant in cervical cancer cell culture on inducing stable M2 phenotype in THP-1 macrophages. Methods: When cervical cancer cell line HeLa cells were cultured on day 3, cell culture supernatant was collected to detect tumor-related cytokines (IL-4, IL-6 and IL-10) and growth factors (angiopoietin-2, hepatocyte growth factor, vascular endothelial growth factor). Macrophages were cultured to logarithmic growth stage and then divided into blank control group (Mθ group), M2 cell induction group (M2 group), and Hela cell culture supernatant treatment group (Mθ+sHeLa group). The M2 group was treated with 20 ng/mL IL-4 and 20 ng/mL IL-10 to polarize M2 cells, and Mθ+sHeLa group was treated with 50% HeLa cell culture supernatant for 3 d, subsequently. Flow cytometry was used to detect the changes of HLA-DR, CD163 and CD206 on the cell surface. Meanwhile, the supernatant of cell culture was collected and the contents of related cytokines, growth factors and TGF-β3 were detected. Finally, the changes of the JAK-STAT6 signaling pathway in macrophages were detected by flow cytometry and Western blot. Results: The content of IL-6, HGF, and VEGF in Hela cell culture supernatant was high. The content of CD163 and CD206 on the surface of macrophages increased after treatment with HeLa cell culture supernatant. Compared with the Mθ group, the levels of related cytokines (IL-4, IL-6 and IL-10), growth factors (ANG-2, HGF, VEGF) and TGF-β3 increased in supernatant of M2 and Mθ+sHeLa group. After removal of the supernatant of HeLa cell culture for 48 h, the content of related cytokines, growth factors and TGF-β3 were still increased in the Mθ+sHeLa group compared with the Mθ group. Finally, flow cytometry and Western blot results showed that the level of pSTAT6 was increased in the Mθ+sHeLa group compared with the Mθ group. Conclusion: Hela cell culture supernatant can promote tumor growth and angiogenesis by activating the polarization of THP-1 macrophages into the M2 via JAK-STAT6 signal pathway.
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