宫颈癌细胞培养上清液诱导THP-1巨噬细胞M2表型

目的：研究宫颈癌细胞培养上清液诱导并维持THP-1巨噬细胞M2表型的作用。方法：培养宫颈癌 细胞系HeLa细胞至第3天，收集细胞培养上清液用以检测肿瘤相关细胞因子（IL-4、IL-6及IL-10）及生长因 子（ANG -2、HGF、VEGF）分泌情况。将培养至对数生长期的巨噬细胞分为空白对照组（Mθ组）、M2细胞诱导组（M2组）以及HeLa细胞培养上清液处理组（Mθ+sHeLa组）。其中M2组予以20ng/mLIL-4、20ng/mLIL-10处理 使其向M2细胞极化，Mθ+sHeLa组加入50%HeLa细胞培养上清液处理培养3d。随后，利用流式细胞术检测 各组细胞表面HLA-DR、CD163、CD206的变化；同时，收集细胞培养上清液并检测其中相关细胞因子、生长 因子及TGF-β3的含量。最后，采用流式细胞术及Westernblot检测巨噬细胞中JAK-STAT6信号通路的变化。 结果：HeLa细胞培养上清液中IL-6、HGF、VEGF含量较多。经过HeLa细胞培养上清液处理后的巨噬细胞表面 CD163和CD206含量升高（ P ＜0.05）。相较于Mθ组，M2组及Mθ+sHeLa组的巨噬细胞培养上清液中的相关细胞 因子（IL -4、IL-6及IL-10）、生长因子（ANG -2、HGF、VEGF）及TGF-β3含量升高（ P ＜0.05）。去除HeLa细胞培 养上清液后48h后，相较于Mθ组，Mθ+sHeLa组分泌相关细胞因子、生长因子及TGF-β3含量升高（ P ＜0.05）。 流式细胞术及Westernblot结果显示，与Mθ组比，Mθ+sHeLa组的pSTAT6水平升高（ P ＜0.05）。结论：HeLa 细胞培养上清液可通过激活JAK-STAT6信号通路诱导的THP-1巨噬细胞向M2表型的极化促进肿瘤的生长和血 管生成。

M2 phenotype in THP-1 macrophages induced by supernatants of cervical cancer cell culture

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 contentof 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.