巨噬细胞对前列腺癌细胞株pc-3作用及机制探讨-effect and mechanism of macrophage on prostate cancer cell line pc - 3.docxVIP

ABSTRACTObjective: In this study, we aim to investigate the effect of four subtypes of macrophages on the proliferation, apoptosis, migration, invasion and angiogenesis of prostate cancer using PC-3 cell lines, and to dress the mechanism underlying these effects.Methods: The human monocytes were obtained from healthy donor by gradient centrifugation using Ficoll, and purified by magnetic activated cell sorting.The monocytes were incubated for 7 days in RPMI1640 supplemented with 10% FBS and 50ng/ml M-CSF in order to generate macrophages. M0 cells were generated by treating the cells with serum-free medium without additional cytokines for 48h. M1 macrophages were polarized by stimulating the cells with LPS (100ng/ml) and IFN-γ (100ng/ml) overnight. M2 macrophages were polarized by stimulating the cells with IL-4 (20ng/ml) overnight. TAM were generated by culturing monocytes for 7 days in RPMI 1640 10% FBS with 50% of CM from PC-3. Characterization of subtypes of macrophages with anti CD-14 FITC and anti CD-163 PE was detected by High Throughput Connotation of Imaging System. The expression levels of IL-1β, IL-6, IL-10, IL-12, IL-23, CXCL9, CCL13 and microRNA let-7 mRNA in four subtypes of macrophages were detected by real-time PCR. The expression levels of TGF-β, TNG-α, IL-10 and IL-12 in the supernate from cultures of four subtypes of macrophages were determined by ELISA. We then carried out MTT, Annexin V-FITC, wound healing, transwell assays to detect the effect of four subtypes of macrophages on the proliferation, apoptosis, migration and invasion ability of PC-3 respectively, we also conducted matrigel assay with HUVEC cell lines to detect the ability of four subtypes of macrophages on the angiogenesis of PC-3.Results: The expression levels of IL-1β, IL-6, IL-12, IL-23, CXCL9 mRNA in M1 cells were detected to be a significant increase as compared with M0, M2 and TAM, while the expression levels of IL-10 mRNA in M2 and TAM showed a significant increase as compare