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目的:探讨孕激素对黑素瘤细胞免疫抑制性分子表达影响及可能的信号传导机制。方法:体外培养黑素瘤细胞系A375及A875,并以不同浓度孕激素、孕激素受体拮抗剂RU486以及PI3-K/Akt抑制剂LY294002进行诱导。RT-PCR、ELISA或蛋白质印迹法分别检测IL-10、TGF-β1及HLA-GmRNA及蛋白表达以及PI3-K/Akt信号途径Akt磷酸化水平。结果:黑素瘤细胞系A375及A875均在mRNA及蛋白水平表达IL-10及TGF-β1。孕激素浓度≥1×10-7mol/L对2株细胞IL-10表达均有显著上调作用;对TGF-β1表达无明显影响;不能诱导HLA-G表达。Akt抑制剂LY294002可消除孕激素对IL-10表达上调效应,而RU486无拮抗效应。孕激素浓度为1×10-7mol/L时上调A375细胞Akt磷酸化水平。结论:高浓度孕激素可体外上调黑素瘤免疫抑制分子IL-10的表达,并与不依赖经典细胞核受体的PI3-K/Akt途径激活有关。
Objective: To investigate the effect of progesterone on immunosuppressive molecules in melanoma cells and the possible signal transduction mechanism. Methods: Melanoma cell lines A375 and A875 were cultured in vitro and induced with different concentrations of progesterone, progesterone receptor antagonist RU486 and PI3-K / Akt inhibitor LY294002. The levels of IL-10, TGF-β1 and HLA-G mRNA and protein as well as Akt phosphorylation of PI3-K / Akt signaling pathway were detected by RT-PCR, ELISA or Western blotting. Results: The melanoma cell lines A375 and A875 both expressed IL-10 and TGF-β1 at the mRNA and protein levels. The concentration of progesterone ≥1 × 10-7mol / L significantly upregulated the expression of IL-10 in two cell lines, but had no significant effect on the expression of TGF-β1 and did not induce the expression of HLA-G. The Akt inhibitor LY294002 abolished the upregulation effect of progesterone on IL-10 expression, whereas RU486 had no antagonistic effect. Progesterone concentration of 1 × 10-7mol / L upregulated Akt phosphorylation in A375 cells. CONCLUSION: High concentrations of progesterone can upregulate the expression of IL-10, a melanoma-related immunosuppressant, in vitro and is related to the activation of PI3-K / Akt pathway, which is independent of classical nuclear receptors.