As_2O_3下调Colon26肿瘤细胞免疫抑制分子分泌的体外研究

Study in vitro of the down-regulating effects of As_2O_3 on secretion of immunosuppressive molecules from Colon26 tumor cells

目的体外研究As2O3下调Colon26肿瘤细胞免疫抑制分子分泌的作用。方法获取体外经As2O3作用后再培养的Colon26培养上清,以不经As2O3作用的Colon26同步培养上清作对照,定量ELISA法测定这些上清中TGF-β1、VEGF、IL-4、IL-6和IL-105种免疫抑制分子的含量,实验研究这些上清对小鼠脾细胞MTT法测定的NK杀伤和Con A诱导转化以及流式细胞计数分析的IL-2Rα、CD3ε+ζ+和CD3ε-ζ+表达5项免疫功能指标的影响。结果不经As2O3作用同步培养的Colon26上清,5种免疫抑制分子均可被测到,以TGF-β1含量最高;该上清对小鼠脾细胞5项免疫功能指标,均有显著抑制作用。As2O3作用后的Colon26,其第一次再培养上清,5种免疫抑制分子含量及对除CD3ε-ζ+表达外的其余4项免疫功能指标的抑制,均明显降低;与第一次再培养上清相比,其第二次再培养上清,TGF-β1、VEGF和IL-4含量及对NK杀伤、Con A诱导转化和CD3ε+ζ+表达的抑制,均明显提高,其余无明显变化。相关分析显示,TGF-β1和IL-10与抑制Con A诱导转化及IL-2Rα表达正相关;IL-4与抑制Con A诱导转化、NK杀伤及CD3ε+ζ+表达正相关;VEGF和IL-6与5项免疫功能指标的抑制不相关。结论As2O3可明显下调Co-lon26肿瘤细胞免疫抑制分子的分泌,下调TGF-β1、VEGF和IL-4分泌的作用可持续48h,下调IL-6和IL-10分泌的作用可持续96h,可能是As2O3抗瘤效应机制之一。

Objective To study in vitro the down-regulating effects of As2O3 on secretion of immunosuppressive molecules from Colon26 tumor ceils. Methods Re-cultured supematants of Colon26 after treated by As2O3 and the corresponding supernatants of Colon26 treated without As2O3 as control were collected. The concentrations of five immunosuppressive molecules（including TGF-βI, VEGF, IL-4, IL-6 and IL-10） indifferent supematants were measured by quantitative ELISA. The effects of different supernatants on five immunofunctions of murine splenocytes （ including NK killing and transformation induced by Con A detected by MTT, and expression levels of IL-2Rα, CD3ε^＋ζ^＋ and CD3ε^-ζ^＋ detected by FCM） were determined. Results For supernatants of Colon26 treated without As2O3, all of five immunosuppressive molecules were found, the concentration of TGF-[31 was highest, and the significant inhibitions of five immunofunctions of murine splenocytes were showed. For Colon26 after treated by As2O3, the concentrations of five immunosuppressive molecules in the first re-cultured supernatant and its inhibition of the four immunofunctions except of CD3ε^-ζ^＋ expression decreased greatly. Compared with the first re-cultured supematant, the concentrations of TGF-β1, VEGF and IL-4 in the second re-euhured supernatant and its inhibition of NK killing, transformation induced by Con A and CD3ε^＋ζ^＋ expression increased highly, and others had no changes. The analysis of correlation indicated the positive correlations existed between TGF-β1 or IL-10 and inhibition of transformation induced by Con A or IL-2Rα expression, between IL-4 and inhibition of NK killing or transformation induced by Con A or CD3ε^＋ζ^＋ expression, and not between VEGF or IL-6 and inhibition of five immunofunctions. Conclusion As2O3 can down-regulate secretion of immunosuppressive molecules by Colon26. The down-regulating effects on secretions of TGF-β1, VEGF and IL-4 can last for 48 h, and on secretions of IL-6 and IL-10 can last for 96 h at least. This effect should be one of anti-tumor mechanisms of As2O3.