【摘要】 目的探讨三氧化二砷在小鼠肝癌细胞H22外泌exosome中的影响。方法MTT法筛选三氧化二砷的合适药物浓度;密度梯度离心和超速离心法提纯exosome,Bradford法对exosome总蛋白进行定量分析;Western blot检测影响小囊泡vesicle分泌的复合体SNARE的重要组成及调节蛋白Syntaxin的表达;结合钙荧光探针Fura-2、荧光倒置定量显微镜测定细胞内游离钙的相对浓度。结果维持小鼠肝癌细胞H2290%存活48h的药物浓度为1.25μmmol/L;药物作用前后,细胞数为5×104ml-1的H22细胞常规培养48h所得细胞悬液200ml,经密度梯度离心和超速离心所得exosome的总蛋白浓度分别为(0.85±0.13)、(0.96±0.10)mg/ml,差异具有统计学意义(P<0.05);三氧化二砷作用前后,H22细胞均有Syntaxin蛋白表达,三氧化二砷处理后,其表达明显增加(P<0.05)。结论SNARE复合体假说也适用于小鼠肝癌细胞H22的exosome外泌过程,三氧化二砷在exosome的外泌过程中有一定的促进作用。更多还原

【Abstract】 Objective To investigate the role of arsenic trioxide(As2O3) in the exocytosis of exosome in mouse hepatoma cell line H22.Methods MTT assay was performed to select the optimal concentration of As2O3.Exosomes produced by H22 cells treated with 1.25 μmol/L As2O3 were extracted by density gradient centrifugation and ultracentrifugation,and total exosome proteins were determined by Bradford’s method.The expression of syntaxin,an important protein of SNARE complex,was detected by Western blotting.The responses of intra-cellular calcium ion to As2O3 were analyzed indirectly by inverted microscope with aid of fluorescence-analyzed software.Results After treated with 1.25 μmol/L As2O3,about 90% of H22 cells survived for 48 h.More total exosome proteins was tested in the supernatants of H22 cells with 1.25 μmol/L As2O3 treatment than in that of without [(0.85±0.13) vs(0.96±0.10)mg/ml,P<0.05].Significantly increased expression of syntaxin was observed in the cells with As2O3 treatment(P<0.05).Conclusion The SNARE hypothesis may be applicable to the exocytosis of exosome.As2O3 may act as a promoter in this process in mouse hepatoma cell line H22.更多还原