三氧化二砷诱导小鼠肝癌细胞（h22）外泌exosome及相关机制研究.docVIP

三氧化二砷诱导小鼠肝癌细胞（H22）外泌exosome及相关机制研究 向自武 沈宜* 汪少华 范维珂 陈黎（重庆医科大学病理生理教研室，重庆 40016） 提要：目的 探讨三氧化二砷在小鼠肝癌细胞(H22)外泌exosome中的影响，对exosome的外泌机制作一初步探讨。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。Western-Blotting法检测影响小囊泡（vesicle）分泌的复合体（SNARE）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(H22)90%存活48h的药物浓度为1.25μmmol/L；药物作用前、后，密度为5×104/ml 的H22细胞常规培养48小时所得细胞悬液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0.76±0.1408mg/ml、0.84±0.1472mg/ml（P0.05具有统计学差异）。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，具有统计学差异(P0.05）。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。 关键词:三氧化二砷 ; exosome; 胞吐作用 ;SNARE假说；syntaxin 中图法分类号:R73-35 Study on arsenic trioxide induced exocytosis of exosome in mouse hepatoma cells(H22) and its molecular mechanism XIANG Zi-wu, SHEN Yi*，WANG Shao-hua, FAN Wei-ke,CHEN Li (Department of Pathophysiology, Chongqing Medical University, Chongqing 400016, China) Abstract： Objective To investigate the exocytosis of exosome induced by Arsenic Trioxide in mouse hepatoma cells (H22) in vitro. Methods Optimal drug concentration was elected by MTT. Exosomes were extracted by density gradient centrifugation and ultracentrifugation. Total exosome proteins were determined by Bradford. The expression of syntaxin, an important protein of SNARE complex, was detected by western blotting. The responses of intra-cellular calcium ion to Arsenic Trioxide were analyzed indirectly by inverted microscope with fluorescence-analyzed software. Results About 90 % of mouse hepatoma cells H22, treated with 1.25umol/L arsenic trioxide, could survive for 48 hours. The total exosome proteins of control group and experimental group, extracted from the same volume of supernatants, were 0.76±0.1408 mg/ml and 0.84±0.1472 mg/ml（P0.05,compared with the control group）, respectively. The expression of syntaxin increased significantly (P0.05). Conclusion The SNARE hypothesis may be applicable to the exocytosis of exosome; Furthmore, arsenic