5-FU和奥沙利铂对结肠癌细胞自噬与外泌体释放的影响及其机制探讨  

Effects of 5-fluorouracil and oxaliplatin on autophagy and exosome release of colon cancer cells and its mechanism

作　　者：施文[1] 何向锋 陈锦鹏 孙永强[1] 周志强 蔡明 邱建 SHI Wen;HE Xiangfeng;CHEN Jinpeng;SUN Yongqiang;ZHOU Zhiqiang;CAI Ming;QIU Jian(Department of Thyroid and Breast Surgery,the Second Affiliated Hospital,Nantong University,Jiangsu Nantong 226001,China)

机构地区：[1]南通大学第二附属医院甲乳外科,江苏南通226001

出　　处：《现代肿瘤医学》2023年第7期1226-1230,共5页Journal of Modern Oncology

基　　金：中国博士后科学基金面上资助项目(编号:2013M530227);江苏省博士后科研资助计划(编号:1301099C);江苏省南通市科学技术局项目(编号:BK2012067,HS2013031);江苏省南通市卫健委面上项目(编号:MB2020008)。

摘　　要：目的:探究5-氟尿嘧啶(5-fluorouracil,5-FU)和奥沙利铂对小鼠结肠腺癌CT26.WT细胞自噬与外泌体释放的影响及两者之间的关联机制。方法:通过蛋白印迹试验(Western blot)检测经正常培养、5-FU、奥沙利铂和饥饿培养处理CT26.WT细胞后自噬相关蛋白以及胞吐调节蛋白Syntaxin的表达情况,利用激光扫描共聚焦显微镜(confocal laser scanning microscope,CLSM)检测CT26.WT细胞内多泡体与自噬小体的相对位置关系以及多泡体与溶酶体的相对位置关系。利用超速离心法提取定量培养各组细胞的培养上清液中的外泌体,用Western blot检测与多泡体的形成和外泌体的释放有关的Syntaxin及外泌体标志蛋白表达情况。结果:5-FU、奥沙利铂和饥饿处理CT26.WT细胞均可诱导细胞自噬的发生,CLSM下可分别观察到部分多泡体同自噬小体,部分多泡体同溶酶体发生位置重合。CT26.WT细胞经饥饿培养后,Syntaxin蛋白较正常对照组表达下调,经5-FU或奥沙利铂处理后,Syntaxin蛋白同正常对照组相比表达无明显变化。5-FU和奥沙利铂处理组的外泌体EGFP-CD63蛋白表达较正常对照组明显下降。结论:5-FU和奥沙利铂不影响CT26.WT细胞内多泡体的形成,但均可诱导CT26.WT细胞发生自噬,并促进多泡体经自噬溶酶体途径降解,从而下调外泌体的释放。Objective:To investigate the effects of 5-fluorouracil(5-FU)and oxaliplatin on autophagy and exosome release of mouse colon adenocarcinoma CT26.WT cells and the correlation mechanism between them.Methods:Western blot was used to detect the expression of autophagy related protein and exocytosis regulatory protein Syntaxin in CT26.WT cells treated with normal culture,5-FU,oxaliplatin and starvation culture.After normal culture,5-FU,oxaliplatin and starvation culture,the relative position relationship between multivesicles and autophagosomes and between multivesicles and lysosomes in CT26.WT cells were detected by confocal laser scanning microscope(CLSM).The exosomes in the culture supernatant of cells in each group were extracted by differential hypervelocity centrifugation,and the expression of Syntaxin and exosome marker protein were detected by Western blot.Results:5-FU,oxaliplatin and starvation treated CT26.WT cells could induce autophagy.Under CLSM,some polyvesicles and autophagosomes coincided,and some polyvesicles coincided with lysosomes.After starvation culture,the expression of syntaxin in CT26.WT cells was lower than that in the normal control group.After treatment with 5-FU or oxaliplatin,the expression of Syntaxin protein had no significant change compared with that in the normal control group.The expression of EGFP-CD63 protein in exosomes of 5-FU and oxaliplatin treatment group was significantly lower than that of normal control group.Conclusion:5-FU and oxaliplatin do not affect the formation of multivesicles in CT26.WT cells,but can induce autophagy in CT26.WT cells and promote the degradation of multivesicles through autophagy lysosomal pathway,so as to down regulate the release of exosomes.

关 键 词：5-氟尿嘧啶 奥沙利铂 结肠腺癌细胞 自噬 外泌体 

分 类 号：R730.5[医药卫生—肿瘤]