【摘要】 目的:研究腺样囊性癌(adenoid cystic carcinoma,ACC)细胞SACC-83来源的外泌体(exosome,EXO)对人正常唾液腺间质成纤维细胞(human normal salivary gland stromal fibroblasts,hSGSFs)表达成纤维细胞活化蛋白(fibroblast activation protein,FAP)的影响。方法:采用超滤管浓缩与EXO提取试剂盒相结合的方法从SACC-83的培养上清中提取EXO,通过透射电镜及Western Blot对所提取的EXO进行鉴定;将SACC-83来源的EXO用荧光染料PKH67标记后与hSGSFs共培养48 h,采用激光扫描共聚焦显微镜(LSCM)观察hSGSFs对于SACC-83来源的EXO的摄取情况;利用qRT-PCR和Western Blot法检测在SACC-83来源的EXO作用下,hSGSFs中FAP的表达变化。结果:SACC-83培养上清中所提取到的微囊泡直径为30~100 nm,EXO的膜蛋白标记物CD63和TSG101的表达为阳性;携带PKH67荧光标记的EXO可被hSGSFs摄取,并可在mRNA和蛋白水平上调hSGSFs中FAP的表达。结论:SACC-83来源的EXO可被hSGSFs摄取,并可显著上调hSGSFs中FAP的表达。提示ACC可能通过EXO途径促进正常唾液腺间质成纤维细胞向癌相关成纤维细胞(cancer-associated fibroblasts,CAF)的转化。更多还原

【Abstract】 Objective:To study the effects of exosomes(EXO) released by adenoid cystic carcinoma SACC-83 cells on the expression of fibroblast activation protein(FAP) in normal human salivary gland stromal fibroblasts(hSGSFs).Methods:ACC exosomes were isolated from SACC-83 cell culture supernatant by using Total Exosome Isolation Reagent.The whole-mount EXO were characterized and assessed by transmission electron microscope and Western Blot.The exosomes were labeled with green fluorescent dye PKH67 and co-cultured with hSGSFs for 48 h,followed by staining with Alexa Fluor 594 Phalloidin and DAPI.Afterwards,exsosomes uptake was observed under a laser scanning confocal microscope.After a 48-hour co-culture of SACC-83 exosomes with hSGSFs,the expression of FAP in SACC-83-EXO-treated hSGSFs was investigated by qRT-PCR and Western Blot.Results:The vesicles isolated from SACC-83 cell culture supernatant had the reported size range of 30- 100 nm,expressed the exosomal marker CD63 and TSG101.After co- culture of hSGSFs with PKH67 labeled SACC-83 exosomes,exosomes were taken up by hSGSFs and FAP expression was elevated in hSGSFs.Conclusion;Exosomes derived from SACC-83 cells can be taken up by hSGSFs and can induce the expression of FAP in hSGSFs.These results suggest that exosomes derived from SACC-83 cells might induce the transformation of normal salivary gland stromal fibroblasts to cancer associated fibroblasts.更多还原