【作者】 宋梦阳； 王方圆； 侯晋； 殷学民；

【Author】 SONG Meng-yang;WANG Fang-yuan;HOU Jin;YIN Xue-min;Department of Stomatology,Nanfang Hospital,Southern Medical University;College of Stomatology,Southern Medical University;

【机构】 南方医科大学南方医院·南方医科大学口腔医学院；

【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,通过透射电镜及蛋白免疫印迹法(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,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q RT-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:透射电镜及Western Blot的检测结果显示,SACC-83培养上清中所提取到的微囊泡直径为30-100nm,且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。该结果提示ACC可能通过EXO途径促进正常唾液腺间质成纤维细胞向癌症相关成纤维细胞(cancer-associated fibroblasts,CAF)的转化。更多还原

【Abstract】 Objective:To determine the effect ofexosomes(EXO) released by adenoid cystic carcinoma(ACC) cells on the expression of fibroblast activation protein(FAP) in normal human salivary gland stromal fibroblasts(h SGSFs). Methods: ACC exosomes were isolated from SACC-83 cell culture supernatants by using Total Exosome Isolation Reagent. The whole-mount EXO were characterized and assessed by Transmission Electron MicroscopeandWestern Blot. The exosomes derived from SACC-83 were labeled with green fluorescent dye PKH67 and co-cultured with h SGSFs for 48 h, followed by staining with Alexa Fluor 594 Phalloidin and DAPI. Afterwards, exsosomes absorption was observed under a laser scanning confocal microscope(LSCM). After a 72-hourco-culture of SACC-83 exosomeswith h SGSFs, the expression of FAP in SACC-83-EXO-treated h SGSFs was investigated byq RT-PCR and Western Blot.Results: TEM and Western Blot results showed that exosomes isolated from SACC-83 cell culture supernatantshad the reported size range of 30–100 nm and expressed the exosomal marker, CD63 and TSG101. Afterco-culture of PKH67 green fluorescently labeled SACC-83 exosomes with h SGSFs, LSCM showed that exosomes were taken up by h SGSFs and FAP expression was elevated in h SGSFs after SACC-83 EXO treatment. Conclusion :Exosomes derived from SACC-83 were taken upby the recipientsh SGSFsand induced the expression of FAP in hS GSFs. These results suggested that exosomes derived from SACC-83 might induce the transformation of normal salivary gland stromal fibroblasts to cancer associated fibroblasts.更多还原