【摘要】：目的:研究腺樣囊性癌(adenoid cystic carcinoma,ACC)細胞SACC-83來源的外泌體(exosome,EXO)對人正常唾液腺間質成纖維細胞(human normal salivary gland stromal fibroblasts,hSGSFs)表達成纖維細胞活化蛋白(fibroblast activation protein,FAP)的影響。方法:采用超濾管濃縮與EXO提取試劑盒相結合的方法從SACC-83的培養(yǎng)上清中提取EXO,通過透射電鏡及Western Blot對所提取的EXO進行鑒定;將SACC-83來源的EXO用熒光染料PKH67標記后與hSGSFs共培養(yǎng)48 h,采用激光掃描共聚焦顯微鏡(LSCM)觀察hSGSFs對于SACC-83來源的EXO的攝取情況;利用qRT-PCR和Western Blot法檢測在SACC-83來源的EXO作用下,hSGSFs中FAP的表達變化。結果:SACC-83培養(yǎng)上清中所提取到的微囊泡直徑為30~100 nm,EXO的膜蛋白標記物CD63和TSG101的表達為陽性;攜帶PKH67熒光標記的EXO可被hSGSFs攝取,并可在mRNA和蛋白水平上調hSGSFs中FAP的表達。結論:SACC-83來源的EXO可被hSGSFs攝取,并可顯著上調hSGSFs中FAP的表達。提示ACC可能通過EXO途徑促進正常唾液腺間質成纖維細胞向癌相關成纖維細胞(cancer-associated fibroblasts,CAF)的轉化。
[Abstract]:Objective: to study the expression of fibroblast activating protein (fibroblast activation protein, (fibroblast activation protein,) in human salivary gland interstitial fibroblasts (human normal salivary gland stromal fibroblasts,hSGSFs) by SACC-83 derived exocrine (exosome,EXO) from adenoid cystic carcinoma (adenoid cystic carcinoma,ACC) cells. FAP). Methods: ultrafiltration tube concentration and EXO extraction kit were used to extract EXO, from the culture supernatant of SACC-83. The extracted EXO was identified by TEM and Western Blot. The EXO derived from SACC-83 was labeled with fluorescent dye PKH67 and co-cultured with hSGSFs for 48 h. The uptake of SACC-83 EXO by hSGSFs was observed by (LSCM) with laser scanning confocal microscope. The expression of FAP in hSGSFs was detected by qRT-PCR and Western Blot under the action of EXO from SACC-83. Results: the expression of CD63 and TSG101, the membrane protein markers with a diameter of 30 ~ 100 nm,EXO extracted from the supernatant of SACC-83 culture, was positive, and the EXO with PKH67 fluorescent labeling could be taken up by hSGSFs, and the FAP expression in hSGSFs could be upregulated at the mRNA and protein levels. Conclusion: EXO derived from SACC-83 can be ingested by hSGSFs and can significantly up-regulate the expression of FAP in hSGSFs. The results suggest that ACC may promote the transformation of normal salivary gland interstitial fibroblasts into cancer-related fibroblasts (cancer-associated fibroblasts,CAF) through EXO pathway.
【作者單位】： 南方醫(yī)科大學南方醫(yī)院口腔頜面外科 南方醫(yī)科大學口腔醫(yī)學院;
【基金】：廣東省科技計劃項目(編號:2014A020212397)
【分類號】：R739.8

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