SACC-83来源的外泌体调节唾液腺间质成纤维细胞表达FAP的实验研究
|
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。
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.
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.
引文
[1]Johnstone RM,Adam M,Hammond JR,et al.Vesicle formation during reticulocyte maturation.Association of plasma membrane activities with released vesicles(exosomes)[J].J Biol Chem,1987,262(19):9412-9420.
[2]Salido-Guadarrama I,Romero-Cordoba S,Peralta-Zaragoza O,et al.MicroRNAs transported by exosomes in body fluids as mediators of intercellular communication in cancer[J].Onco Targets Ther,2014,7:1327-1338.
[3]Milane L,Singh A,Mattheolabakis G,et al.Exosome mediated communication within the tumor microenvironment[J].J Control Relaease,2015,219:278-294.
[4]Li M,Li M,Yi T,et al.Targeting of cancer-associated fibroblasts enhances the efficacy of cancer chemotherapy by regulating the tumor microenvironment[J].Mol Med Rep,2016,16(3):2476-2484.
[5]Colombo M,Raposo G,Thery C,et al.Biogenesis,secretion,and intercellular interactions of exosomes and other extracellular vesicles[J].Annu Rev Cell Dev Biol,2014,30:255-289.
[6]Kourembanas S.Exosomes:Vehicles of intercellular signaling,biomarkers,and vectors of cell therapy[J].Annu Rev Physiol,2014,77:13-27.
[7]Webber J,Steadman R,Mason MD,et al.Cancer exosomes trigger fibroblast to myofibroblast differentiation[J].Cancer Res,2010,70(23):9621-9630.
[8]Paggetti J,Haderk F,Seiffert M,et al.Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts[J].Blood,2015,126(9):1106-1117.
[9]Lee HO,Mullins SR,Franco-Barraza J,et al.FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells[J].BMC Cancer,2011,11:245.
[10]Goodman JD,Rozypal TL,Kelly T.Seprase,a membranebound protease,alleviates the serum growth requirement of human breast cancer cells[J].Clin Exp Metastasis,2003,20(5):459-470.
[11]Lai D,Ma L,Wang F.Fibroblast activation protein regulates tumor-associated fibroblasts and epithelial ovarian cancer cells[J].Int J Oncol,2012,41(2):541-550.
[2]Salido-Guadarrama I,Romero-Cordoba S,Peralta-Zaragoza O,et al.MicroRNAs transported by exosomes in body fluids as mediators of intercellular communication in cancer[J].Onco Targets Ther,2014,7:1327-1338.
[3]Milane L,Singh A,Mattheolabakis G,et al.Exosome mediated communication within the tumor microenvironment[J].J Control Relaease,2015,219:278-294.
[4]Li M,Li M,Yi T,et al.Targeting of cancer-associated fibroblasts enhances the efficacy of cancer chemotherapy by regulating the tumor microenvironment[J].Mol Med Rep,2016,16(3):2476-2484.
[5]Colombo M,Raposo G,Thery C,et al.Biogenesis,secretion,and intercellular interactions of exosomes and other extracellular vesicles[J].Annu Rev Cell Dev Biol,2014,30:255-289.
[6]Kourembanas S.Exosomes:Vehicles of intercellular signaling,biomarkers,and vectors of cell therapy[J].Annu Rev Physiol,2014,77:13-27.
[7]Webber J,Steadman R,Mason MD,et al.Cancer exosomes trigger fibroblast to myofibroblast differentiation[J].Cancer Res,2010,70(23):9621-9630.
[8]Paggetti J,Haderk F,Seiffert M,et al.Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts[J].Blood,2015,126(9):1106-1117.
[9]Lee HO,Mullins SR,Franco-Barraza J,et al.FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells[J].BMC Cancer,2011,11:245.
[10]Goodman JD,Rozypal TL,Kelly T.Seprase,a membranebound protease,alleviates the serum growth requirement of human breast cancer cells[J].Clin Exp Metastasis,2003,20(5):459-470.
[11]Lai D,Ma L,Wang F.Fibroblast activation protein regulates tumor-associated fibroblasts and epithelial ovarian cancer cells[J].Int J Oncol,2012,41(2):541-550.