【摘要】 目的初步探讨人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,hbMSCs)条件培养基(conditioned medium,CM)在人鼻咽癌CNE1及CNE2细胞上皮间质转化(epithelial mesenchymal transition,EMT)中的影响。方法取第3代hbMSCs融合达70%~80%时,换液培养24h后取其上清液(hbMSCs-CM);取对数生长期5×103CNE1及CNE2细胞,分别在普通培养液(对照组)和hbMSCs-CM的培养液(实验组)中培养。使用倒置相差显微镜下观察细胞形态学改变,蛋白质印迹法及q-PCR检测EMT相关蛋白及RNA变化。结果 hbMSCs-CM培养鼻咽癌细胞5d,CNE1和CNE2均观察到细胞间距离增宽,并且折光性强、多量单极或双极梭形状成纤维细胞样细胞,以CNE2细胞最为明显。q-PCR检测结果显示,与对照组相比,hbMSCs-CM培养组细胞上皮细胞标志基因mRNA的相对表达水平下调,差异有统计学意义,P值均<0.001;间质细胞标志基因mRNA相对表达水平上调,差异有统计学意义,P值均<0.001;EMT相关转录因子基因mRNA相对表达量上调,差异有统计学意义,P值均<0.001。蛋白质印迹法检测结果显示,hbMSCs-CM诱导CNE1及CNE2细胞发生EMT并呈一定浓度依赖性。当hbMSCs-CM浓度≥70%,上皮细胞标志蛋白相对表达水平显著下降,差异有统计学意义,P<0.05。当hbMSCs-CM浓度>50%时,CNE1和CNE2细胞间质细胞标志及转录因子蛋白相对表达水平上调,差异有统计学意义,P<0.05。结论 hbMSCs可改变鼻咽癌细胞形态,并下调细胞黏附蛋白及增加细胞间质蛋白水平,可能是参与鼻咽癌上皮间质转化过程的因素之一。更多还原

【Abstract】 OBJECTIVE To investigate the effects of conditioned medium(CM)derived from human bone mesenchymal stem cells(hbMSCs)on the epithelial-to-mesenchymal transition in nasopharyngeal carcinoma cell lines:CNE1and CNE2 cell lines.METHODS hbMSCs were grown in L-DMEM + 10% FBS culture medium,when cells from the third generation of hbMSCs were cultured to confluence of 70%-80%,the supernate was decanted instead of L-DMEM containing 2% FBS culture,incubation for other 24 hand the supernate as conditioned medium(CM)from these cells were harvested and centrifuged and passed through Millipore sterile 20 mL filtration system with 0.22μmol/L polyvinylidene difluoride membrane.CNE1 and CNE2were incubated in fresh medium alone(as the control group)or hbMSCs-CM(as the CM group),the supernate changed every other day.The morphology of CNE1 and CNE2were observed under high magnification microscope,Western blot and q-PCR were used to detect the EMT related protein and RNA levels.RESULTS When nasopharyngeal carcinoma cells exposed in hbMSCs-CM for 5days,it acquired the EMT morphology.Space between cells was widened in both CNE1 and CNE2,and fibroblast-like cell traits,good light refraction,more monopolar and occasional bipolar spindle-shaped cells were observed more easily in CNE2.q-PCR:Compared with the control group,a low level mRNA of epithelial cell markers related genes was detected in the hbMSCs-CM group,P<0.001,and high levels mRNA of mesenchymal cell markers related genes were disposed in the hbMSCs-CM group,P<0.001.The mRNA of EMT related transcription factor genes were increased in the hbMSCs-CM group,P<0.001.Western blot:hbMSCs-CM could triggere CNE1 and CNE2EMT in a certain concentration dependent pattern.When the concentration of hbMSCs-CM reached to 70%,the proteins of epithelial cell markers were decreased obviously(P<0.05),and the concentration of hbMSCs-CM reached to 50%,the proteins of mesenchymal cell markers and EMT related transcription factor were increased respectively,P<0.05.CONCLUSION The products of hbMSCs can trigger epithelial-to-mesenchymal transition in nasopharyngeal carcinoma cell lines:CNE1and CNE2.更多还原