人骨髓间充质干细胞诱导鼻咽癌上皮间质化的初步探讨  

Human bone marrow mesenchymal stem cells triggered epithelial-to-mesenchymal of nasopharyngeal carcinoma cell lines:CNE1 and CNE2

作　　者：蒋东辉[1] 黄博[1] 张月飞[1] 李祥勇[2] 周克元[2] 罗国庆[1] 

机构地区：[1]广东医学院附属医院耳鼻咽喉-头颈外科,广东湛江524000 [2]广东省教育厅医学生物活性分子研究重点实验室,广东湛江524000

出　　处：《中华肿瘤防治杂志》2014年第23期1851-1855,1864,共6页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81272434);广东省科技计划(93036)

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。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：CNE1 and 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 h and the supernate as conditioned medium（CM） from these cells were harvested and centrifuged and passed through Millipore sterile 20 mL filtration system with 0.22 μmol/L polyvinylidene difluoride membrane. CNE1 and CNE2 were 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 CNE2 were 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 5 days, 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 ceils 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 CNE2 EMT 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%0, the proteins of mesenchymal cell markers and EMT related transcript

关 键 词：鼻咽癌 骨髓间充质干细胞 上皮间质转化 条件培养基 

分 类 号：R739.63[医药卫生—肿瘤]