内皮前体细胞与口腔鳞癌细胞自发性融合的实验研究

摘要

目的:探讨骨髓源间充质干细胞之外的其它骨髓来源的细胞是否能够与口腔鳞癌细胞发生自发性融合.方法:5ml外周血经密度梯度离心和EGM-2MV培养基进行诱导培养内皮前体细胞.PKH26标记的内皮前体细胞(EPCs)与CFSE标记的口腔鳞癌细胞系Ca1 27,SCC 4及SCC 9共培养,24h后双阳性且具有多个核的细胞为融合细胞.细胞流式分析检测内皮前体细胞与三种鳞癌细胞系的融合率.免疫荧光双染检测具有上皮标记物(Cytokeratin)及间质细胞标记物(Vimentin)的双阳性细胞.结果:单个核细胞诱导培养8～12天可见内皮样克隆形成；共培养24h能够检测到具有多个核PKH26/CFSE双阳性细胞；细胞流式分析显示内皮前体细胞与不同口腔鳞癌细胞的融合率分别为0.62±0.028,0.59±0.057,0.65±0.099.统计学分析表明融合率之间无显著统计学差异(P=0.706).荧光双染显示共培养体系中存在共表达Cytokeratin/Vimentin的双阳性细胞.结论:内皮前体细胞能够与口腔鳞癌细胞发生自发性融合且融合细胞共表达Cytokeratin/Vimentin,可为从细胞融合角度解释EMT转化及肿瘤侵袭提供基础研究依据.%Objective To investigate whether other bone marrow derived cells besides BMSCs can spontaneous fuse with oral squamous cancer cells.Methods Mononuclear cells (MNCs) were obtained from 5ml fresh human peripheral blood by density gradient fractionation,and resuspended in complete medium (EGM-2 MV) supplemented with 10％ FBS and seeded on 24-well culture plates.PKH26 labeled endothelial progenitor cells were cocultured with CFSE labeled oral squamous cancer cell lines Cal 27,SCC 4 and SCC 9.After 24 h post-coculture,bi-positive cells including two or more nucleus were identified as hybrid cells.Flow cytometry was used to analyze the fusion efficiency of three oral squamous cancer cell lines with EPCs.Immunofluorescence Staining was used to detect the epithelial molecule Cytokeratin and mesenchymal marker Vimentin.Results Single-cell clone appeared after 8～12 days were endothelial-like in appearance and formed a cobblestone-shaped cell monolayer.After 24 h post-coculture,PKH26/CFSE bi-fluorescent cells contained two or more nucleus can be observed in the coculture system.Flow cytometry analysis showed that the fusion efficiency of either oral squamous cancer line with EPCs was 0.62 ±0.028,0.59 ±0.057,0.65 ±0.099,respectively and no significant different was observed between the oral squamous cancer cell lines used (P=0.706).Immunofluorescence Staining revealed that Cytokeratin/Vimentin bi-positive cells contained two different nuclei were existed in the coculture system.Conclusion EPCs can spontaneously fuse with oral squamous cancer cells and the hybrid cells coexpressed the Cytokeratin and Vimentin.The present study provided a novel strategy to understand the EMT and tumor invasion.