小鼠胚胎成纤维细胞对体外培养肺泡上皮Ⅱ型细胞干性的维持作用  

Role of mouse embryonic fibroblast cells in maintaining undifferentiated state of alveolar epithelial type Ⅱ cells in vitro

作　　者：余静[1] 曾灵[2] 李勇[2] 邓婷婷[1] 蒋建新[2] 王继红[1] 

机构地区：[1]重庆医科大学实验教学中心生物化学与分子生物学实验室、分子医学与肿瘤研究中心,重庆400016 [2]第三军医大学大坪医院野战外科研究所,创伤、烧伤、复合伤国家重点实验室第四研究室,重庆400042

出　　处：《重庆医科大学学报》2014年第11期1613-1616,共4页Journal of Chongqing Medical University

基　　金：国家重点基础研究发展计划(973计划)资助项目(编号:2012CB518104)

摘　　要：目的：探讨小鼠胚胎成纤维细胞（mouse embryonic fibroblast cell,MEF）作为滋养细胞对体外培养肺泡上皮Ⅱ型细胞（alveolar epithelial typeⅡcells,ATⅡ）干性的维持作用。方法：通过分散酶（Dispase）、分散酶/胶原酶（colleganse/disapase）和脱氧核糖核酸酶（DNaseⅠ）联合消化小鼠肺组织,制备肺单细胞悬液,通过免疫黏附法纯化ATⅡ。高糖DMEM添加10%FBS、双抗（青霉素20 U/ml,链霉素20 U/ml）;将传代后的ATⅡ接种至丝裂霉素处理的MEF（inactivated MEF,i MEF）培养,并以肺泡表面活性蛋白C（surfactant protein C,SPC）和水通道蛋白5（aquaporins 5,AQP5）为标记蛋白进行鉴定。结果：平均每只成年小鼠可得到总有核细胞数为（1.7-2.0）×10^7。ATⅡ细胞传代接种于滋养细胞前3代都能维持在未分化状态,第4代分化成为肺泡上皮Ⅰ型细胞（alveolar epithelial typeⅠcells,ATⅠ）;而传代后没有接种在滋养细胞上的ATⅡ细胞在第1代就分化成ATⅠ细胞。结论：小鼠胚胎成纤维细胞在一定时段能够维持ATⅡ的干性。Objective：To identify the maintenance of an undifferentiated state of mouse alveolar epithelial type Ⅱ （AT Ⅱ） cells by the co-culture of feeder cells（mouse embryonic fibroblast cells, MEFs）. Methods：The mouse lung single cells were prepared by the combination use of dispase, collagenase and DNase Ⅰ. AT Ⅱ cells were purified by immune adherence. The culture medium was high glucose dulbecco modified eagle medium（HG/DMEM） addicted with 10% fetal bovine serum（FBS）and antibiotics. AT Ⅱ cells were co- cultured with MEFs and were identified by immunofluorescence staining of pulmonary surfactant protein C （SPC） and Aquaporins 5 （AQP5）. Results： （1.7-2.0）×10^7 cells per mouse lung was obtained. ATⅡ cells can maintain their undifferentiated state until the fourth generation when being cultured on feeder cells,but will differentiate into alveolar AT Ⅰ cells when being cultured alone. Conclusion： MEF cells can maintain the undifferentiated state of AT Ⅱ.

关 键 词：滋养细胞 肺泡上皮Ⅱ型细胞 细胞培养 

分 类 号：R781.5[医药卫生—口腔医学]