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

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 type Ⅱ cells，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（DNase Ⅰ）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，iMEF）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）×107。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 type Ⅰ cells，AT Ⅰ）；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。

Abstract:

Objective：To identify the maintenance of an undifferentiated state of mouse alveolar epithelial typeⅡ（ATⅡ） cells by the co-culture of feeder cells（mouse embryonic fibroblast cells，MEFs）. Methods：The mouse lung single cells were prepared by the com－bination use of dispase，collagenase and DNase I. ATⅡ cells were purified by immune adherence. The culture medium was high glu－cose dulbecco modified eagle medium（HG/DMEM） addicted with 10% fetal bovine serum（FBS）and antibiotics. ATⅡ 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）×107 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 ATⅡ.