Purpose:The incidence of acute lung injury(ALI)in severe trauma patients is 48%and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%.Alveolar epithelial type 1 cells(AEC1...Purpose:The incidence of acute lung injury(ALI)in severe trauma patients is 48%and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%.Alveolar epithelial type 1 cells(AEC1 s)and type 2 cells(AEC2s)are the key cells in the repair of injured lungs as well as fetal lung development.Therefore,the purification and culture of AECls and AEC2s play an important role in the research of repair and regeneration of lung tissue.Methods:Sprague-Dawley rats(3-4 weeks,120-150 g)were purchased for experiment.Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells,and then magnetic bead cell sorting was performed to isolate Tla positive cells as AECls from the single-cell suspension by using polyclonal rabbit anti-Tla(a specific AECls membrane protein)antibodies combined with anti-rabbit IgG microbeads.Afterwards,alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining Tla-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads.Cell purity was identified by immunofluorescence staining and flow cytometry.Resii沾••The purity of AECls and AEC2s was 88.3%±3.8%and 92.6%±2.7%,respectively.The cell growth was observed as follows:AECls stretched within the 12-16 h,but the cells proliferated slowly;while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days.Conclusion:AECls and AEC2s sorted by this method have high purity and good viability.Therefore,our method provides a new approach for the isolation and culture of AECls and AEC2s as well as a new strategy for the research of lung repair and regeneration.展开更多
基金the National Natural Science Foundation of China(82020108021 and 81530063)the Projects of the State Key Laboratory of Trauma,Burns and Combined Injury(SKLYQ201901)+1 种基金the Chongqing Science and Technology Talents,Chongqing Special Project for Academicians(cstc2020yszx-jcyjX0004)the Training Plan for Innovation Ability on the Frontiers of Military Medical Research(2019CXJSB014).
文摘Purpose:The incidence of acute lung injury(ALI)in severe trauma patients is 48%and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%.Alveolar epithelial type 1 cells(AEC1 s)and type 2 cells(AEC2s)are the key cells in the repair of injured lungs as well as fetal lung development.Therefore,the purification and culture of AECls and AEC2s play an important role in the research of repair and regeneration of lung tissue.Methods:Sprague-Dawley rats(3-4 weeks,120-150 g)were purchased for experiment.Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells,and then magnetic bead cell sorting was performed to isolate Tla positive cells as AECls from the single-cell suspension by using polyclonal rabbit anti-Tla(a specific AECls membrane protein)antibodies combined with anti-rabbit IgG microbeads.Afterwards,alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining Tla-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads.Cell purity was identified by immunofluorescence staining and flow cytometry.Resii沾••The purity of AECls and AEC2s was 88.3%±3.8%and 92.6%±2.7%,respectively.The cell growth was observed as follows:AECls stretched within the 12-16 h,but the cells proliferated slowly;while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days.Conclusion:AECls and AEC2s sorted by this method have high purity and good viability.Therefore,our method provides a new approach for the isolation and culture of AECls and AEC2s as well as a new strategy for the research of lung repair and regeneration.
