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Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting

Chinese Journal of Traumatology 2022年第3期25卷 138-144页

作者： Di Liu Jian-Hui Sun Hua-Cai Zhang Jian-Xin Jiang Ling Zeng State Key Laboratory of Trauma Bums and Combined InjuriesDoping HospitalArmy Medical UniversityChongqing400042China Medical Center of Trauma and War Injury Daping HospitalArmy Medical UniversityChongqing400042China

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.

关键词： Alveolar epithelial cells type 1 Alveolar epithelial cells type 2 Three-dimensional culture magnetic activated cell sorting

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The mechanism of selective injury of dopaminergic neurons induced by microglial MCP-1 in vitro

The mechanism of selective injury of dopaminergic neurons in...

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中国神经科学学会第十届全国学术会议

作者： Yanhong Wu Ning Yao Lili Ju Yan Zhou Deyi Duan Qunyuan Xu Department of Neuroscience Capital Medical University

objective To investigate the mechanism of selective damage of dopaminergic neurons which is induced by microglia.Methods Primary microglia were sorted by MACS.The cell inhibition rate of MN9D which was co-cultured with microglia in different time points and apoptosis in different cell lines were detected,at the same time,MN9D morphological changes were analyzed.Then the release of inflammatory factors in microglial co-culture with different types of cell lines was investigated.The specific factor which was only high in MN9D and microglia co-cultured system was been found out and added recombinant proteins and neutralization reagent for detection of apoptosis in different cell lines.Knockout animals to validate the above experiments.Results LPS can activate microglia and inhibit the MN9D cell line proliferation.Compared with the NG108-15 and the RMNE6 cell lines,MN9D cells co-cultured with microglia were found to increase MCP-1 expression significantly in protein levels and the apoptosis of MN9D cell line also increased Than the other two groups.The apoptosis bocome higher after joining MCP-1 recombinant proteins,and lower after joining the neutralization reagent.It is interesting that the apoptosis of MN9D cocultured with Microglia inseparated from CCR2 knockout mice bocome lower than any groups.Conclusion activated microglia can obviously damage the MN9D and the release of inflammatory cytokines induced by microglia activation increases significantly.The chemokine MCP-1 may be played an important role in the selective damage to dopaminergic neurons by Increasing apoptosis.

关键词： microglia magnetic activated cell sorting dopaminergic neurons coculture inflammatory factor selective damage apoptosis

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