Improvement Method Of Phenotype Maintenance For Alveolar Type ? Epithelial Cells In Vitro

Objective:Improved isolation and culture method of AEC-?cells from in rats,and To explore a method that can effectively maintain its phenotype in vitro.Methods:The mixture of 0.25%trypsin and 0.1%type I collagenase was infused in rats'lungs continuously via trachea to obtain AEC-?cells from rats'lung tissues..And then AEC-?cells were purified with a series of steps,such as Rats IgG immune adhesion method,differential adhesion.we used trypan blue staining and flow cytometry to detect AEC-?cells vitality.AEC-?cells were identified with bi-immunofluorescence of rats'SP-C and Cytokeratin-8,at the same time,we observed the ultrastructure with electron microscope.Then AEC-?cellsweredivided4groupsafterisolationand purification,group A(SAGM+1%FBS),group B(SAGM+10%FBS),group C(DMEM+1%FBS),group D(DMEM+10%FBS).Then AEC-?cells were collected in the 8th,10th and 12th days after culture,Real-time PCR monitrred the expression of SP-A,SP-B and SP-C to evaluate the phenotype maintenance of AEC-?cells.Results:we can obtain AEC-?cells output at 2×10~7-5×10~7 by infusing digestive enzyme continuously via trachea,the cells'vitality was assessed by trypan blue staining and flow cytometry at(91+1.7)%and(90+1.2)%,SP-C staining assessment of cell purity at(90+1.2)%.In order to study the phenotype maintenance of AEC-II cells,the relative expression levels of SP-A,SP-B and SP-C in four groups of cells were detected by Real-time PCR:(1)At the 8th day of culture,compared with group A cells,the amount of the three substances expressed in group B and group C were significantly reduced(p<0.05).Compared with group B and group C cells,the expression of three substances were significant decrease in group D(p<0.05).(2)Compared with group A cells,the amount of the three substances expressed in group B and group C were significantly reduced(p<0.05).Compared with group B and group C cells,the expression of three substances were significant decrease in group D(p<0.05).(3)At the 12th day of culture,compared with group A cells,the amount of the three substances expressed in group B and group C were significantly reduced(p<0.05).Compared with group B and group C cells,the expression of three substances were significant decrease in group D(p<0.05).Conclusions:1.It's can improve the efficiency of digestion that infuse digestive enzyme continuously to isolate AEC-?cells via trachea from in rats.2.The AEC-?cells are cultured by SAGM+1%FBS can be long time to maintain its phenotype.