| ObjectiveBone marrow mesenchymal stem cells(BMSCs)source wide,easy to train and can induce the differentiation of cell potential,many signaling pathways involved and play an important role in the process of differentiation of BMSCs,and Notch signaling pathway is one of the.The experiments using bone marrow mesenchymal stem cells(BMSCs)and alveolar epithelial cells(II)is a non-contact co culture and in co culture with gamma secretase enzyme inhibitor(dapt)to observe the effect of the production of the BMSCs differentiation,clear gamma-secretase block the enzyme inhibitor(DAPT)Notch signal pathway of BMSCs induced effects in the process of differentiation of alveolar epithelial cells.Method:Bone marrow mesenchymal stem cells were isolated and cultured by the whole bone marrow adherent cell method for 4 weeks,and third generations of bone marrow mesenchymal stem cells were identified by flow cytometry.After the purification of the fourth generation of bone marrow mesenchymal stem cells and alveolar epithelial cells in the Transwell system under non contact co culture,and in the co culture based on the addition of gamma-secreted enzyme inhibitor DAPT.The experiment was divided into three groups: blank control group is low glucose medium cultured alone between bone marrow mesenchymal stem cells,bone marrow mesenchymal stem cells and lung epithelial cells induced by co culture differentiation group,add DAPT20 ng / ml intervention of bone marrow mesenchymal stem cells and lung epithelial cells induced by co culture group.Water channel protein(aquaporin 5 after treatment was observed by light microscope after co culture BMCSs morphology changes,and respectively in co culture of the 10 day extraction of protein and RNA,with Western and RT-q PCR to detect the Notch signaling pathway related genes of Notch1,Hes1 and type II alveolar epithelial cell specific surface active protein(Surfactant protein C,SPC),type I alveolar epithelial cell markers,AQP5)expression levels in order to assess gamma secretase inhibitor dapt inhibited Notch signalingpathway of BMSCs to the role of alveolar epithelial cells.Result:1 after the separation of BMSCs under light microscope can be observed in its morphology is short spindle shape,the flow cytometry identified CD29,CD44 expression positive,CD34,CD45 expression was negative,indicating that the BMSCs.has been successfully extracted2 under the light microscope,the BMSCs of the induced differentiation of co culture was observed to be gradually changed from spindle shape to the shape of the epithelial cells.3 Notch signaling pathway in bone marrow mesenchymal stem cells to alveolar epithelial cell differentiation was activated,and compared to the control group,Notch1 protein expression and HES1 m RNA expression increased(p<0.05),the difference was statistically significant.Begin co culture based on join inhibitor dapt concentration compared to 20 ng / ml,and the co culture group in the BMSCs and mle-12.(p< 0.05)decreased expression of Notch1 protein and HES1 m RNA,the difference has statistical significance,suggesting that the co culture differentiation induced gamma secretase inhibitor dapt to blocking Notch signaling pathway play effect in.4.In the co culture group and DAPT co culture group,the cells were cultured for10 days,SPC,AQP5 protein and m RNA were detected by Western and RT-q PCR.Compared with the blank control group,the expression of SPC,AQP5 protein and m RNA in the co culture group and DAPT co culture group()were significantly higher than that in the control group(p<0.05 P).The expression of SPC,AQP5 protein and m RNA were reduced(p<0.05)in DAPT co culture group and co culture group,the difference was statistically significant.Conclusion:1.Bone marrow mesenchymal stem cells can be induced to differentiate into alveolar epithelial cells by MLE-12.2.Notch signaling pathway is activated in the process of BMSCs induced directional differentiation of alveolar epithelial cells in MLE-123.Inhibition of DAPT signaling pathway in BMSCs induced differentiation ofalveolar epithelial cells may inhibit the secretion of Notch signaling pathway in the process of... |
