The Effect Of Mechanical Stretched Mesenchymal Stem Cells On Pulmonary Microvascular Endothelium Barrier Of ARDS

Part One:Effect of mechanical stretch on bone marrow mesenchymal stem cellsObjective:The effect of mechanical stretch on bone marrow mesenchymal stem cells.Methods:A mechanical stretch unit was introduced in this study to apply the mechanical stretch on MSCs.MSCs were seeded onto a six-well plate with flexible Collagen Type I-coated silicone rubber membranes at the bottom of each well in an incubator of 5%CO₂atmosphere at 95%humidity of 37°C.The MSCs groups were divided as below:(1)Un-MS;(2)MS-10%-24h;(3)MS-10%-48h;(4)MS-20%-24h;(5)MS-20%-48h.After stimulation with mechanical stretch,cells and supernant from the groups were observed and detected as follows:(1)Morphological differences were observed between MSCs groups with Wright-Giemsa staining.(2)MSCs cell surface markers were identified by flow cytometry.(3)Inflammatory mediators in the supernants were detected with enzyme-linked immunosorbent assay(ELISA).(4)MSCs proliferation was tested by cell counting kit-8(CCK-8).Results:(1)MSCs in all groups firmly adhered to the seeding surface.Compared to Un-MS MSCs group,the mechanical stretch MSCs groups showed that cells were in atrophy states appeared thinner,flattened and shrank in a time and magnitude manner.(2)Cell proliferation was found significantly increased in MS-10%MSCs group(P<0.05),but decreased in MS-20%MSCs group(P<0.05).(3)Cell surface markers for MSCs identification demonstrated that CD90 and CD29 were positively expressed nearly 99%on cell surface,and CD45 were negatively expressed lower than 5%in all MSCs groups.(4)TNF-a and IL-6 showed significant differences(P<0.05),but IL-10 did not present significant differences(P>0.05).Conclusion:Mechanical stretch altered the morphological appearance,cell proliferation and inflammatory mediators of mesenchymal stem cells.Part Two: Effect of mechanical stretched MSCs on pulmonary microvascular endothelium barrier in ARDS Objective: To determine the effect of mechanical stretched MSCs on pulmonary microvascular endothelium barrier in ARDS.Methods: ECs were seeded on the upper chambers of a transwell system,and cultured for 2 to 3 days to produce a confluent monolayer in an incubator with an atmosphere of 5% CO2 air and 95% humidity at 37?,and MSCs were seeded in the lower chambers.Before the test of permeability of pulmonary microvascular endothelium barrier,ECs were treated with LPS for 6 hours.The groups were devided as below: ECs group;ECs+LPS group;ECs+LPS+MSCs group;ECs+LPS+MSCs-MS groups.After experiments,cells and supernants from every group were collected and the following parameters were detected:(1)Permeability of ECs barrier was examined by fluorescein isothiocyanate FITC-Dextran;(2)Apoptosis of ECs was detected by flow cytometry;(3)Qualitative analysis of ECs adhesion and gap junction proteins expression was evaluated by western blot;(4)Quantitative analysis of protein expression of VE-cadherin on ECs was examined by confocal laser scanning microscopy with immunofluorescence staining.Results:(1)LPS significantly increased the permeability of pulmonary microvascular endothelium barrier(P <0.05).(2)With the number of MSCs increasing,the MSCs protective effects on elevated ECs barrier permeability induced by LPS significantly enhanced(P <0.05).(3)Compared to the LPS group,MSCs-MS-10% for 24 h and 48 h groups could attenuate the permeability of ECs barrier induced by LPS significantly(P<0.05);and MSCs-MS-20% for 24 h group could also restore the permeability significantly(P<0.05),but for 48 h group the restoration did not show statistic difference(P>0.05).(4)Compared to the LPS group,MSCs-MS-10% groups for 24 h and 48 h decreased the apoptosis of ECs significantly(P<0.05);and MSCs-MS-20% group for 24 h decreased the apoptosis of ECs significantly(P<0.05).But MSCs-MS-20% group for 48 h did not show protective effects(P>0.05).(5)Compared to the LPS group,MSCs-MS-10% for 24 h and 48 h groups restored VE-cadherin and Connexin43 protein expression significantly(P<0.05);and MSCs-MS-20%-24 h group significantly restored the VE-cadherin and Connexin43 protein expression(P<0.05),but MSCs-MS-20%-48 h group did not show therapeutic effects(P>0.05).Conclusion: Mechanical stretched MSCs showed protective effects on the elevated permeability of pulmonary microvascular endothelium barrier,the increased apoptosis of ECs and the decreased expression of cell adherens junction protein induced by LPS,but in line with the time and magnitude.