Effect And Molecular Mechanism Of Bone Marrow Mesenchymal Stem Cells Conditioned Medium On Epithelial–mesenchymal Transition Induced By TGF-beta1

Objectives Using cell experiment in vitro,we established the EMTmodel by TGF-?1-induced alveolar type II epithelial cell damage model.The purpoce of present study is to investigate the effect of BMSCs on EMT,and further explore whether BMSCs-CM inhibit TGF-?1-induced EMT by regulating the activation of Smad3 pathway,finally suppressing fibrosis process.Methods 1 Experimental animal and group: BMSCs were harvested,cultured and passaged from 3-5week SPF male SD rats(100-120 g weight),we collect the conditioned medium of the 3-5 generations,enrichment and cryopreserved.Cultivate type II alveolar epithelial cell line A549 cells,induced by 5 ?g/L TGF-b1 in vitro,randomly divided into four groups: control group,TGF-?1 stimulation group(TGF-?1 group),SIS3 inhibitor group(SIS3 group)and BMSCs-CM intervention group(BMSCs-CM group).2 Detection of index and method:(1)By inverted phase contrast microscope observation the morphological changes of A549 cells and BMSCs;(2)The expression of BMSCs surface markers CD29,CD45,CD54 and CD106 were detected by flow cytometry;(3)The lamellar body of cell were observed by TEM.(4)Immunofluorescence methods were used to determined the location of E-calcium mucins(E-cad),?-smooth muscle actin(?-SMA)and p-Smad3;(5)The expression of E-cad,?-SMA,p-Smad3,Snail1 and type I collagen,type III collagen were detected by Western blot.3 Quantitative assay and Statistical method: PVDF membrane will be for semi-quantitative analysis by Image J medical image analysis system,used by Integral Optical Density values.The average optical density was determined by the medical CMIAS true color image analysis system at the same conditions.For all statistical calculations,we used SPSS17.0 one-way ANOVA to define which group contributed to these differences.There was statistically significant if lateral p<0.05.All data were presented as mean±SD.Results 1 Morphological observation of BMSCs: The morphological characteristics of Primary cell were circles and polygons,nuclear centered and occasional polygonal cells.Cell proliferated mainly in clone mode.BMSCs were purified by changing the medium several times.Following passaging,cell morphology was more uniform and consistent,forming mainly fibroblast like flattened cells.2 The identification results of BMSCs surface marker: The results showed the positive expression rate of CD29 and CD106 were 76.11% and 74.56% respectively,were positive;the positive expression rate of CD45 and CD54 were 3.88% and 5.05% respectively,were negative.3 The morphological observation of A549: A549 cells has an epithelial cobblestone-like morphology,a round or polygonal shape and exhibited very close cell–cell proximity reminiscent of cellular tight-junctions in the control group,while after exposure to TGF-?1,the cells displayed spindle-shaped elongated fibroblast-like morphology.These changes became clearer with increased time.Similar morphological changes were noticeably suppressed in BMSCs-CM group and SIS3 group.4 TEM Results: TEM assay indicated that the A549 cells characteristic of osmiophilic multilamellar body have changed,including degeneration,swelling and vacuoles following TGF-?1 treatment.However,the phenomenon in the BMSCs-CM group showed a reduction.5 The results of immunofluorescence: E-cad is shown as green,a-SMA and p-Smad3 are shown as red.In the control group,the fluorescence expression of E-cad is strong,a-SMA only has a small amount of expression,almost no visible fluorescence.Compared with control group,E-cad fluorescence expression was reduced in the TGF-?1-stimulated cells.At the same time,?-SMA fluorescence expression was increased and uniformly distributed in cytoplasm.The A549 cells also have an obvious nuclear expression of p-Smad3 accompany with spindleshaped morphology changes.Compared with TGF-?1 group,the expression of E-cad in the BMSCs-CM group had less inhibition.In contrast,the BMSCs-CM reduced the intensity of ?-SMA and p-Smad3 fluorescence expression,repaired the spindle-like morphology changes.Cells in the SIS3 group have a similar expression with the BMSCsCM group.6 The results of western blot: In control group,a-SMA,p-Smad3 and Snail1 have a basic amount of expression,and the amount of expression no change at all time points.Compared with control group,the expression of E-cad was significantly decreased(p<0.05),and the expression of ?-SMA,p-Smad3,Snail1 and type I collagen,type III collagen were increased following exposure to 5ng/ml TGF-?1(p<0.05).The SIS3 group and the BMSCs-CM group showed a reduction in TGF-?1-induced up-regulation of ?-SMA,p-Smad3,Snail1 and type I collagen,type III collagen,while a down-regulation of E-cad(p<0.05).Conclusions 1 EMT was activated in alveolar type II epithelial cells suffered from TGF-?1 stimulation,at the same time,it promoted the expression of collagen I,III;2 The Smad3 signaling pathway involved in regulation the process of TGF-?1-induced EMT;3Treatment with BMSCs-CM suppressed the process of EMT in A549,which might be associated with the regulation of TGF-?1/Smad3 pathway.