Effect Of Human Adipose-derived Stem Cells On Human Fibroblasts Function From Different Age In Transwell System

ObjectiveTo investigate the effect of adipose-derived stem cells(ADSCs) onyoung and elderly derived fibroblasts in Transwell system, then makingADSCs used in anti-aging for experimental basis.Methods1.ADSCs were isolated and cultured from human subcutaneous adiposetissue which got from elective operation, then ADSCs were observed withMicroscope.2.Good growth state of the third generation of ADSCs were identifiedcell surface antigen with Flow Cytometer(FCM).3. Fibroblasts were isolated and cultured from young（＜25years old）and elderly(＞60years old) skin tissue which got from elective operation,then fibroblasts were observed with Microscope.4.ADSCs were co-cultured with young and elderly fibroblasts inTranswell chamber, At the same time, non co-culture of young and elderlyfibroblasts were used to as blank control, then non co-culture of young andelderly fibroblasts were observed with Microscope. 5.Fibroblasts proliferation was evaluated by Cell Proliferation andCytotoxicity Assay Kit (MTT).6.Expression of fibroblasts Collagen typeⅠand Matrixmetalloproteinase-1(MMP-1) and β-galactosidase mRNA were evaluatedby RT-PCR.Results1. FCM detection of ADSCs cell surface antigen results: CD44, CD90positive expression, CD34negative expression.2. ADSCs features: the ADSCs cells were long fusiform, primary cellsgrew lower than passage cells.The third passage ADSCs showed a"spiral" distribution under the microscope.3.The features of young and elderly fibroblasts co-cultured withADSCs: the elderly fibroblasts was larger, irregular shape and flat starlike;after co-cultured, the elderly fibroblasts bacame into long shuttle type. Theyoung fibroblasts have long shuttle type, after co-cultured, the features ofyoung fibroblasts did not change.3. The different group of fibroblasts proliferation was evaluated byCell Proliferation and Cytotoxicity Assay Kit (MTT): The proliferation ofyouth experimental group and elderly experimental group were obviouslyhigher than that of young people control group and elderly controlgroup(P=0.000),and the proliferation of youth experimental group is higherthan that of elderly experimental group(P=0.000). 4. The expression of collagen typeⅠand MMP-1andβ-galactosidasemRNA was evaluated by RT-PCR： The expression of CollagentypeⅠandβ-galactosidase mRNA of youth experimental group and elderlyexperimental group were obviously higher than that of youth control groupand elderly control group(P=0.000), and the expression of CollagentypeⅠandβ-galactosidase mRNA of youth experimental group were higherthan elderly experimental group(P=0.002, P=0.000).(3) The expression ofMMP-1mRNA of youth experimental group and elderly experimentalgroup were obviously lower than that of youth control group and elderlycontrol group(P=0.000),but the expression of MMP-1mRNA of youthexperimental group was lower than that of elderly experimentalgroup(P=0.055),the results were no significant, it may be related to theelderly and youth co-culture of fibroblasts proliferate activity and producea few of MMP-1. Conclusion: Cultured with ADSCs can promotefibroblasts proliferation and Collagen secretion.Conclusion1. Primary ADSCs grow lower than passage ADSCs, after passage,ADSCs surface antigen are detected in high purity.2. The young fibroblasts show long shuttle type and grow quickly. Theelderly fibroblast are irregular, grow slower and show spindle or thick cableshaped, there are dark granular material deposited within the cytoplasm.3. Cultured with ADSCs which can promote fibroblasts proliferation and Collagen andβ-galactosidase secretion, reducing the secretion ofMMP-1.