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Objective Comparison of induction time on the proliferation of induced adipose-derived stem cells (ADSC) to differentiate into Schwann-like cells (iSC).Methods From March,2017 to October,2018,ADSCs were isolated from inguinal adipose tissue of healthy adult female SD rats.Flow cytometry was performed to detect ADSC positive markers CD29,CD90 and negative marker CD45.iSC induction medium was used to culture ADSC.S-100 and GFAP were detected by immunofluorescence staining to confirm that ADSC had differentiated into iSC.Morphological changes of cells were observed by inverted microscope on day 1st,4th,7th,10th,13rd,16th and 19th after induction.MTS assay was used to evaluate cell proliferation ability.Tunel staining was applied to assess cell apoptosis.Results Both S100 and GFAP were expressed in iSC.On day 7th,the cell proliferation rate was significantly slower than that before induction (A value was 0.330±0.020 vs.0.400±0.004,P＜0.05).It was negatively correlated with induction time.On day 19th,the proliferation rate of iSC was lower than 50％ of the proliferation rate before induction (A value was 0.016±0.003 vs.0.400±0.004,P＜0.05).Apoptosis of iSC was more obvious than ADSC at the same time point.Conclusion The proliferation ability of ADSC-induced iSC is optimal within 7 days after induction.
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Objective To explore the isolation,amplification methods and adipogenic differentiation under specific culture medium of human keloid-derived precursor cell (KPC) in vitro,in order to study their possibility of being new seed cells of tissue engineering fat.Methods KPCs were isolated from human keloid tissue of 4 different patients in our hospital and were cultured in the modified L-DMEM culture medium.Their cloning efficiency and growth curve were tested.The subcultured cells were tested of the mesenchymal stem cell (MSC)-related gene expression by flow cytometry.In addition,they were cultured in H-DMEM medium (containing 1 μmol/L dexamethasone,0.5 mmol/L 3-isobutyl-1-methyl-xanthine 10 mg/L of bovine insulin,100 mmol/L indomethacin,and 10 ％ FBS)and were later observed in oil red O staining under phase contrast microscope to determine whether lipid droplets generation was formed,using skin-derived precursors (SKP) as control.Results More than 95 ％ KPC expressed many antigens of MSC,such as CD29,CD44,CD90 and CD105 while few of them expressed CD34,CD45(1.0 ％-2.5 ％).And the cells increased in size gradually after inducted the same time,changing from spindle into round or polygonal in shape.The lipid droplets were seen in 72 hours and expressed a positive rate of 78.6 ％ in Day 19 in oil red O staining while the same rate was 54.6 ％ in SKP.Conclusions Human keloid-derived precursor cells can express a variety of MSC-related surface markers without expressing hematopoietic stem cell (HSC) related markers.Furthermore,they can be differentiated into fat cells under certain conditions,which may make them as a new source of seed cells for tissue engineering fat.
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Objective To discuss the possibility of constructing a new kind of active skin substi tutes.Methods The culture of dermal fibroblasts were isolated from foreskin of an infant; preparation of human acellular amniotic extracellular matrix was conducted through the disposition of detergent and enzyme.Surface structure porosity and pore size were detected through scanning electron microscopy.bFGF-chitosan gelatin microspheres were prepared.The features such as the size distribution,drug content,drug encapsulating and in vitro release were studied with scanning electron microscopy,laser grainsize analyzer and ELISA method.MTT test was performed to observe cell proliferation and evaluate the biocompatibility in vitro.Results The cellular layer of amniotic membrane was completely removed but did not damage the collogen scaffolds structure by the disposal of enzyme and detergent.Scanning electron microscopy showed that the scaffolds had criss-cross structure and high porosity,the pore size was irregular and varied from 10 nm to 100 nm.Scanning electron microscopy of bFGF- gelatin-chitosan microspheres surface structure showed that the microspheres had spherical structure,uniform size and smooth surface quality.Controlled release curve showed that the sudden release of bFGF was obvious.Conclusions A new type of active skin substitutes is prepared through culturing fibroblasts on HAAM loaded controlled-released basic fibroblast growth factor from chi tosan-gelatin microspheres.