Objective To investigate the possibility of culturing human oral keratinocyte using autologous serum in order to provide theoretical and technical foundation for clinical application of tissue engineering oral mucosa epithelium.Methods The human oral keratinocytes were cultured by the medium containing different concentrations of autologous serum(10%,20%,30%)and fetalbovine serum (10%), respectively. The growth conditions for the cell and the mucosa epithelium in the groups were observed, the cell growth curves were drawn, and the population doubling time (PDT) was counted. Results The results showed that the human oral keratinocyte could proliferate well in the medium containing autologous serum or fetal bovine serum. The differences in the 24hour clone rate and PDT were not significant. Both the area and the thickness of the cultured oral epithelium increased with the increase of the autologous serum concentration, and the difference between autologous serum and fetal bovine serum was significant, especially with the medium containing 20% autologous serum( P＜0.05) . The human nature of the cultured epithelium was demonstrated by the immunofluorescent mouse anti-HLA antigen. Conclusion The autologous serum can replace the fetal bovine serum to culture the oral keratinocyte well, and the cultured oral mucosa epithelium can be better differentiated in the autologous serum than in the fetal bovine serum.
Objective To explore the effect of basic fibroblast growth factor（bFGF）and epidermal growth factor（EGF）on the growth of muscle derived stem cells(MDSCs). Methods MDSCs were isolated from hindlimb muscle of 15 new born Kunming mice through serial preplates. 2% fetal bovine serum-containing DMEM was used to induce MDSCs to differentiate into skeletal muscle lineage. The expressions of stem cell marker Sca-1 and skeletal musclecell marker αSarcomeric actin were examined by immunocytochemistry. The effect of bFGF and EGF on the proliferation of MDSCs was determined by MTT colorimetric microassay. The solo effect of bFGF or EGF at different concentrations (6.25,12.50, 25.00, 50.00, and 100.00 ng/ml) was examined at 96 h and the combined effect (100.00 ng/ml) was examined at 24,48,72 and 96 h.Results MDSCs were successfully isolated from the hindlimb of neonatal mice. Over 90% of MDSCs showed Sca-1 positive immunoreactivity. MDSCs could give rise to α Sarcomeric actin positive myotubes in differentiation cultures. The proliferative effect of bFGF and EGF on MDSCs increased with the elevated concentration.bFGF began to show significant proliferative effect at 12.50 ng/ml (P＜0.05). The effect increased significantly when the concentration reached 25.00 ng/ml from 12.50 ng/ml (P＜0.01) and reached a saturation point. The effect at 50.00 ng/ml or 100.00 ng/ml showed no significant increase when compared with thatat 25.00 ng/ml. EGF had a similar effect to bFGF except that the saturation concentration was 50.00 ng/ml. EGF showed significant effect at 72 h and bFGF at 96 h (Plt;0.01). When they were applied together, significant effect was shownat 24 h (Plt;0.01) and much higher effect was observed at 48, 72 and 96 h (Plt;0.05). Conclusion Both bFGF and EGF can promote the proliferation of MDSCs. The combined application reacts faster and ber.