自体血清与胎牛血清培养口腔黏膜角质细胞及上皮组织的实验研究

目的探讨自体血清培养人口腔黏膜角质细胞的可行性,为组织工程口腔黏膜用于临床提供理论和技术依据。方法应用自行制备的含10%、20%和30%自体血清的培养液及10%胎牛血清培养液,对人口腔黏膜角质细胞进行培养。对比观察不同浓度自体血清和胎牛血清培养的口腔黏膜角质细胞及上皮组织生长情况,绘制10%自体血清组及10%胎牛血清组细胞生长曲线及计算其倍增时间。并行抗-HLA免疫荧光检测培养上皮。结果各浓度自体血清组和10%胎牛血清组细胞生长及形态无明显差别,10%自体血清组细胞倍增时间为24.02±1.80h,与10%胎牛血清组20.90±0.79h比较,差异无统计学意义(P>0.05)。各组细胞24h克隆形成率比较,差异均无统计学意义(P>0.05);随自体血清浓度升高获得黏膜上皮面积增大,厚度增加,以20%自体血清组最为明显,与10%胎牛血清组培养黏膜上皮比较,差异有统计学意义(P<0.05)。各组培养上皮经抗-HLA免疫荧光检测为阳性。结论制备的自体血清能完全替代胎牛血清对口腔黏膜角质细胞进行培养,且培养的口腔黏膜上皮组织分化优于胎牛血清。

THE EXPERIMENTAL STUDY ON CULTURE OF HUMAN ORAL KERATINOCYTE AND EPITHELIUM USING AUTOLOGOUS SERUM AND FETAL BOVINE SERUM

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 fetal bovine 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 24-hour 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.