表皮细胞体外培养过程中生物学特性改变的观察

The Alteration of the Celluar Properties in the Aging of Normal Human Keratinocytes

目的 通过观察表皮细胞体外增殖与老化规律 ,为选择合适的组织工程化皮肤种子细胞提供依据。方法 取正常年轻人表皮细胞进行传代培养 ,以不同代龄细胞为实验对象 ,采用形态学观察、群体倍增时间(PDT)、免疫细胞化学及β 半乳糖苷酶染色等一系列方法 ,检测表皮细胞老化规律。结果 体外单层培养 9代 ,P2的PDT最短 ,前 5代增殖能力较强 ,P5以后PDT明显延长 ,P8细胞不再增殖 ;随着细胞的连续传代培养 ,SA β Gal表达呈现从弱 (在年轻细胞中占 9% )到强 (在老化细胞中占 6 5 % )的趋势。结论 建立了体外培养正常人表皮细胞的衰老模型 ,第 1～ 5代表皮细胞 (供者年龄 16～ 18岁 )

Objective To investigate the proliferation and aging of keratinocytes isolated from human skin,then to evaluate different passages of keratinocytes as a potential source of seeding keratinocytes in tissue engineered skin.Methods Specimens were obtained for cell culture.The methods of Trypan Blue staining and cell counting were applied to determine the original cell viability.The proliferation of keratinocytes cultured in vitro from P 0 to P 8 was calculated by the population double time (PDT) determination.The aging process was displayed by growth curve of cell line and β-galactosidase histochemical staining.Results The PDT of P 2 was the shortest,and that of P 7 was the longest.Little keratinocyte proliferation could be detected in P 8.The expression of SA-β-Gal tended to be more apparent in old cells than that in young cells,which maintained relatively high as about 65%.Conclusion The human keratinocytes of P 0～P 4 can be used respectively as seeding cells in the engineered skin construction.At the same time we create cellular senescence aging pattern in vitro.