不同质量浓度胰蛋白酶和EDTA细胞消化分离液对人表皮细胞生长的影响

The optimal conditions for bio-engineering of human epidermis

目的:探讨人表皮生物工程的适宜条件。方法:以NIH-3T3细胞作为滋养层,以热溶素分离表皮与真皮,将表皮应用4组不同质量浓度的胰蛋白酶(T)和EDTA(E)的消化液(A组:0.5g/LT加0.2g/LE,B组:2.5g/LT加0.2g/LE,C组:0.5g/LT加1g/LE,D组:2.5g/LT加1g/LE)消化为单个细胞。表皮细胞接种于含8种成分的DMEM-F12的完全培养液中。记录接种表皮细胞的克隆形成率、扩增倍数以及融合时间。结果:接种表皮细胞的克隆形成率和扩增倍数分别是:A组为(10.00±0.09)%和(500.00±9.63)倍,B组为(7.00±0.12)%和(350.00±6.16)倍,C组为(17.00±0.18)%和(850.00±8.83)倍,D组为(14.00±0.19)%和(700.00±9.55)倍,4组间差异均有统计学意义(P<0.05)。接种表皮细胞的融合时间:A组为第(10.00±0.82)天,B组为第(10.00±0.82)天,C组为第(11.50±1.29)天,D组为第(12.00±2.45)天。4组的接种表皮细胞克隆形成率与融合时间无关。结论:同一质量浓度的EDTA作用下,0.5g/L胰蛋白酶组比2.5g/L胰蛋白酶组更有利于表皮细胞克隆的形成和融合。

Aim： To explore the optimal conditions for bio-engineering of human epidermis. Methods：The NIH-3T3 cells were used as a feeder layer. After the epidermis was isolated from dermis with thermolysin. The single epidermal cells were separated by 4 kinds of digesting solutions;0.5 g/L trypsin （T） ＋ 0.2 g/L EDTA （E）for group A,2.5 g/L T ＋ 0.2 g/L E for group B, 0.5 g/LT＋1g/LE for group C and 2.5 g/LT＋1 g/LE for group D. The epidermal cells were plated in DMEM-F12 complete medium containing 8 kinds of contents. The colony formation rate, expanding fold and the confluent time of epidermal cells were recorded. ReSultS ： The colony formation rate and expanding fold of the plated epidermal cells in the digesting solution groups respectively were ： group A, （ 10.00 ± 0.09） % and （ 500.00 ± 9.63） fold ; group B, （7.00±0.12）% and （350.00 ±6. 16） fold; group C,（17.00 ±0. 18）% and （850.00 ±8.83） fold and group D, （ 14.00±0.19） % and （700.00 ± 9.55 ） fold. There was significant difference in all of the 4 groups. The confluent time of the seeded epidermal cells was （ 10. 00 ±0. 82 ） th day in group A, （ 10. 00±0. 82 ） th day in group B, （ 11.50 ±1.29） th days in group C and （12.00 ± 2.45 ）th day in group D. There was no significant difference in correlation between the confluent time and colony formation rate of the seeded epidermal cells in 4 groups. Conclusion ： In comparison of the digesting solution groups with the same concentration of EDTA, the bio-effects of groups with 0.5g/L T were better than those with 2.5g/L T.