以正常人皮肤成纤维细胞滋养层培养人角质形成细胞的研究

Human Keratinocytes Grown in Mitomycin C－treated Human Dermal Fibroblast Feede r Layer

目的探索和建立人皮肤成纤维细胞 (HDF)滋养层培养人角质形成细胞的技 术方法。方法在 HDF滋养层制备中，对丝裂霉素 C的较适浓度、同一株不同代数的 HDF滋养层、滋养层的 细胞密度对正常人表皮角质形成细胞（ NHEK）生长的影响及 NHEK的最低接种密度进行了初 步的探索。结果①终浓度为 10μ g/mL的丝裂霉素 C是制备 HDF滋养层的较适浓度。②同一 株 1～ 20代的 HDF滋养层对不同来源的 NHEK均有较好的促生长增殖作用， 1～ 20代 HDF 的培养代数与 9例 NHEK生长达 80％融合时间 (平均为 11.1 d)之间相关无显著性 (r=－0 .27,P >0.05)。③用 HDF滋养层培养 NHEK可减少原代培养 NHEK的接种量，用 NHEK最小接 种密度是 0.1× 104/cm2经 16 d的培养可达 80％的融合。结论同一株不同代的 HDF适用于不同来源的 NHEK培养。 HDF滋养层可促进 NHEK的生长，这可能 是它减少 NHEK原代培养接种量的原因之一。

Objective To establish a method for the cultivation of keratinocyt es in human dermal fibroblast (HDF) feeder layer. Methods The seeding efficacy, growth and confluence of normal human epidermal keratinocytes (NHEK) in mitomyci n C－treated HDF feeder layers from various passages of same HDF line were obse rved. Results① The concentration of mitomycin C suitable for HDF feeder layer w as 10μ g/mL.② The first to 20th passages of the same HDF line were preferable to establish different allogenic keratinocyte cultures. There was no correlation between the number of passages and periods needed for NHEK to reach 80％ conflu ence (r=－0.27,P >0.05).③ HDF feeder layer could improve the efficient growth of primary keratinocyte culture plated at very low seeding densities (0.1× 104 cells/cm2). Conclusions Different allogenic keratinocytes can grow in HDF feeder layers from various passages of the same HDF line. HDF feeder layer is able to improve the seeding efficacy of human keratinocytes, which may be the reason te culture plated at very low seeding