聚乳酸-羟基乙酸共聚物和胶原膜对体外培养人表皮角质细胞分泌细胞因子的影响

Effects of PLGA Scaffold and Collagen Membrane on Cytokine Secretion of Human Epidermal Keratinocytes in vitro

目的探讨聚乳酸-羟基乙酸共聚物(PLGA)和胶原膜对表皮角质细胞分泌细胞因子的影响及其在创面修复中的可能作用机制。方法实验组分为PLGA组和胶原膜组,表皮角质细胞以3×107cm-2密度分别种植于PLGA和胶原膜上;对照组为单层表皮角质细胞培养。于第7天和第14天扫描电镜下观察细胞生长状况;种植后第1,3,5,7,14天收集细胞培养上清液,检测乳酸脱氢酶(LDH)水平观察细胞损伤程度,应用ELISA法检测培养上清液中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和白细胞介素8(IL-8)的水平。结果表皮角质细胞种植后第7天,扫描电镜下可见细胞粘附于PLGA和胶原膜呈原有三维结构稳定均一生长,第14天有纤维丝状细胞外基质生成。实验组和对照组各时相点LDH水平差别无统计学意义。PLGA组和胶原膜组表皮角质细胞分泌IL-1β、IL-6和IL-8水平与对照组比较均有不同程度上升,其差别具有统计学意义(P<0.05或P<0.01)。结论PLGA和胶原膜在构建组织工程皮肤中作为一种生物支架,增强表皮角质细胞分泌IL-1β、IL-6和IL-8能力,对加速创面愈合可能起着积极作用。

Objoctive To establish in vivo culture of human epidcrmal keratinocytes on biodegrad- able poly-D,L-lactic-co-glycolic acid （PLGA） scaffolds and collagen membrane and explore the influence of cytokihe secretion of keratine, cytes on wound healing. Methods In vitro cultured humaia epidermal kerathaocytes were seeded at the density of 3 10^7cells/cm2 onto the PLGA scaffold：; and collagen membrane to make the cell-polymers construct, which was observed by the scanning electron microscope at 7 d and 14 d. The levels of IL-Iβ, IL-6 and IL-8 in the supernatants of cultured cell-polymer and monolayer kera- tinocytes （as control group） were measured by ELISA and the leakage of lactate dehydrogenase （LDH） was assayed for the evaluation of the cell injvry at 1, 3, 5, 2.：. 14 days after being co-cultured. Results The scanning electron microscope showed that epiderma! keratinocytee; were attached to the PLGA scaffolds and collagen membrane and grew in three-dimensional distribution at 7 d after cell seeding. The extacellular matrix were secreted from scaffolds, inhibiting fibril-formation at 14d. No signlficant changes in the leakage of LDH were observed in the PLGA scaffolds and collagen membrane groups as compared to the control groups at each time point. The statistical analysis showed that the levels of IL-1β（P〈0.05）, IL-6 （P〈0. 01） and IL-8 （P〈0.01）were significantly increased in PLGA scaffolds group and in collagen membrane groups. conclusion The study indicates that the PLGA scaffolds and the collagen membrane are good materials in the tissue engineered skin, which could facilitate epidermal keratinoeytes to secrete some cytokine and provide a favorable environment for wound healing.