人胚胎成纤维细胞作为组织工程皮肤种子细胞的可行性研究|胚胎成纤维细胞

　　[摘要]目的：评估人胚胎成纤维细胞（human embryonic fibroblasts，HEF）作为组织工程皮肤种子细胞的可行性和优越性。方法：取人流产胚胎皮肤、正常儿童皮肤，相同条件下分离培养成纤维细胞，比较两组细胞镜下、超微结构以及增殖特性，异体淋巴细胞混合实验对比其抗原性。以第三代细胞复合鼠尾胶原构建三维培养，ELASA法分别测定两组三维构建培养液中IL-6，TGF-β1含量。结果：与普通成纤维细胞相比，胚胎成纤维细胞扩增后具有更好的细胞形态和功能，生长速度快，分裂指数高，几乎不刺激异体淋巴细胞增殖。在鼠尾胶原支架中成纤维细胞生长状态良好，并且具有一定的组织强度。胎儿成纤维细胞组培养液中的TGF-β1和IL-6在各个时相上分别显著低于和高于普通成纤维细胞组。结论：胚胎成纤维细胞是组织工程皮肤较理想的种子细胞。
　　[关键词]】组织工程；皮肤；无斑痕愈合；胚胎；成纤维细胞
　　[中图分类号]Q813.1[文献标识码]A[文章编号]1008-6455（2011）04-0605-04
　　
　　Investigation on feasibility of constructing engineered skin substitutes with human embryonic fibroblasts
　　LIU Liu1,LI Wu-de1,CAI Guo-bin2
　　（1.Cleft lip and Palate Treatment Center,China Meitan General Hospital,Beijing 100028,China;2.Scar Integrated Treatment Centre,Plastic Surgery Hospital,Chinese Academy of Medical Sciences, Peking Union Medical College,Beijing 100041,China）
　　
　　Abstract:ObjectiveTo assess the biological and functional properties oftissue-engineered three-dimensional (3D) composites seeded with human embryonic fibroblasts （HEF）.MethodsFibroblast cultures were developed from biopsies of human fetus and children. To determine the differences between fetal and adult fibroblasts, the cell shape, ultrastructure, growth cycle were observed. In addition, mixed lymphocyte culture was examined to evaluate the antigenicity. Three-dimensional skin constructs were obtained by seeding cultured cells in rat tail collagen scaffold. The content of IL-6 and TGF-β1 in the dermis culturing supernatant was measured by ELISA method.ResultsIn monolayer culture, HEF displayed more typical pattern and faster growing rate. HEF was poor stimulators of lymphocyte proliferation. In 3D culture, fetal constructs appeared more developed based on gross examination with significantly higher level secretion of IL-6 and lower of TGF-β than adults"(P 　　
　　2结果
　　2.1 二维培养阶段
　　2.1.1光镜下观察：同等培养条件和时间，HEF贴壁覆盖面积大于普通FB，HEF呈梭形、多边形或不规则形，边缘不整齐，极性排列不规则。细胞折光性强。普通细胞更容易呈束状排列（图1.1,图1.2）。
　　2.1.2 透射电镜下观察：HEF（第三代）功能活跃，表现为细胞体积大，胞浆丰富，胞核及核仁增大，胞膜与核膜折皱加深，胞浆内微丝粗大，沿细胞长轴平行排列，未见细胞连接（图2.1，图2.2）。细胞质内含有丰富的粗面内质网、游离核糖体和发达的高尔基复合体。
　　2.1.3 同代(P3)HEF和普通FB细胞生长曲线：在细胞生长前3天，两组细胞增殖速度相似，其后均进入对数生长期，HEF增殖速度超过普通FB，更早进入平台期，而后HEF仍保持着较高的生长密度（图3）。
　　2.1.4 淋巴细胞增殖实验：普通FB：与对照组相比原代～V代均有明显差异。组间比较：P0、P1与其它各代差异明显(P0.05)，P2与P4,P5仍有明显差异(P0.05)。
　　HEF：与对照组相比原代有明显差异（P0.05) (见表1)。
　　2.2 三维培养阶段：两组培养液中TGF-β1和IL-6含量的比较：各组成纤维细胞培养后的第一天，即可检测到一定水平的TGF-β1和IL-6。两组细胞IL-6随着培养时间的延长分泌量增多，到第9天到达稳定高值水平，至第11天于第7天含量比较无统计学意义（P>0.05）；胎儿组IL-6含量在各时间点都高于成人组（P 　　[5]Judith Hohlfeld，et al. Tissue engineered fetal skin constructs for paediatric burns[J].The Lancet,2005,366(3):840-842.
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　　[收稿日期]2010-12-26[修回日期]2011-03-12
　　编辑/张惠娟