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背景:成纤维细胞是构建组织工程化皮肤的种子细胞,通过电镜对各代细胞的超微结构进行了观察,以期为组织工程皮肤选择合适的种子细胞奠定基础。目的:观察正常人成纤维细胞体外培养过程中超微结构的改变。设计:自身对照观察。单位:潍坊医学院整形外科研究所、潍坊医学院附属医院普外科。材料:实验于2000-09/2002-09在潍坊医学院整形外科研究所完成。6-8岁正常男性儿童20例包皮环切术切除的健康包皮组织,均征得监护人同意自愿提供。方法:将正常人二倍体成纤维细胞进行连续传代培养,以不同代次的细胞为实验对象,通过倒置相差显微镜和透射电镜对各代细胞进行形态学及超微结构的观察。主要观察指标:①倒置相差显微镜观察的细胞的形态学改变。②透射电镜观察的成纤维细胞超微结构。结果:①倒置相差显微镜观察的细胞的形态学改变:细胞可传65～70代,成活280～300d。45代以内的细胞生长较迅速,45代后的细胞生长逐渐缓慢,65代细胞几乎无增殖趋势。②透射电镜观察的成纤维细胞超微结构:40代以内细胞的超微结构无明显变化,41～65代细胞出现核膜内折,细胞核/浆比例减小,细胞表面突起和微绒毛减少。结论:体外培养的各代成纤维细胞超微结构改变程度不同,41代后变化明显。提示构建组织工程化皮肤宜选用40代以内的成纤维细胞为种子细胞。
BACKGROUND: Fibroblasts are seed cells for the construction of tissue-engineered skin. The ultrastructures of the cells were observed by electron microscopy in order to lay the foundation for selecting suitable seed cells for tissue engineering skin. Objective: To observe the ultrastructural changes of normal human fibroblasts cultured in vitro. Design: self-control observation. Unit: Weifang Medical College Institute of Plastic Surgery, Weifang Medical College Affiliated Hospital of General Surgery. MATERIALS: Experiments were performed at the Institute of Orthopedic Surgery, Weifang Medical College from September 2000 to September 2002. 20 cases of normal male children 6-8 years old circumcision resected healthy foreskin tissue, both with the consent of the guardian agreed to provide voluntarily. Methods: The normal human diploid fibroblasts were subcultured in succession. Different generations of cells were used as experimental subjects. Morphology and ultrastructure of the cells were observed by inverted phase contrast microscope and transmission electron microscope. MAIN OUTCOME MEASURES: ① Morphological changes observed in inverted phase contrast microscope. ② Transmission electron microscopy of the ultrastructure of fibroblasts. Results: ① Morphological changes of the cells observed by inverted phase contrast microscope: the cells can pass 65 to 70 generations and survive 280 ~ 300d. The cells within 45 generations grew more rapidly, the cells grew slowly after 45 generations, and there was almost no proliferation tendency in 65 generations. ② The ultrastructure of fibroblasts observed by transmission electron microscopy: There was no significant change in the ultrastructure of the cells within 40th generation. The nuclear intima-folds, nucleus / cytoplasm proportion were decreased in 41-65th generation cells, and cell surface processes and microvilli were reduced. CONCLUSION: The ultrastructural changes of fibroblasts cultured in vitro differed in different degrees after 41 generations. Tip construction of tissue engineered skin should use 40 generations of fibroblasts as seed cells.