课题背景：课题为国家自然科学基金资助项目（30772255/C3030308）；教育部博士点基金资助项目（20060533049）；解放军第三军医大学创伤，烧伤，复合伤国家重点实验室开放课题资助项目。上世纪80年代曾有零散的关于成纤维细胞生存率的报道，此后类似的工作一度停滞。受脱细胞真皮的启发，本科近年来开展的“自体薄皮片覆盖变性真皮”手术取得了良好的疗效。因此，实验设计了对热损伤后变性皮肤成纤维细胞恢复情况进行的系列研究，希望为临床治疗提供帮助。

应用要点：本实验结果为进一步研究变性真皮皮肤成纤维细胞功能改变、恢复及相关的保护机制打下了坚实的基础，有利于在细胞生物学层面上深刻理解“自体薄皮片覆盖变性真皮”手术及取得的良好效果。

偏倚或不足：①本文设计上最大的难点就是变性成纤维细胞中“变性”这一概念如何把握？即体外培养的成纤维细胞热损伤到什么程度才能最大限度地反映创面变性真皮皮肤成纤维细胞的形态及功能的变化？目前国外还没有相关的文献报道，国内专家认识也还没有达成一致。同时，与二维培养相比，三维培养成纤维细胞更能模拟皮肤中成纤维细胞的特性，三维培养成纤维细胞损伤模型也更能真实地反映创面变性真皮皮肤成纤维细胞特点。②实验设计中缺乏连续观察细胞周期中S期及凋亡的变化。

摘要
目的:在以往创面愈合研究中，多注意周围正常组织或血液中成纤维细胞的迁入对伤口治愈作用。实验拟验证成纤维细胞热损伤后形态学的变化，为研究“深Ⅱ度烧伤创面自体薄皮覆盖变性真皮”新手术方式提供理论依据。
方法：实验于2006-04/2007-04在解放军第三军医大学创伤，烧伤，复合伤国家重点实验室完成。取门诊手术后废弃的小儿包皮，利用体外细胞培养技术对人真皮成纤维细胞进行传代培养，实验分2组，正常组细胞置于37 ℃ 水浴30 s，实验组细胞采用不同温度（50，51，52，53 ℃）及不同时间（30，60，90，180 s）处理，采用四甲基偶氮唑盐法测定细胞生存率；倒置显微镜及透射电镜下观察不同时间（3 h，1，2，3，4，5，7 d）成纤维细胞损伤后的形态；流式细胞仪技术对烫伤细胞周期进行检测。
结果：不同温度以及同一温度不同时间条件下，各实验组成纤维细胞的生存率与正常组比较，差异均有显著性意义（P < 0.05）。与正常组相比，52 ℃ 30 s成纤维细胞的生存率50%，差异显著（P < 0.05）。透射电镜观察热损伤后成纤维细胞变圆钝，体积增大，表面有较多突起，胞浆中有大量的异常疏松透亮区。热损伤后成纤维细胞的形态有明显改变，7 d 恢复正常。流式细胞仪检测显示成纤维细胞的S期是其热损伤的敏感期，与正常组比，实验组成纤维细胞S期凋亡明显增多（P < 0.01）。
结论：热损伤后变性的成纤维细胞活性明显降低，形态学及细胞周期发生显著改变，7 d 形态基本恢复正常。
关键词：成纤维细胞；热损伤；形态学；变化；皮肤组织构建

李德全，黄晓元，杨兴华．离体人皮肤成纤维细胞热损伤后转归的形态学观察[J].中国组织工程研究与临床康复，2008，12(11):2056-2060 [www.zglckf.com/zglckf/ejournal/upfiles/08-11/11k-2056(ps).pdf]

Morphology of human skin fibroblasts after heat injury in vitro

Abstract

AIM：Previous studies paid more attention to the effect of fibroblast immigration in normal surrounding tissue or blood on wound healing. This study explored the morphological character of fibroblasts after heat injury to support the new operation of autogenous epidermis overlapping denatured dermis.
METHODS: The experiment was performed at the State Key Laboratory of Trauma, Burn and Combined Injury in the Third Military Medical University of Chinese PLA from April 2006 to April 2007. The dermal fibroblast from the infant prepuce disposed after surgery was cultured in vitro, and divided into 2 groups. In normal group, the dermal fibroblasts were put in water bath at 37 ℃ for 30 seconds; the cells in experimental group were stimulated at different temperatures (50, 51, 52, and 53 ℃) for different time (30, 60, 90, and 180 seconds). Cell survival was detected by MTT; morphology of injured cells were observed under inverted microscope and transmission electron microscope at 3 hours, 1, 2, 3, 4, 5, and 7 days; cell cycle was detected by flow cytometry.
RESULTS: At different temperatures and different time, there were significant differences in the survival rate of fibroblast between the experimental groups and normal group (P < 0.05). Compared with the normal group, the survival rate at 52 ℃ for 30 seconds was 50% (P < 0.05). The transmission electron microscopic observation showed after heat injury the fibroblast became round and enlarging with many ecptoma, and many lucent regions were found in kytoplasm until the seventh day. Flow cytometry analysis suggested S was the sensitive period for injured fibroblast. Compared with normal group, apoptosis rate of cells in the experimental groups at S phase was significantly increased (P < 0.01).
CONCLUSION: Fibroblast activity is greatly reduced after heat injury. Morphology and cell cycle alter significantly until 7 days later.

Li DQ, Huang XY, Yang XH. Morphology of human skin fibroblasts after heat injury in vitro.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(11):2056-2060(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-11/11k-2056(ps).pdf]