几丁质膜作为表皮干细胞培养支架的实验研究  

An experimental study of using chitinous membrane as the culture scaffold for epidermal stem cells

作　　者：沈雁 李孝建 梁蓉 李延仓 张琰 梁佩红 戴丽冰 杨小红 谭见容 李叶扬 黄粤 

机构地区：[1]暨南大学附属广州市红十字会医院创伤外科研究所,广州510220 [2]暨南大学附属广州市红十字会医院烧伤科,广州510220

出　　处：《中华烧伤杂志》2009年第3期197-201,共5页Chinese Journal of Burns

基　　金：广东省社会发展领域科技计划（63102）;广州市应用基础研究计划（2008J1-C121）;广州市医药卫生科技重点项目（2005-ZDi-03,2006-ZDi-08,2008-ZDi-08）

摘　　要：目的了解大鼠表皮干细胞（ESC）与几丁质膜共同构建皮肤组织工程覆盖物的可行性。方法采用冷消化法和Ⅳ型胶原贴壁法分离培养大鼠ESC。倒置显微镜观察细胞生长情况，激光共聚焦显微镜检测其DNA和RNA表达，澳玛蓝比色法测定ESC生长曲线，流式细胞仪检测ESC表面标志物CD29、CD71、CD49d和CD34，免疫组织化学法测定细胞角蛋白15（CK15）、CK19和P63阳性表达。检测不同倍比稀释几丁质膜浸出液对细胞的影响，并设常规培养为对照组；观察ESC在载体上的生长情况。结果分离培养的细胞经鉴定确认为ESC，细胞倍增时间为48h。其表面标志物CD29、CD49d为阳性，CD71、CD34为阴性；CK15、CK19、P63为阳性。几丁质膜浸出液培养的细胞与对照组比较，在1：8～1：512等比稀释时，有轻微的促细胞增殖作用但差异无统计学意义（P〉0．05）。ESC在几丁质膜上培养2～4周时，肉眼可见棋盘样细胞集落，显微镜下见大量ESC在纤维上增殖。结论几丁质膜可以作为ESC的培养载体，两者有较好的生物相容性。Objective To investigate the feasibility of constructing a skin tissue engineering covering on chitinous membrane using rat epidermal stern cells （ ESCs）. Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type IV adherent method. Cell colonies were observed with inverted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning eonfoeal microscope. Growth curves of cells were determined with Alamar BlueTM colorimetrie method. Expressions of surface markers of ESCs （CD29, CDTI , CD49d, and CD34） were detected with flow cytometer. Positive expressions of CK15, CK19, and P63 of ESCs were determined by immunohistoehemistry. Influence of original chitinous membrane leaehate in different dilutions on ESCs was observed. Condition of growth of ESCs on the vehicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive; CD71 and CD34 were negative; CK19, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leaehate showed slight cell proliferation when diluted to 1：8 - 1：512 dilutions, but there was no statistically significant difference （ P 〉 0.05）. The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chitinous membrane in 2 - 4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.

关 键 词：皮肤 于细胞 组织工程 仿生材料 几丁质 

分 类 号：R318.0[医药卫生—生物医学工程]