摘要
背景：发育生物学研究表明，表皮干细胞是皮肤及其附属器发生、修复、重建的关键性源泉，如何从皮肤组织中分离获得更多的表皮干细胞，并在体外培养过程中保持干细胞特性是其在皮肤组织工程中应用的重要前提。目的：观察Ⅳ型胶原快速黏附法分选的人胎儿表皮干细胞体外生长状态及克隆形成情况，并与角质细胞进行对照。设计、时间及地点：细胞学体外观察，于2008—01／06在南昌大学第一附属医院烧伤研究所完成。材料：因创伤等原因致意外流产的妊娠24～26周龄胎儿皮肤标本，由南昌大学第一附属医院产科提供。Ⅳ型胶原为美国Sigma公司产品，角质细胞无血清培养基为美国Gibco公司产品。方法：将Ⅳ型胶原用磷酸盐缓冲液稀释成100mg／L，均匀涂被培养板后于超净工作台中风干备用。无菌条件下取胎儿皮肤标本，剪成3．0mm×5．0mm皮条片，4℃胰蛋白酶＋EDTA联合消化8～10h，表皮真皮分离，用角质细胞无血清培养基将表皮反复吹打，获得单细胞悬液接种于预处理好的Ⅳ型胶原培养板，置37℃、体积分数为0．05的CO2饱和湿度培养箱中，20min后吸去培养液和未黏附细胞，加入含体积分数为0.1的胎牛血清、10μg／L表皮生长因子及角质细胞无血清培养基的表皮干细胞培养液，细胞约70％-80％融合后传代扩增；将未黏附细胞接种于另一培养板中，相同条件下作为角质细胞对照培养。主要观察指标：表皮干细胞的形态变化、免疫细胞化学染色鉴定结果及克隆形成率。结果：经贴壁筛选的表皮干细胞接种后呈圆形，折光性较强；1d后细胞略伸展为扁平的多角形，胞质近中央处有圆形核；3d时出现表皮干细胞克隆；5d时克隆数量明显增多，细胞之间紧密衔接，呈铺路石状；14d左右细胞基本融合。分离培养的人表皮干细胞克隆β1整合素、角蛋白19、p63均呈阳性表达。表皮干细胞克隆形成率明显高于角质细胞克隆形成率（t=1．972，P〈0．05）。结论：采用Ⅳ型胶原快速黏附法成功筛选出纯度较高的人胎儿表皮干细胞，且呈明显克隆性生长，具有典型的干细胞生物学特性。
BACKGROUND： The developmental biology studies have shown that epidermal stem cells are the key resources for skin reparation and reconstruction. However, how to isolate more epidermal stern cells, and keep the stem cell characteristics in clinic is an important premise for skin tissue engineering. OBJECTIVE： To observe the morphological features of human fetus epidermal stem cells in vitro which isolated by type Ⅳ collagen attachment method, and compared the results with keratinocyte cells. DESIGN, TIME AND SETTING： The in vitro cytology observation was performed at the Institute of Burn, First Affiliated Hospital of Nanchang University during January to June in 2008. MATERIALS： The skin samples of fetus were taken from the accidental abortion （24-26 weeks gestational age） which provided by the Department of Maternity, the First Affiliated Hospital of Nanchang University. The type Ⅳ collagen was produced by Sigma Corporation of America. METHODS： The type Ⅳ collagen was diluted to 100 mg/L with phosphate buffer saline, spread on culture plate and drying on the super-clean worktable. The fetal skin samples were attained under sterile conditions, cut into pieces with 3.0 mm × 5.0 mm in size, then the pieces were submerged into a culture bottles of Trypsin-EDTA. After 8-10 hours digestion at 4 ℃, the epidermal cells could be separated from dermis completely. The epidermal cells were crashed with the keratinocyte serum free medium （ K-SFM ） repeatedly to harvest epidermal single cell suspension, then the cells were vaccinated in the pretreatment collagen Ⅳ culture plate and placed in an incubator of saturated humidity at 37 ℃, with 0.05 volume faction of CO2 nutritive medium. With 20 minutes cultivation, the cultural fluid and the non-attaching ceil were got rid of and added to nutritive medium which contained 0.1 volume fraction of fetal bovine semm, 10 μg/L epidermal growth factor and keratinocyte serum free medium. When 70%-80% cells were mixed, the cells were subcultured. The non-attaching keratinocyte cells were vaccinated in other culture plate as the control groups. MAIN OUTCOME MEASURES： Morphologic change, identificated result by immunocytochemical stain and clone forming efficiency were investigaged in the study. RESULTS： The epidermal stem cells after vaccination were round with strong light refraction 1 day later, and extended slightly to flat polygons with circular nuclear in the central of cytoplasm. At the 3^rd day, epidermal stem cell clones formed, and the number of cell clones increased significantly at 5^th days, the ceils connected closely with cobblestone features and fused well at the 14^th days.The integrin β1,keratin 19 and p63 -had positive expression in the epidermal stem cells. The clone forming efficiency of epidermal stem cell was significantly higher than that in the keratinocyte group （t=1.972, P 〈 0.05）. CONCLUSION： Type Ⅳ collagen attachment method can screen out human fetus epidemal stem cells, which has the typical biological characteristics of stem cells.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第43期8535-8538,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金(30560058)~~
