摘要
目的:以Ⅳ型胶原黏附法体外快速分离人表皮干细胞,利用其特异性标记物β1整合素及角蛋白19鉴定所培养细胞是否为表皮干细胞。方法:实验于2004-05/2005-03在南昌大学医学院烧伤研究所完成。①包皮来自行包皮环切术的儿童,由南昌大学第一附属医院提供,患者及其家属均签署知情同意书。②取手术刚切下的新鲜包皮,剔除皮下组织,采用DispaseⅡ酶和胰蛋白酶两步法分离出角朊细胞和成纤维细胞。收集处于对数生长期的第2～3代成纤维细胞的上清液,过滤除菌后与角朊细胞无血清培养基等比例混合,再加入体积分数为0.1的胎牛血清、表皮生长因子5μg/L、氢化可的松400μg/L、氯化钙0.05mmol/L、L-谷氨酰胺0.1mol/L、牛垂体提取物1.0mg/L,组成表皮干细胞培养液。分离的表皮片浸入2.5g/L胰蛋白酶-0.2g/L乙二胺四乙酸消化液中5min,所得细胞以表皮干细胞培养液悬浮。③将获得的角朊细胞悬液接种于预铺Ⅳ型胶原的六孔板中,每孔接种约1.5mL,室温静置10min,吸除未贴壁细胞,磷酸盐缓冲液漂洗至无细胞悬浮,即为富集分离的人表皮干细胞。加入人表皮干细胞培养基常规培养,第3天换液,后每隔1d换液一次。以同一标本同一批次培养的角朊细胞作为对照。④通过检测β1整合素、角蛋白19的表达水平及其克隆形成率和克隆维持时间,对其进行鉴定,以角朊细胞作为对照。结果:①人表皮干细胞形态学观察:倒置显微镜下,从角朊细胞中分离出的表皮干细胞体积小,呈单个、圆形贴壁,分散生长;培养12d后呈克隆样生长。②人表皮干细胞的克隆形成率:与同一标本同一批次培养的角朊细胞比较,人表皮干细胞的克隆形成率高[(8.72±0.73)%,(17.04±1.01)%,P<0.01],克隆维持时间更长[(9～10)d,(15～18)d,P<0.01]。③人表皮干细胞β1整合素和角蛋白19的表达:β1整合素及角蛋白19免疫组织化学染色均呈阳性。结�
AIM： To isolate human epidermal stem cells rapidly in vitro by type Ⅳ collagen attachment method and identify epidermal stem cells by 61 integrin and keratin 19. METHODS： The experiment was performed at Institute of Burn of Medical College of Nanchang University from May 2004 to March 2005. （1）Foreskin separated from a child who received circumcision was provided by First Affiliated Hospital of Nanchang University. The patient and family members signed the informed consent. （2）Fresh foreskin was obtained and subcutaneous tissue was removed. Keratinocytes and fibroblasts were separated from foreskin by Dispase Ⅱ and trypsin. Supernatant fluid of generation 2-3 fibroblasts in logarithmic growth phase was collected and mixed with keratinocyte serum-free medium after degerming at the same proportion, and then fetal bovine serum （FBS） of 0.1 volume fraction, 5 μg/L epidermal growth factor, 400 μg/L hydrocortisone, 0.05 mmol/L calcium chloride, 0.1 mol/L L-glutamine and 1.0 mg/L bovine pituitary extract were added in the mixture to form epidermal stem. cell medium. Separated epidermal slices were immersed in 2.5 g/L trypsin-0.2 g/L edetic acid digestive juice for 5 minutes, and obtained cells were suspended in epidermal stem cell medium. （3）Keratinocyte suspension was inoculated in 6-well plate with type Ⅳ collagen, about 1.5 mL in each well at room temperature for 10 minutes. Non-adhered cells were resorbed. The cells were washed with phosphate buffer saline till no cell was suspended, and then enriching separated human epidermal stem cells were seen. Routine human epidermal stem cells medium was added, and the liquid was changed on the 3^rd day and then the liquid was changed once every other day. Homologous keratinocytes were used as control. （4）Expressions of 61 integrin and keratin 19, colony forming efficiency （CFE） and cloning sustaining time （CST） were determined and keratinocytes were as control. RESULTS： （1）Morphological observation of human epidermal stem cells�
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第20期3861-3863,共3页
Journal of Clinical Rehabilitative Tissue Engineering Research
