摘要
目的表皮干细胞是皮肤发生、修复、重建的关键“种子细胞”,因而研究建立人表皮干细胞体外分离、培养及鉴定的方法非常重要。方法通过酶消化法,分离小儿包皮的表皮与真皮层,表皮制成单细胞悬液,以高糖低钙的DMEM培养基,补充100ml/L的干细胞培养专用胎牛血清(FBS),0.05mmol/L氯化钙,10ng/ml表皮生长因子,1×10-6mol/L氢化可的松进行培养。在实验之前用胶原IV包被直径35mm的培养皿,其中有些放置消毒盖玻片以制“细胞玻片,置4℃冰箱中放置30min以上,吸干胶原IV,再将培养皿置室温下自然干燥半小时以上,备用。调整细胞含量为2×106/ml,接种于包被好胶原IV的培养皿中快速黏附10～15min(37℃),弃去未黏附细胞,保留快速黏附细胞继续培养。培养至21d对所培养细胞进行流式细胞仪α6、CD71表型鉴定,角蛋白19(K19)、角蛋白5(K5)免疫细胞化学染色鉴定以及形态学观察、克隆计数。结果在胶原IV包被的培养皿中快速贴壁细胞经继续培养,可见细胞克隆数增多,细胞传代次数达8～10次,培养时间达2个月。K19,K5免疫化学染色阳性,α6briCD71dim细胞占细胞总数的40%左右。结论研究表明用胶原IV快速黏附法可从包皮分离和富集表皮干细胞,用此体外培养体系,可较好地扩增表皮干细胞。
Aim To investigate the method of isolation culture and identification of human epidermal stem cell in vitro.Methods Child foreskin from circumcision procedure was peeled from the subcutaneous tissue gently, cut into small pieces, and then incubated for 3h to 5h in a solution of 0.2 g/L EDTA and 2.5 g/L trypsin at 4 ℃in a dish. The epidermis was separated from the dermis, and shaken for 10min in 2.5 g/L trypsin at 37 ℃to dissociate into single cells. Digestion was inactivated by the addition of 100 g/L serum, and the cells were gently centrifuged and resuspended in the culture medium, which constituted DMEM with high agarose and low calcium, supplemented with 0.05 mmol/L CaCl2,100 g/L fetal calf serum (FBS, for stem cell), 10 ng/ml EGF,1×10-6 mol/L hydrocortisone. 1×106/ml epidermal cells were incubated for 10-15 min at 37 ℃on dishes coated collagen type IV, then the non adherent cells were rinsed off. The adherent cells were grown to confluence. Cells were subcultured in a solution of 0.2 g/L EDTA and 2.5 g/L trypsin for 5-10 min. Once free of the dish, cells were centrifuged, resuspended in fresh medium, and then cultured at 1×105/ml. Cells were examined for phenotypes and colony formation under microscope, the expression of α6 and CD71 were detected with flow cytometry, the expression of K19 and K5 were detected with ABC immunohistochemical methods, then observed under microscope.Results The cells selected by rapid adherence to collagen type Ⅵformed more colonies and were passaged more times than non adherent cells. Approximately 40%cells showed α6 briCD71dim by flow cytometry, 52.9%cells were K19 and 58.2%cells were K5 positive expression by immunohistochemical methods.Conclusion It is suggested that human epidermal stem cells could be separated by our rapid adherent method and expanded in culture conditions.
出处
《中国临床康复》
CSCD
2003年第11期1620-1621,F002,共3页
Chinese Journal of Clinical Rehabilitation
基金
江西省科委重大招标项目资助(E021101)~~
