人皮肤成纤维细胞的培养和鉴定

Culture and identification of human skin fibroblasts

目的 探索和建立人皮肤成纤维细胞体外分离、培养及鉴定的技术方法。方法 通过酶消化法分离人皮肤外分泌汗腺 ,在外分泌汗腺生长的同时 ,成纤维细胞也在生长 ;用质量分数为 0 .2 5 %的胰酶和0 .0 2 %的乙二胺四乙酸 (EDTA)消化分离汗腺细胞和成纤维细胞 ;以 Dulbecco改良的 Eagle培养液 (DMEM)为基础培养基 ,添加胎牛血清 (体积分数 10 % )、青霉素 (10 0 k U/ L)和链霉素 (10 0 mg/ L) ,置 37℃、体积分数为 5 % CO2 、95 %空气、饱和湿度条件下培养。倒置相差显微镜和苏木素伊红染色 ,观察成纤维细胞形态 ,并对培养细胞行波形蛋白免疫组化及染色体鉴定。结果 分离的成纤维细胞可在体外快速贴壁生长、增殖 ,波形蛋白免疫组化染色为阳性 ,染色体分析为 4 6条。结论 该方法所获得的皮肤成纤维细胞可在体外稳定培养 ,为在细胞水平上研究伤口愈合的机制提供了充足、可靠的靶细胞。

Objective To explore the method of isolation, cultivation, and identification of human skin fibroblasts in vitro. Methods By digesting human skin with collagenase type Ⅱ to isolate human eccrine sweat glands. The fibroblasts grew along with the growth of eccrine sweat gland cells,and they were separated by digesting with 0 25% trypsin and 0 02% ethylenediaminetetraacetic acid (EDTA). Dulbecco's modified Eagle's medium(DMEM) was the basic culture medium, being supplemented with fetal bovine serum(10%), penicillin(100 kU/L), and streptomycin(100 mg/L). Fibroblasts were incubated at 37 ℃ in a humidified atmosphere of 5%CO 2 and 95% air in the incubator. Cellular morphologies were observed by inverted phase contrast microscopy and hematoxylineosin staining, and the cultured cells were identified by vimentin immunostaining and chromosome analysis. Results The isolated fibroblasts could grow and proliferate in vitro, and immunostaining of vimentin was positive in cultured fibroblasts and the number of chromosome was 46. Conclusion Acquired human skin fibroblasts can be cultured in stable condition in vitro , and sufficient and reliable target cells can be obtained for the study of the mechanisms of wound healing at molecular level.