人皮肤成纤维细胞的分离培养及鉴定

Separation,culture and characterization of human skin fibroblasts

目的:建立人皮肤成纤维细胞的体外原代及传代培养并鉴定的技术方法。方法:取临床无菌切除的幼儿包皮,利用胶原酶分离表皮和真皮,将真皮剪成肉糜状,再用胰蛋白酶消化,接种于培养皿,加少许含10%小牛血清的DMEM-高糖培养液进行培养,次日补加培养液。原代长满后,进行传代,并对所培养的细胞进行形态学观察及组化鉴定。结果:接种次日倒置镜下可见有长梭形细胞迁出、生长,5-6d进行传代,传代后约5d长满,可长期传代。免疫组化Vimentin表达阳性。结论:该方法可快速高效获得构建组织皮肤真皮所需的成纤维细胞。

Objective： To explore the method of isolation, cultivation and identification of human skin fibroblasts in vitro. Methods： By digesting child foreskin with collagenase type 1I to isolate epidermis and coriurrL The sheafing corium was digesting with trypsin. Dulbecco＇s modified Eagle＇s medium （DMEM） was the basic culture medium, being supplemented with 10 ~ bovine calf ser- um. Cellular morphologies were observed by inverted phase contrast microscopy. The cultured cells were identified by vimentin im- munohistochemical. Results： The spindle shaped fibroblasts could find by inverted phase contrast microscopy in second day. The fibroblasts could passage in fifth day. And immunohistochemical of vimentin was positive in cultured fibroblasts. Conclusion： It is an effective method to cultivate fibroblasts with high productivity and survival rate.