rhFGF-7对体外培养人皮肤角质形成细胞增殖的影响

Effect of rhFGF-7 on the Proliferation of Human Skin Keratinocytes Cultured In Vitro

目的建立一种人皮肤角质形成细胞分离和培养的方法,研究重组人成纤维细胞生长因子7(rhFGF-7)对人皮肤角质形成细胞增殖的影响。方法取环切术后包皮,分别用胰酶和dispaseⅡ酶消化法分离表皮,用无血清培养基进行无滋养层培养,采用免疫细胞化学方法对培养的细胞进行鉴定,并用细胞计数法测定细胞的生长曲线,MTT法检测rhFGF-7对人皮肤角质形成细胞增殖的影响。结果与胰酶消化分离表皮相比,dispaseⅡ酶消化获得的表皮,其细胞贴壁率高,增殖速度快,且成纤维细胞污染少;荧光显微镜下细胞胞浆呈黄绿色,为角蛋白阳性染色;培养的角质形成细胞可持续增殖至第8代,10ng/mL的rhFGF-7促角质形成细胞增殖作用最显著。结论所建立的人皮肤角质形成细胞分离和培养方法,可获得高纯度的人皮肤角质形成细胞,rhFGF-7可促进体外培养人皮肤角质形成细胞的增殖。

Objective To establish a method for isolation and cultivation of human skin keratinocytes, and to investigate the effect of the recombinant human fibroblast growth factor 7 （ rhFGF-7 ） on the proliferation of human skin keratinocytes. Methods Trypsin and dispase Ⅱ were used to isolate the epidermis from the foreskin. The keratinocytes were cultured in serum-free medium without feeder layer. Immunocytochemistry was applied to identify the cultured cells. The growth curve of the cells was assessed with haemocytometer. MTT method was used to detect the effect of rhFGF-7 on the proliferation of the keratinocytes. Results Compared with that of the epidermis isolated by trypsin, the epidermis isolated by dispase Ⅱ contained cells with higher plating efficiency,greater proliferation rate, and less fibroblast contamination. The cytoplasm of the cells presented Kelly fluorescence under the fluorescent microscope, indicating that the cells were keratin positive. The keratinocytes could be passaged for 8 times. 10ng/mL of rhFGF-7 had the most significant effect on the keratinocyte growth. Conclusions The established method for isolation and cultivation of human skin keratinocytes could be used to obtain the keratinocytes with high purity, rhFGF-7 could stimulate the proliferation of human skin keratinocytes in vitro.