人皮肤真皮组织中角朊干细胞样细胞的分离与培养

Isolation and cultivation of keratinocyte stem-like cells from human skin dermis

背景:皮肤真皮组织是皮肤损伤修复时除表皮基底层以外可形成表皮层结构的重要干细胞来源。目的:探索从人皮肤真皮组织中分离培养角朊干细胞样细胞的新方法。方法:将一定大小的成人腹部全层皮肤组织用Dispase过夜消化后去除表皮层结构,然后用0.1%Ⅰ型胶原酶过夜消化,离心分离以获取真皮来源总细胞。将分离的细胞用含有体积分数1%N2,2%B27,20μg/L表皮生长因子和40μg/L碱性成纤维细胞生长因子的DMEM/F12(1:1)无血清培养液悬浮后以低密度接种于培养皿进行培养。采用RT-PCR和免疫细胞荧光染色法对其进行分析。结果与结论:RT-PCR分析结果显示所分离的真皮细胞中含有表达Lgr5和Lirg1等特异基因的毛囊来源干细胞;免疫细胞荧光分析结果显示其表达细胞角蛋白8和细胞角蛋白14。在无血清条件下培养6d时,可观察到表达细胞角蛋白14和整合素α6的"铺路石"样细胞克隆。进行传代培养后P1细胞表达角朊干细胞标志物CD29、细胞角蛋白14和P63。传代培养至P3,4时大部分细胞分化,不能继续传代。结果证实,以无血清培养液结合低密度培养法可直接从人真皮组织中有效地培养增殖活跃、细胞角蛋白14和P63阳性的角朊干细胞样细胞。

BACKGROUND： Skin dermis is an essential stem cell source, except basal layer of epidermis, for forming epidermis structure during the process of skin wound healing. OBJECTIVE： To explore a new method for isolating and culturing keratinocyte-stem like cells from human skin dermis.METHODS： Definitive sizes of full skins from adult abdomen were digested with dispase overnight and the epidermal structure was discarded. The remaining dermis were digested with 0.1% type I collagenase overnight. Thereafter, the digested cells were collected by centrifugation, suspended in serum-free Dulbecco＇s modified Eagle＇s medium/nutrient mixture F12 （1：1 ） containing 1% N2, 2% B27, 20 μg/L epidermal growth factor, 40 μg/L basic fibroblast growth factor, and cultured at a low cell density. The isolated and cultivated cells were analyzed by reverse transcription-polymerase chain reaction and immunofluorescence analysis. RESULTS AND CONCLUSION： The reverse transcription-polymerase chain reaction analysis showed that, the cells isolated from dermis contained hair follicle stem cells expressing Lgr5 and Lirg 1. The immunofluorescence analysis showed that, the cells isolated from dermis expressed cytokeratin 8 and cytokeratin 14. After cultivated in serum-free media for 6 days, some cytokeratin 14 and integrin-α6 positive cell colonies appeared Upaving stone＂ like morphology. The passage 1 cells expressed keratinocyte stem cell markers, such as cytokeratin 14, CD29 and P63. The cell proliferation rate was declined with passage times and almost all cells were terminally differentiated at passage 3 or 4. Experimental findings indicate that, keratinocyte stem-like cells expressing CD29 cytokeratin 14 and P63 can be successfully isolated from human skin dermis by directly cultured at a low density and in serum-free media.