猴表皮干细胞的分离和培养

Isolation and culture of rhesus putative epidermal stem cells

目的:探索猴表皮干细胞分离纯化和培养方法。方法:将猴皮肤标本剪成皮条,加入0.25%胰酶浸泡过夜;然后去除角质层,刮上皮面获取表皮细胞进行培养。取第2-4代的细胞,经消化后,用Ⅳ型胶原黏附法获得为快吸附的表皮细胞。对快吸附的表皮细胞行流式细胞仪、免疫组化和RT-PCR检测。结果:快吸附细胞从mRNA转录水平及蛋白表达水平均呈现表皮干细胞的特性:β1整合素、K15和α6整合素阳性,而CD71和K1/K10阴性。结论:所采用的分离纯化方法和培养条件适合猴表皮干细胞的分离纯化及生长。

AIM： Our study was designed to look for an easy and feasible approach to isolate and culture rhesus epidermal stem cells. METHODS： Skin specimens were cut into strips and immersed into 0.25% trypsin overnight. Then transparent cuticular layeres were striped off with ophthalmic microscopic forceps. The epithelial layers were scratched, harvested and transferred to culture in skin epithelium media. The cells in 2nd - 4th passages were harvested by 0.25 % trypsin and relayed in Ⅳ collagen plate at 37 ℃, 5 % CO2 for 20 min to harvest rapid attaching cells. Flow cytometric analysis, immunohistology and RT - PCR were conducted to identify rapid attaching cells. RESULTS： The specific protein and mRNA of epidermal stem cells （a6 integrin, K15 and β1 integrin） were identified in rapid attaching cells. No K1/K10 （marker of terminally differentiated cells） and CD71 expression were found. CONCLUSION： Our method of isolation and culture can apply for rhesus epidermal stem cells.