成人表皮干细胞和汗腺细胞的体外分离和培养

摘要

Background Research on the culture and identification of human Epidermal Stem Cells (ESCs)and Sweat Gland Cells(SGCs)In vitro,Which can provide the foundation for the feasibility of Epidermal Stem Cells(ESCs)to regenerate Sweat Gland. Objective To study the effective method on isolation and culture of sweat gland cells(SGCS)and human Epidermal Stem Cell(ESCs)in vitro. Methods Foreskin tissue with different ages were collected by Urology Department,after being fully cleaned and disinfected,we removed the subcutaneous tissue;then We trimmed left tissue to 0.5cm × 0.5cm size;with the help of Ⅳ collagen to purify and enrich adult's Epidermal stem cells(ESCs). Epidermal Stem Cells'form were observed by inverted phase contrast microscope:Immunofluorescence staining to analysis adult's epidermal stem cells'phenotype;CCK-8 was used to detect proliferation curve;We used collagenase digestion to isolate sweat gland from intact skin,and than amplified and identified Sweat Gland Cells(SGCs). Results Using inverted phase contrast microscope to observe isolated adult Epidermal Stem Cells,finding out that Epidermal Stem Cells were cultured like oval,and closely linked among each other,like cobblestone;Immunofluorescence stain was used to find that Epidermal Stem Cells expressed CK19,β1-integrin.We observed that Sweat Gland Cells were cultured like flat polygonal and expressing markers like CK7,CK18,CK19 and CEA. Conclusion In a word,it is feasible to separate and culture Epidermal Stem Cells with adherent method and Sweat Gland Cells with collagenase digestion method.%背景：人表皮干细胞和汗腺细胞的分离培养及鉴定，为探讨人表皮干细胞再生汗腺的可行性打下基础。目的：探讨体外分离培养人表皮干细胞及汗腺细胞的有效方法。方法：采集不同年龄段的泌尿外科患者术后包皮组织，充分清洗消毒后去除皮下组织，将其修剪成0.5cm×0.5cm的皮片，用Ⅳ型胶原纯化、富集成人表皮干细胞，倒置相差显微镜观察细胞形态：使用免疫荧光染色对成人表皮干细胞表型进行分析；CCK-8检测细胞的增殖曲线；采用胶原酶消化法从人全层无损伤皮肤中分离提取汗腺细胞，并进行扩增和鉴定。结果：倒置相差显微镜下见分离培养的成人表皮干细胞呈卵圆形、细胞之间紧密相连，呈铺路石状，免疫荧光染色显示细胞表达CK19、β1整合素。倒置相差显微镜下见汗腺细胞呈扁平多角形，表达汗腺细胞标志细胞CK7、18、19及CEA。结论：本实验结果说明胶原酶消化法分离培养成人表皮干细胞及汗腺样细胞是可行的。