人皮肤表皮干细胞外泌体的分离培养与鉴定

Isolation,culture and identification of human skin epidermal stem cellexosomes

目的通过优化技术流程,探究人皮肤表皮干细胞(EPSCs)体外分离培养及表皮干细胞外泌体(EPSCs-Exo)分离纯化的方法。方法首先通过优化人源EPSCs的分离酶,运用改良的无血清培养液,添加10种因子刺激EPSCs生长,促进其分泌EPSCs-Exo,同时维持EPSCs干性和增殖性,延缓EPSCs的分化和成熟。进一步优化差速离心的条件,高效高纯度提取人源的EPSCs-Exo。对人皮肤组织进行免疫荧光染色溯源EPSCs;对分离的EPSCs进行Western blot和免疫荧光染色检测其表面多个标志物;对EPSCs-Exo从形态、粒径大小、多个胞内和膜上标志物进行鉴定。结果人皮肤组织分层表达表皮细胞的标志物,EPSCs高表达整合素α6(integrin-α6)、整合素β1(integrin-β1)、P63蛋白(P63)、细胞角蛋白19(CK19)等标志物,EPSCs上清液超速离心后的透射电镜显示茶托状结构,粒径大小检测直径介于30~150 nm,蛋白印迹显示Tsg101、CD9、CD63(溶酶体相关膜蛋白3)呈阳性,Calnexin、GAPDH呈阴性。结论该研究成功在体外分离培养了人源EPSCs并获得人源EPSCs-Exo。

Objective To explore method for isolating and culturing human epidermal stem cells(EPSCs)in vitro and isolating and purifying epidermal stem cell exsomes(EPSCs-Exo)by optimizing the technical process.Methods Firstly,the improved separating enzyme was used to isolate the EPSCs derived from human skin tissue.Then,an improved serum-free culture medium and 10 specific factors were combined to construct optimized 2D culture medium which could stimulate the growth of EPSCs,promote the secretion of EPSCs-Exo,maintain the stemness and proliferation of EPSCs,and delay the differentiation and maturation of EPSCs.Further,the conditions of differential centrifugation was optimized,and then the human EPSCs-Exo was successfully extracted with high efficiency and high purity.Results The human skin tissue was confirmed with the expressions of markers for epidermal cells.EPSCs were verified with high expression levels of integrin-α6,integrin-β1,P63 and CK19 by immunofluorescence staining and Western blot.The nanoparticle tracking analysis results showed the particles separated for EPSCs supernatant was saucepan with the detected diameter between 30-150 nm.The Western blot results showed the positive expression of membrane markers Tsg101,CD9 and CD63 and the negative expression of intracellular markers Calnexin and GAPDH.Conclusion The results show that the human-derived EPSCs have been successfully isolated and cultured in vitro,and the EPSCs-Exo have been successfully isolated and identified.