HaCaT条件培养基联合全反式维甲酸诱导人脂肪干细胞向表皮细胞分化的初步研究

Investigation on HaCaT cell condition medium combined with all-trans-retinoic acid initiating differentiation of human adipose-derived stem cells into epidermal cells

目的探讨HaCaT条件培养基能否促进全反式维甲酸(all-trans retinoic acid,ATRA)诱导人脂肪干细胞向表皮细胞的分化。方法体外分离培养脂肪干细胞,通过流式细胞术检测CD34、CD45、CD73、CD90、CD105蛋白的表达以及成脂成骨诱导分化鉴定脂肪干细胞。Transwell小室构建气液界面培养模型,配制含ATRA、表皮生长因子(epidermal growth factor,EGF)、角质细胞生长因子(keratinocyte growth factor,KGF)的诱导培养基A以及含50%HaCaT上清液、ATRA、EGF、KGF的诱导培养基B。实验分3组,即诱导培养基A组、诱导培养基B组及常规培养未诱导组,气液界面诱导培养12 d,流式细胞术检测诱导后细胞CK14、15、16、19广谱细胞角蛋白(pan cytokeratin,Pan-CK)的表达。免疫荧光法检测CK14的表达。结果脂肪干细胞成表皮诱导培养后,流式细胞术检测结果显示诱导组B Pan-CK表达率大于诱导组A,两组均大于未诱导组[分别为(22.0±3.5)%、(11.9±2.7)%、(1.1±0.3)%,P<0.01],免疫荧光法检测结果显示诱导组均具有CK14荧光表达,诱导组B CK14表达率大于诱导组A[分别为(19.5±7.0)%、(10.8±5.7)%,P<0.01],未诱导组未见CK14荧光表达。结论 HaCaT条件培养基可加强ATRA诱导人脂肪干细胞向表皮细胞的分化的能力。

Objective To investigate the possibility of enhancing the inducing rate of adipose-derived stem cells （ASCs） into epidermal cells in the medium containing all-trans retinoic acid （ATRA） by supplementing with HaCaT condition medium. Methods ASCs were isolated and identified by detecting the expression of CD34, CD45, CD73, CD90, and CD105 with flow cytometry and differentiating into adipose and osteoblast lineage in the induction medium. The air-liquid interface cell culture model was established with the Transwell Room. The induction medium A contained ATRA, epidermal growth factor （EGF）, and keratinocyte growth factor （KGF）, while the induction medium B contained ATRA, EGF, KGF, and HaCaT condition medium. Experiment was divided into three groups cultured for 12 days： induction medium A （group A）, induction medium B （group B）, basic medium （group C）. The epidermal cell surface markers： cytokeratin （CK） 14, 15, 16, 19 （Pan-CK） were detected by flow cytometry and CK14 were identified by immunofluorescence stain. Results After induction for 12 days, flow cytometry showed that the positive rate of Pan-CK in group B [（22.0±3.5）%] was higher than that in group A [（11.9±2.7）%], which were both higher than that in group C [（1.1±0.3）%], and the differences were statistical significantly （P〈0.01）. Immunofluorescence stain showed that the positive rate of CK14 in group B was higher than that in group A [（19.5±7.0）%vs. （10.8±5.7）%, P〈0.01], and the expression of CK14 was negative in group C. Conclusion HaCaT condition medium can enhance the ability of ASCs differentiation into epidermal cells in the culture medium containing ATRA.