华西医学

华西医学

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

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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.

关键词: 人脂肪干细胞; 表皮细胞; 全反式维甲酸; HaCaT 细胞

Key words: Human adipose-derived stem cells; Epidermal cells; All-trans-retinoic acid; HaCaT cell

引用本文: 林文韬, 刘晓雪, 刘勇, 陈俊杰, 岑瑛. 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. 华西医学, 2018, 33(3): 325-331. doi: 10.7507/1002-0179.201611154 复制

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