TGF-β1诱导瘢痕疙瘩表皮细胞发生上皮-间质转化的研究

In vitro study of TGF-β1-induced epithelial-mesenchymal transition of keloid epithelial cells

目的 通过在体外应用TGF-β1诱导瘢痕疙瘩表皮细胞,探讨其是否发生上皮-间质转化,以及上皮-间质转化对表皮细胞中干细胞表面标志的影响.方法 采集耳部瘢痕疙瘩标本培养表皮细胞,以培养基中终浓度为1 ng/ml的TGF-β1诱导瘢痕疙瘩表皮细胞5d作为实验组,以未诱导的瘢痕疙瘩表皮细胞作为阴性对照组,每组样本均为3例.分别采用免疫荧光染色、Real-time PCR和Western blot检测上皮-问质转化相关标志和基因的表达,并采用Real-time PCR检测表皮干细胞表面标志的表达.采用独立样本t检验对检测结果进行统计学分析.结果 上皮-间质转化相关基因Snail2的mRNA表达从诱导前的0.91±0 23上升至诱导后的1.69 ±0.10,间质细胞标志波形蛋白由诱导前5.86±2.07明显增加至诱导后的24.29 ±5.39（P ＜0.05）,而上皮标志E-钙粘蛋白的mRNA表达由诱导前的1.06 ±0.19下降为诱导后的0.65 ±0.09,表达减弱;于细胞表面标志CD29和Lgr6的mRNA表达分别由诱导前0.55±0.14和1.61±0.31增加至诱导后1.19 ±0.12和3.84±0.62,均明显升高（P＜0.05）.免疫荧光染色和Western blot均证实细胞E-钙粘蛋白减少,波形蛋白增加,同时p-Smad3表达增加.结论 TGF-β1通过诱导Snail2基因上调启动瘢痕疙瘩表皮细胞发生上皮-间质转化,此过程有TGF-β1/Smad3信号通路参与,而瘢痕疙瘩表皮细胞中干细胞的表面标志发生改变.

Objective To construct and characterize the TGF-β1 induced epithelial-mesenchymal transition （EMT） model of keloid epithelial cells in vitro,and to investigate the expression of epithelial stem cells related surface markers in keloid epithelial cells during EMT induction.Methods The epithelial cells from 3 keloid samples of ears were cultured in vitro and induced by transforming growth factor beta1 （TGF-β1,1 ng/ml） for 5 days,which was the experimental group,the same cells untreated were considered as the negative control group.The expressions of EMT-associated markers and regulative genes were detected using immunofluorescence staining,real-time PCR and western blot analysis.Then the surface markers of cpithelial stem cells were detected using real-time PCR.Statistical significance was determined using Independent-Samples t Test,a p value less than 0.05 was considered statistically significant.Results The mRNA expression of transcription factor snail2 and mesenchymal-specific marker vimentin increased significantly in TGF-β1 induced keloid epithelial cells （P 〈 0.05）,in which snail2 increasing from 0.91 ±0.23 to 1.69 ±0.10,and vimentin from 5.86 ±2.07 to 24.29 ±5.39.Whereas the mRNA expression of epithelial-specific marker E-cadherin decreased from 1.06 ± 0.19 to 0.65 ± 0.09.The mRNA expression of CD29 and Lgr6,two surface markers of epithelial stem cells,significantly increased after induction of the TGF-β1 （P 〈 0.05）,from 0.55 ± 0.14 and 1.61 ± 0.31 to 1.19 ± 0.12 and 3.84 ± 0.62 respectively.In induced cells,the immunofluorescence results showed staining of E-cadherin became faint,but the number of positive staining cells of vimentin increased.Western blot confirmed the protein expression of E-cadherin weakened,and the vimentin and p-Smad3 enhanced （P 〈0.05）.Conclusions TGF-β1 initiated EMT in keloid epithelial cells by inducing the up-regulation of snail2,and TGF-β1/Smad3 signaling pathway was involved in EMT.EMT could change the phenotype of epitbelial stem cells in keloid.