鞘氨醇激酶在人瘢痕疙瘩成纤维细胞中的表达及其作用

Expression and function of sphingosine kinases 1 and 2 in human keloid fibroblasts

目的探讨鞘氨醇激酶（SphK）1和SphK2在人瘢痕疙瘩成纤维细胞中的表达及其作用。方法取12例瘢痕疙瘩患者手术切除的瘢痕疙瘩及其周围正常皮肤标本，原代组织块法培养成纤维细胞，分为正常皮肤组、瘢痕疙瘩组和瘢痕疙瘩加转化生长因子β1（TGF—β1）组（瘢痕疙瘩＋TGF-β1组）。用免疫荧光法观察各组细胞中SphK1和SphK2蛋白的分布；实时PCR法检测SphK1和SphK2mRNA的表达；蛋白质印迹法检测SphK1和SphK2蛋白的表达。结果Sphk1蛋白在3组成纤维细胞中均主要分布于细胞核；而Sphk2蛋白在细胞核和胞质中均有表达。瘢痕疙瘩组SphK1 mRNA和蛋白表达（分别为0．0608±0．0190、0．8308±0．1093）明显高于正常皮肤组（分别为0．03834±0．0147、0．68004±0．1126，均P〈0．05），但明显低于瘢痕疙瘩＋TGF-β1组[分别为0．07904±0．0280（P〈0．05）、1．4267±0．1938（P〈0．01）]；SphK2 mRNA和蛋白在3组中的表达差异无统计学意义（均P〉0．05）。结论SphK1在瘢痕疙瘩成纤维细胞增殖过程中具有重要作用。TGF-β1可促进SphK1在瘢痕疙瘩成纤维细胞中的表达，提示SphK1可能参与了TGF-β信号转导通路的调节。

Objective To study the expression and function of sphingosine kinase （SphK）1 and SphK2 in human keloid fibroblasts. Methods Specimens of keloid and surrounding normal skin were collected from 12 patients with keloid during operation. Primary fibroblasts were isolated, cultured, and randomly divided into 3 groups：normal skin group, keloid group, and keloid with transforming growth factor （TGF）-β1 group cultured with TGF-β1 for 48 h. Immunofluorescence technique was used to detect the location of SphK1 and SphK2 protein. Real-time PCR and Western blotting were used to measure the mRNA and protein expression levels of SphK1 and SphK2. Results Sphk1 protein was localized primarily in the nuclei of the fibroblasts, and Sphk2 protein was detected both in the cytoplasm and nuclei in the 3 groups. The mRNA and protein levels of Sphkl in the keloid group were （0. 0608 ± 0. 0190） and （0. 8308 ± 0. 1093 ） respectively,both significantly higher than those of the normal skin group [ （0. 0383 ±. 0147） and （0. 6800 ±0. 1126 ） respectively, both P 〈 0. 05 ] , but significantly lower than those of the keloid fibroblasts with TGF-β1 group [ （0. 0790 ±0. 0280 ） , P 〈 0. 05, and （ 1. 4267 ±0. 1938 ）, P 〈 0. 01 ]. There was no significant differences in the Sphk2 mRNA and protein levels among these 3 groups （all P 〉 0. 05 ）. Conclusions Sphkl plays a leading role in keloid pathogenesis. The SphK1 mRNA and protein levels are increased by TGF-β1 stimulation in keloid fibroblasts, perhaps indicating that Sphkl is involved in TGF-β signal transduction pathway.