甲基化酶抑制剂5氮杂-2-脱氧胞苷对瘢痕疙瘩成纤维细胞TGF-β／smad信号通路的影响  

Effect of the methylation enzyme inhibitors of 5-aza-2-deoxycytidine on the TGF-β/smad signal transduction pathway in human keloid fibroblasts

作　　者：邹崎葩[1] 杨娥[1] 张恒术[1] 

机构地区：[1]重庆医科大学附属第一医院整形美容外科,重庆400016

出　　处：《中华整形外科杂志》2013年第4期285-289,共5页Chinese Journal of Plastic Surgery

基　　金：重庆市教委科技项目（KJ090317）

摘　　要：目的探讨甲基化酶抑制剂5-氮杂-2-脱氧胞苷对瘢痕疙瘩成纤维细胞TGF-β／smad信号通路的影响。方法收集瘢痕疙瘩及正常皮肤组织各15例，免疫组化检测磷酸化smad2（P-smad2）和磷酸化smad3（p-smad3）在瘢痕疙瘩和正常皮肤中的阳性表达率；将瘢痕疙瘩分为实验组和对照组，实验组以5，氮杂-2-脱氧胞苷（5×10^-5mmol／L）干预，对照组用等量的DMEM培养液，采用组织块法培养人瘢痕疙瘩成纤维细胞，流式细胞仪分析5-氮杂-2-脱氧胞苷（5×10^-5mol／L）对瘢痕疙瘩成纤维细胞周期及凋亡的影响；Westernblot检测各组p-smad2、p-smad3、TGF-β1和smad7的表达变化；细胞免疫荧光染色法观察各组瘢痕疙瘩成纤维细胞内p-smad2和p-smad3蛋白的影响。结果瘢痕疙瘩组织中p-smad2和p-smad3阳性表达率明显高于正常皮肤组织中p-smad2和P-smad3阳性表达。流式细胞仪显示5-氮杂-2-脱氧胞苷干预瘢痕疙瘩成纤维细胞后，细胞停滞于G0／G1期比例增加并且细胞凋亡率增加，瘢痕疙瘩成纤维细胞中p-smad2和p-smad3蛋白的表达减少，同时，TGF—β1蛋白表达减少，smad7蛋白表达回升。此外，5-氮杂-2-脱氧胞苷抑制smad2和smad3磷酸化及核转移。结论甲基化酶抑制剂5-氮杂-2-脱氧胞苷可抑制瘢痕疙瘩成纤维细胞的增殖及促进其凋亡，其作用机制可能与抑制TGF-β／srnad信号通路有关。Objective To investigate the effect of 5-aza-2-deoxycytidine on the TGF-β/smad signal transduction pathway in human keloid fibroblasts（KFSs）. Methods Firstly, immunohistochemical method was used to detect the positive expression rate of phoshpo-smad2 and phoshpo-smad3 in the specimens of 15 cases of keloid and 15 cases of normal skin. The keloid fibroblasts were cultured in vitro with 5-aza-2-deoxycytidine（experimental group） or with DMEM （control group）. The effect of 5-aza-2- deoxycytidine on the cell cycle and apoptosis of fibroblasts was analysed with flowcytometry （ FCM ）. Transforming growth factor （TGF）-β, Smad7, phoshpo-smad2 and phoshpo-smad3 were analyzed by Western Blot, and Immunofluorescence. Results It was found that the positive expression of phoshpo- smad2 and phoshpo-smad3 in keloid were higher than those in normal skin. The FCM showed that the proportion of cells in G0/G1 stage was increased, and so does the proportion of apoptosis cells in keloid fibroblasts intervented by 5-aza-2-deoxycytidine. The expression of TGF-β ,phoshpo-smad2 and phoshpo- smad3 protein were significantly suppressed while the expression of smad7 protein increased in keloid fibroblasts with 5-aza-2-deoxycytidine. In addition, 5-aza-2-deoxycytidine reversed phosphorylation and nuclear translocation of smad2 and smad3. Conclusions 5-aza-2-deoxycytidine, methylaze inhibitors, inhibits cell proliferation and promotes apoptosis of KFSs, which may be associated with the suppression of TGF-β/smad signal pathway.

关 键 词：瘢痕疙瘩 成纤维细胞 5-氮杂-2-脱氧胞苷 TGF-Β SMAD通路 

分 类 号：R6[医药卫生—外科学]