Wnt／β连环蛋白信号在人真皮成纤维细胞表型转化中的作用及机制  

Effects of Wnt/β-catenin signaling on the phenotype change of human dermal fibroblasts and its mechanism

作　　者：刘佳琦 潘庆[2] 王耘川 刘洋[1] 王耀军 白丽 白晓智 胡大海 

机构地区：[1]第网军医大学西京医院全军烧伤中心、烧伤与皮肤外科,西安710032 [2]解放军第二炮兵工程学院门诊部

出　　处：《中华烧伤杂志》2012年第4期282-287,共6页Chinese Journal of Burns

摘　　要：目的了解Wnt／β连环蛋白信号在人正常真皮Fb向肌成纤维细胞（MFb）表型转化中的作用及机制。方法采用酶消化法分离培养人难常皮肤真皮Fb。（1）实验1。按随机数字表法将细胞分为4组：对照组，采用无血清DMEM培养液（以下简称培养液）培养；TGF-β1组，用含10ng／ml,氧组人TGF-β1（浓度下同）的培养液培养；Wnt3a组，用含150ng／mL重组人Wnt3a（浓度下同）的培养液培养；TGF-β1＋Wnt3a组，用含重组人TGF-β1和重组人Wnt3a的培养液培养。48h后，分别采用实时荧光定量PCR法和蛋白质印迹法，检测Fbβ连环蛋白和α平滑肌肌动蛋白（α-SMA）的mRNA及蛋白表达水平：（2）实验2。按照随机数字表法将细胞分为4组：对照组、TGF-β1组，处理方法均同实验l；SB415286（糖原合酶激酶3β阻断剂）组，用含10μmol／LSB415286（浓度下同）的培养液培养；TGF-β1＋SB415286组，用含重组人TGF-β1和SB415286的培养液培养。48h后，分别采用实时荧光定量PCR法和蛋白质印迹法检测Fb α-SMA的tuRNA和蛋白表达水平；用免疫荧光细胞化学法检测α-SMA阳性表达情况。各项检测重复3次，对数据进行方差分析及LSD-t检验。结果（1）实验1：①β连环蛋白的mRNA表达水平：对照组、TGF-β1组、Wnt3a组和TGF-β1＋Wnt3a组Fb组间比较，差异无统计学意义（F=0．302，P=0．823）。②β连环蛋白的蛋白表达水平：4组比较，差异l有统计学意义（F=16.713，P=0．001），与对照组表达水平（0．34±0．11）相比，TGF-β1组、Wnt3a组均显著上调（0．73±0．12、0．82-0．17，t值分别为3．028、3．727，P〈0．05或P〈0．01）。TGF-β1＋Wnt3a组（1．23±0．21）显著高于其余3组（t值分别为6．911、3．883、3．184，P值均小于0．01）。③α-SMAmRNA表达水平：4组比较，差异有统计学意义（F=31．830，P=0．001）；与对照组相比，TGF-β．组硅著上调，wnt3a组下Objective To study the role of Wnt/β-catenin signaling in the phenotype change of normal skin fibroblasts （NFb） into myofibroblasts and the underlying mechanism. Methods NFb were isolated by collagenase digestion and cultured. （ 1 ） Experiment one. NFb were divided into four groups according to the random number table. Cells in control group were cultured with serum-free DMEM nutrient solution （briefly called nutrient solution）. Cells in TGF-β1 group were cultured with nutrient solution containing 10 ng/mL recombinant human TGF-β1（the same concentration for following experiments）. Cells in Wnt3a group were cultured with nutrient solution containing 150 ng/mL Wnt3a （ the same concentration for following experiments）. Cells in TGF-β1 ＋ Wnt3a group were cultured with nutrient solution containing TGF-β1 and Wnt3a. The mRNA and protein expression levels of β-catenin and α-smooth muscle actin （α-SMA） were determined by real-time fluorescent quantitative PCR and Western blotting at post culture hour （PCH） 48. （2） Experiment two. NFb were divided into four groups according to the random number table. Cells in control group and TGF-β1 group were treated as those in the corresponding groups in experiment one. Cells in SB415286 （glycogen synthase kinase-3β inhibitor） group were cultured with nutrient solution containing 10 μmol/L SB415286 （the same concentration for following experiments）. Cells in TGF-β1＋ SB415286 group were cultured with nutrient solution containing TGF-β1 and SB415286. The mRNA and protein expres- sion levels of α-SMA were determined by real-time fluorescent quantitative PCR and Western blotting, and the α-SMA-positive myofibroblasts were detected by immunofluorescence cytochemical staining at PCH 48. The experiments were all repeated for three times. Data were processed with analysis of variance and LSD- t test. Results （1） Experiment one. There was no statistically significant difference among four groups in β-catenin mRNA level （ F = 0.

关 键 词：成纤维细胞 转化生长因子1 WNT蛋白质类 表型转化 Β连环蛋白 肌成纤维细胞 

分 类 号：R363[医药卫生—病理学]