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正常皮肤真皮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10 ng/mL重组人TGF-β1(浓度下同)的培养液培养;Wnt3a组,用含150 ng/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.48 h后,分别采用实时荧光定量PCR法和蛋白质印迹法,检测Fb β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1;SB415286(糖原合酶激酶3β阻断剂)组,用含10 μmol/L SB415286(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.48 h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mRNA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-SMA mRNA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-β1组显著上调,Wnt3a组下调(t值分别为6.759、2.535,P＜0.05或P＜0.01);TGF-β1+Wnt3a组低于TGF-β1组(t=4.532,P＜0.01).④α-SMA蛋白表达水平:对照组、TGF-β1组、Wnt3a组、TGF-β1+ Wnt3a组分别为0.83±0.17、1.43±0.20、0.53±0.12、0.89±0.14(F=16.597,P=0.001).与对照组相比,TGF-β1组显著上调,Wnt3a组下调(t值分别为4.582、2.291,P＜0.05或P ＜0.01);TGF-β1 +Wnt3a组低于TGF-β1组(t =4.123,P＜0.01).(2)实验2.①α-SMA mRNA表达水平:4组比较,差异有统计学意义(F=34.101,P=0.001).SB415286组明显低于对照组(t =2.511,P＜0.05),TGF-β1+SB415286组明显低于TGF-β1组(t=3.587,P＜0.01).②α-SMA蛋白表达水平:4组细胞比较,差异有统计学意义(F =11.381,P=0.003).SB415286组显著低于对照组(t=2.364,P＜0.05),TGF-β1+ SB415286组低于TGF-β1组(t=2.556,P＜0.05).③免疫荧光细胞化学法检测显示,对照组Fb α-SMA表达微弱;TGF-β1组α-SMA表达较对照组显著增强(t =11.198,P＜0.01);SB415286组表达较对照组减少,TGF-β1+ SB415286组表达较TGF-β1组显著减少(t=5.902,P＜0.01).结论 Wnt/β连环蛋白信号可能参与Fb细胞表型转化,并且对TGF-β1所介导的促转化效应起负性调控作用.

abstractsObjective 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 μ mol/L SB415286 ( the same concentration for following experiments).Cells in TGF-β1 + SB415286group were cultured with nutrient solution containing TGF-β1 and SB415286.The mRNA and protein expression 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 =0.302,P =0.823 ).There were statistically significant differences among four groups in β-catenin protein level ( F =16.713,P =0.001 ).The protein level of β-catenin was higher in TGF-β1 group (0.73 ±0.12) and Wnt3a group (0.82 ±0.17) than in control group (0.34 ±0.11,with t values respectively 3.028,3.727,P ＜ 0.05 or P ＜ 0.01 ).The protein level of β-catenin in TGF-β1 +Wnt3a group ( 1.23 ± 0.21 ) was higher than that of the other three groups ( with t values respectively 6.911,3.883,3.184,P values all below 0.01 ).There were statistically significant differences among four groups in α-SMA mRNA level ( F =31.830,P =0.001 ).Compared with that of control group,the expression level of α-SMA mRNA was up-regulated in TGF-β1 group and down-regulated in Wnt3a group (with t values respectively 6.759,2.535,P ＜0.05 or P ＜0.0l ).The expression level of α-SMA mRNA in TGF-β1 + Wnt3a group was lower than that of TGF-β1 group ( t =4.532,P ＜ 0.01 ).The protein levels of α-SMA in control,TGF-β1,Wnt3a,and TGF-β1 + Wnt3a groups were respectively 0.83 ±0.17,1.43 ±0.20,0.53±0.12,and0.89 ±0.14 ( F =16.597,P =0.001).Compared with that of control group,the protein level of α-SMA was up-regulated in TGF-β1 group and down-regulated in Wnt3a group ( with t values respectively 4.582,2.291,P ＜ 0.05 or P ＜ 0.01 ).The protein level of α-SMA in TGF-β1 + Wnt3a group was lower than that of TGF-β1 group ( t =4.123,P ＜0.01 ).(2) Experiment two.There were statistically significant differences among four groups in α-SMA mRNA level ( F =34.101,P =0.001 ).The α-SM A mRNA level in SB415286 group was lower than that of control group ( t =2.511,P ＜ 0.05 ).The α-SMA mRNA level in TGF-β1 + SB415286 group was lower than that of TGF-β1 group ( t =3.587,P ＜0.01 ).There were statistically significant differences among four groups in α-SMA protein level ( F =11.381,P =0.003).The α-SMA protein level was lower in SB415286 group than in control group ( t =2.364,P ＜0.05 ).The α-SMA protein level was down-regulated in SB415286 + TGF-β1 group as compared with that of TGF-β1 group ( t =2.556,P ＜ 0.05 ).There were few α-SMA-positive fibroblasts in control group.Compared with that of control group,the expression of α-SMA was significantly increased in TGF-β1 group ( t =11.198,P ＜ 0.01 ),and the expression of α-SMA was down-regulated in SB415286 group.Meanwhile,the expression of α-SMA in TGF-β1 + SB415286 group were significantly lower than that of TGF-β1 group ( t =5.902,P ＜0.01 ). Conclusions The Wnt/β-catenin signaling might be involved in the fibroblasts-myofibroblasts transition,and it negatively regulate the TGF-β1-mediated profibrotic effects.