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.
目的 瞭解Wnt/β連環蛋白信號在人正常真皮Fb嚮肌成纖維細胞(MFb)錶型轉化中的作用及機製.方法 採用酶消化法分離培養人正常皮膚真皮Fb.(1)實驗1.按隨機數字錶法將細胞分為4組:對照組,採用無血清DMEM培養液(以下簡稱培養液)培養;TGF-β1組,用含10 ng/mL重組人TGF-β1(濃度下同)的培養液培養;Wnt3a組,用含150 ng/mL重組人Wnt3a(濃度下同)的培養液培養;TGF-β1+Wnt3a組,用含重組人TGF-β1和重組人Wnt3a的培養液培養.48 h後,分彆採用實時熒光定量PCR法和蛋白質印跡法,檢測Fb β連環蛋白和α平滑肌肌動蛋白(α-SMA)的mRNA及蛋白錶達水平.(2)實驗2.按照隨機數字錶法將細胞分為4組:對照組、TGF-β1組,處理方法均同實驗1;SB415286(糖原閤酶激酶3β阻斷劑)組,用含10 μmol/L SB415286(濃度下同)的培養液培養;TGF-β1+ SB415286組,用含重組人TGF-β1和SB415286的培養液培養.48 h後,分彆採用實時熒光定量PCR法和蛋白質印跡法檢測Fb α-SMA的mRNA和蛋白錶達水平;用免疫熒光細胞化學法檢測α-SMA暘性錶達情況.各項檢測重複3次,對數據進行方差分析及LSD-t檢驗.結果 (1)實驗1.①β連環蛋白的mRNA錶達水平:對照組、TGF-β1組、Wnt3a組和TGF-β1+Wnt3a組Fb組間比較,差異無統計學意義(F=0.302,P=0.823).②β連環蛋白的蛋白錶達水平:4組比較,差異有統計學意義(F=16.713,P=0.001).與對照組錶達水平(0.34±0.11)相比,TGF-β1組、Wnt3a組均顯著上調(0.73±0.12、0.82±0.17,t值分彆為3.028、3.727,P＜0.05或P＜0.01).TGF-β1+Wnt3a組(1.23±0.21)顯著高于其餘3組(t值分彆為6.911、3.883、3.184,P值均小于0.01).③α-SMA mRNA錶達水平:4組比較,差異有統計學意義(F=31.830,P=0.001);與對照組相比,TGF-β1組顯著上調,Wnt3a組下調(t值分彆為6.759、2.535,P＜0.05或P＜0.01);TGF-β1+Wnt3a組低于TGF-β1組(t=4.532,P＜0.01).④α-SMA蛋白錶達水平:對照組、TGF-β1組、Wnt3a組、TGF-β1+ Wnt3a組分彆為0.83±0.17、1.43±0.20、0.53±0.12、0.89±0.14(F=16.597,P=0.001).與對照組相比,TGF-β1組顯著上調,Wnt3a組下調(t值分彆為4.582、2.291,P＜0.05或P ＜0.01);TGF-β1 +Wnt3a組低于TGF-β1組(t =4.123,P＜0.01).(2)實驗2.①α-SMA mRNA錶達水平:4組比較,差異有統計學意義(F=34.101,P=0.001).SB415286組明顯低于對照組(t =2.511,P＜0.05),TGF-β1+SB415286組明顯低于TGF-β1組(t=3.587,P＜0.01).②α-SMA蛋白錶達水平:4組細胞比較,差異有統計學意義(F =11.381,P=0.003).SB415286組顯著低于對照組(t=2.364,P＜0.05),TGF-β1+ SB415286組低于TGF-β1組(t=2.556,P＜0.05).③免疫熒光細胞化學法檢測顯示,對照組Fb α-SMA錶達微弱;TGF-β1組α-SMA錶達較對照組顯著增彊(t =11.198,P＜0.01);SB415286組錶達較對照組減少,TGF-β1+ SB415286組錶達較TGF-β1組顯著減少(t=5.902,P＜0.01).結論 Wnt/β連環蛋白信號可能參與Fb細胞錶型轉化,併且對TGF-β1所介導的促轉化效應起負性調控作用.
목적 료해Wnt/β련배단백신호재인정상진피Fb향기성섬유세포(MFb)표형전화중적작용급궤제.방법 채용매소화법분리배양인정상피부진피Fb.(1)실험1.안수궤수자표법장세포분위4조:대조조,채용무혈청DMEM배양액(이하간칭배양액)배양;TGF-β1조,용함10 ng/mL중조인TGF-β1(농도하동)적배양액배양;Wnt3a조,용함150 ng/mL중조인Wnt3a(농도하동)적배양액배양;TGF-β1+Wnt3a조,용함중조인TGF-β1화중조인Wnt3a적배양액배양.48 h후,분별채용실시형광정량PCR법화단백질인적법,검측Fb β련배단백화α평활기기동단백(α-SMA)적mRNA급단백표체수평.(2)실험2.안조수궤수자표법장세포분위4조:대조조、TGF-β1조,처리방법균동실험1;SB415286(당원합매격매3β조단제)조,용함10 μmol/L SB415286(농도하동)적배양액배양;TGF-β1+ SB415286조,용함중조인TGF-β1화SB415286적배양액배양.48 h후,분별채용실시형광정량PCR법화단백질인적법검측Fb α-SMA적mRNA화단백표체수평;용면역형광세포화학법검측α-SMA양성표체정황.각항검측중복3차,대수거진행방차분석급LSD-t검험.결과 (1)실험1.①β련배단백적mRNA표체수평:대조조、TGF-β1조、Wnt3a조화TGF-β1+Wnt3a조Fb조간비교,차이무통계학의의(F=0.302,P=0.823).②β련배단백적단백표체수평:4조비교,차이유통계학의의(F=16.713,P=0.001).여대조조표체수평(0.34±0.11)상비,TGF-β1조、Wnt3a조균현저상조(0.73±0.12、0.82±0.17,t치분별위3.028、3.727,P＜0.05혹P＜0.01).TGF-β1+Wnt3a조(1.23±0.21)현저고우기여3조(t치분별위6.911、3.883、3.184,P치균소우0.01).③α-SMA mRNA표체수평:4조비교,차이유통계학의의(F=31.830,P=0.001);여대조조상비,TGF-β1조현저상조,Wnt3a조하조(t치분별위6.759、2.535,P＜0.05혹P＜0.01);TGF-β1+Wnt3a조저우TGF-β1조(t=4.532,P＜0.01).④α-SMA단백표체수평:대조조、TGF-β1조、Wnt3a조、TGF-β1+ Wnt3a조분별위0.83±0.17、1.43±0.20、0.53±0.12、0.89±0.14(F=16.597,P=0.001).여대조조상비,TGF-β1조현저상조,Wnt3a조하조(t치분별위4.582、2.291,P＜0.05혹P ＜0.01);TGF-β1 +Wnt3a조저우TGF-β1조(t =4.123,P＜0.01).(2)실험2.①α-SMA mRNA표체수평:4조비교,차이유통계학의의(F=34.101,P=0.001).SB415286조명현저우대조조(t =2.511,P＜0.05),TGF-β1+SB415286조명현저우TGF-β1조(t=3.587,P＜0.01).②α-SMA단백표체수평:4조세포비교,차이유통계학의의(F =11.381,P=0.003).SB415286조현저저우대조조(t=2.364,P＜0.05),TGF-β1+ SB415286조저우TGF-β1조(t=2.556,P＜0.05).③면역형광세포화학법검측현시,대조조Fb α-SMA표체미약;TGF-β1조α-SMA표체교대조조현저증강(t =11.198,P＜0.01);SB415286조표체교대조조감소,TGF-β1+ SB415286조표체교TGF-β1조현저감소(t=5.902,P＜0.01).결론 Wnt/β련배단백신호가능삼여Fb세포표형전화,병차대TGF-β1소개도적촉전화효응기부성조공작용.
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 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.
