Liu Jia-Qi, Pan Qing, Wang Yun-Chuan, Liu Yang, Wang Yao-Jun, Bai Li, Bai Xiao-Zhi, Hu Da-Hai
Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Zhonghua Shao Shang Za Zhi. 2012 Aug;28(4):282-7.
To study the role of Wnt/beta-catenin signaling in the phenotype change of normal skin fibroblasts (NFb) into myofibroblasts and the underlying mechanism.
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-beta1 group were cultured with nutrient solution containing 10 ng/mL recombinant human TGF-beta1 (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-beta1 + Wnt3a group were cultured with nutrient solution containing TGF-beta1 and Wnt3a. The mRNA and protein expression levels of beta-catenin and alpha-smooth muscle actin (alpha-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-beta1 group were treated as those in the corresponding groups in experiment one. Cells in SB415286 (glycogen synthase kinase-3beta inhibitor) group were cultured with nutrient solution containing 10 micromol/L SB415286 (the same concentration for following experiments). Cells in TGF-beta1 + SB415286 group were cultured with nutrient solution containing TGF-beta1 and SB415286. The mRNA and protein expression levels of alpha-SMA were determined by real-time fluorescent quantitative PCR and Western blotting, and the alpha-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.
(1) Experiment one. There was no statistically significant difference among four groups in beta-catenin mRNA level (F = 0.302, P = 0.823). There were statistically significant differences among four groups in beta-catenin protein level (F = 16.713, P = 0.001). The protein level of beta-catenin was higher in TGF-beta1 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 beta-catenin in TGF-beta1 + 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 alpha-SMA mRNA level (F = 31.830, P = 0.001). Compared with that of control group, the expression level of alpha-SMA mRNA was up-regulated in TGF-beta1 group and down-regulated in Wnt3a group (with t values respectively 6.759, 2.535, P < 0.05 or P < 0.01). The expression level of alpha-SMA mRNA in TGF-beta1 + Wnt3a group was lower than that of TGF-beta1 group (t = 4.532, P < 0.01). The protein levels of alpha-SMA in control, TGF-beta1, Wnt3a, and TGF-beta1 + Wnt3a groups were respectively 0.83 +/- 0.17, 1.43 +/- 0.20, 0.53 +/- 0.12, and 0.89 +/- 0.14 (F = 16.597, P = 0.001). Compared with that of control group, the protein level of alpha-SMA was up-regulated in TGF-beta1 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 alpha-SMA in TGF-beta1 + Wnt3a group was lower than that of TGF-beta1 group (t = 4.123, P < 0.01). (2) Experiment two. There were statistically significant differences among four groups in alpha-SMA mRNA level (F = 34.101, P = 0.001). The alpha-SMA mRNA level in SB415286 group was lower than that of control group (t = 2.511, P < 0.05). The alpha-SMA mRNA level in TGF-beta1 + SB415286 group was lower than that of TGF-beta1 group (t = 3.587, P < 0.01). There were statistically significant differences among four groups in alpha-SMA protein level (F = 11.381, P = 0.003). The alpha-SMA protein level was lower in SB415286 group than in control group (t = 2.364, P < 0.05). The alpha-SMA protein level was down-regulated in SB415286 +TGF-beta1 group as compared with that of TGF-beta1 group (t = 2.556, P < 0.05). There were few alpha-SMA-positive fibroblasts in control group. Compared with that of control group, the expression of alpha-SMA was significantly increased in TGF-beta1 group (t =11.198, P < 0.01), and the expression of alpha-SMA was down-regulated in SB415286 group. Meanwhile, the expression of alpha-SMA in TGF-beta1 + SB415286 group were significantly lower than that of TGF-beta1 group (t = 5.902, P < 0.01).
The Wnt/beta-catenin signaling might be involved in the fibroblasts-myofibroblasts transition, and it negatively regulate the TGF-beta1 -mediated profibrotic effects.
研究Wnt/β-连环蛋白信号通路在正常皮肤成纤维细胞（NFb）向肌成纤维细胞表型转化中的作用及其潜在机制。
采用胶原酶消化法分离培养NFb。（1）实验一。将NFb按随机数字表法分为四组。对照组细胞用无血清DMEM培养液（简称培养液）培养。转化生长因子β1（TGF-β1）组细胞用含10 ng/mL重组人TGF-β1的培养液培养（后续实验浓度相同）。Wnt3a组细胞用含150 ng/mL Wnt3a的培养液培养（后续实验浓度相同）。TGF-β1 + Wnt3a组细胞用含TGF-β1和Wnt3a的培养液培养。于培养后48小时采用实时荧光定量PCR和蛋白质印迹法检测β-连环蛋白和α-平滑肌肌动蛋白（α-SMA）的mRNA和蛋白表达水平。（2）实验二。将NFb按随机数字表法分为四组。对照组和TGF-β1组细胞处理同实验一相应组。糖原合酶激酶-3β抑制剂（SB415286）组细胞用含10 μmol/L SB415286的培养液培养（后续实验浓度相同）。TGF-β1 + SB415286组细胞用含TGF-β1和SB415286的培养液培养。于培养后48小时采用实时荧光定量PCR和蛋白质印迹法检测α-SMA的mRNA和蛋白表达水平，并采用免疫荧光细胞化学染色法检测α-SMA阳性肌成纤维细胞。实验均重复3次。数据采用方差分析和LSD-t检验进行处理。
（1）实验一。四组β-连环蛋白mRNA水平差异无统计学意义（F = 0.302，P = 0.823）。四组β-连环蛋白蛋白水平差异有统计学意义（F = 16.713，P = 0.001）。TGF-β1组（0.73±0.12）和Wnt3a组（0.82±0.17）β-连环蛋白蛋白水平高于对照组（0.34±0.11，t值分别为3.028、3.727，P < 0.05或P < 0.01）。TGF-β1 + Wnt3a组β-连环蛋白蛋白水平（1.23±0.21）高于其他三组（t值分别为6.911、3.883、3.184，P值均< 0.01）。四组α-SMA mRNA水平差异有统计学意义（F = 31.830，P = 0.001）。与对照组相比，TGF-β1组α-SMA mRNA表达水平上调，Wnt3a组下调（t值分别为6.759、2.535，P < 0.05或P < 0.01）。TGF-β1 + Wnt3a组α-SMA mRNA表达水平低于TGF-β1组（t = 4.532，P < 0.01）。对照组、TGF-β1组、Wnt3a组和TGF-β1 + Wnt3a组α-SMA蛋白水平分别为0.83±0.17、1.43±0.20、0.53±0.12和0.89±0.14（F = 16.597，P = 0.001）。与对照组相比，TGF-β组α-SMA蛋白水平上调，Wnt3a组下调（t值分别为4.582、2.291，P < 0.05或P < 0.01）。TGF-β1 + Wnt3a组α-SMA蛋白水平低于TGF-β1组（t = 4.123，P < 0.01）。（2）实验二。四组α-SMA mRNA水平差异有统计学意义（F = 34.101，P = 0.001）。SB415286组α-SMA mRNA水平低于对照组（t = 2.511，P < 0.05）。TGF-β1 + SB415286组α-SMA mRNA水平低于TGF-β1组（t = 3.587, P < 0.01）。四组α-SMA蛋白水平差异有统计学意义（F = 11.381，P = 0.003）。SB415286组α-SMA蛋白水平低于对照组（t = 2.364，P < 0.05）。与TGF-β1组相比，SB415286 + TGF-β1组α-SMA蛋白水平下调（t = 2.556，P < 0.05）。对照组α-SMA阳性成纤维细胞较少。与对照组相比，TGF-β1组α-SMA表达显著增加（t = 11.198，P < 0.01），SB415286组α-SMA表达下调。同时，TGF-β1 + SB415286组α-SMA表达显著低于TGF-β1组（t = 5.902，P < 0.01）。
Wnt/β-连环蛋白信号通路可能参与成纤维细胞向肌成纤维细胞的转化，并对TGF-β1介导的促纤维化作用起负性调节作用。
