Smurf2对增生性瘢痕TGF-β1信号通路负向调节因子Smad7的影响及调控机制

Effect of Smurf2 on Smad7,a negative regulator of TGF-β1 signaling pathway in hypertrophic scar and its mechanism

目的探讨Smurf2对增生性瘢痕成纤维细胞TGF-β1/Smad信号转导通路负向调节因子Smad7的影响及调控机制。方法标本来自暨南大学附属广州市红十字会医院2014年1月至10月收治的8例需要手术的增生性瘢痕患者,年龄1岁8个月至7岁,瘢痕增生的时间在2~12个月,以同一患者术中剩余正常皮肤作为对照。采用组织块培养法分离后传代培养人增生性瘢痕成纤维细胞和正常皮肤成纤维细胞,取第3~5代细胞进行实验。①采用蛋白质印记法检测2组细胞中Smad7的蛋白表达水平;②加入外源性TGF-β1(10ng/ml)作用0min、5min、15min、30min、1h、2h、12h后,采用RT-PCR和蛋白质印记法分别检测2组细胞中Smad7的mRNA和蛋白表达水平;③收集增生性瘢痕成纤维细胞和正常皮肤成纤维细胞的细胞裂解液,分别加入泛素化混合物于37℃孵育1h、2h、6h,采用蛋白质印记法检测2组细胞中Smad7的体外降解水平;④收集增生性瘢痕成纤维细胞的细胞裂解液,同时加入泛素化混合物及蛋白酶体抑制剂MG132/MG115(50μmol/L∶50μmol/L),研究其对Smad7体外降解的抑制作用;⑤利用免疫共沉淀技术,检测增生性瘢痕成纤维细胞中Smad7与E3泛素连接酶Smurf2是否存在相互作用;⑥采用siRNA干扰技术沉默增生性瘢痕成纤维细胞中Smurf2的基因表达,研究沉默Smurf2后,增生性瘢痕成纤维细胞中Smad7蛋白在TGF-β1刺激下的表达水平。实验数据采用两样本t检验、单因素方差分析进行统计分析,组内两两比较采用SNK法,以P<0.05认为差异有统计学意义。结果在正常皮肤及增生性瘢痕成纤维细胞中,Smad7的蛋白表达水平无明显差异(t=0.763,P=0.46);②在外源性TGF-β1刺激下,正常皮肤成纤维细胞中Smad7mRNA和蛋白表达水平均呈时间依赖性增高均P<0.05,增生性瘢痕成纤维细胞中Smad7mRNA表达呈时间依赖性增加(除5min时P>0.05,其余各时间点与未刺激细胞比较,P值均<0.05),蛋白水平无明显变化(P>0.05);③正常皮肤成纤维细胞中,Smad7的体外降解无明显变化(P=0.162),增生性瘢痕成纤维细胞中,Smad7的体外降解增强(各时间点Smad7蛋白表达水平与对照组及上一个时间点比较,除2h与1h比较时P值为0.012,其余P值均为0.000);④蛋白酶体抑制剂MG132/MG115可以阻断增生性瘢痕成纤维细胞中Smad7的体外降解;⑤在用Smurf2抗体免疫共沉淀下来的蛋白复合物中检测到Smad7,表明增生性瘢痕成纤维细胞中Smurf2与Smad7存在相互作用;⑥增生性瘢痕成纤维细胞中,加入TGF-β1刺激,Smad7的蛋白表达水平无明显变化;沉默Smurf2基因后,Smad7蛋白在TGF-β1刺激下表达增高。结论在烧伤后儿童增生性瘢痕成纤维细胞中,Smurf2通过泛素化降解Smad7,削弱了Smad7对TGF-β1信号转导的抑制作用,从而增强了TGF-β1/Smad信号转导,可能参与了增生性瘢痕的形成。

Objective To investigate the effect and regulatory mechanism of Smurf2 on the negative regulator Smad7 of TGF-β1/Smad signaling pathway in hypertrophic scar fibroblasts.Methods From January to October 2014, 8 patients with hypertrophic scar after burn were admitted. The age of patients ranged from 1 year 8 months to 7 years, and the time of scar hyperplasia ranged from 2 to 12 months. The residual normal skin of the same patient was used as the control. The fibroblasts were isolated from hypertrophic scar and normal skin respectively and cultured. The third to fifth passage cells were used for the experiments. ① The protein expression of Smad7 in the two groups was detected by Western Blot. ② Hypertrophic scar fibroblasts and normal skin fibroblasts were treated with exogenous TGF-β1 at concentration of 10 ng/ml for 0 min, 5 min, 15 min, 30 min, 1 h, 2 h and 12 h. The expression of Smad7 protein and mRNA were detected by Western Blot and RT-PCR, respectively. ③ The cell lysates of the two groups were collected and incubated with the ubiquitin mixture for 1 h, 2 h and 6 h at 37℃, respectively. The degradation level of Smad7 protein was detected by Western Blot. ④ The cell lysate of hypertrophic scar fibroblasts was collected and incubated with ubiquitin mixture with or without proteasome inhibitor (MG132: MG115=1: 1) to study its inhibitory effect on the degradation of Smad7 in vitro. ⑤ Immunoprecipitation (IP) technique was used to detect the interaction between Smad 7 and E3 ubiquitin ligase Smurf2 in hypertrophic scar fibroblasts. ⑥ The expression of Smad7 protein in hypertrophic scar fibroblasts stimulated by TGF-β1 after Smurf2 silencing by small interference RNA (siRNA) technique were detected by Western blot.Results① There was no significant difference in the expression level of Smad7 protein between hypertrophic scar and normal skin fibroblasts(t=0.76, P=0.46). ② Expressions of Smad7 mRNA and protein in normal skin fibroblasts stimulated by exogenous TGF-β1 gradually increased in a time-dependent manner(P<0.05). The expression of Smad7 mRNA in the hypertrophic scar fibroblasts increased at all-time points except at 5min , (P<0.05), while there was no significant difference in the expression level of Smad7 protein at all-time points with or without TGF-β1 stimulation(P>0.05). ③ Degradation of Smad7 protein was enhanced in hypertrophic scar fibroblasts (the expression level of Smad 7 protein at each time point was compared with that of the control group and the last time point, P<0.05), while there was no significant difference in Smad7 protein degradation in normal skin fibroblasts(P=0.162). ④ Enhanced degradation of Smad7 in hypertrophic scar fibroblasts was blocked by the addition of the proteasome inhibitors MG132/MG115. ⑤ In hypertrophic scar fibroblasts, the Smurf2-Smad7 complex was detected, which indicated the interaction between Smurf2 and Smad7 in hypertrophic scar fibroblasts. ⑥ The expression of Smad7 protein was not increased in the hypertrophic scar fibroblasts stimulated by TGF-β1, whereas the stimulation of TGF-β1 increased the expression of Smad7 protein after silencing of Smurf2 gene expression.Conclusions In the hypertrophic scar fibroblasts, Smurf 2 attenuates the inhibitory effect of Smad 7 on TGF-β1 signaling pathway through the degradation of Smad7 by ubiquitination, which may be involved in the formation of hypertrophic scar.