Effects of mechanical tension on the formation of hypertrophic scars in rabbit ears and transforming growth factor-β1/Smad signaling pathway
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摘要:
目的 探讨机械张力对兔耳增生性瘢痕的形成及转化生长因子β1（TGF-β1）/Smad信号通路的影响。 方法 采用实验研究方法。取6只3~5个月龄雌雄不拘新西兰大白兔，于每侧兔耳腹面制作5个全层皮肤缺损创面。观察术后0（即刻）、7、14、21、28 d所有兔耳创面外观。术后28 d，计算瘢痕形成率。将每只兔左耳的3个成熟瘢痕纳入张力组并采用螺旋扩弓器持续扩弓，将每只兔右耳的3个成熟瘢痕纳入假张力组并仅缝合螺旋扩弓器不扩弓，每组共18个瘢痕。经机械张力处理（以下简称处理）40 d，观察2组兔耳瘢痕组织颜色、质地。处理40 d，观察并计算瘢痕增生指数（SEI），分别行苏木精-伊红染色观察组织形态、Masson染色观察胶原形态，采用实时荧光定量反转录PCR法检测瘢痕组织中TGF-β1、Smad3、Ⅰ型胶原、Ⅲ型胶原、α平滑肌肌动蛋白（α-SMA）的mRNA表达，采用蛋白质印迹法检测瘢痕组织中TGF-β1、Ⅰ型胶原、Ⅲ型胶原、α-SMA的蛋白表达和Smad3磷酸化水平。以上实验各组样本数均为3。对数据行独立样本t检验。 结果 术后0 d，所有兔耳均形成5个新鲜创面；术后7 d，可见创面结痂；术后14 d，大部分创面已上皮化；术后21 d，可见全部创面上皮化；术后28 d，形成明显的增生性瘢痕。术后28 d，瘢痕形成率为75%（45/60）。处理40 d，张力组的兔耳瘢痕组织凸起较假张力组明显，瘢痕组织较硬，颜色较红润；张力组兔耳瘢痕的SEI为2.02±0.08，明显高于假张力组的1.70±0.08（t=5.07，P<0.01）。处理40 d，与假张力组相比，张力组兔耳瘢痕组织角质层变厚，真皮层可见大量新生的毛细血管、炎症细胞和成纤维细胞；胶原排列更加紊乱，呈结节状或旋涡状分布。处理40 d，张力组兔耳瘢痕组织中TGF-β1、Smad3、Ⅰ型胶原、Ⅲ型胶原、α-SMA的mRNA表达量分别为1.81±0.25、5.71±0.82、7.86±0.56、4.35±0.28、5.89±0.47，分别明显高于假张力组的1.00±0.08、1.00±0.12、1.00±0.13、1.00±0.14、1.00±0.14（t值分别为5.36、9.82、20.60、18.26、17.13，P值均<0.01）；张力组兔耳瘢痕组织中TGF-β1、Ⅰ型胶原、Ⅲ型胶原、α-SMA的蛋白表达和Smad3磷酸化水平分别为0.865±0.050、0.895±0.042、0.972±0.027、1.012±0.057、0.968±0.087，分别明显高于假张力组的0.657±0.050、0.271±0.029、0.631±0.027、0.418±0.023、0.511±0.035（t值分别为5.08、21.27、15.55、16.70、8.40，P值均<0.01）。 结论 机械张力会刺激瘢痕增生，抑制真皮层胶原纤维的正常排列，加剧胶原纤维的沉积，从而对兔耳增生性瘢痕的消退起抑制作用，其机制可能与机械张力激活TGF-β1/Smad信号通路有关。 Abstract:Objective To explore the effects of mechanical tension on the formation of hypertrophic scars in rabbit ears and transforming growth factor-β1 (TGF-β1)/Smad signaling pathway. Methods The experimental research method was adopted. Six New Zealand white rabbits, male or female, aged 3-5 months were used and 5 full-thickness skin defect wounds were made on the ventral surface of each rabbit ear. The appearance of all rabbit ear wounds was observed on post surgery day (PSD) 0 (immediately), 7, 14, 21, and 28. On PSD 28, the scar formation rate was calculated. Three mature scars in the left ear of each rabbit were included in tension group and the arch was continuously expanded with a spiral expander. Three mature scars in the right ear of each rabbit were included in sham tension group and only the spiral expander was sutured without expansion. There were 18 scars in each group. After mechanical tension treatment (hereinafter referred to as treatment) for 40 days, the color and texture of scar tissue in the two groups were observed. On treatment day 40, the scar elevation index (SEI) was observed and calculated; the histology was observed after hematoxylin eosin staining, and the collagen morphology was observed after Masson staining; mRNA expressions of TGF-β1, Smad3, collagen Ⅰ, collagen Ⅲ, and α-smooth muscle actin (α-SMA) in scar tissue were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction; and the protein expressions of TGF-β1, collagen Ⅰ, collagen Ⅲ, and α-SMA, and phosphorylation level of Smad3 in scar tissue were detected by Western blotting. The number of samples of each group in the experiments was 3. Data were statistically analyzed with independent sample t test. Results On PSD 0, 5 fresh wounds were formed on all the rabbit ears; on PSD 7, the wounds were scabbed; on PSD 14, most of the wounds were epithelialized; on PSD 21, all the wounds were epithelialized; on PSD 28, obvious hypertrophic scars were formed. The scar formation rate was 75% (45/60) on PSD 28. On treatment day 40, the scar tissue of rabbit ears in tension group was more prominent than that in sham tension group, the scar tissue was harder and the color was more ruddy; the SEI of the scar tissue of rabbit ears in tension group (2.02±0.08) was significantly higher than 1.70±0.08 in sham tension group (t=5.07, P<0.01). On treatment day 40, compared with those in sham tension group, the stratum corneum of scar tissue became thicker, and a large number of new capillaries, inflammatory cells, and fibroblasts were observed in the dermis, and collagen was more disordered, with nodular or swirling distribution in the scar tissue of rabbit ears in tension group. On treatment day 40, the mRNA expressions of TGF-β1, Smad3, collagen Ⅰ, collagen Ⅲ, and α-SMA in the scar tissue of rabbit ears in tension group were respectively 1.81±0.25, 5.71±0.82, 7.86±0.56, 4.35±0.28, and 5.89±0.47, which were significantly higher than 1.00±0.08, 1.00±0.12, 1.00±0.13, 1.00±0.14, and 1.00±0.14 in sham tension group (with t values of 5.36, 9.82, 20.60, 18.26, and 17.13, respectively, all P<0.01); the protein expressions of TGF-β1, collagen Ⅰ, collagen Ⅲ, and α-SMA, and phosphorylation level of Smad3 in the scar tissue of rabbit ears in tension group were respectively 0.865±0.050, 0.895±0.042, 0.972±0.027, 1.012±0.057, and 0.968±0.087, which were significantly higher than 0.657±0.050, 0.271±0.029, 0.631±0.027, 0.418±0.023, and 0.511±0.035 in sham tension group (with t values of 5.08, 21.27, 15.55, 16.70, and 8.40, respectively, all P<0.01). Conclusions Mechanical tension can inhibit the regression of hypertrophic scars in rabbit ears through stimulating the hyperplasia of scars, inhibiting the normal arrangement of dermal collagen fibers, and intensifying the deposition of collagen fibers, and the mechanism may be related to the activation of TGF-β1/Smad signaling pathway by mechanical tension. -
Key words:
- Cicatrix /
- Transforming growth factor beta1 /
- Smad proteins /
- Mechanical tension
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参考文献
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2 兔耳增生性瘢痕模型的构建及经机械张力处理40 d的2组兔耳瘢痕的大体外观。2A.术后0 d（即刻），可见5个直径为1 cm的圆形新鲜创面；2B.术后7 d，可见各创面已结痂；2C.术后14 d，可见部分创面上皮化；2D.术后21 d，可见全部创面上皮化；2E.术后28 d，形成明显的增生性瘢痕，外观明显凸起、颜色红润，其增生范围不超过原创缘；2F、2G.分别为处理前假张力组、张力组瘢痕，均明显凸起；2H、2I.分别为处理40 d假张力组、张力组瘢痕，图2I瘢痕凸起更加明显且颜色红润
3 经机械张力处理40 d的2组兔耳增生性瘢痕组织形态观察。3A.假张力组可见少量新生毛细血管 HE×20；3B.图3A中方框处放大图，可见少量炎症细胞和Fb HE×100；3C.张力组可见角质层较图3A变厚，真皮层可见大量新生的毛细血管 HE×20；3D.图3C中方框处放大图，可见大量炎症细胞和Fb HE×100；3E.假张力组可见少量胶原，排列较紊乱 Masson×20；3F.图3E中方框处放大图，可见少量排列较紊乱的胶原 Masson×100；3G.张力组可见胶原排列图较图3E更加紊乱，呈结节状或旋涡状分布 Masson×20；3H.图3G中方框处放大图，可见大量纤维化和胶原，胶原排列更加紊乱 Masson×100
注：HE为苏木精-伊红，Fb为成纤维细胞，胶原Masson阳性染色为蓝色
4 蛋白质印迹法检测经机械张力处理40 d的2组兔耳增生性瘢痕组织中TGF-β1、Ⅰ型胶原、Ⅲ型胶原、α-SMA的蛋白表达和Smad3磷酸化水平
注：TGF-β1为转化生长因子β1，α-SMA为α平滑肌肌动蛋白，条带上方1、2分别指假张力组、张力组
表1 采用实时荧光定量反转录PCR法检测兔耳增生性瘢痕组织中TGF-β1、Smad3、Ⅰ型胶原、Ⅲ型胶原、α-SMA的mRNA表达的引物序列及产物大小
基因名称 引物序列（5'→3'） 产物大小（bp） GAPDH 上游：GCTTCTTCTCGTGCAGTGCTA下游：ATGACCAGCTTCCCGTTCTC 239 TGF-β1 上游：CCAAGTGGACATCAACGGGA下游：CTCTGTGGAGCTGAAGCAGT 175 Smad3 上游：AGACCTGACATCCGAGACGA下游：CATCAGGACAGCCGAACAGT 105 Ⅰ型胶原 上游：GCAAGAACGGAGATGACGGA下游：TTGGCACCATCCAAACCACT 151 Ⅲ型胶原 上游：CCGAACCGTGCCAAATATGC下游：AACAGTGCGGGGAGTAGTTG 158 α-SMA 上游：AACAATGTGCTCTCAGGGGG下游：TCTGGAGGGGCAATTATCTTGA 116 注：GAPDH为3-磷酸甘油醛脱氢酶，TGF-β1为转化生长因子β1，α-SMA为α平滑肌肌动蛋白 
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