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  • Title: [Effects of silencing Smad ubiquitination regulatory factor 2 on the function of human hypertrophic scar-derived fibroblasts].
    Author: Zhang Z, Kuang F, Liu CL, Chen B, Tang WB, Li XJ.
    Journal: Zhonghua Shao Shang Za Zhi; 2017 Mar 20; 33(3):145-151. PubMed ID: 28316163.
    Abstract:
    Objective: To explore the effects of silencing Smad ubiquitination regulatory factor 2 (Smurf2) on the secretion of transforming growth factor beta 1 (TGF-β(1)), alpha-smooth muscle actin (α-SMA), and collagen type Ⅰ by human hypertrophic scar-derived fibroblasts. Methods: The human normal skin-derived fibroblasts and hypertrophic scar-derived fibroblasts were cultured with explant culture technique from the normal skin and hypertrophic scar tissue, which was obtained from 9 patients with hypertrophic scars after burn. Two kinds of fibroblasts of the third passage were both divided into 6 groups according to the random number table, with 9 wells in each group. Fibroblasts in blank control group were cultured for 72 h without transfection of any small interfering RNA (siRNA), fibroblasts in negative control group were for cultured for 72 h after transfected with non-target siRNA, fibroblasts in Smurf2 siRNA group were cultured for 72 h after transfected with 100 nmol/L Smurf2 siRNA, fibroblasts in blank control+ TGF-β(1) group were cultured for 72 h without transfection of any siRNA and then treated with 10 ng/mL TGF-β(1) for 6 h, fibroblasts in negative control+ TGF-β(1) group were cultured for 72 h after transfected with non-target siRNA and then treated with 10 ng/mL TGF-β(1) for 6 h, fibroblasts in Smurf2 siRNA+ TGF-β(1) group were cultured for 72 h after transfected with Smurf2 siRNA and then treated with 10 ng/mL TGF-β(1) for 6 h. (1) The protein and mRNA expression levels of Smurf2 of the two kinds of cells in blank control group, negative control group, and Smurf2 siRNA group were assessed by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR), respectively. (2) The content of TGF-β(1) in the cell culture supernatant of the two kinds of cells in blank control group and Smurf2 siRNA group was determined by enzyme-linked immunosorbent assay (ELISA). (3) The protein expression levels of α-SMA of the two kinds of cells in the 6 groups were assessed by Western blotting. The content of collagen type Ⅰ in the cell culture supernatant of the two kinds of cells in the 6 groups was determined by ELISA. (4) The mRNA expression levels of α-SMA and collagen type Ⅰ of the two kinds of cells in the 6 groups were assessed by RT-PCR. The sample numbers of each group in the above experiments were all 9. Data were processed with analysis of variance of factorial design and Bonferroni test. Results: (1) The protein and mRNA expression levels of Smurf2 of the two kinds of cells in Smurf2 siRNA group were significantly lower than those in blank control group and negative control group (with P values below 0.05). The protein and mRNA expression levels of Smurf2 of the two kinds of cells in blank control group and negative control group were close (with P values above 0.05). (2) The content of TGF-β(1) in the cell culture supernatant of hypertrophic scar-derived fibroblasts in blank control group and Smurf2 siRNA group was respectively (4.34±0.56) and (2.14±0.28) pg/mL, which was significantly higher than (1.52±0.20) and (1.41±0.18) pg/mL of normal skin-derived fibroblasts respectively (with P values below 0.05). In hypertrophic scar-derived fibroblasts, the content of TGF-β(1) in the cell culture supernatant in Smurf2 siRNA group was significantly lower than that in blank control group (P<0.05). In normal skin-derived fibroblasts, the content of TGF-β(1) in the cell culture supernatant in Smurf2 siRNA group was close to that in blank control group (P>0.05). (3) The protein expression levels of α-SMA and content of collagen type Ⅰ in the cell culture supernatant of the two kinds of cells in blank control+ TGF-β(1) group were significantly higher than those in blank control group (with P values below 0.05). The protein expression levels of α-SMA and content of collagen type Ⅰ in the cell culture supernatant of the two kinds of cells in negative control+ TGF-β(1) group were significantly higher than those in negative control group (with P values below 0.05). The protein expression levels of α-SMA and content of collagen type Ⅰ in the cell culture supernatant of the two kinds of cells in Smurf2 siRNA group were close to those in blank control group and negative control group (with P values above 0.05). The protein expression levels of α-SMA and content of collagen type Ⅰ in the cell culture supernatant of the two kinds of cells in Smurf2 siRNA+ TGF-β(1) group were significantly lower than those in blank control+ TGF-β(1) group and negative control+ TGF-β(1) group (with P values below 0.05). (4) The mRNA expression levels of α-SMA and collagen type Ⅰ of the two kinds of cells in blank control+ TGF-β(1) group were significantly higher than those in blank control group (with P values below 0.05). The mRNA expression levels of α-SMA and collagen type Ⅰ of the two kinds of cells in negative control+ TGF-β(1) group were significantly higher than those in negative control group (with P values below 0.05). The mRNA expression levels of α-SMA and collagen type Ⅰ of the two kinds of cells in Smurf2 siRNA group were close to those in blank control group and negative control group (with P values above 0.05). The mRNA expression levels of α-SMA and collagen type Ⅰ of the two kinds of cells in Smurf2 siRNA+ TGF-β(1) group were significantly lower than those in blank control+ TGF-β(1) group and negative control+ TGF-β(1) group (with P values below 0.05). Conclusions: Silencing Smurf2 in human hypertrophic scar-derived fibroblasts can reduce the autocrine of TGF-β(1) and inhibit the TGF-β(1)-induced α-SMA expression and collagen type Ⅰ synthesis. 目的： 探讨沉默Smad泛素化调节因子2(Smurf2)基因对人增生性瘢痕Fb分泌TGF－β(1)、α平滑肌肌动蛋白(α－SMA)、Ⅰ型胶原的影响。 方法： 取9例烧伤后增生性瘢痕患者的正常皮肤组织和瘢痕组织，用组织块法培养人正常皮肤Fb和增生性瘢痕Fb，将2种第3代Fb均按随机数字表法分成6组，每组9孔。空白对照组，不加任何小干扰RNA培养72 h；阴性对照组，转染非靶向的小干扰RNA培养72 h；Smurf2小干扰RNA转染组，转染100 nmol/L的Smurf2小干扰RNA培养72 h；空白对照＋TGF－β(1)组，不加任何小干扰RNA培养72 h后加入10 ng/mL TGF－β(1)刺激6 h；阴性对照＋TGF－β(1)组，转染非靶向的小干扰RNA培养72 h后加入10 ng/mL TGF－β(1)刺激6 h；Smurf2小干扰RNA转染＋TGF－β(1)组，转染Smurf2小干扰RNA培养72 h后加入10 ng/mL TGF－β(1)刺激6 h。(1)分别采用蛋白质印迹法、RT－PCR法检测2种细胞空白对照组、阴性对照组、Smurf2小干扰RNA转染组Smurf2的蛋白和mRNA表达。(2)采用ELISA法检测2种细胞空白对照组和Smurf2小干扰RNA转染组细胞培养上清液中TGF－β(1)含量。(3)采用蛋白质印迹法检测2种细胞6组α－SMA的蛋白表达，ELISA法检测2种细胞6组细胞培养上清液中Ⅰ型胶原含量。(4)采用RT－PCR法检测2种细胞6组α－SMA和Ⅰ型胶原的mRNA表达。以上实验中各组样本数均为9。对数据行析因设计方差分析、Bonferroni法检验。 结果： (1)2种细胞Smurf2小干扰RNA转染组中Smurf2的蛋白和mRNA表达量均明显低于空白对照组和阴性对照组(P值均小于0.05)，空白对照组和阴性对照组中Smurf2的蛋白和mRNA表达量均相近(P值均大于0.05)。(2)增生性瘢痕Fb空白对照组和Smurf2小干扰RNA转染组细胞培养上清液中TGF－β(1)含量分别为(4.34±0.56)、(2.14±0.28)pg/mL，均明显高于正常皮肤Fb的(1.52±0.20)、(1.41±0.18)pg/mL(P值均小于0.05)。增生性瘢痕Fb中，Smurf2小干扰RNA转染组细胞培养上清液中TGF－β(1)含量明显低于空白对照组(P<0.05)；正常皮肤Fb中，Smurf2小干扰RNA转染组细胞培养上清液中TGF－β(1)含量与空白对照组相近(P>0.05)。(3)2种细胞空白对照＋TGF－β(1)组α－SMA蛋白表达量和细胞培养上清液中Ⅰ型胶原含量均明显高于空白对照组(P值均小于0.05)，阴性对照＋TGF－β(1)组α－SMA蛋白表达量和细胞培养上清液中Ⅰ型胶原含量均明显高于阴性对照组(P值均小于0.05)，Smurf2小干扰RNA转染组α－SMA蛋白表达量和细胞培养上清液中Ⅰ型胶原含量均与空白对照组和阴性对照组相近(P值均大于0.05)，Smurf2小干扰RNA转染＋TGF－β(1)组α－SMA蛋白表达量和细胞培养上清液中Ⅰ型胶原含量均明显低于空白对照＋TGF－β(1)组和阴性对照＋TGF－β(1)组(P值均小于0.05)。(4)2种细胞空白对照＋TGF－β(1)组α－SMA和Ⅰ型胶原的mRNA表达量均明显高于空白对照组(P值均小于0.05)，阴性对照＋TGF－β(1)组α－SMA和Ⅰ型胶原的mRNA表达量均明显高于阴性对照组(P值均小于0.05)，Smurf2小干扰RNA转染组α－SMA和Ⅰ型胶原的mRNA表达量均与空白对照组和阴性对照组相近(P值均大于0.05)，Smurf2小干扰RNA转染＋TGF－β(1)组α－SMA和Ⅰ型胶原的mRNA表达量均明显低于空白对照＋TGF－β(1)组和阴性对照＋TGF－β(1)组(P值均小于0.05)。 结论： 沉默人增生性瘢痕Fb中Smurf2基因，可减少TGF－β(1)的自分泌及抑制TGF－β(1)诱导的α－SMA表达和Ⅰ型胶原合成。.
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