皮肤桥蛋白通过转化生长因子β1促进皮肤纤维化的机制研究

Mechanisms of dermatopontin promoting skin fibrosis through transforming growth factor β1

目的探讨皮肤桥蛋白在转化生长因子β1介导的成纤维细胞活化促进皮肤纤维化过程中的作用机制。方法2022年3月至2023年7月,实验在上海交通大学医学院附属第九人民医院开展。于体外培养C57BL/6J小鼠皮肤成纤维细胞,用小干扰RNA进行皮肤桥蛋白的敲除并筛选沉默效率最高的小干扰RNA序列。确认转化生长因子β1刺激成纤维细胞的活化的最佳作用浓度。在体外建立敲除皮肤桥蛋白的小鼠皮肤成纤维细胞模型并加入转化生长因子β1促进其纤维化,利用CCK-8实验、流式细胞术等方法观察转化生长因子β1刺激后对成纤维细胞增殖、凋亡和细胞周期调控的影响,通过实时荧光定量反转录聚合酶链式反应检测纤连蛋白、α-平滑肌肌动蛋白、Ⅰ型胶原蛋白α1链的RNA表达水平。结果小干扰RNA序列-3为沉默皮肤桥蛋白效率最高的序列(F=80.77,P<0.05)。转化生长因子β1刺激成纤维细胞活化的最佳浓度为10 ng/ml。CCK-8实验显示,敲除皮肤桥蛋白可显著抑制转化生长因子β1促进的成纤维细胞增殖(F=71.06,P<0.05)。流式细胞仪结果显示,敲除皮肤桥蛋白可阻止成纤维细胞进入G2/M期,差异均有统计学意义(F=11.15、8.30,均P<0.05),但对成纤维细胞凋亡无明显影响,显示皮肤桥蛋白主要通过加快细胞周期进程促进转化生长因子β1介导的细胞增殖。实时荧光定量反转录聚合酶链式反应的结果显示敲除皮肤桥蛋白后,纤连蛋白和Ⅰ型胶原蛋白α1链的表达下降,差异均有统计学意义(F=109.30、8.82,均P<0.05),α-平滑肌肌动蛋白的表达变化差异无统计学意义,证明皮肤桥蛋白可促进转化生长因子β1介导的成纤维细胞纤维化指标的表达。结论皮肤桥蛋白在转化生长因子β1介导的成纤维细胞活化引起的皮肤纤维化过程中起着重要的调控作用。

Objective To observe the mechanism of dermatopontin in transforming growth factor β1-mediated fibroblast activation for skin fibrosis by silencing their expression.Methods The experiment was conducted from March 2022 to July 2023 at the Ninth People′s Hospital of Shanghai Jiaotong University School of Medicine.Firstly,C57BL/6J mouse skin fibroblasts were cultured in vitro.Small interfering RNAs were used to knockdown dermatopontin,and the sequence with the highest silencing efficiency was screened.Then,the optimal concentration of transforming growth factor β1 used to stimulate the activation of fibroblasts was confirmed.Finally,a mouse skin fibroblast model with knockdown of dermatopontin was established in vitro and transforming growth factorβ1 was added to promote its fibrosis.CCK-8 assay and flow cytometry were utilized to explore the effects of transforming growth factorβ1 stimulation on fibroblast proliferation,apoptosis and cell cycle regulation.By real-time fluorescence quantitative reverse transcription polymerase chain reaction,the effects of fibronectin,α-smooth muscle actin and collagen typeⅠalpha 1 chain expression levels were detected.Results The small interfering RNA sequence-3 was the most efficient sequence for silencing dermatopontin(F=80.77,P<0.05).The optimal concentration of transforming growth factor β1 to stimulate fibroblast activation was 10 ng/ml.CCK-8 assay demonstrated that knockdown of dermatopontin significantly inhibited transforming growth factor β1-promoted fibroblast proliferation(F=71.06,P<0.05).The results of flow cytometry showed that knockdown of dermatopontin significantly prevented fibroblasts from entering the G2/M phase(F=11.15,P<0.05;F=8.30,P<0.05),but had no significant effect on fibroblast apoptosis,which proved that dermatopontin promoted transforming growth factor β1-mediated cell proliferation mainly through accelerating cell cycle progression.The results of real-time fluorescence quantitative reverse transcription polymerase chain reaction showed that knockdown of dermatopontin resulted in decreased expression of fibronectin and collagen type Ⅰ alpha 1 chain(F=109.30,P<0.05;F=8.82,P<0.05),and there was no significant difference in the change of α-smooth muscle actin expression,demonstrating that dermatopontin could promote the expression of fibrotic indicators by fibroblasts mediated by transforming growth factor β1.Conclusions In conclusion,this study demonstrats that dermatopontin plays an important regulatory role in the process of skin fibrosis induced by transforming growth factor β1-mediated fibroblast activation.