CAJ | 학술논문

目的观察整合素连接激酶(ILK)在转化生长因子-β1(TGF-β1)/Smad3信号通路中的作用,探讨ILK对瘢痕成纤维细胞分化影响的作用机制.方法体外分离培养瘢痕成纤维细胞后,实验分为4组:对照组、ILK小分子干扰RNA(siRNA)组、TGF-β1组、ILK siRNA+TGF-β1组.采用荧光标记的免疫细胞化学法检测各组成纤维细胞中ILK的表达变化,应用实时荧光定量聚合酶链反应(FQ-PCR)检测各组成纤维细胞中ILK mRNA、Smad3 mRNA及α-平滑肌肌动蛋白(α-SMA) mRNA的表达.结果免疫荧光图像显示,经siRNA沉默后,成纤维细胞的ILK表达水平下调;经TGF-β1(5μg/L)刺激后,ILK表达水平明显上调.FQ-PCR检测结果显示:经ILK siRNA转染后,成纤维细胞的ILK mRNA(0.522±0.113)、Smad3 mRNA (0.222±0.089)及α-SMA mRNA(0.118±0.080)表达水平同时下降;应用TGF-β1刺激后,成纤维细胞的ILK mRNA (2.233±0.511)、Smad3 mRNA(2.891±0.475)及α-SMA mRNA(2.581 ±0.826)表达水平重新上升,与对照组及ILK siRNA组比较差异有统计学意义(P＜0.05).结论在瘢痕成纤维细胞中,Smad3及α-SMA的mRNA水平可以被ILK上调,而TGF-β1又可以诱导ILK的表达,ILK可能参与Smad蛋白的调控,对TGF-β1/Smad3信号通路诱导的促成纤维细胞分化作用产生影响.
목적 관찰정합소련접격매(ILK)재전화생장인자-β1(TGF-β1)/Smad3신호통로중적작용,탐토ILK대반흔성섬유세포분화영향적작용궤제.방법 체외분리배양반흔성섬유세포후,실험분위4조:대조조、ILK소분자간우RNA(siRNA)조、TGF-β1조、ILK siRNA+TGF-β1조.채용형광표기적면역세포화학법검측각조성섬유세포중ILK적표체변화,응용실시형광정량취합매련반응(FQ-PCR)검측각조성섬유세포중ILK mRNA、Smad3 mRNA급α-평활기기동단백(α-SMA) mRNA적표체.결과 면역형광도상현시,경siRNA침묵후,성섬유세포적ILK표체수평하조;경TGF-β1(5μg/L)자격후,ILK표체수평명현상조.FQ-PCR검측결과현시:경ILK siRNA전염후,성섬유세포적ILK mRNA(0.522±0.113)、Smad3 mRNA (0.222±0.089)급α-SMA mRNA(0.118±0.080)표체수평동시하강;응용TGF-β1자격후,성섬유세포적ILK mRNA (2.233±0.511)、Smad3 mRNA(2.891±0.475)급α-SMA mRNA(2.581 ±0.826)표체수평중신상승,여대조조급ILK siRNA조비교차이유통계학의의(P＜0.05).결론 재반흔성섬유세포중,Smad3급α-SMA적mRNA수평가이피ILK상조,이TGF-β1우가이유도ILK적표체,ILK가능삼여Smad단백적조공,대TGF-β1/Smad3신호통로유도적촉성섬유세포분화작용산생영향.
Objective To explore the role of integrin-linked kinase (ILK) in the transforming growth factor-β1 (TGF-β1)/Smad3 signal pathway,and the action mechanism of ILK on the differentiation of fibroblasts in hypertrophic scar.Methods The fibroblasts of human hypertrophic scar were isolated and cultured in vitro.The cells were divided into 4 groups:control group,ILK small interfering RNA (siRNA) group,TGF-β1 group,and ILK siRNA + TGF-β1 group.The ILK expression levels were detected by using immunofiuorescence.The mRNA expression levels of ILK,Smad3 and α-smooth muscle actin (α-SMA) were detected by using real-time fluorescent quantitative polyrnerase chain reaction (FQ-PCR).Results The immunofluorescence showed that ILK expression was decreased after transfection of siRNA,and increased again by stimulation with TGF-β1 (5 μg/L).FQ-PCR revealed that the mRNA expression of Smad3 (0.222 ± 0.089) and α-SMA (0.118 ± 0.080) was decreased with ILK (0.522 ± 0.113) after transfection of ILK siRNA,and then the mRNA expression of Smad3 (2.891 ± 0.475) and α-SMA (2.581 ± 0.826) was decreased with ILK (2.233 ± 0.511) increased significantly after stimulation of TGF-β1 as compared with that in control group and ILK siRNA group (P ＜ 0.05).Conclusion The mRNA level of Smad3 and α-SMA could be up-regulated by ILK,which could be stimulated by TGF-β1.ILK may be an important upstream gene for Smad proteins to affect the differentiation of fibroblasts in hypertrophic scar via TGF-β1/Smad3 signal pathway.