Modulation of collagen synthesis in keloid fibroblasts by silencing Smad2 with siRNA
Modulation of collagen synthesis in keloid fibroblasts by silencing Smad2 with siRNA
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DOI：
10.1097/01.prs.0000239537.77870.2c
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发表时间：
2006-11-01
影响因子：
3.6
通讯作者：
Jin, Youxin
中科院分区：
文献类型：
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作者：
Gao, Zhongyu;Wang, Zimin;Jin, Youxin
背景：瘢痕疙瘩是皮肤创伤后的一种失调反应，导致细胞外基质过度沉积，尤其是I型和III型胶原。转化生长因子-β通过诱导和维持瘢痕疙瘩成纤维细胞的激活，在纤维化的发病机制中发挥核心作用。然而，人们对其潜在的机制知之甚少。在这项研究中，作者利用小干扰RNA(SiRNA)检测了新近鉴定的转化生长因子-β信号转导的细胞内效应分子Smad2在瘢痕疙瘩成纤维细胞中的功能。方法：设计3对针对人Smad2的siRNA双链，从中选出最有效的用于进一步研究。结果：siRNA下调瘢痕疙瘩成纤维细胞Smad2基因的表达，并呈剂量和时间依赖关系。免疫荧光实验证实Smad2基因在蛋白水平表达下调。SiRNA下调Smad2后，瘢痕疙瘩中I型和III型前胶原的mRNA水平也明显降低。结论：Smad2在转化生长因子-β诱导的瘢痕疙瘩纤维化中起重要作用。下调瘢痕疙瘩成纤维细胞中Smad2的表达可显著降低前胶原基因的表达。靶向Smad2的siRNA是减少细胞外基质沉积、延缓纤维化进程的有效试剂。通过阻断转化生长因子-β信号通路，有望成为促进皮肤创面愈合和抑制纤维化进展的一种新的治疗方法。
Background: Keloids represent a dysregulated response to cutaneous wounding that results in an excessive deposition of extracellular matrix, especially types I and III collagen. Transforming growth factor (TGF)-beta plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of keloid fibroblast. However, the underlying mechanisms are poorly understood. In this study, the authors examined the function of Smad2, a recently characterized intracellular effector of TGF-beta signaling, in keloid fibroblasts using small interfering RNA (siRNA).Methods: Three pairs of siRNA duplexes targeting human Smad2 were designed; the most efficient one was selected and used for further research. Keloid fibroblasts were treated with or without Smad2 siRNA, and the expression levels of related genes were examined by reverse-transcriptase polymerase chain reaction and immunofluorescence.Results: The down-regulation of Smad2 by siRNA led to a significant decrease in mRNA levels of Smad2 in both a dose-dependent and time-dependent manner. The knockdown of Smad2 expression in protein level was confirmed using immunofluorescence. The mRNA levels of types I and III procollagen were also significantly and uniquely decreased following the reduction of Smad2 by siRNA.Conclusions: The results indicate that Smad2 plays an important role in TGF-beta-induced fibrosis in keloids. Down-regulation of Smad2 expression in keloid fibroblasts can significantly decrease procollagen gene expression. Also, siRNA targeting Smad2 was an efficient reagent with which to reduce extracellular matrix deposition and attenuate process of fibrosis. It could be a new, promising therapeutic approach for improving skin wound healing and inhibiting progression of fibrotic conditions by interrupting the TGF-beta signaling pathway.