Chen J-J, Zhao S, Cen Y, Liu X-X, Yu R, Wu D-M
Department of Burns and Plastic Surgery, West China Hospital, Sichuan University, No. 37 GuoXueXiang, Chengdu 610041, Sichuan, China.
Br J Dermatol. 2007 Jun;156(6):1188-95. doi: 10.1111/j.1365-2133.2007.07898.x.
Keloid is characterized by excessive collagen accumulation, but the mechanism of keloid formation remains unknown, and none of the treatment modalities are consistently effective. Heat shock protein (HSP) 47, known as a collagen-specific molecular chaperone, plays a critical role in collagen biosynthesis. Our previous research has demonstrated that HSP47 is highly expressed in keloid compared with normal skin tissues, which indicates that there might be a close relationship between overexpression of HSP47 and excessive collagen accumulation in keloid formation.
To further investigate whether overexpression of HSP47 might promote excessive collagen deposition in keloid formation, we examined the alteration of intracellular and extracellular collagen expression, following inhibition of HSP47 expression in keloid fibroblast cells by the RNA interference technique.
Three constructed psiRNA-hH1neo plasmids, carrying three pairs of related HSP47-shRNA (small hairpin RNA), respectively, were transfected into keloid fibroblast cells and compared with three control groups. After transfection, the mRNA and protein expression of HSP47 and collagen type I were detected by quantitative real-time polymerase chain reaction and Western blot; the content of extracellular secreting collagen was assessed by hydroxyproline assay; and the MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] method was adopted to examine the proliferation of keloid fibroblast cells.
Both the mRNA and protein levels of HSP47 in keloid fibroblast cells decreased dramatically 48 h after post-transfection of three related HSP47-shRNA plasmids, compared with control groups. Following the downregulation of HSP47, we found that the expression of intracellular and extracellular collagen was correspondingly reduced. On the other hand, the MTT assay showed that transfection of HSP47-shRNA plasmids did not influence the growth of keloid fibroblast cells.
Combined with our previous histological results, we propose that overexpression of HSP47 in keloid fibroblast cells could induce excessive collagen accumulation by enhancing synthesis and secretion of collagen, which not only presents a possible mechanism of keloid formation, but also offers a therapeutic potential of RNA interference to HSP47 for the treatment of keloid and other fibroproliferative disorders.
瘢痕疙瘩的特征是胶原蛋白过度积累，但瘢痕疙瘩形成的机制尚不清楚，且目前没有一种治疗方法能始终有效。热休克蛋白（HSP）47作为一种胶原蛋白特异性分子伴侣，在胶原蛋白生物合成中起关键作用。我们之前的研究表明，与正常皮肤组织相比，HSP47在瘢痕疙瘩中高表达，这表明HSP47的过表达与瘢痕疙瘩形成过程中胶原蛋白的过度积累之间可能存在密切关系。
为了进一步研究HSP47的过表达是否可能促进瘢痕疙瘩形成过程中胶原蛋白的过度沉积，我们通过RNA干扰技术抑制瘢痕疙瘩成纤维细胞中HSP47的表达，检测细胞内和细胞外胶原蛋白表达的变化。
将分别携带三对相关HSP47短发夹RNA（shRNA）的三种构建好的psiRNA-hH1neo质粒转染到瘢痕疙瘩成纤维细胞中，并与三个对照组进行比较。转染后，通过定量实时聚合酶链反应和蛋白质印迹法检测HSP47和Ⅰ型胶原蛋白的mRNA和蛋白表达；通过羟脯氨酸测定评估细胞外分泌胶原蛋白的含量；采用MTT [3-(4, 5-二甲基噻唑-2)-2, 5-二苯基四氮唑溴盐]法检测瘢痕疙瘩成纤维细胞的增殖情况。
与对照组相比，转染三种相关HSP47-shRNA质粒后48小时，瘢痕疙瘩成纤维细胞中HSP47的mRNA和蛋白水平均显著降低。随着HSP47表达下调，我们发现细胞内和细胞外胶原蛋白的表达相应减少。另一方面，MTT分析表明，转染HSP47-shRNA质粒不影响瘢痕疙瘩成纤维细胞的生长。
结合我们之前的组织学结果，我们提出瘢痕疙瘩成纤维细胞中HSP47的过表达可通过增强胶原蛋白的合成和分泌诱导胶原蛋白过度积累，这不仅提出了瘢痕疙瘩形成的一种可能机制，也为RNA干扰HSP47治疗瘢痕疙瘩和其他纤维增生性疾病提供了治疗潜力。
