Cha Jisun, Kwak Taehee, Butmarc Janet, Kim Tae-Aug, Yufit Tatyana, Carson Polly, Kim Seong-Jin, Falanga Vincent
Department of Dermatology and Skin Surgery, Roger Williams Medical Center, Providence, RI 02908, USA.
J Dermatol Sci. 2008 Apr;50(1):15-23. doi: 10.1016/j.jdermsci.2007.10.010. Epub 2007 Dec 19.
Increasing evidence shows persistent phenotypic alterations in fibroblasts from non-healing human chronic wounds, which may result in faulty extracellular matrix deposition and keratinocyte migration. We have previously shown that these cells are characterized by morphological changes, low proliferative potential and unresponsiveness to TGF-beta1, and down regulated phosphorylation of Smad 2/3 and p42/44 MAPK from decreased expression of the TGF-beta type II receptor.
To identify genes and proteins that may be differentially expressed in chronic wounds and their cultured fibroblasts.
Differential display analysis with 120 random primer sets was used in fibroblasts from human venous ulcers and acute wounds created on the ipsilateral thighs of the same patients. Positive differential results were confirmed by RT-PCR. Immunohistochemistry of cultured fibroblasts and tissues was used to determine the expression of differentially expressed proteins.
A total of 16 differentially expressed genes were identified and cloned. The only candidate gene that was differentially expressed in all patients and confirmed by repeated differential display testing and RT-PCR was beta ig-h3, a TGF-beta-induced gene involved in cell adhesion, migration, and proliferation. Decreased expression of beta ig-h3 in chronic wounds and their fibroblasts was further confirmed by Western blot and immunostaining.
These findings point to beta ig-h3 as an important gene characterizing the abnormal phenotype of chronic wound fibroblasts. Corrective measures to increase the expression of this protein might have therapeutic potential.
越来越多的证据表明，人类慢性难愈合伤口的成纤维细胞存在持续的表型改变，这可能导致细胞外基质沉积异常和角质形成细胞迁移障碍。我们之前已经表明，这些细胞具有形态改变、增殖潜力低、对转化生长因子-β1（TGF-β1）无反应的特征，并且由于TGF-βⅡ型受体表达降低，Smad 2/3和p42/44丝裂原活化蛋白激酶（MAPK）的磷酸化水平下调。
鉴定在慢性伤口及其培养的成纤维细胞中可能差异表达的基因和蛋白质。
对来自人类静脉溃疡和同一患者同侧大腿急性伤口的成纤维细胞，使用120套随机引物进行差异显示分析。通过逆转录聚合酶链反应（RT-PCR）确认阳性差异结果。利用培养的成纤维细胞和组织的免疫组织化学方法确定差异表达蛋白质的表达情况。
共鉴定并克隆出16个差异表达基因。在所有患者中差异表达且经重复差异显示测试和RT-PCR确认的唯一候选基因是βig-h3，这是一个受TGF-β诱导、参与细胞黏附、迁移和增殖的基因。蛋白质免疫印迹法和免疫染色进一步证实了慢性伤口及其成纤维细胞中βig-h3表达降低。
这些发现表明βig-h3是表征慢性伤口成纤维细胞异常表型的一个重要基因。增加该蛋白表达的纠正措施可能具有治疗潜力。
