Corallo Claudio, Santucci Annalisa, Bernardini Giulia, Figura Natale, Leoncini Roberto, Riolo Giulia, Montella Antonio, Chirico Chiara, Nuti Ranuccio, Giordano Nicola
From the Department of Medicine, Surgery, and Neurosciences; the Department of Biotechnology, Chemistry and Pharmacy; and the Department of Medical Biotechnology, University of Siena, Siena, Italy.
C. Corallo, PhD, Department of Medicine, Surgery and Neurosciences, University of Siena; A. Santucci, PhD, Department of Biotechnology, Chemistry and Pharmacy, University of Siena; G. Bernardini, PhD, Department of Biotechnology, Chemistry and Pharmacy, University of Siena; N. Figura, MD, Department of Medicine, Surgery, and Neurosciences, University of Siena; R. Leoncini, PhD, Department of Medical Biotechnology, University of Siena; G. Riolo, PhD, Department of Medical Biotechnology, University of Siena; A. Montella, MD, Department of Medicine, Surgery, and Neurosciences, University of Siena; C. Chirico, MD, Department of Medicine, Surgery, and Neurosciences, University of Siena; R. Nuti, MD, Department of Medicine, Surgery, and Neurosciences, University of Siena; N. Giordano, MD, Department of Medicine, Surgery, and Neurosciences, University of Siena.
J Rheumatol. 2017 Jan;44(1):40-48. doi: 10.3899/jrheum.160736. Epub 2016 Dec 1.
To identify using proteomic analysis the proteins of altered abundance in the affected and unaffected limited cutaneous systemic sclerosis (lcSSc) skin fibroblasts.
Excision biopsies (3 mm) were obtained from the affected and unaffected skin of 5 patients with lcSSc. Dermal fibroblasts were isolated enzymatically. Two-dimensional gel electrophoresis was used to separate and define proteins in affected and unaffected fibroblast lysates. Proteins of altered abundance were identified by mass spectrometry. Differences among skin samples were confirmed also by immunohistochemistry (IHC) and by quantitative real-time PCR (qRT-PCR) for type I collagen (Col-1) and vimentin (VIM).
Proteomic analysis revealed different expressions of proteins involved in cytoskeleton organization (27%), extracellular matrix remodeling (11%), response to oxidative stress (22%), energy metabolism (19%), protein metabolism (5%), cellular homeostasis (5%), signal transduction (3%), and protein transcription, synthesis, and turnover (8%). IHC analysis showed that SSc-affected epidermis is thickened and the dermis is strongly reactive to Col-1 and VIM (typical markers of activated myofibroblasts) compared to SSc-unaffected skin, whose stainings are comparable to those of control healthy skin. Overexpression of Col-1 and VIM mRNA levels in affected lcSSc fibroblasts compared to unaffected lcSSc ones was confirmed by qRT-PCR.
Consistent with previous studies, these findings are important for 2 reasons: first, because they reveal the opposite behavior of dermal fibroblasts in the unaffected and affected skin areas of the same patient with lcSSc; second, because they demonstrate the histological/histochemical similarities between unaffected skin from patients with lcSSc and healthy control skin.
通过蛋白质组学分析确定局限性皮肤型系统性硬化症（lcSSc）患者受累及未受累皮肤成纤维细胞中丰度改变的蛋白质。
从5例lcSSc患者的受累及未受累皮肤获取切除活检标本（3毫米）。通过酶解法分离真皮成纤维细胞。采用二维凝胶电泳分离并鉴定受累及未受累成纤维细胞裂解物中的蛋白质。通过质谱鉴定丰度改变的蛋白质。还通过免疫组织化学（IHC）以及针对I型胶原蛋白（Col-1）和波形蛋白（VIM）的定量实时聚合酶链反应（qRT-PCR）来确认皮肤样本之间的差异。
蛋白质组学分析显示，参与细胞骨架组织（27%）、细胞外基质重塑（11%）、氧化应激反应（22%）、能量代谢（19%）、蛋白质代谢（5%）、细胞稳态（5%）、信号转导（3%）以及蛋白质转录、合成和周转（8%）的蛋白质存在不同表达。免疫组织化学分析表明，与未患SSc的皮肤相比，患SSc的表皮增厚，真皮对Col-1和VIM（活化肌成纤维细胞的典型标志物）呈强烈反应，未患SSc的皮肤染色与健康对照皮肤相当。qRT-PCR证实，与未受累的lcSSc成纤维细胞相比，受累的lcSSc成纤维细胞中Col-1和VIM mRNA水平过表达。
与先前研究一致，这些发现具有重要意义，原因有二：其一，它们揭示了同一lcSSc患者未受累及受累皮肤区域真皮成纤维细胞的相反行为；其二，它们证明了lcSSc患者未受累皮肤与健康对照皮肤在组织学/组织化学上的相似性。
