文章發表：J Pharm Biomed Anal. 2010 Feb 5;51(3):784-94.
大綱
顯微再植手術及血管重建常用於挽救因交通事故而發生嚴重下肢骨折的血管受損受害者。但是，由於組織壞死與敗血症，有些患者需要在血管重建成功後一周內進行第二次截肢。需去了解潛在分子機制且此類事件是必要且應深入探討。因此，蛋白質體學在這項研究中用來評估氧化壓力於一系列嚴重道路交通事故中倖存人類之顯微手術後急性損傷中的應用。使用雙向電泳（2-DE）和免疫墨點法分析，可以觀察到蛋白質體的水平變化及蛋白質羰基化伴隨的含量。由於某些急性期蛋白質的氧化，不僅使其喪失抗氧化劑的功能，而且還啟動了調節細胞激素及趨化激素反應的細胞內壓力信號途徑，也過蛋白質排列分析評估細胞激素表現與氧化壓力的關係。據觀察，生長調節的致癌基因蛋白家族（GRO），IL-6，IL-8，IL-10和單核細胞趨化蛋白1（MCP-1）的範圍，血漿中負責嗜中性白血球和單核球聚集及隨後細胞毒性作用在被截肢者中明顯升高，在挽救的患者組中，趨化激素將白血球吸收到炎症位點（RANTES）的水平降低了。我們的結果表明，血行重建過程中的嚴重氧化損傷擾亂了正常的氧化還原平衡並促使特定蛋白質的羰基化，從而激活促炎因子，導致嚴重的組織損傷。此處揭示了不同的2-DE蛋白和細胞激素可能反映導致組織氧化損傷的不同病因，並且可以作為局部壞死和隨後需要再次截肢的關鍵指標。我們認為蛋白質組學和細胞因子結合的資料顯示，在本研究中提供了更可靠的信息，並在臨床實踐上定義了比當前使用的 C反應蛋白水平(CRP)或白血球(WBC)更複雜的標準，以預測需要肢體挽救手術之病人的二次截肢需求。
Microsurgical replantation and revascularization are frequently performed to salvage devascularized severe lower-extremity fractures in the human victims of road traffic-related injuries. However, some patients require secondary amputation within 1 week of successful revascularization due to tissue necrosis and sepsis. Enhanced efforts to understand the underlying molecular mechanism of such events are needed and should characterized in depth. Thus, functional proteomics were applied in this study to evaluate the role of oxidative stress in acute injury following microsurgery in a set of human subjects surviving serious road traffic accidents. Changes in the levels of protein volume and the accompanying content in protein carbonylation were visualized using two-dimensional electrophoresis (2-DE) and immunoblot analysis. Since oxidation of some acute-phase proteins not only causes them to lose their function as antioxidants but also initiates the intracellular stress signaling pathway that regulates cytokine and chemokine responses, how cytokine expression correlated with oxidative stress was also evaluated via protein array assays. It was observed that the growth-regulated oncogene protein family (GRO), the range of IL-6, IL-8, IL-10 and monocyte chemotactic protein-1 (MCP-1), which are responsible for neutrophil and monocyte aggregation with subsequent cytotoxic effects, were significantly elevated in the plasma of amputees subjects, whilst the level of chemokine recruiting leucocytes into inflammatory sites (RANTES) was diminished in the salvaged group of patients. Our results suggest that severely oxidative injury during revascularization perturbs the normal redox balance and induces carbonylation of specific proteins, thereby activating pro-inflammatory factors leading to severe tissue damage. The dissimilar 2-DE protein and cytokine profiles revealed here might reflect distinct etiologies resulting in oxidative damage to tissues and may serve as pivotal indicators of local necrosis and the subsequent need for secondary amputation of limbs. We believe that the combination of proteomic and cytokine profile results presented in this work offers more reliable information and defines more sophisticated criteria in clinical practice than currently used C-reactive protein levels (CRP) or white blood cells counts (WBC) for predicting secondary amputation requirements in patients requiring limb salvage surgery.
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