探究血液發炎參數與自然免疫反應的變化在臨床急性闌尾炎的預測價值與敗血性腹膜炎動物實驗的角色

Predictive Value of Inflammatory Markers and Expression in Proinflammatory Markers of the Innate Immunity System in Patients with Perforated Appendicitis and in Rats with Polymicrobial Septic Peritonitis Induced by Cecal Ligation and Puncture

吳漢屏(Han-Ping Wu)

指導教授 ： 林 清 淵

長榮大學/健康科學學院/醫學研究所/碩士(2007年)

https://doi.org/10.6833/CJCU.2007.00051

摘要

急性闌尾炎不僅是急診室最常見的腹部急症之一，也是急診室裡需要接受緊急腹腔手術的病患中最常見的病因之一。單純性(非破裂性)闌尾炎可以進展到破裂性闌尾炎，而破裂性闌尾炎將會導致更嚴重的敗血性腹膜炎與敗血症，倘若未積極治療，甚至會有致死的可能性。在臨床上，如果能夠在及早正確的診斷急性闌尾炎，則可以避免其進展到闌尾破裂而導致腹膜炎的發生；同時，早期診斷可以減少因延遲處置所造成的醫療資源耗損。然而，要在早期正確診斷急性闌尾炎並不容易，使得目前臨床上急性闌尾炎的誤診率及闌尾破裂率居高不下。

為了能提高急性闌尾炎的診斷正確性，本研究採回溯性分析急性闌尾炎病患之C-反應蛋白在臨床診斷上的價值與應用。再者，第二部份研究使用前瞻性分析方法來探究不同時段的血液發炎參數的變化是否可以預測急性闌尾炎的存在。第三部份則分析前發炎標記在被切除的闌尾上的病理免疫表現；最後，第四部份我們利用敗血性腹膜炎大老鼠的動物模式，來探究前發炎標記在急性腹症病程進行中的表現差異

本研究首先收集568位從民國90年至民國93年因疑似急性闌尾炎至彰化基督教醫院就診的病患資料，並將病患症狀持續的天數（包括第1天，第2天及第3天）與其C-反應蛋白的數值做分析，探究此二者之關聯性。第二部份研究自民國92年12月至民國93年12月期間，前瞻性收集225位因「疑似急性闌尾炎」而至彰化基督教醫院就診的病患，將二次血液檢測後有意義改變的參數與症狀持續的天數進行ROC（receiver operating characteristic）分析以探究其對急性闌尾炎之區辨能力。第三部份我們篩選自民國95年5月至民國95年12月共86位因臨床診斷為急性闌尾炎而至彰化基督教醫院就診並接受闌尾切除手術的病患，將切除的闌尾進行myeloid活化因子88（MyD88）與TRIF-related adaptor molecule（TRAM）蛋白的免疫組織化學（IHC）分析。第四部份我們將大老鼠使用盲腸結紮戮孔（CLP）手術引發敗血性腹膜炎，並進一步分析MyD88依賴性與非依賴性路徑在病程中的變化與扮演的角色。

本研究結果顯示，急性闌尾炎病患之C-反應蛋白較非闌尾炎之病患之C-反應蛋白呈現有意義的上升（P＜0.001）；而破裂性闌尾炎病患之C-反應蛋白數值則會比單純性闌尾炎病患C-反應蛋白數值來得高（P＜0.001）。我們找出C-反應蛋白在第一天為15mg/L，第二天為40 mg/L，第三天為105 mg/L，可以做為區辨急性闌尾炎與其他腹症之參數; 而在破裂性闌尾炎方面，我們定出C-反應蛋白的數值在為第一天33mg/L，第二天85mg/L，第三天120 mg/L可做為區辨破裂性闌尾炎之參數。第二部份，根據多變項邏輯回歸分析得出二次抽血檢驗之間有意義改變的參數：第一天為嗜中性白血球（P=0.005）；第二天為C-反應蛋白（P=0.024）；第三天亦為C-反應蛋白（P=0.032）。第一天的嗜中性白血球在二次檢驗後的數值比第一次數值上升達3.2％；第二天的C-反應蛋白的改變增加9.5 mg/L；第三天C-反應蛋白的改變增加17.0 mg/L，則可用來區辨急性闌尾炎與其他腹部疾病。第三部份結果顯示MyD88蛋白在單純性闌尾炎早期階段的表現比正常闌尾以及破裂性闌尾炎都來得顯著，但在晚期階段則無差異。相對地，TRAM蛋白的表現在破裂性闌尾炎的呈現，無論是早期階段或晚期階段，都比單純性闌尾炎來得顯著。在第四部份的結果，敗血性腹膜炎CLP組大白鼠於術後6小時的MyD88 mRNA與蛋白質之表現比起對照組有明顯增加的情形，而在術後24小時的表現則相對地減少；但TRAM蛋白則是在術後24小時及術後72小時的表現較術後6小時有明顯增加的情形，而非MyD88依賴性路徑的下游因子的表現亦是如此。

關鍵字

C-反應蛋白 ； 血液發炎參數 ； myeloid活化因子88 ； TRIF-related adaptor molecule（TRAM）C-反應蛋白 ； 血液發炎參數 ； myeloid活化因子88 ； TRIF-related adaptor molecule（TRAM ； 急性闌尾炎 ； CLP動物實驗。 ； 急性闌尾炎 ； CLP動物實驗。

並列摘要

Acute appendicitis is the most common abdominal surgical emergency. As evidenced by high misdiagnosis and perforation rates, the diagnosis of appendicitis is still difficult and probably the most common problem in clinical surgery. Diagnostic difficulties lead surgeons to perform unnecessary appendectomies while misdiagnosis can lead to perforation, and abscess formation. Perforated appendicitis may carry septic peritonitis and mortality. The aims of this study were to det1ermine whether the different cutoff values of C-reactive protein (CRP) based on the first 3 days after the onset of patient’s symptoms can be used to early predict acute appendicitis, and to determine whether the change between primary and repeated serum inflammatory markers measured 6 to 10 hours later could improve diagnostic accuracy in appendicitis. Furthermore, we intended to investigate whether proinflammatory markers can aid in the differentiation of perforated appendicitis, and to determine the time points of expressions of myeloid differentiation factor 88 (MyD88)-dependent and MyD88–independent pathways in septic peritonitis animal model. We first analyzed retrospectively from 2001 to 2004 the hospital records of 568 patients who underwent appendectomies for suspected appendicitis. From 2003 to 2004, our prospectively study next comprised 225 patients who presented to hospital with equivocal signs of appendicitis. According to the period from the onset of symptoms to admission, the receiver operating characteristic (ROC) curves were used to determine the cutoff values of CRP concentration in predicting acute appendicitis, and to determine the cutoff values of the change between primary and repeated laboratory examinations in diagnosing appendicitis. From 2005 to 2006, 86 patients with suspected appendicitis were enrolled in further analysis. The protein expressions of tissue MyD88 and TRIF-related adaptor molecule (TRAM) in appendicitis were studied by immunohistochemistry (IHC) staining of excised appendices from appendectomies. Furthermore, a randomized animal study was investigate to clarify the roles of MyD88-dependent and MyD88-independent pathways in ninety male Wistar rats with septic peritonitis induced by cecal ligation and puncture (CLP).
ROC analysis has shown that CRP measurement can increase the diagnostic accuracy in acute appendicitis. The cutoff values of CRP concentration taken as the first (Day 1), second (Day 2) and third (Day 3) days after onset of symptoms which distinguish acute appendicitis from other acute abdominal diseases were 15 mg/L, 40 mg/L and 105 mg/L respectively; the values which distinguish perforated appendicitis from other acute abdominal diseases were 33 mg/L (Day 1), 85 mg/L (Day 2) and 120 mg/L (Day 3). ROC analysis also revealed that the cutoff values for the change in total neutrophil count on Day 1 (3.2%), and in CRP concentration on Day 2 and Day 3 (9.5 mg/L and 17.0 mg/L, respectively) were significant parameters for acute appendicitis. Based on the results of IHC staining of excised appendices, tissue MyD88 protein expression was predominant in early-stage nonperforated appendicitis, but TRAM could be highly expressed in perforated appendicitis in early and late stages. In our experimental animal model, CLP septic peritonitis increased liver MyD88 mRNA and protein expressions compared to controls at 6 hours after surgery, but decreased expressions after 24 hours. In contrast, CLP septic peritonitis increased liver TRAM protein expression 24 hours after surgery. Also, the downstream factors of the MyD88-independent pathway elevated 24 hours after surgery. In conclusions, serum inflammatory biomarkers may serve as a kind of useful predictors for nonperforated and perforated appendicitis. However, the expression of proinflammatory markers, including MyD88 and TRAM may aid in early differentiating perforated appendicitis from nonperforated appendicitis. Furthermore, during CLP septic peritonitis, the MyD88-dependent pathways can be activated early and subsequently the MyD88-independent pathways will be induced to enhance signal transduction in experimental rats.

並列關鍵字

C-reactive protein ； serum inflammatory markers ； myeloid differentiation factor 88 ； TRIF-related adaptor molecule (TRAM) ； acute appendicitis ； cecal ligation and puncture (CLP).

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