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標題: | 從動物模型中建立實驗性心肌梗塞之病理診斷準則:
心臟組織之早期損傷鑑定 Establishment of Pathological Criteria for Experimental Myocardial Infarction in Animal Model: Assessment of the Early Damage of the Myocardium |
作者: | He-Shiuan Liang 梁赫烜 |
指導教授: | 王汎熒 |
關鍵字: | 心衰竭,早期診斷,病理檢查,特殊染色, myocardial infarction,early diagnosis,pathology,IHC, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 傳統心肌的組織學檢查,在透過蘇木紫(hematoxylin)及伊紅(eosin)的染色後,對於心肌細胞的死亡可以由下列標準(本研究中定為方法A)來判定:1) 核濃縮(pyknosis)、核破裂(karyorrhexis)及核溶解(karyolysis);2) 肌漿斷裂、棉絮化(flocculation)及/或出現收縮帶(contraction band);3) 炎症細胞的浸潤。然而,多數以上變化並無法在心肌細胞死亡後六小時內觀察到。本研究中,使用多種方式實驗性的致使心肌壞死或衰竭,包括:1) 莫能菌素中毒(monensin intoxication);2) 左冠狀動脈前降枝永久性30分鐘結紮(ligation of the left anterior descending (LAD) coronary artery for 30 min without reperfusion); 3) 左冠狀動脈迴旋枝永久性30分鐘結紮(ligation of the left circumflex (LCX) branch of coronary artery for 30 min without reperfusion)及4) 鈣離子阻斷劑併用乙型交感神經拮抗劑之生理性衰竭(application of calcium blocker combined with beta antagonist)。除了上述傳統壞死標準外,本研究中加入了另外三種組織學診斷標準(歸納為方法B),專門針對早期心肌傷害或壞死,包括:1) 彎曲變形或萎縮濃染的細胞核(wavy or condensed nucleus); 2) 過度嗜伊紅性的肌漿伴有細胞腫脹或萎縮(hypereosinophilia of sacroplasm with either swelling or atrophy in diameter); 3) 核旁疑似胞器腫脹(粒線體或肌內質網)混濁腫脹的空泡(perinuclear cloudy vacuolation likely results from swollen organelles such as mitochondria and/or sarcoplasmic reticulum)。 使用免疫組織化學染色針對心肌內結構性蛋白(structural protein)、功能性蛋白(functional protein)、胞器內酵素型蛋白(organelle-binding enzymatic protein)及第九號補體(complement 9)。將上述染色結果依標準量化並以統計比對,結果顯示對於心肌早期壞死及傷害,方法B有著和cardiac troponin I(cTnI)及telethonin (T-cap)相同的有效診斷能力,且強過本實驗中其他早期診斷性標的蛋白(biomarker)。另外,本實驗染色結果中,T-cap 從壞死區的流失現象比現今的黃金標準cTnI略為顯著且廣泛。基質金屬蛋白酶的染色顯示(matrix metalloproteinase 2, MMP2)此酵素不僅同時出現在胞內肌漿及胞外基質,還在部分受損心肌細胞中輕微增加,此耐人尋味的結果意謂著此酵素有潛力做為一心肌壞死標的物。其餘標的物則沒有上述數種蛋白顯著或僅有相似的診斷能力。至於C9在本實驗中則無法提供早期診斷能力(無明顯沉積)。 Traditionally histological criteria used to interpret necrotic cardiomyocytes on hematoxylin and eosin (H&E) stained tissue sections (designated as “method A” in this study) are: 1) nuclear pyknosis, karyorrhexis, and karyolysis; 2) sarcoplasmic fragmentation, flocculation, and/or the presence of contraction band; 3) inflammatory infiltration. However, these changes are usually unremarkable until at least 6 hours postnecrotic. In this study, myocardial necrosis were experimentally induced by various models: 1) monensin intoxication; 2) ligation of the left anterior descending (LAD) coronary artery for 30 min without reperfusion; 3) ligation of the left circumflex (LCX) branch of coronary artery for 30 min without reperfusion; and 4) application of calcium blocker combined with beta-antagonist to induce functional left heart failure. Three additional criteria (designated as “method B”) were also proposed in this study to interpret early cardiomyocyte necrosis or myocardial injuries on H&E stained tissue sections. They were: 1) wavy or condensed nucleus; 2) hypereosinophilia of sacroplasm with either swelling or atrophy in diameter; 3) perinuclear cloudy vacuolation likely results from swollen organelles such as mitochondria and/or sarcoplasmic reticulum. Immunohistochemistry (IHC) using antibodies against structural proteins, functional proteins, organelle-binding enzymatic proteins together with complement 9 were also employed. The IHC stained section were scored with a known system and compared statistically with methods A and B for their sensitivity or superiority in detecting early MI in tissue specimens. It revealed that method B was as sensitive as cardiac troponin I (cTnI) and telethonin (T-cap), which were more superior to other biomarkers used in detecting early necrotic events in cardiomyocytes. In instances T-cap was slightly superior to cTnI, the current gold standard. The staining for matrix metalloproteinase 2 (MMP2), an intracellular and extracellular proteins, revealed intriguing results and suggested early activation and early diagnostic potential of this molecule for necrotic events. Other biomarkers were either fair or insensitive (such as deposition C9) for detecting early necrosis in the models used here in this study. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62743 |
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顯示於系所單位: | 獸醫學系 |
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