從心肌炎組織切片探討常見致病RNA腸病毒的盛行率

Abstract

急性心肌炎所造成的心因性猝死，一直都是法醫學的重要研究議題。心肌炎的致病因子，包括：微生物感染、自體免疫疾病與毒藥物等。在北美洲與歐洲地區，病毒感染是心肌炎的重要致病因子。然而，臺灣本土性心肌炎的致病病毒盛行率至今仍不明確。近年來，福馬林固定石蠟包埋組織蠟塊的核酸萃取技術與分子生物學診斷技術日益成熟。因此，有越來越多的研究文獻開始使用心肌蠟塊作為實驗材料，進行心肌炎的流行病學研究。本篇論文的研究目的是藉由診斷為心肌炎的心肌蠟塊作為實驗材料，進行臺灣本土性心肌炎的腸病毒盛行率研究。本研究所使用的心肌蠟塊總共十一件，九件是經心導管採樣的心肌蠟塊，一件是心臟移植病人的左心室心肌蠟塊以及一件法醫解剖案例的心肌蠟塊。利用腸病毒VP1單株抗體進行免疫組織化學染色，並針對腸病毒的5’ NTR基因片段進行反轉錄聚合酶鏈鎖反應，用以檢測心肌蠟塊有無受到腸病毒的感染。實驗結果發現，在十一件心肌蠟塊中，有七件心肌蠟塊的腸病毒免疫組織化學染色呈現陽性結果。此七件腸病毒免疫組織化學染色呈陽性結果的心肌蠟塊，經由反轉錄聚合酶鏈鎖反應檢測後，發現共有四件心肌蠟塊檢測出腸病毒5’ NTR基因的存在。本研究證實，臺灣心肌炎中約有36.4% 與腸病毒感染有關。除此之外，本研究也發現萃取自心肌蠟塊的RNA片段多已降解至152 ~ 470 nt之間，因此在使用反轉錄聚合酶鏈鎖反應檢測組織蠟塊中的RNA表現時，應選擇產物片段大小為200 bp左右的引子組較為適合。本研究所獲得的心肌炎腸病毒盛行率僅是臺灣心肌炎流行病學的一小部分，期望未來能藉由臺灣法醫解剖案件中，所保存下來的心肌蠟塊繼續進行大規模本土心肌炎致病病毒盛行率的研究。

The etiological factor of sudden cardiac death due to acute myocarditis has long been an important issue in forensic medicine. Due to the progression of molecular technique, there are increasing researches about the etiology of myocarditis. Although the cause of myocarditis in any given pations, systemic diseases, drugs, and toxins have been associated with the development of this disease. Viruses are an important cause of myocarditis in North America and Europe. The prevalence of viral myocarditis is still unclear in Taiwan. The purpose of this study was to investigate the prevalence of myocarditis infected by RNA enterovirus in Taiwan. The formalin fixed paraffin embedded myocardial tissue blocks of myocarditis were obtained from endomyocardial biopsy (9 samples), heart transplantation (1 sample) and forensic autopsy specimen die of myocarditis (1 sample). Tissue blocks were studied by using immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) for enterovirus. This study showed that histological section from 7 of 11 myocarditis cases were positive for the viral capsid protein VP1 by immunohistochemical staining. In 4 of 7 VP1 immunohistochemical positive staining specimens, viral genome of enterovirus was detected by RT-PCR using 5’ NTR genomic fragment. These results showed most RNA fragment recovered by RT-PCR which fall between 152-470 nt. The product of RT-PCR probe should less than 200 bp which is the best candidate in these recovery. Also the study provided data of rough enteroviral prevalence in myocarditis (more than 36.4%). Further evidence of prevalence about enterovirus involvement in myocarditis needs more large scale of such cases using the method provided in the study to get in Taiwan.