| Abstract: | 廣東住血線蟲(Angiostrongylus cantonensis)其幼蟲寄生於宿主腦中，發育成熟後移行至肺動脈中。在太平洋及東南亞地區，大部份罹患嗜伊紅性腦膜灸的患者是因此寄生蟲寄生於腦部所引發，其感染途徑是因人攝食了感染廣東住血線蟲第三期幼蟲的蝸牛所造成，此幼蟲會穿過消化道移行至腦、脊髓及眼部而引起疾病，至今造成此種腦膜炎的致病機轉尚不十分清楚。蛋白分解酵素是指可分解Peptide bonds的酵素，其可出現在小到病毒大至人類的物種中，若以其作用部位的化學基來分類可將其分為四大類蛋白分解酵素，分別是Serine、Metallo、Thiol及Aspartyl；蛋白分解酵素可催化許多重要的生物反應，包括荷爾蒙原的代謝過程、血液凝結與血纖分解、蛋白代謝、免疫反應及組織復原，因此不難想像蛋白分解酵素在寄生蟲感染疾病中，可扮演很重要的角色，如協助寄生蟲侵入宿主組織、消化宿主蛋白、躲避宿主免疫系統攻擊及防止血液凝結；蛋白分解酵素的功能除了可幫助寄生蟲感染之外，對於研究寄生蟲發育過程中，基因調控方面的研究也可提供極易偵測的標的，此外其也可作為寄生蟲感染的免疫治療及化學治療藥物作用的對象，甚至可作為免疫診斷時偵測的標的。然而對廣東住血線蟲這種本土性的寄生蟲而言，至今仍無這類研究其蛋白分解酵素的報告。利用Gelatin及Casein-substrate zymography分析廣東住血線蟲第一期、第二期、第四期及第五期幼蟲和成蟲蟲體本身及各時期蟲體所分泌的蛋白酵素，再進一步以EDTA、Leupeptin及PMSF等不同類型蛋白質抑制劑加以分辨其蛋白酵素之種類，實驗結果得知：第一期和第三期幼蟲本身具有之酵素分子量為94kDa之MMP-9，而同時期蟲體亦可分泌相同之酵素但活性表現強度較弱。在第四、五期幼蟲及成蟲之蟲體及分泌並無發現此酵素。實驗結果顯示其與蟲體侵入宿主之機轉有關。
Angiostrongylus cantonensis, also named rat lung worm, was originally described by Chen in 1935 from the Rattus norvegius and R. rattus caught in Canton, China. It was found to mature in the pulmonary arteries of rats after migration from their brains. In the areas of Pacific and Southeast Asia, eosinophilic meningitis or meningoencephalitis is caused mainly by invasion of the human central nervous system by the lung worm. Human infection is from the ingestion of snails infected with third-stage larvae that will migrate into the brain, spinal cord, and eyes and cause disease. The pathogenesis of this parasitic meningitis is not fully understood until now. In Taiwan, people living in rural and mountain areas frequently collect and eat snails infected with larvae of A. cantonensis. Therefore, cases continue to occur yearly despite public educational programs. Physical diagnosis for A. cantonensis infection is difficult and inconsistent. Recently, some different immunodiagnostic methods for A. cantonensis infection in human have been achieved. Although immunodiagnosis is more convenient, the sensitivity and specificity is not good enough in these methods. On the other hand, effective therapy for A. cantonensis infection is still lacking. Proteases are enzymes that catalyze the hydrolysis of peptide bonds. They are found in species from viruses to humans. On the basis of the important chemical groups in their active site, proteases are separated into four major classes-serine, metallo, thiol and aspartyl. Protease catalyze a broad spectrum of important biological reactions, including prohormone processing, blood coagulation and fibrinolysis, protein metabolism. Immune reactions, and tissue remodeling. It is not suprising, therefore, that proteases have been found to play a number of critical roles in the pathogenesis of parasitic diseases. Parasitic proteases facilitate invasion of host tissues, allow parasites to digest host proteins, help parasites evade the host immune response, and prevent blood coagulation. Aside from the functions that proteases perform for parasites, they have been immensely useful in providing researchers with easily detectable probes for studying developmental regulation of parasite genes. Parasite-derived proteases have also been proposed as potential targets for immunotherapeutic and chemotherapeutic agents and, in some cases, serodiagnostic reagents for detection of parasite diseases. To date, no information is available on the proteases present in or secreted by the different developmental stages of A. cantonensis. The purpose of this study was to analyze proteases of first, third, fourth and fifth stage larvae of Angiostrongylus cantonensis, as well as proteases secreted or excreted from larvae into the cultured fluid by utilizing Gelatin and Casein-substrate Zymography. Moreover, different types of proteases inhibitors were distinguished through EDTA, Leupeptin and PMSF. The result of this study showed the first and third stage larvae carried protease which molecular weight was MMP-9 of 94kDa. Besides, exact the same protease with weaker presenting intensity was also found in the cultured fluid of first and third stage larvae No protease was found in either the fourth and fifth stage larvae, or in the same stage of cultured fluid. The finding of this study suggests the relationship between proteases and the mechanism of how the parasites attack the host environment. |
