- 學位論文
沙漠黑蛇蛇毒鋅蛋白酶分子及非抗血清蛇咬救治之探討
Study on a metalloproteinase of Walterinnesia aegyptia venom and snakebite treatment
摘要
鋅蛋白酶普遍存在於各種出血性蛇毒內，一般認為是造成患者大量出血、水腫甚至是死亡的主要成分，因此被研究較為透徹，在神經性的蛇毒中則較少研究。根據蛋白質一級結構，鋅蛋白酶可區分成P-I、P-II、P-III class 三大類，本文發現一種全新之 P-III class 鋅蛋白酶存在於沙漠黑蛇(W. aegyptia)蛇毒當中，利用選殖定序完成其胺基酸序列，透過與其他蛇毒內鋅蛋白酶一級胺基酸序列的比較和種屬演化樹的分析，推測此酵素可能屬於非出血性 ( non-hemorrhage ) 的鋅蛋白酶，其胺基酸序列與Naja kaouthia 蛇毒內的 kaouthiagin有較高的相似性。 自從1970年代，中國大陸已有科學家利用注射胰蛋白酶的方式作為急救治療蛇咬傷，但對於出血毒蛇咬傷的治癒效果較差。我們試圖找尋更好的水解酶來做替代，並尋求抗蛇毒血清之外的急救配方。在in vitro實驗選擇馬來蝮蛇、台灣眼鏡蛇和草原響尾蛇做為研究對象，利用SDS-PAGE比較所選擇的角蛋白酶 (STC) 和胰蛋白酶水解蛇毒之能力，結果顯示STC水解蛇毒的效力大於胰蛋白酶，特別是EDTA也同時存在的清況下 ; 另外也發現蛇毒內之鋅蛋白酶能抵抗外來水解酶的破壞。 在in vivo實驗上利用馬來蝮蛇毒和小鼠作為模擬，研究小鼠預先注射蛇毒再注射EDTA ± STC後的效果及疼痛感，我們發現蛇毒內的鋅蛋白酶很可能是造成小鼠不適的成分之一。當注射蛇毒15分鐘內注射急救藥劑可降低死亡率，5分鐘內注射急救藥劑則可明顯降低皮膚潰爛的程度和小鼠的疼痛感。數據顯示此急救藥劑有潛力成為有用的蛇咬傷急救包，但急救藥劑內容未來可能要做些微調整，例如降低STC的劑量或是改變配方，由EDTA和hyaluronidase inhibitor搭配做治療。
並列摘要
The snake venom metalloproteinases (SVMPs) are abundant in snake venoms and have been considered as the key toxins involved in snake venom-induced pathogenesis, such as hemorrhage, edema and mortality. According to their primary structures, SVMPs can be classfied into three categories. Here I cloned a novel P-III class SVMP from the cDNA prepared from the Walterinnesia aegyptia venom glands and determined its cDNA sequence and full amino acid sequence. By phylogenetic analysis this P-III was found to be a non-hemorrhagic SVMP, and is most similar to the P-III SVMPs from Asian and African cobra venom. Since 1970, Chinese researchers have developed the First-Aid Kit injecting trypsin solution to the bite area for snakebite treatments.The treatment has been ineffective against viper’s snakebites, Here I tried to find better protease to replace trypsin for snakebite treatment. I also looked into possible improvement of the non-antiserum or preliminary snakebite treatment. For in vitro analyses using SDS-PAGE, I have chosen crude venom of three medically important species as the substrates : Calloselasma rhodostoma, Crotalus viridis viridis and Naja atra, and compared the efficiencies of trypsin and Subtilisin Carlsberg (STC) to degrade these venom. Results of these in vitro experiments showed that STC was better than trypsin in the hydrolysis of snake venom, especially when was also included EDTA. Besides, we found that SVMPs in viper snake venom were rather resistant to trypsin digestion. Furthermore, we used mice as a model to study the pain level and protection effect of STC and EDTA after injecting C. rhodostoma venom (CRV). It was found that SVMPs in CRV may be the source of pain. When first aid treatment by EDTA together with various level of STC was given within 15 min after envenomation, the lethality of venom was reduced, and skin necrosis and pain level of the mice were reduced when EDTA and/or STC was given within 5 min after the venom injection. These data confirm that the first-aid treatment have potential to be useful for snakebite treatment. Further refinement of antidose, e.g. EDTA and lower dose of STC; or EDTA plus hyaluronidase inhibitor.