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Title: | 蛇毒蛋白 Trimpase引起內皮細胞凋亡及影響血小板凝集作用機轉之研究 The Effects of Trimpase, a Metalloproteinase of Trimeresurus mucrosquamatus, on Endothelial cell Apoptosis and Platelet Aggregation |
Authors: | Yi-Hung Shih 史一宏 |
Advisor: | 黃德富 |
Keyword: | 蛇毒金屬依賴性蛋白酶,細胞凋亡,液相層析,血小板凝集,纖維蛋白原, snake venom metalloproteinase,apoptosis,liquid chromatography,platelet aggregation,fibrinogen, |
Publication Year : | 2007 |
Degree: | 碩士 |
Abstract: | 蛇毒金屬依賴性蛋白酶 ( SVMP )是一種在結構上和存在體內的ADAM ( a disintegrin and a metalloproteinase) 相似的蛋白酶。蛇毒金屬依賴性蛋白酶可分為四大類,分別為 N-I ~N-IV 及 P-I ~P-IV,其中 N及P分別表示核苷酸及蛋白質。本實驗中從台灣龜殼花蛇毒中所分離出的trimpase 其還原或非還原態的分子量,則大約在16kDa,是一個分子量較小的 P-I 蛇毒金屬依賴性蛋白酶。Trimpase 可抑制由 thrombin或collagen所引起的血小板凝集,並且其抑制活性可被EDTA前處理所抑制。根據MTT檢測、DNA片段化檢測及流式細胞儀分析細胞週期之DNA含量皆顯示它會引起人類臍靜脈內皮細胞的凋亡。進一步實驗顯示 trimpase引起的內皮細胞凋亡,首先是經由分解細胞外基質(如:纖維蛋白原)而抑制內皮細胞的附著及加速細胞的脫落。Trimpase 主要會裂解人類纖維蛋白原的 α 鏈,但隨著處理的濃度及時間增加時也會裂解 β 鏈。Trimpase會抑制內皮細胞附著至人類纖維蛋白原上,也會造成已貼附在人類纖維蛋白原內皮細胞的脫落,這些作用會因為前處理EDTA而被抑制。相對上,trimpase 不會抑制細胞附著至膠原蛋白上。除了纖維蛋白原會被分解,我們同時發現 adherens junction中的α-catenin 和β-catenin會被裂解,而另外VE-cadherin和γ-catenin 則沒有明顯的影響。另一方面,我們也發現 Bcl-2 的表現量減少,然而 Bax 的表現量增加,這兩者的改變都會促進細胞凋亡,綜合以上所述,我們發現一個小分子量的 P-I 龜殼花蛇毒金屬依賴性蛋白酶 trimpase ,主要利用其酵素的活性而抑制內皮細胞附著和裂解 adherens junction 使得內皮細胞產生細胞凋亡。而trimpase 會藉由裂解ECMs而抑制血小板凝集。 Snake venom metalloproteinase (SVMP), which is structurally similar to ADAM ( a disintegrin and a metalloproteinase ), is Zn2+ -dependent proteinase and have a catalytic sequence, i.e. HEXXHXXGXXH. Based on their domain structures, SVMPs are divided into four classes, N-I to N-IV and P-I to P-IV, where N and P represent nucleotide and protein, respectively. The molecular weights of P-I SVMPs are about 20~30kDa. However, the reduced or non-reduced molecular weight of trimpase, a SVMP purified from Trimeresurus mucrosquamatus in this study, migrates as a molecule of 16kDa. Trimpase inhibits platelet aggregation induced by thrombin and collagen, and its inhibitory effect could be blocked by pretreating with EDTA. According to MTT assay, DNA fragmentation, and cell cycle analysis with flow cytometer, all results demonstrated that trimpase can induce endothelial cell apoptosis. Further experiments reveal that the mechanism of apoptosis induced by trimpase was through degradation of extracellular matrices and inhibiting endothelial cell adhesion and inducing endothelial cell detachment. Trimpase preferentially cleaved α chain of fibrinogen and cleaved β chain after a longer incubation or at a higher concentration. Trimpase inhibited endothelial cell adhesion to immobilized fibrinogen and induced endothelial cell detachment. The effects on endothelial cell were abolished when trimpase was pretreated with EDTA. However, trimpase did not inhibit endothelial cell adhesion to immobilized collagen. The adherens junctions protein including α-catenin and β-catenin were cleaved by trimpase, but VE-cadherin and γ-catenin were not affected. On the other hand, we also found that the expression of Bcl-2 decreased and that of Bax increased in trimpase treated HUVECs, suggesting induction of cell apoptosis. In summary, we found a P-I SVMP with a smaller molecular weight, named trimpase, and it can inhibit endothelial cell adhesion and cleave adherens junction to induce endothelial cell apoptosis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/29364 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 藥理學科所 |
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