中文摘要
Vasostatin為calreticulin之N端片段(1-180 amino acids)，可有效且專一的抑制內皮細胞的增生，使新生血管無法生成，進而抑制腫瘤生長，但其抑制血管增生之機制尚未明確。因此，本論文朝(1) Fas /FasL pathway； (2) 氧化壓力； (3) 一氧化氮…等方向來研究vasostatin引起細胞凋亡的機制。利用流式細胞儀分析發現vasostatin處理過的BAEC (bovine aortic endothelial cells)，其pre-G0/G1之細胞週期百分比明顯上升；將vasostatin處理過的BAEC進行Hoechst 33258染色，於螢光顯微鏡下可觀察到細胞核縮小、核染色質濃集、皺縮及凋亡小體的產生；vasostatin處理過的BAEC有DNA fragmentation之現象，這些實驗都可用來說明vasostatin會引發內皮細胞進行細胞凋亡。以西方墨點法分析vasostatin處理凋亡蛋白之表現，發現Fas、FADD表現量明顯增加。而Fas經刺激後會形成death-inducing signaling complex (DISC)，FADD/MORT1會將訊息傳給caspase-8，造成caspase-8 oligomerization而相互活化，並且活化較下游的caspase，例如：caspase-3。進行caspase-3、caspase-8的活性及西方墨點法分析，都可發現隨著vasostatin濃度的增加，活化現象越明顯。然而caspase-3、-8的抑制劑僅能部分抑制由vasostatin所誘導的細胞凋亡，因此可能還有其他途徑的參與。再氧化壓力部分，因vasostatin處理而增加WST-1轉換成formazan之現象，因此推測vasostatin處理會促進superoxide的產生。我們以cytochrome c方法測定O2-，發現vasostatin 確實使BAEC培養液中superoxide含量顯著上升；抗氧化劑NAC、GSH、BHA可部分減緩vasostatin的毒殺作用； xanthine oxidase 的抑制劑allopurinol，對vasostatin所造成的細胞毒殺有很好的抑制作用，由此可知氧化壓力也是vasostatin造成BAEC凋亡的原因之一。以EMSA(electrophoretic mobility shift assays)的方法可測到轉錄因子NFκB的DNA結合能力增加，並以西方墨點法證實NFκB的量增加、IκB受到降解。此外，也可藉由EMSA分析得知，vasostatin處理會增加AP-1與DNA的結合能力；於西方墨點分析可看到c-Fos的表現，而這些轉錄因子的活化可能是藉由氧化壓力或其他原因所造成的。vasostatin處理後，可於培養液中測到一氧化氮(nitric oxide；NO)代謝物的增加，並由西方墨點分析中看到eNOS的增加，這可能與AP-1的活化有關；給予一氧化氮合成酶抑制劑L-NAME、NO清除劑，皆可稍微的減緩vasostatin的毒殺作用。此外，我們也發現vasostatin處理BAEC有劑量性增加nitrotyrosinlation之現象，推測可能是NO與O2-形成ONOO-，攻擊細胞內蛋白質，造成細胞損害。由以上結果推測vasostatin能由多重途徑引起內皮細胞的凋亡，而這些途徑之間的關係仍待進一步的研究。

Abstract
Vasostatin, the N-terminal 180 amino acids domain of calreticulin, induces apoptosis in endothelial cells and inhibits angiogenesis. However, the mechanism underlying the apoptosis induce by vasostatin remains elusive. In the present study, we investigated the role of (1) Fas /FasL pathway, (2) oxidative stress, and (3) nitric oxide (NO) in the apoptotic mechanism of vasostatin in endothelial cells. Recombinant vasostatin was generated and shown to induce apoptosis of bovine aortic endothelial cells (BAEC) as demonstrated by flow cytometry analysis, nucleus staining, and DNA fragmentation assay. Vasostatin elevated the levels of Fas and its adaptor, FADD, in BAEC. Furthermore, vasostatin treatment increased the activities as well as the expression of active form of caspase-8 and caspase-3 in BAEC. However, pretreatment with either caspase-3 inhibitor or caspase-8 inhibitor alone was not sufficient to blockade the vasostatin-mediated apoptosis, suggesting the involvement of other pathways. Extensive screening using an array of caspase inhibitors further supported such notion. Oxidative stress is frequently involved in the apoptosis of endothelial cells. Previous studies indicated that vasostatin enhanced WST-1-derived formazan formation despite its cytotoxic effect, suggesting vasostatin treatment might enhance the production of superoxide. By measuring the level of superoxide anion in cultured media by cytochrome c reducing test, it was found that vasostatin treatment increased the production of superoxide anion in endothelial cells. Antioxidants such as NAC, GSH, BHA partially attenuated the vasostatin-mediated cytotoxicity and cell death in endothelial cells. Noteworthingly, adding allopurinol, inhibitor of xanthine oxidase, but not other oxidase inhibitors abrogated the cytotoxicity of vasostatin, indicating that xanthine oxidase could be the source of ROS produced by vasostatin relate with apoptosis. The elecctrophoretic mobility shift assays (EMSA) suggested that vasostatin treatment increased the NFκB DNA binding activity. Western blot analysis indicated vasostatin increased the levels of NFκB but decreased IκB level, which seemed to coincide with the EMSA findings. NO plays an important role in endothelial function. To investigate the role of NO in the cytotoxicity by vasostatin, analyzed the levels of NO metabolites in cultured media of endothelial cells and found that vasostatin treatment increased NO release in time- dependent manners. The expression of eNOS, but not iNOS, in endothelial cells was upregulated by vasostatin. Besides, vasostatin treatment also increased the AP-1 binding activities. Moreover, NOS inhibitor, L-NAME, or NO scavenger, carboxy-PTIO, slightly attenuated the cytotoxic effects of vasostatin in endothelial cells. In addition to direct cytotoxicity, NO may react with superoxide (O2-) to form peroxynitrite (ONOO-), which attacked the intracellular protein and caused the cell damage. Indeed, we also detected a dose-dependent increment in the nitrotyrosination of cellular protein by vasostatin treatment. Taking together, these results indicate that vasostatin induces apoptosis in endothelial cells via multiple pathways. The interactions between these distinct pathways remain to be elucidated in the future.

目錄
第一章 前言
1-1血管增生(angiogenesis)…………………………………………….. 1
1-2血管增生抑制因子…………………………………………………..2
1-3 vasostatin……………………………………………………………..2
1-4血管增生和腫瘤細胞轉移(angiogenesis and metastasis) …………..4
1-5 apoptosis……………………………………………………………...5
1-6 Reactive oxygen species (ROS)…………………………………….. 9
1-7研究目的……………………………………………………………14
1-8研究目標……………………………………………………………14
第二章 材料與方法
2-1細胞培養……………………………………………………………16
2-2 vasostatin重組蛋白………………………………………………...16
2-3西方墨點分析………………………………………………………17
2-4細胞增殖分析
a. 抗氧化劑對處理vasostatin細胞之細胞增殖分析………………19
b. 過氧化物合成抑制劑對處理vasostatin細胞之細胞增殖分析…20
c. NO inhibitor、NO donor對處理vasostatin細胞之細胞增殖分析..21
d. caspase inhibitor對處理vasostatin細胞之細胞增殖分析……….21
2-5 caspase-3、-8活性分析…………………………………………….22
2-6 cytochrome c的還原偵測superoxide anion試驗…………………23
2-7 NO分析……………………………………………………………..23
2-8 DNA fragmentation分析…………………………………………...24
2-9 Hoechst 33258螢光染色...…………………………………………25
2-10 caspase inhibitors、抗氧化劑、過氧化物合成抑制劑、NO
inhibitor、NO donor於處理vasostatin細胞之細胞流式儀分析….25
2-11電泳移動偏移分析
a. 細胞核蛋白萃取……………………………………………26
b. 雙股核酸探針放射標定…………………………………...27
c. 電泳移動偏移分析…………………………………………27
第三章 結果
3-1 Fas /FasL pathway於vasostatin誘發內皮細胞凋亡之研究
a. vasostatin改變內皮細胞的型態、細胞核，並提高pre-G0/G1 level…………………………………………….29
b. vasostatin增加Fas、FADD表現，並活化caspase-3、caspase-8……………………………………………………29
c. Bcl-2、Bcl-xL可見減少的趨勢，並可看到Bax 的cleavage form p18……………………………………………………...30
d. caspase-1、-2、-3、-5、6、-8、-9、pan-inhibitor能部分減緩vasostatin抑制細胞的作用，尤以pan- inhibitor最為明顯……………………………………………………………30
3-2 vasostatin 誘導BAEC 產生過氧化物
a. vasostatin誘導BAEC產生O2-…………………………..31
b . 抗氧化劑能部分減緩vasostatin抑制細胞的作用…….31
c. xanthine oxidase inhibitor，allopurinol可減緩vasostatin抑制細胞的作用…………………………………………….32
d. vasostatin增加BAEC的XO轉換與NADPH oxidase的表現；XO的轉換可受allopurinol 影響……………………….33
e. vasostatin 處理增加BAEC內 NFκB的活性…….…….33
f. NFκB的表現量增加、IkB的表現量減少………………..34
3-3 vasostatin 誘導BAEC 產生NO
a. 培養液中可測到NO代謝產物NO2的量上升…………..34
b. vasostatin處理增加內皮細胞eNOS的表現……………..34
c. vasostatin 處理增加BAEC內AP-1的活性…………….35
d. vasostatin處理增加 c-Fos的表現……………………….35
e. NO inhibitor 能部分減緩vasostatin抑制細胞的作用；NO donor則稍微加強vasostatin抑制細胞的作用…………..35
f. vasostatin處理增加內皮細胞蛋白質nitrotyrosinylation
程度………………………………………………………….36
第四章 討論……………………………………………………………37
第五章 圖表……………………………………………………………44
第六章 參考文獻………………………………………………………76

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