利用抗血管生成 (antiangiogenesis) 的方式來治療癌症，已經引起許多人強烈的興趣，主要是因為他們廣泛的作用、低毒性以及直接以內皮細胞做為治療目標 (較不易引起細胞抗藥性的發生)。在新進增加種類的抗血管生成抑制劑中，endostatin是最受到注目的一種，同時它也是許多爭議的焦點。如同angiostatin或vasostatin抗血管生成抑制劑，endostatin是細胞外膠原蛋白XVIII (collagen XVIII)經過蛋白質水解脢作用後的一個片段 (~20 kDa)。它能有效地抑制內皮細胞的增生與血管的生成，但是對癌細胞本身卻不具任何毒殺效果。先前實驗利用遺傳工程生產出His-tagged endostatin並定期注射治療帶有腫瘤生長的老鼠，能導致腫瘤的萎縮並使腫瘤細胞處於休眠期 (dormancy），而這整個治療過程並不會導致抗藥性的發生。然而，我們對於endostatin抑制內皮細胞增生的機制仍然不清楚。我們利用RT-PCR從老鼠的肝臟中取得endostatin的cDNA，經過DNA定序確定後，將它殖入載體並送入E.coli中表現和產生大量的重組蛋白質 (GST-endostatin)。GST-endostatin可溶於水，而且可以20 nM的濃度抑制HUVEC, EA.hy926和BAEC的生長速率達百分之五十，這是和His-tagged endostatin的最大不同點。利用流式細胞儀分析細胞週期之變化，發現GST-endostatin可以誘導EA.hy926細胞的凋亡 (apoptosis)。而且0.5 nM的GST-endostatin，對於EA.hy926移向chemoattractant – bFGF的實驗，就可以達到IC50的抑制效果。在雞蛋絨毛尿囊膜的實驗中，GST-endostatin能夠抑制膜上的血管生成過程，而在帶有Lewis lung carcinoma的老鼠實驗中，GST-endostatin也能夠抑制腫瘤的生長。經過了這些功能的分析研究後，我們決定採用GST-endostatin及EA.hy926細胞來當作研究endostatin其抑制機制的模型。在使用fura-2螢光探針後，我們發現GST-endostatin能夠提高細胞內鈣離子的濃度，而這些鈣離子主要是由細胞外進入細胞內，且會隨著GST-endostatin處理濃度的增加而提高。利用EGTA去螫和細胞外的鈣離子，或是利用鈣離子通道抑制劑nifedipine去抑制離子通道，均能有效減緩GST-endostatin對內皮細胞的毒殺效果，由此可以看出GST-endostatin所導致的細胞內鈣離子增加，可能在抑制機制中扮演著重要的角色。而且GST-endostatin也會刺激Bkca channel的活動，更進一步支持GST-endostatin會導致一連串內皮細胞膜上離子通道活動的變化。呼吸脢的活動和內生ATP的合成均會被GST-endostatin嚴重地抑制，這兩個現象告訴我們：GST-endostatin能夠耗盡內皮細胞的能量來源。總之，截至目前的研究指出：GST-endostatin能夠引起內皮細胞電生理特性的改變和減少內生ATP的合成，而這可能在抑制機制中佔有重要的地位。

Antiangiogenic tomor therapies have attracted intense interest for their broad-spectrum action, low toxicity, and in the case of direct endothelial targeting, an absence of drug resistance. Among the growing list of antiangiogenic agents, endostatin has attracted most attention and been under the spotlight of numerous debates. Like other angiogenesis inhibitors, endostatin is also a proteolytic fragment (~20 kDa) from an extracellular protein, collagen XVIII. It potently inhibits endothelial cell proliferation and angiogenesis, but has no cytotoxic effects on cancer cells. Above all, therapy of experimental cancer with endostatin in rodents leads to tumor dormancy and does not induce resistance. However, the exact mechanism on how endostatin inhibited endothelial cells proliferation remains largely unknown. We have cloned mouse endostatin cDNA from mice liver by RT-PCR. After verification by DNA sequencing, endostatin cDNA was subcloned in to E.coli expression vector to express and generate large quantities of recombinant GST-fused endostatin (GST-endostatin). Unlike His-tagged endostatin, GST-endostatin is soluble and capable of inhibiting various endothelial cell lines including HUVEC, EA.hy926 and BAEC with IC50 ~ 20 nM. Flow cytometry analysis indicated GST-endostatin induced apoptosis in EA.hy926 cells. GST-endostatin also inhibited the cell migration of EA.hy926 cells toward chemoattractant bFGF with IC50 ~ 0.5 nM. Further more, GST-endostatin inhibited in vivo angiogenesis in chicken chorioallantoic membrane and suppressed tumor growth in mice bearing Lewis lung carcinoma cells. After functional characterization of GST-endostatin, we decided to use GST-endostatin and EA.hy926 cells as a model system to study the inhibitory mechanism of endostatin in endothelial cells. By using fura-2 fluorescence probe, GST-endostatin was shown to elevate the cytosolic calcium in dose-dependent manner from extracellular source. Chelation of extracellular Ca2+ by EGTA or inhibition of calcium channel by nifedipine abolished the cytotoxic effect endostatin, suggesting the calcium rise by endostatin play an important role in its inhibitory mechanism. Besides, endostatin also stimulated activity of a large- conductance calcium-activated potassium (Bkca) channel, further supporting endostatin initiated serial changes in ion channels activities in endothelial cells. Respiratory enzyme activities and endogenous ATP synthesis in endothelial cells were significantly inhibited by GST-endostatin treatment, indicating GST-endostatin depleted the energy source for endothelial cells. In summary, present study demonstrated GST-endostatin caused dramatic changes in electrophysiologic properties and decreased endogenous ATP synthesis in endothelial cells, which may participate in its inhibitory mechanism.

誌謝 1
中文摘要 3
英文摘要 5
目錄 7
緒論 14
Angiogenesis 14
Angiogenesis inhibitor and endostatin 15
材料與方法 19
材料 19
細胞株 19
藥品 20
購自Sigma藥廠 20
購自J. T. Baker藥廠 21
購自Merck藥廠 21
購自GibcoBRL藥廠 21
購自Perkin Elmer藥廠 21
購自Kodak公司 22
購自Boehringer Mannheim公司 22
購自Qiagen公司 22
購自RBI公司 22
購自Pierce公司 22
購自Amersham Pharmacia Biotech公司 22
購自CalBiochem公司 22
其它 23
方法 24
Fusion Protein 24
由老鼠（mice）的肝臟抽取純化RNA 24
以RT-PCR來選殖endostatin基因 24
DNA的瓊脂醣凝膠電泳 24
DNA定序分析 25
在大腸桿菌中表現並純化重組的endostatin 26
蛋白質濃度分析 27
蛋白質電泳 28
西方墨點法 29
產生endostatin的抗體 31
細胞培養 31
細胞數目之測定 32
蛋白質功能分析 33
細胞增生分析 33
Chorioallantoic Membrane Assay 34
Lewis Lung Carcinoma in BL57/6 Animal Model 34
Mechanism 34
流式細胞儀分析細胞週期之變化 34
Chemotaxis Assay 36
Zymography and Activity 37
Patch Clamp 38
細胞內鈣離子濃度測定 39
細胞內外ATP含量的變化 41
結果 42
合成Fusion Protein (GST-endostatin) 及測試其抑制效果 42
GST-endostatin造成細胞死亡的機制 46
討論 52
參考文獻 58
圖表 63
表一：內生性調控血管生成 (angiogenesis) 的物質。………..63
圖一：血管生成 (angiogenesis) 與抑制血管生成 (anti-angiogenesis) 的過程。 64
圖二：Endostatin的結構。 65
圖三：DNA電泳分析endostatin之RT-PCR產物。 66
圖四：選殖endostatin之DNA定序分析。 67
圖五：GST fusion protein與載體的簡易說明圖。 68
圖六：蛋白質之SDS-PAGE與Native Gel。 69
圖七：WST-1分裂為formazan之機轉。 70
圖八：內皮細胞株EA.hy926細胞經過不同濃度GST-endostatin或GST處理48小時後的型態變化。 71
圖九：Endostatin對EA.hy926細胞增生的抑制之劑量效應分析。 72
圖十：Endostatin對雞胚Chorioallantoic Membrane之新生血管的效應研究。 73
圖十一：Chorioallantoic Membrane Assay結果統計圖。 74
圖十二：Endostatin對Lewis Lung Carcinoma 在老鼠之生長效應測試。 75
圖十三：GST-endostatin對EA.hy926細胞48小時後之細胞週期分析。 77
圖十四：Boyden Chamber的結構圖。 79
圖十五：GST-endostatin對內皮細胞移行之效應。 80
圖十六：GST-endostatin對內皮細胞移行之劑量效應統計圖。 81
圖十七：GST-endostatin對內皮細胞MMPs分泌之效應。 82
圖十八：EA.hy926細胞經GST-endostatin處理1小時內的細胞型態變化。 83
圖十九：EA.hy926細胞經GST-endostatin處理24小時後的細胞型態變化。 85
圖二十：EA.hy926細胞經GST-endostatin處理48小時後的細胞型態變化。 87
圖二十一：FURA-2於不同隻鈣離子濃度時的激光光譜圖。 89
圖二十二：GST-endostatin對內皮細胞鈣離子濃度變化之效應。 90
圖二十三：GST-endostatin誘發胞外鈣離子進入內皮細胞。 92
圖二十四：Patch Clamp技術說明。 94
圖二十五：GST-endostatin刺激BKca Channel的活動。 96
圖二十六：GST-endostatin增加EA.hy926細胞膜上BKca Channel的開啟頻率。 97
圖二十七：GST-endostatin對BKca Channel電流和電壓關係的效果。 98
圖二十八：GST-endostatin對EA.hy926細胞ATP生成或釋放之變化。 99
圖二十九：EGTA對內皮細胞EA.hy926受到GST-endostatin處理的增生影響。 101
圖三十：QUIN-2/AM對內皮細胞EA.hy926受到GST-endostatin處理的增生影響。 103
圖三十一：Nifedipine對內皮細胞EA.hy926受到GST-endostatin處理的增生影響。 105
圖三十二：Endostatin對內皮細胞作用的簡單機制圖。 107
作者簡歷 108

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