臺灣博碩士論文加值系統

基於腫瘤生長及其轉移的發生是必須依賴血管生成的發現，且由於抗血管生成的腫瘤療法具有不易產生抗藥性的特點，利用阻止血管的生成來抑制腫瘤及轉移的發生是近年來積極研究的方向。Angiostatin和endostatin分別是人體中plasminogen和collagen XVIII經蛋白質水解水解後的片段，已有研究指出，這兩個蛋白質片段能有效地抑制內皮細胞增生及腫瘤生長。在腫瘤位置長期維持這些抗血管生成因子的量及濃度是為了讓腫瘤限制在一定的大小之下且處於〝休眠〞的狀態，而欲達到這樣的目的，基因療法無疑是最好的選擇。
基於這樣的理由，我們分別構築了帶有人類angiostatin (HA)、endostatin(HE)和老鼠endostatin(ME) cDNA的重組反轉錄病毒載體。利用這些載體將抗血管生成因子的cDNA轉殖入GP7TB和RT2兩腫瘤細胞株中。首先我們以西方墨點分析法確定這些抗血管生成因子是可以分泌到細胞外表現。接著我們利用共同培養的方法和in vivo腫瘤生長性的實驗測試這些因子的生物活性。在in vitro實驗中雖然只有在會表現HA和HE的腫瘤細胞組中看到抑制內皮細胞增生的現象，但是在in vivo的腫瘤生長性實驗中，我們可以確定這三個抗血管生成因子都是具有生物活性的。
為了以後能直接進行in vivo的治療，我們也構築了帶有HA和ME的重組腺病毒(Ad-HA和Ad-ME)。在以重組腺病毒感染HUVEC細胞來測試這兩個抗血管生成因子的生物活性時，雖然我們只看到Ad-ME能抑制HUVEC細胞增生，並不表示Ad-HA就沒有這樣的效果。因為我們在選殖重組反轉錄病毒和重組腺病毒時所用的cDNA片段來源是一樣，理論上在重組反轉錄病毒中可看到效果，在重組腺病毒中也應該有一樣的效果。
此外，我們也構築了一個特定只在肝癌細胞中表現腺病毒E1蛋白質的質體，並確定此質體確實只在肝癌細胞中表現E1蛋白質以供給重組腺病毒複製所需，而並非是在子宮頸癌細胞中表現。
在本篇論文中，我們製備了帶有抗血管生成因子的重組反轉錄病毒和重組腺病毒，希望能將這些抗血管生成之基因療法的材料和腫瘤的免疫療法搭配使用，以達到治療的加成性。

Based on the observation that solid tumor growth and metastasis are angiogenesis dependent, antiangiogenic tumor therapies have recently attracted intense interest for their broad-spectrum action, low toxicity, and, in the case of direct endothelial target, an absence of drug resistance. To promote tumor regression and to maintain dormancy, anti-angiogenic agents need to be chronically administered. Gene therapy offers a potential way to achieve sustained therapeutic release of potent antiangiogenic substances. Angiostatin and endostatin , the proteolytic fragments of plasminogen and collagen XVIII, respectively, have recently been shown to potently inhibit endothelial proli-feration in vitro and tumor growth in vivo.
As a step toward this goal, we have generated re-combinant retroviral vectors that carry genes coding for human angiostatin(HA), human endo-statin(HE), and mouse endostatin(ME). These retroviral vectors successful-ly transduced angiostatin and endostatin cDNA in two rat tumor cell lines, GP7TB and RT2. We confirmed by Western blotting analysis that the anti-angiogenic factors could be secreted out of the cells. In vitro bioassays indicated that only the tumor cells expressing HA and HE could inhibit HUVE cells proliferation. Nevertheless, all three anti-angiogenic factors exhibited the potentials of regressing tumor in vivo.
We also generated recombinant adenoviral vectors which carried genes coding for HA or ME(Ad-HA or Ad-ME). Using the recombinant adenoviruses to infect HUVE cells at an MOI of 300, the proliferation of HUVE cells were inhibited by Ad-ME, but not by Ad-HA. However, we can't negate the biological activity of Ad-HA currently, because the cDNA fragment was obtained from the same source as that in construction of recombinant retroviral vectors.
We also constructed the pShuttle-AFP-E1 plasmid which could specifically express E1 protein in hepatoma cell lines. We have confirmed that expression of AFP-E1 could limit the replication of adenoviruses in hepatoma cells. Next step we will generate recombinant adenoviruses containing the AFP-E1 fragment. Our purpose is to construct a conditionally replicative adenovirus in order to improve the effects of adenoviral gene therapy.
In conclusion, in this study, we have generated recombinant retroviral and adenoviral vectors carrying antiangiogenic factor cDNAs. These vectors can provide a potential use for tumor gene therapy in combination with immunogene therapy.

【中文摘要】 ---------------------------------------------------------------------------------------------------- i
【英文摘要】 ---------------------------------------------------------------------------------------------------- iii
【縮寫表】 ------------------------------------------------------------------------------------------------------- vi
【導論】 ----------------------------------------------------------------------------------------------------------- 1
壹、血管生成(Angiogenesis)之機制與調控 ------------------------------------------- 1
貳、血管生成和腫瘤生長及轉移(Metastases) ---------------------------------------- 3
參、腫瘤之抗血管生成療法(Antiangiogenesis) --------------------------------------- 6
肆、抗血管生成因子之一---Angiostatin ------------------------------------------------ 7
伍、抗血管生成因子之二---Endostatin ------------------------------------------------- 12
陸、抗血管生成的基因療法 ------------------------------------------------------------- 14
【材料與方法】 ------------------------------------------------------------------------------------------------- 17
材料 --------------------------------------------------------------------------------------------- 17
方法 --------------------------------------------------------------------------------------------- 28
壹、抗血管生成因子(Antiangiogenic factors) cDNA之選殖 --------------- 28
貳、抗血管生成因子在大腸桿菌中的表現 --------------------------------- 39
參、反轉錄病毒的製備 ----------------------------------------------------------- 46
肆、重組腺病毒的製備(recombinant adenoviruses) -------------------------- 56
【結果】 ---------------------------------------------------------------------------------------------------------- 64
壹、抗血管生成因子cDNA之選殖與序列分析 ------------------------------------ 64
貳、在大腸桿菌系統表現、純化抗血管生成因子及其抗體之製備 -------- 65
參、抗血管生成因子穩定表現細胞株之建立及細胞株釋
放之抗血管生成因子的活性測試 ----------------------------------------------- 67
肆、利用重組腺病毒載體表現抗血管生成因子 ---------------------------------- 72
伍、提供特定情況下重組腺病毒複製能力之測試 ------------------------------- 73
【討論】 ---------------------------------------------------- 76
【總結】 ---------------------------------------------------- 83
【圖表】 ---------------------------------------------------- 84
【參考文獻】 ---------------------------------------------- 107

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