臺灣博碩士論文加值系統

最近許多研究報導指出，可藉由蛋白傳送區域 (PTDs) 例如愛滋病毒 (human immunodeficiency virus) 之TAT (transcription activator) 的蛋白傳送區、第一型單純疱疹性病毒 (herpes simplex virus-1)的VP22 (DNA-binding protein) 與想傳送各種分子 (如合成性小分子、胜肽、或蛋白質) 進行基因融合，幫助物質送入活體內外標的處。多種細胞中測試，藉由TAT或VP22傳送蛋白質的過程效率非常高且毒性低。本實驗中，首先想比較植物毒素A胜肽鏈(Abrin-A chain; ABR-A)及雞貧血病毒蛋白 (Apoptin)兩種天然毒素的差異性。由植物(Abrus precatoriusa)種子萃取出的植物毒素(Abrin)，含有具毒性的A胜肽鏈和能與細胞結合的B胜肽鏈，兩者藉由雙硫鍵結合。缺少B胜肽鏈，植物毒素A (ABR-A) 能有效抑制抑制蛋白質轉譯且不具細胞毒性。Apoptin衍生自雞貧血病毒，許多實驗指出此蛋白對正常細胞不會有作用，只會針對腫瘤細胞進行毒殺作用，且大多出現於腫瘤細胞的細胞核。所以有人推測Apoptin 的腫瘤特異性可能與他坐落於細胞核的位置有相關性。我們將此兩種抗腫瘤蛋白，ABR-A和Apoptin與第一型單純疱疹性病毒的蛋白傳送區域(VP22)進行基因融合，希望能增加天然毒素(ABR-A或Apoptin)進入腫瘤細胞的傳送率，因而提升抗腫瘤效果。首先，我們分別製造含有表現螢光(GFP)、控制組(HA)，以及蛋白傳送區域(VP22)的植物毒素A(ABR-A)及雞貧血病毒蛋白(Apoptin)的表現載體，以西方點墨法 (western blotting) 確定蛋白質表現。分別將各種表現載體將送入細胞，評估天然毒素(ABR-A或Apoptin) 在非轉型細胞(NIH3T3) 以及轉型細胞(HeLa；人類子宮頸癌細胞、A357；黑色素瘤細胞)基因傳送效果。以細胞增生分析及Hoechest染色發現基因傳送ABR-A會抑制非轉型及轉型細胞的生長。但Apoptin 基因傳送僅毒殺腫瘤細胞。與蛋白傳送區域(VP22) 融合的ABR-A及Apoptin 細胞毒殺效果更顯著。另外，我們也探討雞貧血病毒蛋白的結合蛋白1 (Apoptin associated protein 1; APAP1)，是否會影響Apoptin 在腫瘤細胞的毒殺性。結果顯示，在人類子宮頸癌細胞中缺少雞貧血病毒蛋白的結合蛋白1 (Apoptin associated protein1; APAP1)，會降低雞貧血病毒蛋白(Apoptin) 在腫瘤細胞的毒殺性。新的蛋白傳送區域結合毒素載體(PTD-toxin vectors) 抗腫瘤效果將應用於細胞培養以及動物實驗。我們希望這個研究能展開癌症治療的新紀元。

Protein transduction domains (PTDs), such as TAT from human immunodeficiency virus (HIV) or VP22 from herpes-simplex-virus-1, have been shown to deliver a myriad of molecules, including synthetic small molecules, peptides and proteins in vivo and in vitro. The protein transduction processes mediated by TAT or VP22 are highly efficient and occur in many types of cells with low toxicity. The anti-tumor proteins to be investigated are abrin A chain (ABR-A) and Apoptin. ABR-A is the toxophoric subunit of plant toxin abrin from the seeds of Abrus precatoriusa. ABR-A is a potent inhibitor of translation, but not toxic to cells due to its lack of the cell-binding B chain. Apoptin is a protein derived from chicken anemia virus and has been proved to be selectively cytotoxic to various tumor cells but not to normal cells. The tumor-specific activity of Apoptin is correlated with its nuclear localization in tumor. In this study, we employed VP22 PTDs to promote the entry of natural toxins, such as ABR-A or Apoptin, into tumor cells, thereby to enhance their anti-tumor effects. We generated and characterized green fluorescent protein (GFP)-, hemagglutini (HA)-, and VP22-fused expression constructs for ABR-A and Apoptin, to evaluate the gene delivery effect of ABR-A/Apoptin genes in non-transformed NIH3T3 cells and tumor cells, including Hela and A375 melanoma cells. Gene delivery of ABR-A led to growth inhibition by 50~70% in transformed and non-transformed cells. In contrast, Apoptin gene delivery exhibited cytotoxicity only in tumor cells. The cytotoxicity of ABR-A and Apoptin gene delivery was enhanced when fused with VP-22. Furthermore, the depletion of APAP1 reduced the cytotoxic effect of Apoptin gene delivery. In the future, the anti-tumor effect of these novel PTD-toxin vectors will be explored in cell culture as well as animal model. We hope these studies will open a new avenue for cancer therapy.

Abstract----------------------------------------------------------------------------------------I
摘要--------------------------------------------------------------------------------------------II
Key words-----------------------------------------------------------------------------------IV
Introduction---------------------------------------------------------------------------------1
ABR-A (Abrin-a A chain) --------------------------------------------------------------2
Apoptin-----------------------------------------------------------------------------------5
APAP1------------------------------------------------------------------------------------7
VP22--------------------------------------------------------------------------------------8
Specific Aims------------------------------------------------------------------------------10
Materials and methods------------------------------------------------------------------11
Results-------------------------------------------------------------------------------------17
Discussion --------------------------------------------------------------------------------21
References--------------------------------------------------------------------------------23
Figures and legends---------------------------------------------------------------------29
Table---------------------------------------------------------------------------------------44
Index---------------------------------------------------------------------------------------46

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