中文摘要

自從1982年以來，癌症一直高居台灣十大死亡原因之首。其中腦瘤是15-35歲族群的主要癌症死亡原因。 針對無法切除的腦部腫瘤，一般只能使用放射治療或化學療法。然而到目前為止，這些治療方法的效果都不甚理想。目前我們所使用的癌症化療藥物皆無法穿透血腦屏障，所以對腦瘤幾乎沒有任何療效。近年來由於台灣人口加速老化，腦瘤與腦部退化性疾病的發生率正不斷的增加，因此研發新一代能穿透血腦屏障以治療腦部腫瘤及退化性疾病的藥物實為當務之急。
目前使用的癌症化療藥物都是利用破壞癌細胞的去氧核醣核酸(DNA)以達到療效。然而近年來科學家們發現，能破壞癌細胞核醣核酸(RNA)的核醣核酸酶(RNase)可能是一個更好的選擇。其中胞外分泌型人類嗜伊紅球陽離子蛋白(ECP)及嗜伊紅球神經毒蛋白(EDN)已被證實對癌細胞有毒殺作用因而被命名為抗癌核醣核酸酶。
在本研究中，我們使用重組基因技術製造這兩種抗癌核醣核酸酶‐即：以大腸桿菌製備成熟人類嗜伊紅球陽離子蛋白(rECP)及成熟嗜伊紅球神經毒蛋白(rEDN)，以研究其活體細胞穿透機制及其應用於治療腦部腫瘤等疾病的可能性。我們的研究結果發現rECP 和rEDN 兩種基因重組的抗癌核醣核酸酶都對呼吸道及腸道柱狀上皮細胞具有高度的親和性與細胞穿透性。在我們的進一步研究中更發現這種細胞穿透作用可能是透過與細胞膜上的Heparan sulfate受器結合而達成的。同時在本研究中發現雖然rECP和 rEDN 都無法穿透血腦屏障，但是如果我們把rECP或 rEDN 注射入腦脊液中，這兩種人工合成的抗癌核醣核酸酶都對腦中的大型神經元細胞（pyramidal neuron及 Purkinje neuron）具有高度的親和性與細胞穿透性。
未來如果能利用基因工程搭配定點突變的人工合成方法改變rECP或rEDN的分子結構以加強其專一性及減少其副作用，便能進一步提升這兩種抗癌核醣核酸酶成為癌症化療藥物的潛力;甚至可以當作特洛伊木馬分子以協助治療腦部腫瘤及退化性疾病，或呼吸道及腸道相關的其他疾病。

Abstract
Cancer has been the leading cause of death in Taiwan since 1982. Among which brain tumors are the leading factors of cancer deaths in the population of 15-35 years old. Traditionally therapeutic strategies for unresectable brain tumors were radiation therapy and chemotherapy. However, most of these strategies are not satisfactorily successful. Meanwhile, due to the progressive aging of our society, there is increasing incidence of brain tumors and degenerative diseases. The development of new generation brain targeting drugs for brain tumors and degenerative diseases is very important.
Traditional chemotherapeutic strategies for cancer are developed mainly based on DNA damaging mechanism. However, recently scientists found that RNA damage caused by ribonucleases (RNases) could be an important alternative. Among these, the secretory human eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) have been shown to be effective for some cancers and are classified as antitumor RNases.
In the study, recombinant DNA technology was applied to produce the two antitumor RNases, i.e. recombinant mature human eosinophil cationic protein (rECP) and recombinant mature human eosinophil-derived neurotoxin (rEDN). The cell entry ability and potential roles of these human eosinophil Rnases in the therapy of brain tumor and other diseases were investigated. It was found that both rECP and rEDN internalized the columnar epithelium of respiratory and intestinal tracts efficiently. Further study proved that such cell entry effect occurred very likely through the binding with some special type of membrane bound heparan sulfate receptors on the cell surface. Although neither rECP nor rEDN could directly penetrate through blood brain barrier (BBB), once they were forcefully injected into the subarachnoid spaces both showed highly selective cell binding and cell entry effect to the pyramidal neurons and Purkinje neurons.
Further modification of the rECP and rEDN molecules by genetic engineering techniques such as chimeric or site-directed mutagenesis can be carried out in the future to improve the selectivity and decrease possible side effects of the two recombinant RNases, the modified eosinophil RNases can be further used as potential chemotherapeutic agents or even as molecular Trojan horses for the therapy of brain tumors, brain degenerative diseases, and other special diseases related to respiratory and digestive organs.

目錄
Chapter 1:General Background ……………………P1-7
Figures of Chapter 1…………………………P8-10
Chapter 2:The Acute Stage in vivo Biodistribution and Cell
Entry Ability of rECP and REP-24 peptide…P11-27
Figures of Chapter 2………………………P28-42
Chapter 3:The Chronic Stage in vivo Biodistribution and
Cell Entry Ability of rECP and rEDN………P43-53
Figures of Chapter 3………………………P54-68
Chapter 4:The Study About Membrane Bound Receptor and Cell
Entry Ability of rECP and rEDN………………P69-74
Figures of Chapter 4………………………P75-83
Chapter 5:Osmotic Opening of BBB to Facilitate the Entry
of Antitumor RNases into CNS System …………P84-87
Figures of Chapter 5…………………………P88-90
Chapter 6:Direct Intrathecal Injection of rECP and rEDN to
Observe Their Cell Entry Ability Within CNS
System…………………………P91-95
Figures of Chapter 6………………………P96-99
Chapter 7:Final Discussion…………………………P100-103
Figures of Chapter 7………………………P104
References………………………………………………P105-110
Appendices………………………………………………P111-122
Abbreviations…………………………………………………P123

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