臺灣博碩士論文加值系統

近年來，斑馬魚成為研究人類白血病及其他血液疾病的重要模式生物。斑馬魚的優勢在於：繁殖週期短，產生後代數量多；胚胎透明，且體外發育，易於觀察；易於飼養，生長迅速。且斑馬魚造血作用與人類及哺乳動物相似，有兩個造血高峰期。斑馬魚造血遺傳體系與人類的在演化上具有高度保守性，多種人類重要的造血調控轉錄因子，斑馬魚都有其同源類似物。
在之前的對急性骨髓性白血病的病人檢體進行研究時，發現7個病人出現相同的TALDO1-DSN1融合基因。人類TALDO1是轉醛醇酶的編碼蛋白，轉醛醇酶對磷酸戊糖途徑有至關重要的調節作用。而在過往研究中，TALDO1的異常與一系列不同的自體免疫疾病和惡性腫瘤有重要相關。人類DSN1編碼了動粒蛋白，在細胞分裂中起重要作用。在乳癌和結腸直腸癌研究中，DSN1與癌症的發生和轉移有關。
本研究欲通過用spi1啟動子引導的髓系特定表達TALDO1-DSN1融合基因的斑馬魚Tg(pTolCG-spi1:TALDO1-DSN1-PA/CG)，觀察其生命週期中，造血系統是否出現異常，以確認該融合基因在造血作用中扮演的角色。
我們篩選出穩定遺傳目的基因的轉基因斑馬魚進行研究。對於發育5天以內的幼魚，用qRT-PCR檢測造血作用中特定轉錄因子的表達情況。結果發現多個基因表達異常，其中，以gata1的增多、mpo的減少最為顯著。因此，我們採用整體原位雜交技術，進行雙重確認。
對6至12個月的成魚，取其周邊血及腎髓製作血液抹片進行細胞分型及計數。發現在成魚中，有部分轉基因斑馬魚的周邊血中出現未成熟白血球，伴隨腎髓中的芽細胞異常增多，而成熟白血球減少的現象。

In acute myeloid leukemia（AML），a hematologic malignancy, there is an urgent need for molecular understanding as well as targeted therapy. Recently, zebrafish has become an important model organism for the study of AML and other blood diseases, due to many advantages of zebrafish, such as short reproductive cycle, a large number of offspring, transparently and observable embryos, easy to breed and grow rapidly. Like higher vertebrates, zebrafish have two major waves of hematopoiesis, the primitive and definitive waves. The hematopoietic genetic system of zebrafish is highly conserved with humans in evolution. Various important human hematopoietic regulatory transcription factors are conservative.

In previous studies, seven patients, who suffer from acute myeloid leukemia (AML), had the same TALDO1-DSN1 fusion gene. Human TALDO1 encodes an enzyme, transaldolase, which is a crucial regulator in pentose phosphate pathway. In previous studies, the abnormality of TALDO1 was associated with the development of different autoimmune diseases and malignancies. Human DSN1 encodes kinetochore proteins which plays an irreplaceable role in cell division. In breast cancer and colorectal cancer, DSN1 is related to the occurrence and metastasis of cancer.

In this study, we use the transgenic zebrafish line, Tg (pTolCG-spi1: TALDO1-DSN1-PA/CG), to display that the human TALDO1-DSN1 fusion gene driven by spi1 (a myeloid-specific promoter) can lead to abnormal hematopoiesis during its life cycle.

We screened transgenic zebrafish for genetically stable transgenes. For the embryo less than 5 days-post-fertilization (dpf), we detected the expression of specific transcription factors in hematopoiesis by qRT-PCR. The result demonstrated that various genes expressed abnormal. Interestingly, gata1, the erythropoiesis transcription factor, surged at several points in time, as well as the granulocyte marker mpo reduced significantly, and subsequently, we use the whole-mount in situ hybridization technique for double confirmation.

For adult fishes aged from 6 to 12 months, peripheral blood and kidney marrow were taken, and followed by made for blood smears. We taken the blood morphological examination by cell typing and counting. The results displayed that in some transgenic adult zebrafish, immature myelo/monocytes appeared in the peripheral blood. In kidney marrow, the percentage of blast cells and myelo/monocytes increased abnormally.

致謝 I
目 錄 II
圖目錄 V
表目錄 VI
縮寫表 VII
摘要 VIII
Abstract IX
第一章 前言 1
1.1 模式生物—斑馬魚 1
1.2 斑馬魚的造血系統（附圖一） 1
1.3 斑馬魚的造血調控因子 2
1.4 蛋白編碼基因TALDO1 4
1.4.1 TALDO1之簡介 4
1.4.2 轉醛醇酶之功能 4
1.4.3 TALDO1突變導致之病症（附表一） 5
1.4.4 TALDO1與癌症關聯性之研究 5
1.5 蛋白編碼基因DSN1（附圖二） 6
1.6 TALDO1-DSN1融合基因 7
第二章 研究目的 8
第三章 材料與方法 9
3.1 材料 9
3.1.1 斑馬魚 9
3.1.2 儀器設備 9
3.1.3 藥品 10
3.1.4 抗體 11
3.1.5 酵素與試劑 12
3.1.6 生物試劑組 12
3.1.7 藥品與試劑配置 12
3.2 方法 13
3.2.1 斑馬魚飼育 13
3.2.2 基因分型（Genotyping） 14
3.2.3 基因表達量分析 15
3.2.4 原位雜交技術（Whole-mount in situ hybridization， WISH） 18
3.2.5 血球與組織分析 20
3.2.6 影像處理 21
3.2.7 統計方法 21
第四章 實驗結果 22
4.1 TALDO1-DSN1轉基因斑馬魚的篩選與觀察 22
4.2 融合基因對造血相關轉錄因子的影響 22
4.2.1 原始造血轉錄因子的異常表達 22
4.2.2 永久造血轉錄因子的異常表達 23
4.3 成魚血球染色分析 24
4.3.1 6月齡成魚的血球分析 24
4.3.2 12月齡成魚的血球分析 25
第五章 討論 26
第六章 參考文獻 30
表 35
圖 39
附圖 61
附表 62

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