臺灣博碩士論文加值系統

Progranulin (PGRN)是一種具有多功能的生長因子，具有促進細胞增生，參與癌症形成、抗細胞凋亡、神經細胞存亡，抑制發炎，組織修復與發育等功能。在哺乳類的基因體裡只存在1個pgrn基因，其編譯7個具有12個cysteins保守結構granulin (GRN)之PGRN。PGRN會被嗜中性球所產生的絲氨酸蛋白質酶裂解為GRN胜肽而誘導促發炎反應的細胞激素IL-8表現。相較於哺乳類只有單一個PGRN基因，在魚類基因體中具有不只一個pgrn基因。在吳郭魚 (Oreochromis mossambicus)先前的研究中從肝臟發現到1個具有2個GRN單元 (GRN-A及GRN-B)的pgrn基因能與IGF-1同時被生長激素所誘導而表現。本研究利用基因體南方墨點雜交分析偵測到吳郭魚的基因體中具有6個pgrn基因。本研究從吳郭魚genome選殖到pgrn-1基因，表現pgrn-1轉錄本及編碼先前發現的PGRN-1。另外本實驗還從吳郭魚中找到1個利用79個核苷酸的intron 1之選擇性剪接產物，造成氨基酸轉譯序列提前終止，而產生在C端較短的只具有41個氨基酸的GRN胜肽將之命名為GRN-41。此GRN-41胜肽在N端的30個氨基酸是與GRN-A相同，而其餘11個氨基酸產生變異。除此之外，本研究也選殖到一個新的pgrn-2基因其轉錄本編碼的PGRN-2具有與PGRN-1差異4個氨基酸而產生一個新的GRN-C胜肽 (與GRN-A變異2個氨基酸)，pgrn-2會在所有的組織表現。本研究利用肌肉專一性啟動子結合雙向表現TetOff載體系統建立表現GRN-41、GRN-A、GRN-C及PGRN-1、PGRN-2之基因轉殖斑馬魚，藉此以探討吳郭魚不同的PGRN及GRN胜肽的功能。從肌肉表現GRN-41及GRN-C斑馬魚中，發現到其具有能明顯誘導IL-8、IL-1β、IL-8R1與c3b等免疫相關基因的表現，並且同時也向上調控MyoD與myogenin等肌肉生長調節因子的表現。由此五個具有表現吳郭魚PGRN或GRN的基因轉殖斑馬魚品系中發現，在表現小單元吳郭魚GRN的品系相較於表現全長型PGRN，在先天免疫機制與肌肉發育有較好的反應機制。本研究提供魚類GRN在活化先天免疫及肌肉生長調節因子基因的新發現，在未來希望在養殖產業應用上，GRN能提供作為一個抗病能力或是肌肉發育促進能力上的新角色。

Progranulin (PGRN) is a multi-functional growth factor in regulating cell proliferation, tumorgenesis, anti-apoptosis, neuron survival, anti-inflammation, would healing and development. Only one progranulin (pgrn) gene encoding PGRN composed of 7 granulin (GRN) units with 12 conserved cysteines exists in mammalian genome. Progranulin undergoes proteolysis by neutrophil serine protease into small GRN peptides to induce proinflammatory cytokine IL-8. In contrast to mammals, teleosts have more than one pgrn genes in genome. We previously identified one tilapia pgrn gene with two GRN units (GRN-A and GRN-B) was co-induced with IGF1 by growth hormone in the liver of tilapia (Oreochromis mossambicus). In this study, six progranulin genes were identified in tilapia genome by genomic Southern hybridization. I found PGRN-1 gene expresses both PGRN-1 transcript encoding PGRN-1 and a novel alternative splicing pgrn transcript in all tested tissues of tilapia. The alternative splicing pgrn transcript use 79-nucleotide intron 1 as exon sequence resulting in in-frame stop codon to generate a C-terminal truncated secreted form GRN with 41-amino acid named as GRN-41. GRN-41 shares identical N-terminal 30 a. a. with GRN-A and a variant 11-a.a. C-terminal. In addition, I also isolated a new progranulin pgrn-2 gene and transcript encoding PGRN-2 with 4 amino acid variations compared with PGRN-1 to produce a new GRN-C (2 a. a. different from GRN-A) and GRN-B peptides was also expressed in all tissues of adult tilapia. In this study, I established GRN-41, GRN-A, GRN-C and PGRN-2, PGRN-1 transgenic zebrafish lines by muscle-specific promoter and bi-directional TetOff regulatory system to study functions of variant tilapia PGRN and GRN peptides. Muscle-specific overexpression of the novel tilapia GRN-41 and GRN-C peptides in zebrafish not only can induce IL-8, IL-1β, IL-8R1 and c3b etc. innate immune related genes but also upregulat MyoD and myogenin. In the five PGRN or GRN transgenic zebrafish, I found that expression of small tilapia GRN peptide has better response in innate immunity or myogenesis than tilapia PGRN. This study indicates immune response and myogenic regulatory factors genes can be activated by specific GRN peptides from teleost and suggestst GRN can serve a new role in disease-resistant and/or growth promotion in Aquaculture.

謝誌 i
中文摘要 iii
Abstract v
目錄 vii
圖目錄 ix
表目錄 xi
壹、 前言 1
貳、 實驗材料與方法 11
一、 實驗動物 11
二、 實驗材料 12
三、 實驗方法 16
1. 吳郭魚progranulin基因選殖 16
2. 基因轉殖質體構築 19
3. 建立基因轉殖斑馬魚 22
4. 吳郭魚不同組織progranulin基因表現 27
5. 吳郭魚基因體progranulin基因數量分析 27
6. 表現吳郭魚PGRN-1、PGRN-2、GRN-41、GRN-A、GRN-C基因轉殖斑馬即時定量聚合酶鏈鎖反應分析 32
7. LPS腹腔注射誘導吳郭魚三種pgrn-1、pgrn-2、Grn-41基因表現與兩類Genomic Pgrn的表現模式 33
參、 實驗結果 35
一、 吳郭魚progranulin基因之選殖 35
二、 建立表現PGRN-1、PGRN-2、GRN-41、GRN-A、GRN-C基因轉殖斑馬魚 36
三、 吳郭魚基因體之progranulin基因選殖 36
四、 吳郭魚progranulin基因在不同組織之表現模式 37
五、 吳郭魚progranulin基因在幼魚時期表現模式 38
六、 LPS誘導下吳郭魚progranulin基因之表現模式 38
七、 吳郭魚基因體progranulin基因數量 39
八、 表現吳郭魚PGRN-1、PGRN-2、GRN-41、GRN-A、GRN-C基因轉殖斑馬魚影響肌肉相關基因表現模式 39
九、 表現吳郭魚PGRN-1、PGRN-2、GRN-41、GRN-A、GRN-C基因轉殖斑馬影響先天免疫相關基因表現模式 40
肆、 討論 42
伍、 引用文獻 46
圖 56
表 86
附錄 90

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