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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48627

Title: 肌肉抑制素a與b在單基因及雙基因剔除斑馬魚之角色研究
Study on roles of Myostatin a and b in single and double gene knockout zebrafish
Authors: Tsai, Yi-Ting
蔡宜庭
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 肌肉抑制素 a;肌肉抑制素 b;常間回文重複序列叢集/常間回文重複序列叢集關聯蛋白系統;基因剔除;肌纖維增生;細胞凋亡
Myostatin a;Myostatin b;CRISPR/Cas9;Gene knock out;Hyperplasia;apoptosis
Date: 2017
Issue Date: 2018-08-22T06:06:50Z
Abstract: 本研究使用 CRISPR/Cas9 靶向基因組編輯技術於斑馬魚剃除 (KO) 兩個不同的肌肉抑制素基因-mstna 和 mstnb。因 mstna 和 mstnb 在肌肉及非肌肉組織中皆有表達,藉由基因剔除以了解 MSTN 扮演在肌肉生長和其他未知之功能及角色。成功建立 mstna KO、mstnb KO 單基因剃除斑馬魚及 mstna/mstnb 雙基因剃除斑馬魚家系的 F2 後代,並將其體表型與野生型斑馬魚進行比較。結果顯示,在 90 dpf的 mstna&mstnb (mstna/b) 雙基因剃除斑馬魚比 mstnb 基因剃除斑馬魚肌肉生長明顯增加,並且可觀察到明顯突起的背部肌肉。組織切片分析結果顯示,與野生型對照組相比,mstna/b 雙基因剃除斑馬魚皮下的肌細胞呈現中度增生,而 mstnb基因剃除斑馬魚的肌肉纖維雖面積較小但均勻增生。然而,比較 150 dpf 之 mstna/b 雙基因剃除、mstna 基因剃除和 WT 斑馬魚之體重,結果顯示三者並無顯著差異,但 mstnb 基因剃除斑馬魚體重相較於 WT 斑馬魚則顯示出 40% 的體重增加。我們接著比較在 150 dpf 每組的形態,發現在 mstna/b 雙 KO 斑馬魚有部分的魚隻出現身體畸形、佝僂的徵狀,且 mstna/b 雙基因剃除斑馬魚會出現突然性的死亡,且受精卵死亡率高。而 150 dpf 之組織切片分析結果顯示 mstna/b 雙基因剃除斑馬魚之肌肉組織產生嚴重的肌細胞增生,而 mstnb KO 之肌纖維則增長的更大。此外,為了瞭解 MSTN 如何影響肌肉生長,檢測肌肉生長相關基因的 mRNA 表現量。在 mstnb 基因剃除斑馬魚,MRFs (MyoD, Mrf4, Myf5, Myogenin) 和 pax7 基因皆上調控,而 mstna/b 雙基因剃除斑馬魚僅 MyoD 高度活化。我們進一步以 TUNEL 檢測與分析細胞凋亡相關基因的 mRNA 表現量來了解 mstna/b 雙基因剃除斑馬魚的猝死和身體畸形現象,結果顯示,僅在 mstna/b 雙基因剃除斑馬魚表現出因 DNA 降解而產生強烈的細胞凋亡訊號,且細胞凋亡相關 TNFa 和 BAD 基因皆顯著上調控。根據本研究結果,我們推測雙基因剃除mstna和 mstnb不僅影響斑馬魚胚胎發育,甚至可能調控老化、繁殖及免疫力。在斑馬魚主要肌肉生長的負調節因子為 mstnb 而非 mstna。推測 mstna 和 mstnb 在參與衰老、繁殖和免疫之功能可以在 mstna 或 mstnb 單基因剃除斑馬魚中相互補償。本研究表明,透過 CRISPR/Cas9 技術剃除主要負調節肌肉生長的 mstnb 基因可以建立新的雙倍肌肉硬骨魚品系,獲得明顯的肌肉增強效果而沒有觀察到副作用。
In this study, two distinct myostatin genes, mstna and mstnb were knockout (KO) by CRISPR/Cas9 targeted genome editing technology in zebrafish to understand their roles in muscle growth and unknown functions due to their extended expression in non-muscle tissues. F2 offspring of mstna KO, mstnb KO and mstna/mstnb double KO zebrafish lines were established to compare their phenotypes with wild-type zebrafish. The muscle growth was most significantly enhanced in mstna & mstnb (mstna/b) double KO zebrafish at 90 dpf, which exhibiting obvious phenotype with bulging muscles on the back. The muscle growth of mstnb KO zebrafish was better than that of mstna KO zebrafish and wild-type zebrafish. The muscle histological analysis showed moderate hyperplasia muscle cells under subcutaneous tissue of mstna/b double KO zebrafish. Mstnb KO zebrafish section shows muscle fibers hyperplasia evenly with smaller areas compared with the wild-type control fish. However, the 150 dpf mstna/b double KO and mstna KO zebrafish showed just slightly higher body weight than WT, but mstnb KO increased to 1.4-fold in body weight. Compare morphology of each groups, some mstna/b double KO zebrafish got rickets and have high mortality rate in adult fish and fertilize eggs. The muscle section shows marvelous hyperplasia in mstna/b double KO zebrafish. Conversely, the originally smaller hyperplasia mstnb muscle fibers grow bigger comparable to WT. Moreover, to understand how MSTN affect muscle growth, mRNA levels of muscle-growth-relate genes were measured. In mstnb KO zebrafish, MRFs and pax7 genes were all up regulated, whereas only high activation of MyoD was observed in mstna/b double KO zebrafish. We further examined the mRNA expression levels of apoptosis-related genes and TUNEL assay in the skeletal muscle to understand the sudden death and body deformity phenomenon in mstna/b double knockout zebrafish. Result shows strong apoptosis signal by DNA degradation and upregulation of apoptosis-related TNFa and BAD genes in mstna/b double KO zebrafish. According to our results, we speculate that mstna/b double knockout affects not only myogenesis but also aging, reproduction and immunity. Zebrafish mstnb not mstna is the major negative regulator involved in muscle growth. The functions of mstna and mstnb involved in aging, reproduction and immunity can be compensated each other in mstna or mstnb gene single KO zebrafish. Our study suggest that new double muscle teleost fish strains can be achieved by major mstnb gene knockout with CRISPR/Cas9 technology to obtain obvious muscle enhancement without observed side effect.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010533003.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48627
Appears in Collections:[水產養殖學系] 博碩士論文

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