臺灣博碩士論文加值系統

腦源性神經營養因子（brain-derived neurotrophic factor, BDNF）被歸類於神經營養因子家族，此家族包含NGF、NT-3、NT-4/5和NT-6。BDNF基因具有多重啟動子(promoter)控制其在週邊組織與神經系統不同之表現部位，與神經的生長與存活密切相關。本實驗首先設計引子對利用RT-PCR方法增幅出斑馬魚BDNF第二外顯子，以此基因序列當探針，從斑馬魚基因體基因庫中篩選出三株選殖株，分析其基因體結構後，利用其中一株含有斑馬魚BDNF基因啟動子區域20 Kb的選殖株，設計引子對利用PCR方式增幅出約4 Kb的啟動子片段命名為BDNF-1C，並將此片段構築於pEGFP-1報告載體上命名為pBDNF-1C EGFP。將此載體轉殖到石斑魚心臟細胞株(GH)以及石斑魚腦細胞株(GB3)中，可以觀察到綠色螢光的表現；而將pBDNF-1C EGFP利用顯微注射方式注入斑馬魚受精卵中，在72 hr斑馬魚胚胎中可觀察到綠色螢光蛋白表現在肌肉、腸道、表皮細胞和體節等部位。以第二顯子為探針進行整體原位雜合反應(Whole mount in situ hybridization)，可以觀察到訊號表現在頭部、體節兩側之肌肉、尾部和主靜脈等部位。

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family that includes NGF, NT3, NT4/5 and NT6. BDNF play a critical role in the regulation of neuron survival and growth. BDNF gene has a complex structure with multiple regulatory elements and multiple promoters that are differentially expressed in central or peripheral nerve tissues. In this study, we have designed primers for the amplification of BDNF exon 2 and used this DNA fragment as a probe to screen the zebrafish genomic library. We obtained three BDNF clones from zebrafish genomic library. After genomic structure analysis, we found that one of the BDNF clones included 20 kb insert, which contained the BDNF multiple promoter regions. To analyze the promoter activity of BDNF-1C, we designed primers to amplify a 4 kb promoter region and ligated this DNA fragment into pEGFP-1 vector. The resulting plasmid was named pBDNF-1C EGFP. After transfection of pBDNF-1C EGFP into grouper brain (GB3) and grouper heart (GH) cells, the expression of EGFP was observed, which revealed BDNF-1C promoter functions in the two cells. By microinjection of pBDNF-1C EGFP into zebrafish fertilized eggs, the expression of EGFP was also observed in muscle, gut, epidermis cell and somite of 72-hr embryos. Whole mount in situ hybridization was performed to further confirm the expression of BDNF during zebrafish development. The expression of BDNF was observed in brain, tail, muscle and common cardinal vein.

目錄
摘要…………………………………………………………………………I
Abstract……………………………………………………………….……II
謝誌……………………………………………………………….………III
目錄………………………………………………………………...……...V
圖表目次……………………………………………………………..….VII
第ㄧ章、前言……………………………………….…….………1
第二章、材料與方法………………………………….…….……6
ㄧ、實驗材料……………………………………………………………6
二、核酸探針之製備…………………………………………...………19
三、斑馬魚BDNF基因之篩選………………………………..………20
四、Lambda 噬菌體重組基因之純化…………………………………22
五、BDNF基因分離與鑑定……………………………………………23
六、pBDNF-1C EGFP報告載體之構築………………………...….…25
七、pBSK BDNF-exon2載體之構築………………………..…………31
八、pBDNF-1C EGFP報告載體之轉殖………………………………32
九、pBDNF-1C EGFP斑馬魚胚胎顯微注射…………………………33
十、整體原位雜合反應……………………………………..…………33
第三章、結果…………………………………………....………42
ㄧ、斑馬魚BDNF基因之篩選………………………………...………42
二、斑馬魚BDNF 1C啟動子區域之選殖………………….…………43
三、BDNF promoter 1C螢光蛋白報告載體之構築………………..…43
四、pBDNF-1C EGFP報告載體在細胞株之表現 …………….….…44
五、斑馬魚胚胎顯微注射與pBDNF-1C EGFP轉殖基因表現………44
六、斑馬魚胚胎整體原位雜合之分析…………………………...……45
第四章、討論……………………………………………………46
第五章、參考文獻………………………………………………70

圖表目次

圖一、pBSK BDNF-exon2載體之製備示意圖…………….…….....……49
圖二、斑馬魚BDNF基因體選殖株之南方墨點分析…………...………51
圖三、斑馬魚BDNF三株選殖株相對位置……………………………...53
圖四、pBDNF-1C EGFP報告載體之構築示意圖………………………55
圖五、斑馬魚BDNF-1C在GH以及GB-3細胞中的表現情形………57
圖六、綠色螢光蛋白在斑馬魚胚胎發育期之表現………………...……59
圖七、Sense以及Antisense probe之製備………………………………..61
圖八、全體原位雜合反應RNA訊號表現位置…………………………63
圖九、BDNF promoter 1C序列轉錄因子結合位之預測………….……65

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