斑馬魚髓鞘鹼性蛋白之基因表現與功能研究

Abstract

髓鞘鹼性蛋白（Myelin basic protein,MBP）是髓鞘的主要組成蛋白，髓鞘可緊密保裹神經纖維，也因為髓鞘的絕緣性質，使得神經傳導訊息可以進行有效率的傳送。許多組成髓鞘的蛋白質如MAG、OMGP、NOGO等，在許多研究已被指出具有抑制神經纖維生長的能力，因此本研究欲探討MBP是否也具有可使神經纖維外生長程度減弱的能力。首先，利用生物資訊方法與分子生物技術選殖出兩種不同形式的MBP(MBP1、MBP2)，並以全胚胎原位雜合法探討其mRNA在斑馬魚胚胎的表現分佈情形，結果顯示MBP1、MBP2表現位置相同，但表現的時期則有差異。隨後構築MBP之表現質體，利用神經膠細胞專一性GFAP啟動子以表達MBP，以顯微注射技術將該MBP表現質體與可促進神經纖維外生長因子，如HBNF、NGF等共同注入斑馬魚單細胞魚卵，結果發現MBP具有導致神經纖維外生長減弱的現象。此外，為探討MBP減弱神經纖維生長的能力是否為EPO所影響，我們將MBP、HBNF和EPO共同注入斑馬魚的胚胎中，研究結果顯示，MBP減弱神經纖維生長的能力會受到 EPO 的影響。此外，為了探討MBP對於斑馬魚胚胎發育之影響，我們將針對 MBP 所設計的反義寡核苷酸（Morpholino oligonucleotide, MO）注射到斑馬魚胚胎中，結果發現，斑馬魚胚胎會出現生長遲緩、脊索發育不正常、魚體不規律顫抖等現象，表示MBP在斑馬魚發育過程中，在神經系統扮演相當重要的角色。最後，我們成功培育出MBP基因轉殖魚，並用以佐證前實驗中所得到MBP影響神經纖維生長的結果。

Myelin basic protein（MBP） is the major constitutional component in vertebrate myelin, which wraps axon rigidly and forms multilayered compact myelin sheath. The insulation nature of myelin sheath makes neuronal conduction impulse more efficient and faster. Many proteins which make up myelin including MAG, OMGP and NOGO, have been shown to be able to inhibit neurite outgrowth. In this study, we cloned zebrafish MBP genes and investigate whether fish MBP has similar function or not. First, by using bioinformatics and PCR technology, we cloned two zebrafish MBP isoforms: MBP1 and MBP2, and then analyzed the temporal and spatial expression pattern of MBP1 and MBP2 during zebrafish development by whole-mount in situ hybridization. MBP1 and MBP2 had similar expression pattern, but MBP1 was expressed in earlier stage than MBP2. Furthermore, in order to investigate the roles of MBP1 and MBP2 in HBNF-induced neurite outgrowth in zebrafish embryos, we co-injected MBP1/MBP2 and HBNF into zebrafish embryos. It was found that the degree of neurite outgrowth was decreased, and this decrease effect could be rescued by co-injecting with a neuroprotective factor, EPO. However, the reduction level of neurite outgrowth caused by MBP1 and MBP2 are different. In addition, by virtue of MO knockdown technology, we found some zebrafish embryos manifested developmental delay and died the next day after injecting MBP1-MO, and some MBP1 MO-injected embryos showed abnormal shake behaviors. In the case of MBP2-MO knockdown, we found obvious defect in the notochord formation during embryonic development. Finally, we generated a transgenic zebrafish line expressing MBP-GFP, and we utilized these MBP transgenic fishes to confirm above results of microinjection.