斑馬魚胚胎發育中 serbp1 基因之表現及功能分析

中山醫學大學機構典藏 CSMUIR > 醫學科技學院 > 生物醫學科學學系暨碩士班 > 博碩士論文 > Item 310902500/4139

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Title:	斑馬魚胚胎發育中 serbp1 基因之表現及功能分析 Developmental expression and functional analysis of serbp1 gene in zebrafish
Authors:	張佩芯 Pei-Hsin,Chang
Contributors:	中山醫學大學;醫學科技學院;生物醫學科學學系碩士班;李娟
Keywords:	斑馬魚;胚胎發育 serbp1;zebrafish
Date:	2011
Issue Date:	2011-10-25T07:02:30Z (UTC)
Abstract:	SERBP1 全名為 SERPINE1 mRNA binding protein 1，也被稱為 PAI-RBP1 或 CGI-55。SERBP1 可與 pai-1 mRNA 3’-UTR 的 cyclic nucleotide-responsive sequences (CRS) 區域結合，並推測可藉此調控 pai-1 mRNA 的穩定性。另外 serbp1在卵巢癌上皮細胞中大量表現，且在不同階段的腫瘤細胞中，表現量會有差異。而目前已知許多基因在腫瘤的形成與轉移及胚胎發育過程扮演重要角色，在此，我們利用斑馬魚作為模式動物，對 serbp1 在胚胎發育過程中的表現及功能作進一步的研究。serbp1胺基酸序列在人類、老鼠、爪蟾及斑馬魚中高度保留。在 RT-PCR 實驗中，我們發現 serbp1 在斑馬魚成魚組織中廣泛的分佈，並無組織專一性；而在斑馬魚胚胎中，無論是 RNA 或蛋白，serbp1 皆從胚胎發育早期即開始表現且持續到發育後期。另外，我們利用全胚體原位雜交的實驗發現：serbp1 在 24 hpf 時主要表現在體節及腦部，在 48 hpf則主要表現在腦部交界及胸鰭芽，而在 72 hpf 則表現在胸鰭及咽弓處。接著我們利用 morpholino oligonucleotide (MO) 抑制 serbp1 的蛋白表現，而serbp1 morphants 會有較小的頭部且肌肉與體軸會有不正常的發育。同時，我們也在胚胎的頭部及體軸發現有細胞凋亡的現象。更進一步利用標示基因對 serbp1 morphants 發育異常處進行分析，我們發現抑制 serbp1 後，在胚胎前、中腦及中後腦交界處有輕微缺陷，且會造成胚胎 presomitic mesoderm (PSM) 不正常的發育；另外 serbp1 morphants 脊索較短及 margin 較大，但其胚胎內胚層的 forerunner cell 不會有明顯異常。接著，我們針對影響肌肉發育的標示基因 (myf5、myod、myogenin) 作進一步的分析，抑制 serbp1 後，會使上述的基因在體節發育時期表現量下降，也會造成胚胎發育過程不正常的體軸發育，因此，根據我們的研究，我們認為 serbp1 在胚胎肌肉發育過程可能扮演重要角色。 SERPINE1 mRNA binding protein 1 (SERBP1) also known as PAI-RBP1 or CGI-55, can bind to the 3’- UTR of pai-1 mRNA to regulate its stability. Furthermore, expression of serbp1 is significantly elevated in tumor epithelial cells and its expression correlates with the advanced disease stage in ovarian cancer. As many genes involved in tumor formation also play important roles in embryonic development, we use zebrafish as a model system to study the expression and functional of serbp1 during embryogenesis. The amino acid sequence of SERBP1 is highly conserved in human, mouse, xenopus and zebrafish. RT-PCR analysis revealed that serbp1 is universally expressed in adult tissues of zebrafish. Besides, the serbp1 is expressed at both mRNA and protein level in zebrafish embryos from early to late developmental stages. Moreover, we analyzed its expression by whole mount in situ hybridization (WISH) and detected serbp1 expression in somites at 24 hpf, brain boundary and pectoral fin bud at 48 hpf, and pectoral fin and splanchnocrania at 72 hpf. We then knocked down serbp1 by morpholino oligonucleotide (MO), and serbp1 morphants showed small head and abnormal muscle and defective somite patterning. At the same time, we detected cell apoptosis at somites and brain of serbp1 morphants. Moreover, inhibition of serbp1 resulted in mild defects in forebrain, midbrain and mid-hindbrain boundary (MHB), abnormal presomitic mesoderm (PSM) formation, shorter notochord and larger margin, but no significant effects on the forerunner cells of endoderm. Further analyses with specific marker genes (myf5, myoD and myogenin) revealed decreased expression as well as abnormal expression patterns of these marker genes during segmentation period. Based on our findings, serbp1 may play an important role during myogenesis of embryonic development.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/4139
Appears in Collections:	[生物醫學科學學系暨碩士班] 博碩士論文

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