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標題: | 斑馬魚KLF8於消化器官發育功能性之研究 Functional Analyses of Zebrafish KLF8 on Digestive Organ Development |
作者: | Ming-Yuan Tsai 蔡銘原 |
指導教授: | 曾萬年(Wann-Nian Tzeng) |
關鍵字: | 斑馬魚,KLF8,消化器官,細胞凋亡,p53, zebrafish,KLF8,Digestive Organ,apoptosis,p53, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 本實驗室先前選殖到一斑馬魚KLF8基因,可轉譯出具有348個胺基酸的蛋白質,並與人類的KLF8有63%的胺基酸相似度(similarity)。斑馬魚KLF8蛋白質C端有3個krüppel -type zinc finger domain,而N端有一個repressor motif (PVALS/T)。當利用KLF8反意寡核酸抑制KLF8之功能,在morphant胚胎外觀上可觀察到在24 hpf時中後腦交界模糊,48 hpf時心臟looping不正常,72 hpf時圍心膜與腦室腫大、眼睛變小及尾巴彎曲,而在96 hpf morphant胚胎可由外觀看出前腸空腔較小之情況。另外,利用組織切片觀察消化道器官方面,則發現96 hpf morphant胚胎其外分泌胰臟細胞萎縮、肝藏發育不全和腸道上皮細胞缺乏極化(polarization)之情形。因此本論文的主要目的在研究斑馬魚KLF8 基因在消化道發育所扮演的功能。經DNA微矩陣試驗分析出,在morphant胚胎中p53和Cyclin G1相對72 hpf野生種表現量上升,繼而藉由cDNA末端快速擴增技術找出表現量上升的是為P53 isoform的△113p53。進一步由即時定量聚合酶連鎖反應指出,△113p53和Cyclin G1在72 hpf表現量分別上升7.76和2.88倍,而在30 hpf胚胎則為11.86倍和6.41倍,而p53其他下游基因如p21與Mdm2,以及促細胞凋亡基因Noxa和Puma表現量亦有上升情況。同時以全覆式原位雜合反應分析亦顯示出p53在30 hpf、48 hpf及72 hpf morphant胚胎中,大量分佈於眼、腦、脊椎和前腸之中。而在96 hpf不論是morphant或野生種之胚胎p53皆無太多表現。利用acridine orange染色觀察到25 hpf morphant胚胎的眼、腦與脊椎部位有大量細胞凋亡情況。為了探討KLF8是否經由p53而影響消化道之發育,我們利用注射p53裁切或p53起始反意寡核酸於KLF8 morphant胚胎中來拯救不同消化器官之發育。在同時注射KLF8-MO1和p53裁切反意寡核酸到胚胎中,與單獨注射KLF8-MO1之胚胎相比較,觀察到有16%之34 hpf morphant胚胎表現FoxA3之肝及胰芽生缺陷可以被拯救,而有34%之72 hpf morphant胚胎其IFABP在腸道之表現區域有增加之情形。相反地,一同注射p53起始反意寡核酸並無任何拯救morphant中肝胰芽生缺失及IFABP表現區域之效果,其中單獨注射p53裁切或p53起始反意寡核酸則不影響IFABP之表現區域。因此由上述實驗結果顯示抑制斑馬魚KLF8之表現會促使△113p53上升,進而促進前腸細胞凋亡而影響肝、胰芽之budding及後續腸道的形態形成。 Previously we have isolated a KLF8 cDNA from zebrafish and it encodes 348 amino acids long polypeptide which sharing high (63%) amino acid sequence similarity with human KLF8. There are three tandem krüppel-type Cys2His2 zinc-finger motifs at the carboxyl terminus and a repressor motif (PVALS/T) located at the N-terminus of zebrafish KLF8. To explore the function of KLF8 on zebrafish development, we used KLF8 specific antisense morpholino oligomer (MO) to knock down the expression of KLF8. We observed defect in midbrain/hindbrain boundary formation in 24 hours post fertilization (hpf) morphant embryos. We also detected abnormal heart looping morphology with swollen pericardiac chamber, brain edema, small eyes, and malformed tail in 48hpf and 72hpf morphant embryos, respectively. In 96hpf morphant embryos,.we also found defects including lack of folds in intestinal epithelium and small intestinal lumen In addition, paraffin sectioning on 96hpf morphant embryos showed hypoplastic development of liver and exocrine pancreas, as well as the presence of cuboidal intestinal epithelium lacking of polarization. Therefore, the main goal of this thesis is to investigate function of KLF8 in zebrafish gut development. Results from DNA microarray revealed that up-regulation of p53 and Cyclin G1 expression in 72hpf morphant embryos. We also confirmed that △113p53 was the P53 isoform showing up-regulated expression in 72 hpf morphant embryos by Rapid amplification of cDNA ends. Real time Q-PCR was conducted to confirm some up-regulated genes in morphant embryos. Expression of △113p53 and Cyclin G1 were increased 7.76 and 2.88 fold respectively in 72hpf morphant embryos, but they were respectively increased 11.86 and 6.41 fold in 30hpf morphant embryos. Some p53 downstream genes including p21, Mdm2, Noxa and Puma were also up-regulated. Whole-mount in situ hybridization indicated p53 gene was abundantly expressed in the eyes, brain, spinal cord and gut in morphant embryos during 30 to 72 h of development. However no significant difference in p53 expression was detected between wild type and KLF8 morphant embryos at 96 hpf. We used acridine orange staining and found a significantly increased apoptosis in the eyes, brain and spinal cord in 25hpf morphant embryos. To explore how KLF8 affects p53 to regulate development of the digestive system, we co-injected KLF8-MO1 either with p53 splicing MO or with p53 ATG MO into 1-2 cells and examining their effects on foxa3 expressions in the liver and pancreas buds at 34hpf and on IFABP expression domain in the intestinal bulb at 72 hpf injected embryos. Approximately 16% 34hpf morphant embryos showing defects in liver and pancreas budding can be rescued by co-injection with p53 splicing MO but not with p53 ATG MO. However, approximately 34% 72hpf morphant embryos that have been co-injected with p53 splicing MO showing increased IFABP expression domain in the intestinal bulb regions while no improvement of IFABP expression domain can be detected when co-injecting with p53 ATG MO. Whereas both foxa3 and IFABP expression domains in embryos that either have been injected with p53 splicing MO or p53 ATG MO alone were the same as wild type 34hpf and 72 hpf embryos. Overall, these results suggest that up-regulation of △113p53 caused by loss of KLF8 function may promote apoptosis in the fore gut and affect liver and pancreas budding as well as subsequent intestinal morphogenesis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28801 |
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顯示於系所單位: | 漁業科學研究所 |
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