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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/17122

Title: Tbx5基因缺損對斑馬魚(Danio rerio)胚胎時期細胞週期及細胞凋亡之影響
The impacts of tbx5 gene knockdown on cell cycle and apoptosis during zebrafish (Danio rerio) embryonic development
Authors: Shu-Yu Yeh
葉書優
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 斑馬魚;細胞凋亡;細胞週期
zebrafish;apoptosis;cell cycle
Date: 2010
Issue Date: 2011-06-30T08:51:25Z
Abstract: Tbx5屬於T-box家族之轉錄因子,表現於脊椎動物胚胎時期的心臟、前肢和眼睛。以專一反意股寡核苷酸抑制斑馬魚胚胎tbx5基因表現,除了會造成胚胎心臟缺損,也會造成心臟肌肉生成基因cmlc2、amhc、vmhc及調控胚胎心臟發育之轉錄因子tbx5, dHAND, nkx2.5的表現量下降,另外也會造成斑馬魚前肢發育不全以及軀幹彎曲等現象。由TUNEL assay結果可發現,在tbx5被弱化後,斑馬魚魚體上可發現到大量的細胞凋亡的細胞,而進一步在心臟及胸鰭組織上同樣可發現較多的細胞凋亡細胞,推測tbx5 基因被弱化後造成缺損的原因之一為促進細胞凋亡;在免疫螢光染色結果顯示,不論是心臟或者是胸鰭組織上,Bcl2, Bad, Cdk2, P27等基因的表現都有增加的情形,推測tbx5 基因被弱化後造成缺損的原因可能為促進細胞凋亡及抑制細胞週期進行。而在半定量RT-PCR中可發現tbx5 基因被弱化後,其細胞凋亡及細胞週期相關基因:Bcl2, Bad, Bax,Cdk2, Pcna, P27, P57等基因的表現量都有顯著的上升(p<0.05),推測tbx5與細胞凋亡及細胞週期相關基因有直接或間接的關連。在半定量RT-PCR實驗中,tbx5-MO與生長荷爾蒙(growth hormone, GH)同步注射下,其細胞週期及細胞凋亡相關基因:Bcl2, Bad, Bax,Cdk2, Pcna, P27, P57等表現量會較tbx5-MO處理組低。在TUNEL assay實驗上,tbx5-MO與生長荷爾蒙同步注射處理組可發現斑馬魚魚體及心臟、胸鰭組織上的細胞凋亡細胞有減少的情形。免疫螢光染色實驗中,tbx5-MO與生長荷爾蒙同步注射處理組的心臟以及胸鰭組織上,BCL2, BAD, CDK2, P27等基因的表現量也比tbx5-MO處理組來的弱。推測添加生長荷爾蒙後,可使細胞週期及細胞凋亡相關基因其mRNA及蛋白質表現量下降,進而改善斑馬魚心臟及胸鰭上的缺損。以斑馬魚DNA 晶片偵測tbx5基因弱化後斑馬魚胚胎在24、30及48hpf基因表現情形,在1.5倍條件篩選下,表現量上升的基因在24hpf有2956個,在30hpf為2638個,而在48hpf有861個。其中有233 個基因會同時表現在24&30hpf ,有40 個基因會同時表現於30&48hpf;而這些基因跟抑制生長、免疫接收子、加強腫瘤的生成、細胞凋亡、細胞週期等功能有相關。另外表現量下降的基因在24hpf有3372個,在30hpf有2951個,在48hpf有916個。其中有346 個基因會同時表現於24&30hpf,有66個基因會同時表現於30&48hp;這些基因跟膠原蛋白、肌肉的調節、生長因子、轉錄因子等功能有相關。由晶片分析結果我們推測,tbx5基因弱化後可能是因為影響這些基因進而導致斑馬魚的缺損。透過此一結果我們可了解tbx5基因弱化後與其他基因之間不論是直接或間接可能的調控機制與作用路徑,可做為日後找尋疾病生成原因之依據,進一步找出解決之方法。
Tbx5 is a member of the T-box family of transcription factors which expresses in the heart, fore limbs/pectoral fins and eyes of vertebrate animals during embryonic development. Tbx5 gene knockdown by morpholino antisense RNA can cause heart defect, and decrease cardiomyogenesis genes expression, such as: cmlc2, amhc, vmhc ; transcription factors, such as: tbx5, dHAND, nkx2.5. Pectoral fin defect and trunk defect was also induced by tbx5 gene knockdown. The result of TUNEL assay indicated that apoptosis cells was induced in the heart and pectoral fin tissue of tbx5 knockdown embryos. Presumed that the inducing of apoptosis might be a reasons of these defects. Immunohistochemistry staining results indicated that the expression of apoptosis and cell cycle-related genes increased in tbx5 gene knockdown treatment, such as: BCL2, BAD, CDK2, P27. Suggested that the inducing of apoptosis and restraining cell cycle might be another reason is causing heart, fin defect. The results of semi-quantitative RT-PCR analysis indicated mRNA expression of apoptosis and cell cycle-related genes increased, such as: bcl2, bad, bBax, cdk2, pcna, p27, p57 in tbx5 gene knockdown zebrafish embryos. The results of semi-quantitative RT-PCR analysis indicated that co-injection of tbx5-MO and growth hormone(GH) at the same time could restore the gene expression of bcl2, bad, bax, cdk2, pcna, p27, p57. The TUNEL assay revealed fewer number of apoptosis cells were become observed in tbx5-MO and GH co-injected treatment only tbx5-MO treatment group. Immunohistochemistry staining result indicated that BCL2, BAD, CDK2, P27 genes expression were reduced at heart and fins tissue in tbx5-MO and GH co-injection group. In present study, DNA microarray was employed to study the gene-gene interaction of tbx5 knockdown zebrafish. The results of DNA transcriptomic revealed 2956 genes at 24hpf embryos were detected effectively from up-regulated genes of total screening process, 2638genes at 30 hpf embryos were detected effectively from up-regulated genes of total screening process, 861genes at 48hpf embryos were detected effectively from up-regulated genes of total screening process on tbx5 gene knockdown zebrafish embryos. Also indicated that there were 233 genes at 24&30hpf and 40 genes at 30&48hpf were up-regulated on tbx5 gene knockdown zebrafish embryos which were related to growth inhibition, immunoreceptor, tumor enhancer, apoptosis, cell cycle and etc. Furthermore DNA microarray data revealed 3372 genes at 24hpf embryos were detected effectively from down-regulated genes of total screening process, 2951genes at 30 hpf embryos were detected effectively from down-regulated genes of total screening process, 916genes at 48hpf embryos were detected effectively from down-regulated genes of total screening process on tbx5 gene knockdown zebrafish embryos. Also indicated that there were 346 genes at 24&30hpf and 66 genes at 30&48hpf were down-regulated on tbx5 gene knockdown zebrafish embryos which were related to collagen, muscle regulation, growth factor, transcription factor and etc. DNA microarray screening of tbx5 gene knockdown embryos elucidated the genes interaction and regulation network during zebrafish development, and it might provide possible therapy protocol.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M97330006
http://ntour.ntou.edu.tw/ir/handle/987654321/17122
Appears in Collections:[水產養殖學系] 博碩士論文

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