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Title: 斑馬魚胚胎發育期間Survivin/Birc5a與能量代謝關係
Survivin/Birc5a and energy metabolism in zebrafish embryos
Authors: Ho, Chia-Yin
何佳穎
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: Survivin/Birc5a;糖解作用;神經發育;斑馬魚
Neural developmental;Survivin/Birc5a, -5b;2-deoxyglucose;Parkin
Date: 2017
Issue Date: 2018-08-22T06:31:28Z
Abstract: Survivin是BIRC細胞抗凋亡蛋白家族中最小的成員,在斑馬魚內有兩個相關基因,而在發育過程中以birc5a的表現為主。過去的研究發現Survivin在胚胎血管新生、血球生成以及心臟發育之過程當中都擔任非常重要的角色。在斑馬魚的神經前驅細胞中,Survivin/Birc5a, -5b亦對神經細胞的生存與分化扮演重要的功能,但其機制並不清楚。許多文獻指出許多快速生長的癌細胞以及未分化的幹細胞多進行有氧糖解代謝(亦稱瓦氏效應),近年來發現神經母細胞瘤中Survivin參與了能量代謝轉換的角色。過量表現的Survivin會促使細胞內粒線體片斷化,並抑制粒線體電子傳遞鏈活性,降低細胞內ROS過量累積,因而使癌細胞的能量代謝由氧化磷酸化反應轉變成有氧糖解作用。以糖解作用抑制劑2-DG(2-deoxyglucose)處理後,會活化神經母細胞瘤內的PINK1激酶及下游的Parkin泛素連接酶作用,促使Survivin水解,並使粒線體進行自噬作用,造成腫瘤細胞最終走向細胞凋亡。在本論文中,主要在探討斑馬魚胚胎發育時期以2-DG抑制糖解作用對Birc5a/Survivin1以及神經分化的影響,藉以瞭解Survivin控制神經發育的機制。實驗結果顯示在發育早期以2-DG抑制糖解作用會造成胚胎死亡率上升,顯示在胚胎發育初期糖解作用扮演非常重要的功能。利用Acridine Orange 染色觀察發現2-DG抑制糖解作用後會引起胚胎中樞神經系統 (central nerve system, CNS)的細胞凋亡。利用神經細胞標識基因HuC觀察斑馬魚胚胎體內神經發育的情形,發現早期2-DG處理會造成斑馬魚神經發育受到抑制,因此,推測神經細胞分化的前期需要進行糖解作用。一旦糖解作用被阻斷,會誘發神經細胞凋亡。進一步以神經先驅細胞標識基因sox3分析,發現隨著2-DG浸泡的濃度增加,未分化的神經先驅細胞有逐漸增加的現象,顯示抑制糖解作用後造成斑馬魚神經發育異常的原因之一是神經細胞停留於前驅細胞未分化的階段所造成。由於在神經母細胞瘤中,2-DG抑制糖解作用會透過PINK1-Parkin的作用導致Survivin降解,本實驗利用西方點墨分析發現隨著2-DG濃度增加, Survivin的表現量有下降的情形。利用RNA原位雜交分析發現birc5a mRNA表現並未改變,而敲落park2基因會減緩2-DG所引起的Survivin表現象降的情況,顯示2-DG處理所造成的Survivin表現量下降,可能與神經母細胞瘤類似,是透過PINK激酶與Parkin泛素連接酶的活性造成Survivin的水解。值得注意的是將park2敲落雖然維持2-DG處理胚胎內Survivin蛋白質的表現,但是並無法恢復中樞神經的發育,顯示Survivin蛋白質的表現與否並非是決定神經細胞分化的關鍵或唯一條件,細胞的能量代謝路徑對神經細胞的分化應有重要的作用。
Survivin is the smallest member of the of inhibitor of apoptosis (IAP) family. There are two homologous genes, birc5a and birc5b, in zebrafish and birc5a is the predominant one expressed in the developing embryos. In the developing embryos, Survivin plays critical roles in vasculogenesis, angiogenesis, neurogenesis, cardiogenesis and hematopoiesis. Survivin is also required for neuronal survival and differentiation, but the detailed mechanism is still unclear. It was shown that rapidly growing cancer cells and stem cells are more likely to undergo aerobic glycolysis (also known as Warburg's effect). Recent studies suggested that Survivin plays a key role in mitochondrial metabolism in neuroblastoma. Overexpression of Survivin promotes mitochondrial fissions and inhibits oxidative respiration to prevent ROS accumulation. As a result, the energy metabolism in cancer cells is mostly accomplished by aerobic glycolysis. Inhibition of glycolysis by 2-deoxyglucose (2-DG) induces Survivin degradation via PINK-Parkin ubiquitin E3-ligase and enhances mitochondrial autophagosis and cell death. In this study, I would like to investigate the biological functions of Survivin in neural differentiation by examining the correlations between glycolysis and Survivin expression. It appears that inhibition of glycolysis by 2-DG in the early stages of development increased embryonic mortality, indicating the importance of glycolysis in the early stages of development. Acridine orange staining revealed that 2-DG treatment induced cell death in the developing embryos. The status of neural development in 2-DG treated embryos was revealed by pan neuronal marker, HuC. It appears that inhibition of glycolysis by 2-DG impaired neuronal differentiation, suggesting that glycolysis plays critical functions in neuronal progenitor cells differentiation. Inhibition of glycolysis elicited neuronal cell death. With increased amount of 2-DG treatment, the transcription of sox3, a neuronal progenitor cell marker, was enhanced, suggesting that impaired neural development in 2-DG treated embryos was caused partly by failure of neuronal differentiation. Western blot analysis revealed that 2-DG treatment decreased the protein level of Survivin. Nevertheless, birc5a transcription was not changed by 2-DG. Knockdown park2 ameliorated 2-DG-induced Survivin degradation, suggesting that the Survivin reduction in 2-DG treated embryos is probably mediated by PINK-Parkin ubiquitin E3 ligase activity, like what observed in neuroblastoma. It was noted that knockdown park2 did not ameliorate CNS development in 2-DG treated embryos, suggesting that Survivin expression is not the only criteria for neuronal differentiation. A proper energy metabolism is required for neuronal survival and differentiation. My study confirm that glycolysis plays critical functions in neuronal differentiation and Survivin is sensitive to the status of cellular metabolism. Inhibition of glycolysis elicited Survivin degradation via PINK-Parkin system.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G001043B008.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48804
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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