Integration and Evolution of Hypoxia Response Network: Systems Biology and Evolution Approach (III)

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基本資料

Project title

Code/計畫編號

MOST104-2627-B019-002

Translated Name/計畫中文名

低氧訊息網路的系統與演化生物學研究---細胞在缺氧時耗氧代謝途徑的代謝變化與生理反應之探討---以斑馬魚血色素氧化酶相關代謝途徑為例(子計畫3)( III )

Project Coordinator/計畫主持人

Han-Jia Lin

Funding Organization/主管機關

National Science and Technology Council

Co-Investigator(s)/共同執行人

鄒文雄

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=11596524

Year

2015

Start date/計畫起

01-08-2015

Expected Completion/計畫迄

01-07-2016

Co-Investigator(s)

Wen-Shyong Tzou

Bugetid/研究經費

1380千元

ResearchField/研究領域

海洋科學

血色素 (Heme) 是血紅素中負責與氧分子結合的輔基 (prosthetic group)，但是自由型態存在的血色素卻會產生自由基，而對正常細胞造成氧化壓力，因此必須透過血紅素氧化酶 (heme oxygenase, 簡稱 HO) 將之移除。在哺乳類動物中有發現Ho1 和 Ho2 兩種同源基因。以小鼠為模式的實驗顯示兩種同源基因之間有組織表現、基因調控以及生化特性的差異。除了代謝血色素之外，兩種同源基因也被發現與鐵離子平衡和抗氧化等反應相關。尤其是 Ho 基因也被證實對於發育中的胚胎扮演重要的角色，顯示血色素的代謝可能有更複雜的生理意義。本研究室在斑馬魚基因體中發現有 4 種 ho，分別稱為 ho1a、 ho1b、 ho2a 和 ho2b。因為斑馬魚是較原始的脊椎動物，加上體外授精的特性，十分合適研究 ho 相關基因功能的演化以及在胚胎發育中所扮演的角色。在本計畫的第一、二年首先針對 4種 ho 進行原位雜交以及 Q-RT-PCR 等方法，分析各基因在受精後 24 至 120 小時的胚胎中的表現位置與表現量。結果顯示在胚胎發育過程中 ho1a 以及 ho2b 分別在卵黃囊 (yolk sac) 和中樞神經中會有較明顯的表現。由於在哺乳類中，ho1 的表現受到缺氧的調控，因此我們也嘗試將班馬魚卵進行缺氧模擬條件的處理，也觀察到胚胎中的 ho1a 基因表現有增加的現象。然後我們繼續針對斑馬魚 ho1a 啟動子區域進行研究，以 5’RACE 確定轉錄起始位置，並選殖不同片段的啟動子區域進行啟動子區域活性分析。結果顯示在啟動子 -1100 bp~-1200 bp 的區域似乎具缺氧反應因子 (hypoxia response element, 簡稱HRE) 的存在。由計畫研究顯示，ho1 在卵黃囊的表現與受缺氧誘導的特性，可能早在脊椎動物演化的初期就已經出現了，其功能可能只是針對血色素的代謝。而有趣的是ho2 表現在中樞神經的現象，其功能可能不只是代謝血色素，而是積極地與神經的發育有關。在第三年的計畫中，我們採用了次世代定序的方法，並以生理缺氧的實驗模式，建立了斑馬魚成魚大腦在缺氧以及嚴重無氧之下的基因調控網絡，證實 ho1a 基因確實會在輕微缺氧之下受到誘導，但在嚴重缺氧時反而表現量下降。此外，我們也嘗試以最新的基因編輯技術建立 ho 基因欠損的動物模式，並建立微流道斑馬魚生理監控系統。在未來可以利用這些跨領域的研究技術，把相關實驗推展到更深入的層次。 Heme is a prosthetic group that binds to oxygen molecules in hemoglobin, but the free form of heme is very toxic and causes oxidative stress on normal cells. Therefore, free heme must be metabolize by a special enzyme, heme oxygenase (HO). Ho1 and Ho2 are two homologous genes found in mammals. Experiments in mouse models showed that Ho1 and Ho2 are different in their expression patterns and profiles. In addition to metabolic hemoglobin, two homologous genes were also found to be associated with iron balance and antioxidant responses. In particular, the Ho gene has also been shown to play an important role in developing embryos, suggesting that the metabolism of hemoglobin may have more complex physiological implications. Our laboratory found four kinds of ho in the zebrafish genome, named ho1a, ho1b, ho2a and ho2b, respectively. Because zebrafish are external fertilization vertebrate, they are very suitable for ho-related gene function evolution and the role played in embryonic development. In the first and second years of this project, we performed in situ hybridization and Q-RT-PCR to analyze the expression and expression of genes in embryos from 24 to 120 hours after fertilization. The results showed that the expression of ho1a and ho2b in yolk sac and central nervous system were more obvious in embryonic development. Since the expression of ho1 was regulated by hypoxia in mammals, we also attempted to treat the eggs in hypoxic conditions, and observed an increase in ho1a gene expression in the embryos. We then proceeded to study the promoter region of zebrafish ho1a, determined the transcriptional start position by 5‘RACE, and selected the promoter regions of different fragments to carry out the promoter region activity analysis. The results showed that there was hypoxia response element (HRE) in the promoter region of -1100 bp to -1200 bp. It has been shown by research studies that the performance of ho1 in the yolk sac and the hypoxia-induced trait may have occurred early in the evolution of the vertebrate, and its function may be directed solely at hemoglobin metabolism. We found that ho1a were induced under mild hypoxia, however, it declined under anoxia. In addition, we also established the animal model of ho gene knockout with the latest genome editing technology and applied the microfluid zebrafish physiological monitoring system. These interdisciplinary works may help us to bring our research to the next level in the future.

Keyword(s)

血色素氧化酶
基因調控
斑馬魚
缺氧
gene regulation
heme oxygenase
hypoxia
zebrafish

DSpace CRIS

Integration and Evolution of Hypoxia Response Network: Systems Biology and Evolution Approach (III)

基本資料

Description