探討Sirt1對Ngn3所誘導的內分泌細胞分化之影響

Modulation of Ngn3-mediated endocrine differentiation by Sirt1

張哲瑋(Che - Wei Chang)

指導教授 ： 江明格

國立中正大學/理學院/生命科學系生物醫學研究所/碩士(2015年)

摘要

胰臟 (Pancreas)，在維持內分泌 (endocrine)、外分泌 (exocrine)系統中皆扮演重要角色。有研究顯示在誘導胰臟發育成熟的過程中需要多種的轉錄因子 (transcription factors)調控，例如PDX1、P48 (Ptf1a)以及Ngn3。在些轉錄因子中，Ngn3為鹼性-螺旋-環-螺旋家族 (basic helix-loop-helix family)成員之一，在誘導胰臟內分泌細胞發育過程中擁有重要的地位，內分泌先驅細胞會先表現出Ngn3，接著才會誘導分化出其餘內分泌細胞，如α、β、δ細胞以及PP細胞。
先前實驗室利用酵母菌雙雜交系統 (Yeast two-hybrid assay)篩選出會與Ngn3交互作用的蛋白質，Sirtuin (Sirt1)。Sirt1在酵母菌中有同源性非常高的蛋白質Sir2 (Silent mating type information regulation 2 homolog S.cerevisiae)，在哺乳類動物中Sirt1是依賴NAD+才會具有去乙醯化活性的能量調控者。
在我的研究中想了解Sirt1既然與Ngn3之間有直接的交互作用，那Sirt1對於Ngn3的作用為何?首先，我利用shSirt1在穩定表達Ngn3的PANC1 Clone當中將Sirt1基因沉默 (Knockdown)，以及利用對Sirt1具Stimulators或Inhibitors的藥物在穩定表達Ngn3的PANC1 Clone當中處理過後，透過RT-PCR (Reverse transcription Polymerase chain reaction)以及Q-PCR (Real-time polymerase chain reaction)觀察在Sirt1被沉默或抑制後對於Ngn3所啟動的下游基因表現是否受到影響。實驗結果發現，Ngn3下游基因表現是會受到Sirt1沉默或活性被抑制的影響而增強，而當Sirt1活性受到藥物促進時，Ngn3下游基因表現是會減弱的。再來也利用螢光素酶報告實驗確認當Sirt1同時與Ngn3表達時則會降低Ngn3下游基因NeuroD啟動子所驅動的冷光蛋白的表現。另外先前文獻提到Sirt1具有去乙醯化的活性，這裡我想進一步了解Ngn3是否可能有會被乙醯化的現象，由螢光素酶基因檢測初步得知，P300存在的情況下會使Ngn3活性有加強的現象，由此推測Ngn3可能會被乙醯化。除了上述實驗，我也做了CHIP實驗證實在Sirt1被沉默的情況下，Ngn3結合到下游基因NeuroD 啟動子的能力也明顯增強。

關鍵字

內分泌細胞

並列摘要

Pancreas is a glandular organ in human gastrointestinal tract, which plays a crucial role in maintaining endocrine and exocrine. Previous studies have shown that pancreas maturation requires functions of several transcription factors, such as PDX1、P48(Ptf1a) and Ngn3. Neurogenin3(Ngn3) is a member of the basic helix-loop-helix family. It has been shown to be important for the development of endocrine cells of pancreas, such α cells、β cells、δ cells and pancreatic polypeptide cells. Previously, we utilized the yeast two-hybrid assay to find proteins that can interact with Ngn3, and Sirtuin1 (Sirt1) was identified from that screening. Sirt1 was originally found as the mammalian homologue of yeast Sir2. In human, Sirt1 constitutes an energy sensors, which mediate NAD+-dependent deacetylation. Besides, sirt1 regulates various functions such as metabolism, aging and apoptosis by modulating target proteins via deacetylation, such as FOXA2, P53. We utilized shRNA to knockdown Sirt1 expression in PANC1-Ngn3 cells and used drugs like β-NAD, Resveratrol and Nicotinamide to promote or repress Sirt1 activity, and then to observe their effects on the expression of Ngn3 downstream genes. In addition, the CHIP assay was also used to further test the effects of Sirt1 on the interactions between Ngn3 and the promoter regions of its downstream target genes.

並列關鍵字

Sirt1

參考文獻

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