研究Dnmt1在Pdx1所調控的胰臟β細胞分化中所扮演的角色

Studying the Role of Dnmt1 in Pdx1-mediated β Cell Differentiation

張益彰(Yi-Chang Chang)

指導教授 ： 江明格

國立中正大學/理學院/生命科學系分子生物研究所/碩士(2013年)

摘要

胰臟得發育需要許多因子例如Pdx1、Ptf1a、Ngn3…等。這些因子會形成網路調控不同細胞系的分化。先前研究已指出在老鼠胚胎表現產生胰臟芽(pancreatic buds)以及維持β cell形成和功能都是需要Pdx1的表現。先前我們實驗室利用酵母菌雙雜交系統(yeast two-hybrid assay)發現Dnmt1(DNA methyltransferase 1) 胺基酸序列1509~1590片段蛋白能夠與Pdx1交互作用。在發育過程中，藉由基因甲基化修飾(DNA methylation)確保細胞專一的分化，而整個調控過程中Dnmt1是扮演一個核心角色之一。此外，最近研究指出在斑馬魚胚胎中抑制Dnmt1表現造成晚期胰臟分化失敗，另外Dnmt1失去催化活性可能會影響β前驅細胞(β cell progenitors)分化或其他細胞轉分化成β細胞的路徑也會受到影響。此研究目的是用GST-pull down assay證明Dnmt1和Pdx1之間的交互作用，也要用共同免疫沉澱(co-immunoprecipitation)證明Dnmt1和Pdx1會在哺乳細胞中進行交互作用。此外，我也利用shDnmt1慢病毒(lentivirus)成功抑制大鼠胰島細胞株Dnmt1表現，並且發現當Dnmt1表現量降低時，調控胰島素基因:Glut2、insulin、Pdx1表現量也跟著下降，另外所製備shDnmt1慢病毒也能成功抑制人類肝臟細胞以及胰臟細胞Dnmt1的表現。接著，除了運用人類胰臟細胞探討Dnmt1對於胰臟發育晚期的影響外，未來也會在肝臟細胞異位表達Pdx1使其轉分化成胰臟細胞，並且再抑制Dnmt1表現，探討Dnmt1對於胰臟早期發育基因的影響，利用這三個細胞株模組能更加清楚Dnmt1在胰臟細胞分化所扮演的角色。

關鍵字

胰腺和十二指腸同源性盒子1 ； DNA 甲基化轉移酶1 ； 胰臟發育

並列摘要

Proper Pancreas development requires many regulatory factors, such as Pdx1, Ptf1a, Ngn3. These factors form networks that regulate the differentiation of various cell lineages , Previous studies have shown that Pdx1 is required for both formation of the pancreatic buds in the embryo and the subsequent β cell formation and function. Our laboratory has utilized the yeast two-hybrid assay to find that truncated Dnmt1 protein(aa1509~1590) can interact with Pdx1. Dnmt1 (DNA methyltransferase 1)plays a central role in the epigenetic regulation of gene expression during development and disease. In addition, recent studies have shown that Knockdown of Dnmt1 in zebra fish embryos caused defects in terminal differentiation of the pancreas, furthermore, losing catalytic activity of Dnmt1 may influence the differentiation of pancreatic β cell progenitors or the reprogramming of cells toward the pancreatic beta cell fate. The object of this study is to prove the interaction between Dnmt1 and Pdx1 by pull down assay and also show that Pdx1 and Dnmt1 interact with each other within mammalian cells by co-immunoprecipitation. To further identify the region of Dnmt1 responsible for interacting with Pdx1, a series of GST-tagged Dnmt1 truncated protein have been purified, and will be tested for their interactions with Pdx1 through pull-down assay. In addition, I have utilized the shDnmt1 lentivirus to knockdown the expression of Dnmt1 successfully in liver and pancreatic cell lines. Next I will overexpress Pdx1, together with the knockdown of Dnmt1 in liver cell line to determine the biological function of Dnmt1 during pancreatic cell trans-differentiation.

並列關鍵字

DNA methyltransferase 1 ； pancreas development ； pancreatic and duodenal homeobox 1

參考文獻

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