- 學位論文
探討Zfp280d調控PDX1所驅使的肝臟轉分化為胰臟的能力
Investigating the Ability of Zfp280d to Modulate PDX1-mediated Liver-Pancreas Transdifferentiation
摘要
胰臟是人類的第二大腺體器官，它掌控了血糖的穩定。關於胰臟發育已研究出一些轉錄因子(transcription factors)在發育過程中有其重要性，這些轉錄因子有 PDX, Ptf1a, Ngn3。其中PDX1掌控內胚層中原腸腔(primitive gut)發育成胰臟，因此PDX1 基因缺陷會導致小鼠胰臟無法發育，也有研究指出PDX1 基因也對 β細胞的功能相當重要。為了進一步了解PDX1，我們實驗室利用酵母菌雙雜交系統(yeast two–hybrid assay)尋找10.5天胚胎胰臟芽基因庫(E10.5 pancreatic bud cDNA library)當中，能與PDX1 交互作用的蛋白質。我們篩選出許多基因，其中一個是鋅模組蛋白Zinc finger protein 280d（Zfp280d），而它的功能尚未被研究。Zfp280d包含九個鋅模組和DUF4195區域，DUF4195是一個保守性但功能尚未知的序列。我們實驗室先前利用蛋白質交互沉澱(GST-pull down assay)和共同免疫沉澱(co-immunoprecipitation)證實Zfp280d是透過DUF4195與PDX1有交互作用。此外，我們還進一步作共同免疫螢光染色(co-immunofluorescence)證實Zfp280d和PDX1共同座落在細胞核。為了知道Zfp280d對胰臟發育的功能為何，實驗中我利用NIT-1(小鼠β細胞株)減少Zfp280d的表現量，結果發現Zfp280d減少會影響內分泌細胞基因如Pax6、Glut2、Isl-1、Nkx6.1的表現量都有下降，這一項結果顯示Zfp280d對β細胞的功能維持扮演了一個重要的角色。另一方面，我也利用BNL(小鼠的肝臟細胞)與HepG2、Huh7(人類肝癌細胞)做轉分化成胰臟的實驗，利用shRNA將Zfp280d表現降低後再轉染 PDX1、Ngn3及Mafa，這三個基因對胰臟發育都是關鍵基因，接著利用RT-PCR分析有關胰臟基因的表達，而在這方面尚無法確定Zfp280d對轉分化過程如何影響，因此未來我們會利用初級肝臟細胞(primary hepatocytes)作為轉分化實驗的細胞。
並列摘要
The pancreas is a glandular organ in humans, it controls blood glucose stability. Recent studies about pancreas development have been focused on some transcription factors that are important for the developmental process. These transcription factors include PDX1, Ptf1a, Ngn3. Among them, PDX1 plays a crucial role in defining the region of the primitive gut that will form the pancreas; as a result PDX1-deficient mice fail to develop pancreatic tissue. Besides, it has been shown that PDX1 is also important for β cell function. To further understand the action of PDX1, we have used the yeast two-hybid assay to screen an E10.5 pancreatic bud cDNA library using PDX1 as a bait. We identified several genes, and one of the identified gene is zinc finger protein 280d (Zfp280d) whose function has not been described before. Zfp280d contains nine zinc finger domains and a DUF4195 domain. Our lab previously used GST-pull down assay and co-immunoprecipitation to confirm that Zfp280’s interaction with PDX1 is through its DUF4195. In addition, we also used co-immunofluorescence staining to confirm that Zfp280d and PDX1were co-localized in the nucleus. In order to know the function of Zfp280d in pancreas development, in my experiments I knocked down the expression of Zfp280d in NIT-1 by using lentivirus, preliminary results show that knockdown Zfp280d down regulated the expression of several endocrine cell specific genes, such as Pax6, Glut2, Isl1 and Nkx6.1. On the other hand, I performed transdifferentiation experiments in BNL, HepG2, and Huh7 cells. I utilized shRNA to knockdown Zfp280d and then transfected the cells with plasmids for overexpression of PDX1, Ngn3 and Mafa, because these transcription factors are important for pancreas development. Subsequently, I analyzed the expression of pancreatic genes by using RT-PCR. However, the results still could not fully determine the biological function of Zfp280d in the process of transdifferentiation. So in the future, primary liver cells may be more suitable for the transdifferentiation experiment.
參考文獻
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