探討原致癌基因MDM2在斑馬魚肝臟生長及器官型態形成的調控功能

Abstract

在許多癌症中均發現MDM2(mouse double minute 2)基因有過量表現之情形，這是因為MDM2基因可以抑制腫瘤抑制基因p53的表現量。除此以外，目前的研究也發現MDM2可以參與其他獨立於p53的功能，其在細胞週期調控、分化、細胞命運的決定、DNA的修補、基本轉錄調控及其他方面的功能上均扮演重要角色。此外，在不同的模式動物如轉殖鼠、果蠅跟斑馬魚之研究顯示過量表現MDM2會抑制生長及影響細胞分化。因此本實驗中我們利用肝臟發綠螢光的轉基因斑馬魚，以過量表現MDM2造成的抑制功能來探討其對肝臟發育的影響。同時，我們將利用肝臟脂肪酸結合蛋白( L-FABP)的啟動子來驅動mdm2使其專一表現在肝臟。開始實驗前注射各種濃度的構築體以測試死亡率以及作用效果，我們選擇了200ng/μl為最適當注射濃度。過量表現MDM2在斑馬魚肝臟的結果顯示會抑制肝臟生長並導致其發育不全。在第四天的組織切片中顯示，與過量表現MDM2蛋白的肝臟相比較，正常的肝細胞顯的相當的完整，接著第十天的MDM2過量表現肝臟出現小空泡的累積。分析相關的MDM2下游基因，發現細胞週期調控的相關基因如RB-1、β-catenin、 TGF-β1跟SP1以及肝臟生長的相關調控基因如Hnf-1α, Hnf4α 跟Ntl表現量受到調控導致表現量下降，因此推測MDM2肝臟的發育上可能經由抑制細胞週期所需的Sp-1基因或是肝臟發育所需要的β-catenin及Ntl基因而導致肝臟發育不全。本實驗模式系統可提供來探討MDM2與肝臟發育相關的分子機制之研究。

Mouse double minute-2 (mdm2) gene has been shown to be overexpressed in many cancers that presumably lead to the inactivation of tumor suppressor protein p53. In addition,MDM2 also has p53-independent functions and plays significant roles in cell cycle control, differentiation, cell fate determination, DNA repair, basal transcription, and other processes. Moreover, MDM2 overexpression has been demonstrated to have growth arrest function in different animal model such as mice, drosophila and zebrafish . In the present study we made an attempt to study the inhibitory effect of MDM2 overexpression in the growth of liver. We had already developed a liver-GFP zebrafish transgenic line, a unique tool that can be used to examine liver morphogenesis. Here we used, liver fatty acid binding protein (L-FABP) promoter to drive the mdm2 expression in a liver-specific manner. “Various concentractions of DNA were microinjected and tested, 200 nanogram/microliter was chosen for the next experiments”. The results revealed that overexpression of MDM-2 cause the inhibition of liver growth and ultimately resuled in hypoplasia of the liver. Histopathological studies using HE staining in 4 dpf zebrafish showed the intact hepatocytes in the wild type liver tissue when compared to the MDM2 transient expressed zebrafish. Using semi-quantitative PCR, we found that some genes involved in the regulation of the cell cycle such as RB-1, β-catenin,Sp1 and TGF-β and liver growth genes such as Hnf-1α, Hnf4α and Ntl have been down-regulated,suggesting the possible role of MDM2 in cell cycle and liver development. The hypoplasic liver may have resulted from cell cycle disorder.Hence, zebrafish liver provides a unique experimental model to explore the molecular interactions of MDM2 in liver morphogenesis.