PTEN在斑馬魚胚發育過程中之基因表現及功能研究

Abstract

中文摘要 細胞增殖、分化及移動在胚發育過程中具有極其重要的意義，已知有許多蛋白質分子涉及調控這些細胞生理活動，而近來的研究更著重於細胞凋亡(apoptosis)、腫瘤發生(tumorigenesis)及癌症等方面之探討。磷酸肌醇磷酸酶PTEN/MMAC1為一種腫瘤抑制蛋白質，其藉由將磷脂醯肌醇三磷酸酯(phosphatidylinositol 3,4,5-trisphosphate)去磷酸化，進而抑制細胞之生存、增殖及移動，許多研究指出PTEN在調控哺乳類及昆蟲胚正常發育上扮演重要的角色，但在魚類胚發育過程中之研究則付之闕如，因此為研究PTEN在硬骨魚類胚發育過程之基因表現及其功能，本研究選擇以斑馬魚作為本論文之實驗物種。 已知斑馬魚之PTEN有四型，分別為PTEN A群中之AY398668及AY398669以及PTEN B群中之AY398670及AY398671，我們已選殖到其中AY398668，AY398669及AY398670三型之基因。根據不同物種間的序列分析比對，斑馬魚的四型PTEN蛋白質和其他物種有超過74%之相似度，並且皆具有相同或相似的功能區域。在基因表現方面，斑馬魚的PTEN於各個胚發育時期及成體組織中皆有程度不同之表現，而在胚發育晚期時特別以頭部區域表現量較高。當過度表現(overexpression)此兩型PTEN時，發現其並不影響胚之發育，但於降低此四型PTEN的轉譯(translation)時，發現PTEN A但非PTEN B的轉譯降低會造成原腸化(gastrulation)時期魚胚之細胞移動受阻並嚴重影響其發育，綜合以上之研究結果，顯示PTEN A之正常表現對斑馬魚胚原腸化時期之發育及調控細胞移動是必須的。

The tumor suppressor, PTEN (also known as MMAC-1), is a lipid phosphatase which counteracts the effects of phosphoinositide 3-kinase (PI3-kinase) by dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate (PIP3). In this way, PTEN downregulates the protein kinase B (PKB) and downstream pathways affecting cell growth, migration and survival. It has also been demonstrated to be essential for embryonic development in human, mouse and Drosophila. In zebrafish, four PTEN isoforms have been registered in the NCBI database, but no descriptive or function assay has been reported at our knowledge. Here, the expression patterns of zebrafish PTENs as well as their functional characterizations during early embryonic development were presented. The results of RT-PCR and whole-mount in situ hybridization analyses showed that PTENs expressions occur ubiquitously in the early developmental stages and decrease during the epiboly periods. The zygotic expression turn on after 24 hour post fertilization and gradually accumulate in the head region afterward. In adult fish, PTENs expressed in all tissues examined with stronger expression in brain, eye, ovary, kidney and blood. Overexpressions of two PTENs, AY398669 and AY398670 had no effect on the early development of zebrafish embryos. Knockdown of PTEN A (AY398668 and AY398669), but not PTEN B (AY398670 and AY398671) disturbed the cell movements during gastrulation, caused abnormal development and resulted in embryonic death. These results suggest that PTEN A is essential for the embryonic development of the zebrafish, presumably via the inhibition on embryonic cell migration.