斑馬魚selectins的選殖、表現與功能分析

Abstract

選擇蛋白(selectin)是一種黏著分子，在哺乳類的研究中顯示其可調節白血球滾動、復位和再循環。選擇蛋白有三種，分別是：L-selectin以游離形式出現在血清中，也以跨膜蛋白的形式出現在嗜中性白血球、單核球、Ｔ細胞、Ｂ細胞、自然殺手細胞及未成熟的造血細胞膜上；P-selectin表現在被活化的血小板及內皮細胞膜上；E-selectin表現在內皮細胞膜上。斑馬魚在最近被用來研究白血球在傷口的聚集作用，然而我們對斑馬魚選擇蛋白的所知有限。因此本研究選殖出斑馬魚的選擇蛋白，並嘗試釐清其可能的功能與表現。我選殖了三種選擇蛋白，並利用整體原位雜交方法觀察選擇蛋白在斑馬魚的表現位置。結果顯示這三種選擇蛋白的表現位置有部分重疊。我們亦利用morpholino削弱選擇蛋白在魚體中的表現，於受精後三天將魚尾切除以製造傷口，利用螢光顯微鏡在切除魚尾8.5小時後觀察其嗜中性球的遷移情形。我發現削弱L及E選擇蛋白的表現會抑制嗜中性球遷移到傷口處，削弱Ｐ選擇蛋白的表現則會在製造傷口後前兩小時促進嗜中性球的遷移。因此我們推測斑馬魚的選擇蛋白在調節白血球於傷口的聚集作用上，有與哺乳類選擇蛋白類似的功能。

Selectins are adhesion molecules known to mediate leukocyte rolling, homing and recirculation. There are three types of selectins: L-selectin is in a soluble form in serum and a transmembrane protein on the membrane of neutrophils, monocytes, T and B cells, natural killer cells and immature hematopoietic cells; P-selectin is expressed in activated platelets and endothelial cells; E-selectin is expressed on endothelium cell membranes. Zebrafish is a newly established model for leukocyte recruitment study, but we know very little about selectins in zebrafish. Therefore, I aimed to investigate the roles of selectins in zebrafish. I cloned three selectins and studied their expression patterns by RT-PCR and whole-mount in situ hybridization (WISH). Zebrafish selectins share sequence homology to their respective homologs. WISH analysis showed overlapped expression patterns among three types of selectins. To characterize their roles in neutrophil recruitment, I cut the tails of Tg(MPO::EGFP)114 zebrafish at 3 days post-fertilization (dpf) and observed neutrophil recruitments at 8.5 hours post-injury (hpi) by epifluorescent microscope. Knockdown of L– or E-selectin inhibits neutrophil recruitment whereas knockdown of P-selectin enhances neutrophil recruitment at first 2 hours after injury. It suggests that zebrafish selectins may play a conserved role in mediating neutrophil recruitment in zebrafish.