我們假設CRSBP-1和它的結合子不只能調節組織間液流動，也參與淋巴管新生。由於淋巴管新生機制包含淋巴管內皮細胞移動、分化，我們便利用細胞移動實驗 (in vitro scratch assay) 測定CRSBP-1專一性結合子調節淋巴管內皮細胞(SVEC4-10 細胞) 之分子機制。我們則證明CRSBP-1專一性結合子 (PDGF peptide和VEGF peptide) ， 促發炎反應細胞激素 (TNF-α)， 及PDGF-BB 會刺激SVEC4-10細胞移動。並且證明PDGF peptide 或 VEGF peptide 是經由與 PDGF-BB不同機制而進而促進淋巴管內皮 (LEC) 細胞移動。除此之外，PDGF peptide在細胞有TNF-α存在下，會保護CRSBP-1不被TNF-α 抑制並且PDGF peptide依然能夠促進細胞移動。最後，我們在斑馬魚當中找出人類CRSBP-1的直系同源蛋白 (orthologue)，是由328個胺基酸組成。衍生斑馬魚CRSBP-1胺基酸序列與人類有26% 相同性，其存在斑馬魚7號染色體上，含有6個外顯子，5個內顯子。斑馬魚CRSBP-1的基因構造和在脊椎動物內的直系同源蛋白相似程度高。我們利用morpholino降低 (knock-down) 該基因在斑馬魚的表現並且得到初步的結果表示CRSBP-1在淋巴管新生分子機制中扮演一個重要角色。 We hypothesize that CRSBP-1 and its ligands not only regulate interstitial-lymphatic flow but also play a role in lymphangiogenesis. Since lymphangiogenesis involves cell migration and proliferation of LECs, the 1st goal of this thesis work is to determine the role of specific CRSBP-1 ligands in regulation of cell migration in SVEC4-10 cells (lymphatic endothelial cells) by using an in vitro cell migration assay. Here we demonstrate that specific CRSBP-1 ligands (PDGF peptide, VEGF peptide), PDGF-BB and TNF-α stimulate cell migration in SVEC4-10 cells, that specific CRSBP-1 ligands, PDGF-BB and TNF-α stimulate cell migration in these cells through different mechanisms and that PDGF peptide protects cell-surface CRSBP-1 from TNF-α-induced down-regulation and suppresses TNF-α-stimulated cell migration in these cells. The 2nd goal of this thesis is to identify zebrafish CRSBP-1 and its function in zebrafish. Here we demonstrate that the deduced amino acid sequence and genomic structure of zebrafish CRSBP-1 identified are similar to those of other vertebrate orthologues. The preliminary results from experiments using morpholino down-regulation support the hypothesis that CRSBP-1 plays a role in lymphangiogenesis.
