凝血酶調節素(Thrombomodulin, TM)是一種表現於血管內皮細胞表面的醣蛋白，是生理上重要的抗凝血因子，其蛋白質結構從氮端依序被分為lectin-like結構區(TMD1), EGF-like 結構區(TMD2), serine/ threonine-rich結構區(TMD3), transmembrane結構區(TMD4)及碳端的cytoplasmic結構區(TMD5)。其中，TM的氮端lectin-like結構區與調節免疫反應的C-type lectins具有序列相似性。本論文主要採用兩種不同表現系統製備TM重組蛋白，並以此重組蛋白探討凝血酶調節素lectin-like結構區對於細胞的增殖、移動以及抗發炎反應等等方面的影響。首先利用酵母菌表現系統表現TMD1重組蛋白質，在TM重組蛋白的碳端接有腸激酶切割序列，c-myc epitope以及His-tag等融合蛋白；篩選高表現量的酵母菌株後，誘導重組蛋白大量表現，培養液經由鎳離子親和性樹脂(nickel-chelating Sepharose)管柱，可純化出高純度的TMD1重組蛋白，並利用此重組蛋白自兔子中製備TMD1抗體。人類胚胎腎臟細胞株(HEK293 cell line)則是第二種表現系統，含有重組蛋白的細胞培養液藉由通過Q Sepharose及nickel-chelating Sepharose管柱可達到純化TM重組蛋白的目的。利用相同的哺乳動物細胞表現系統，我們成功地表現TMD12, TMD123, TMD23, TMD2, TMD1+HP等其他五個不同片段的TM重組蛋白，並且證實這些重組蛋白具有活化protein C的功能，將進一步被應用於探討TM不同結構區的生物活性。

　　關於TMD1的功能，我們發現重組TMD1蛋白並不影響牛主動脈內皮細胞(BAEC)的生長；不過，重組TMD1蛋白可以抑制BAEC的移動。由於細胞與基質間的交互作用對於細胞移動是很重要的，初步推測可溶性TMD1片段可能透過影響基質間的交互作用而干擾細胞的附著而達到抑制細胞移動的效果。此外，由此兩種表現系統所表現的TMD1重組蛋白會抑制人類腫瘤壞死因子α (TNFα)誘導人類臍靜脈內皮細胞(HUVEC)的訊息因子ERK的磷酸化，以及抑制LPS刺激巨噬細胞釋放TNFα。本實驗初步證實TMD1可以透過抑制細胞激素的分泌而達到抗發炎反應的效果，然而其調控發炎反應的機制則有待進一步研究。

　　Thrombomodulin (TM) is a membrane glycoprotein, which functions as a potent anticoagulant factor. It contains five structure domains: N-termianal lectin-like domain (TMD1), EGF-like domain (TMD2), serine and threonine-rich domain (TMD3), transmembrane domain (TMD4) and C-terminal cytoplasmic domain (TMD5). The extracellular N-terminal lectin-like domain of TM has homology to C-type lectins that are involved in immune regulation. In this study, we explored the role of lectin-like domain of TM in modulating cell proliferation, migration and anti-inflammation. Recombinant human lectin-like domain of TM, TMD1 was prepared by different expression systems. First, human TMD1 gene ligated with enterokinase cutting site, c-myc epitope and His-tag was constructed into the pPICZalpha A vector, and then transformed into Pichia pastoris expression X-33 cells. The yeast-expressed TMD1 was purified with nickel-chelating Sepharose and then recognized with anti c-myc antibody. Polyclonal antibody against the TMD1 protein was generated from TMD1 immunized-rabbit. HEK293 cells-based mammalian expression system was the second tool to prepare recombinant TMD1. TMD1 released in the conditioned medium was purified by Q Sepharose and nickel-chelating Sepharose columns. The other recombinant TM fragments (TMD12, TMD123, TMD1+HP, TMD2, TMD23) were also expressed successfully by HEK293 cells-based mammalian expression system. The anti-coagulant function of these recombinant proteins was determined by in vitro protein C activation test. The biological functions of different functional domains will further be studied using these recombinant proteins.

　　Recombinant TMD1 had no effect on the proliferation of bovine aortic endothelial cells (BAEC), but it inhibited cell migration. Given that properly regulated cell-substratum interaction is important to the process of cell migration, we propose that soluble TMD1 may interfere cell adhesion by interaction with extracellular matrix. Moreover, TMD1 purified from both expression systems inhibited TNFα-induced ERK phosphorylation in human umbilical vein endothelial cells, and also inhibited Lipopolysaccharide-induced TNFalpha secretion in human monocytic leukaemia U937 cell line. This result suggested that TMD1 had anti-inflammatory properties through suppression of the release of cytokines. The molecular mechanism involved in the anti-inflammatory properties of TMD1 needs further investigation.

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