人類凝血脢調節素(Thrombomodulin , 簡稱：TM)是血管內皮細胞表面的醣蛋白，目前研究已知其主要功能是調節血液的凝結反應。當TM與凝血脢(thrombin)結合形成複合體時，可以活化Protein C，而被活化的Protein C可以分解凝血因子Ⅴa與Ⅷa，中斷凝血機制。而目前也有研究指出，TM的第二及第三結構體(Domain),TMD23,與細胞的增殖有關。
　　本實驗室構築出可以製造人類TMD23蛋白的酵母菌，利用大量發酵槽來培養酵母菌，及表現TMD23蛋白，再進一步的對TMD23蛋白的特性和功能做分析。
　　本實驗發現TMD23蛋白具有幫助scu-PA(single-chain urokinase-type plasminogen activator)活化plasminogen的功能。而本實驗室發現隨著劑量增加的關係，TMD23蛋白促進scu-PA活化plasminogen的速率可增加4~6倍。因此TM蛋白除了調控凝血機制的功能外，也有可能藉著調控plasminogen活化的機制，而直接或間接參與細胞內其他作用。
　　另外有文獻指出，TM蛋白與A549(human lung cancer cell line)細胞的增殖有關，而本實驗發現TMD23對BAEC (bovine arterial endothelia cells)也有刺激增殖的效果，我們進一步利用細胞免疫螢光染色法研究，發現A549細胞以TMD23處理一小後，再以共軛焦顯微鏡觀察，可發現TMD23已進入細胞質內，4小時後TMD23已大量的分布於細胞質，20小時後，則可發現TMD23已逐漸減少。接著我們以lysotracker標定lysosome，可以發現TMD23在1個小時到4個小時逐漸進入lysosome的現象，以上結果顯示，TMD23會進入A549細胞內，並可能在lysosome 被分解。

　　Thrombomodulin (TM), a thrombin receptor protein found on the endothelial cell surface, plays an important role in regulation of blood coagulation. By forming a 1:1 complex with thrombin, TM alters the procoagulant activity of thrombin and activates protein C that can inactivate blood coagulation factorⅤa and Ⅷa. Moreover, TM has several biological functions, including proliferation of many cell lines.
　　In this study, recombinant human TM peptide fragment, TMD23, containing six EGF-like motifs and serine/threonine-rich domain (residues 224-479) and a tag of six histidine was expressed in Pichia pastoris, and was purified by a Ni-chelating affinity column. The purified protein was used to study the biological function of TM.
　　We demonstrated that TMD23 could enhance plasminogen activation by single chain urokinase-type plasminogen activator . It implicated that TM might enhance activation of plasminogen to plasmin and be involved in the regulation of variuos biological functions such as fibrinolysis, degradation of extracellular matrix, ovulation, tumor matastasis and cellular differentiation.
　　In addition, we found that TMD23 could stimulate the proliferation of several cell lines, including bovine arterial endothelia cells (BAEC), and human lung cancer cell line (A549). The cells were incubated with FITC-labeled TMD23 and location of the fluorescent labeled protein was observed with a confocal microscope. The labeled TMD23 was engulfed and internalized into cytoplasm in 1 h. After 4 h incubation, the fluorescence of FITC was distributed over the entire cytoplasm and especially near peri-nuclear region. The intensity of intracellular fluorescence slowly decreases in 20 h incubation. Using lysosomal fluoro-staining, we demonstrated that internalized TMD23 and lysosome were colocalized in 1 h and the colocalization become more obvious in incubation for 4 h. The results suggested that the soluble TMD23 might internalize into cells and was degraded in lysosome. Therefore we proposed that TMD23 might interact with the cellular receptor and be internalized. At mean time, it might trigger the signal transduction for cell proliferation.

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