凝血脢調節素(Thrombomodulin，TM)以下簡稱TM。廣泛存在於細胞表面如單核球、嗜中性血球、內皮細胞、平滑肌細胞、及腫瘤細胞，在血液凝結的調控上扮演一個很重要的角色。文獻報導指出，human umbilical vein endothelial cells (HUVEC)處理cytokines如endotoxin、tumor necrosis factor 及interleukin-1 β會使TM mRNA的表現量及活性下降。而tumor-promoting phorbol esters (PMA)、histamine、retinoic acid 或是活化cAMP的藥物會促使TM mRNA表現上升。報導指出，TM啟動區-1531至-1536bp上RARE (retinoid acid responsive element)及-123至-135bp上兩個Sp1結合位置對retiond acid調控TM之mRNA的表現是重要的。
　　報導指出PMA對TM的影響有部份是透過活化cyclooxygenase-2 (COX-2)的表現。但是對於PMA如何調控TM基因還不清楚。COX-2受到抑制後，PMA對TM的誘導也會被抑制。於是在本篇論文中，主要是探討PMA對TM基因調控。
　　由北方墨點法實驗結果顯示，PMA誘導TM呈現時間及劑量相關性的增加，5 nM的PMA處理HeLa細胞在24小時內TM mRNA達到最高表現。另一方面，用一系列不同長度的5’端段切的TM啟動區報導基因進行分析，分別將TM-181/+2、TM-119/+2、TM-55/+2、TM-27/+2與TM-245/+2比較其報導基因活性分別下降64%、88%、93%、97% 顯示出-245至-55bp區域含有調控TM 基因表現的活性。點突變報告基因分析發現，此區域所包含的第三個Sp1 site (-135至-141bp)是最重要的，其次是第二個Sp1 site (-201至-207bp)，且TM啟動區-245至-119bp上三個Sp1 site對基因表現的調控具有協同作用。而在PMA的調控方面，-245至-55bp區間有2.4倍的PMA誘導倍率。利用點突變報導基因分析，發現只有同時將三個sp1 site同時突變，才會對PMA誘導有輕微抑制。此外，凝膠電泳DNA位移測定實驗的結果顯示出在-245至-119bp區間上者三個Sp1 site皆有Sp1蛋白質的結合，而我們所設計的Sp1 site點突變後，Sp1的結合也消失了。這也顯示出PMA調控TM表現的機制中，Sp1不是主要調控的因子。但是在TM的基楚調空中，Sp1扮演非常重要的角色。

　Thrombomodulin (TM), a glycoprotein expressed on the endothelial cell surface, plays an important role in the regulation of blood coagulation. It was also demonstrated that TM was widely distributed on monocytes, neutrophils, epithelial cells, smooth muscle cells and tumor cells. TM mRNA expression is down-regulated in human umbilical vein endothelial cells (HUVEC) when they are incubated with endotoxin, tumor necrosis factor or interleukin-1 β and up-regulated with tumor-promoting phorbol esters, histamine, retinoic acid (t-RA) and reagents which increase intracellular cAMP. Moreover, recent study indicated that two genomic DNA regions, the DR4 site (-1531 to –1516bp) and the Sp1-binding site (-119 to -135bp) are required for t-RA-dependent augmentation of TM gene expression.

　Some reports indicate that PMA mediates TM function by activating COX2 but the gene controlling its expression is to be defined. Once COX2 is suppressed, the induction effect of PMA to TM expression is also inhibited. In this study, the regulation mechanism of PMA-induced TM gene expression in HeLa cells was stuied.

　By Northern blot analysis, we found that the TM mRNA expression pattern has a dose- and time-dependent manner after PMA treatment in HeLa cells. PMA(5nM) increased TM mRNA expression to its maximal level after 24th hours treatment. Analysis of the 5’ deletion mutants revealed that the 5’flanking region of TM promoter sequence between –245bp and –55bp was involved in gene expression. Reporter activities of TM-181/+2, TM-119/+2, TM-55/+2 and TM-27/+2 were decreased by 36%, 12%, 7% and 3% respectively compared with TM-245/+2(100%). Furthermore, analysis of point mutation of TM promoter indicated that Sp1 site (-135 to -141bp) was the most important site for TM basal activity, the by the Sp1 site (-201 to -207bp) was the next important. Three Sp1 sites in the TM promoter region from–245 to -119bp have a synergetic effect for TM basal activity. Finally, TM promoter region (-245 to -119bp) was found to have contribution for PMA induction up to 2.4-folds. However, from the results of point mutation experiment, we found only double Sp1 site mutant has minor effect on PMA-dependent TM gene expression. Beside, the result of EMSA indicated that Sp1 protein binds to three Sp1 site between–245 to -119bp, and point mutant of Sp1 site also disrupted this binding. This result all indicated that Sp1 is not very important in the regulation mechanism of PMA-induced TM expression. But Sp1 plays a major role in the basal transcriptional regulation of TM.

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