凝血酶調節素(Thrombomodulin；簡稱TM)，廣泛地存在於體內不同組織細胞中，如：上皮細胞、平滑肌細胞、角質細胞及內皮細胞等等。TM是身體中重要的抗凝血分子，其主要功能是藉著結合凝血酶，進而限制凝血酶的促凝血活性及生理活性。本實驗室最近的研究也證明，TM參與細胞間的相互黏著，而此過程需要lectin-like domain的參與。但是對於TM表現的調控機轉仍不清楚。因此，我們針對TM表現與細胞增生間的關係加以研究。首先，培養相同細胞數量的牛動脈內皮細胞(BAECs)，並以MTT測試每天細胞的生長速度及西方點墨法去分析TM表現量的變化；再利用流式細胞儀去分析TM的變化是否與細胞的生長有關。藉由這些實驗結果，我們初步認為細胞表面的TM，其表現量會隨著細胞的增生而增加，當細胞培養到第三天時，在培養皿上的細胞數量約接近飽和，此時TM的表現量會最高，在細胞培養超過三天之後，TM的表現量便逐漸減少。接著，我們試著去解開TM表現量增加及減少的原因。當我們用VEGF去刺激BAEC增生時，亦發現TM的表現增加。我們也發現，TM表現量的減少，並不是透過內吞作用(internalization)或是蛋白酶的分解作用，而是當細胞長滿後，TM基因在轉錄過程中受到影響，導致TM 的表現量減少。為了更進一步檢測細胞生長與TM表現量的關係，我們將不同細胞數的BAECs種入培養皿中，經過15小時後，測定TM的蛋白量，結果發現細胞數目未多至飽和之前，TM表現量並無明顯差異，當細胞數過多時，則TM便會減少，表示細胞密度較低時並不會影響TM表現，細胞密度過高時，則抑制TM的表現。另外，在人類的角質細胞上，我們也觀察到相同的結果，其TM表現量會隨著細胞生長而增加。由上述的實驗結果可得知，當細胞快速生長時，TM的表現量會增加；而在細胞長滿後，TM的表現量便會減少。因此，我們推論TM蛋白及mRNA的表現量會受到細胞增生及密度的調控。

　　Thrombomodulin(TM) is widely distributed in a number of different cell types, such as epithelial cells, smooth muscle cells, keratinocytes, and endothelial cells. TM is a glycoprotein receptor and cofactor for thrombin’s activity in a physiologically important natural anticoagulant system. Our recent study has demonstrated that TM may mediate cell-cell adhesion through its lectin-like domain. However, the mechanisms of regulating TM expression are still unclear. In this report, the correlation of TM expression and cell proliferation was investigated. Bovine aortic endothelial cells (BAECs) were incubated for various time intervals and the growth rate and TM expression level were analyzed by MTT assay and Western blot, respectively. Furthermore, flow cytometry was used to determine TM expression levels on cell surface. The results demonstrated that the TM level was increased with cell proliferation and reached maximum at day 3, in which cells grew to confluent monolayer. The TM level was decreased when BAECs were cultured longer than three days. In order to further examine the effect of cell density on TM expression, BAECs with different density were plated onto culture dishes, and TM expression was determined after 15 hr incubation to eliminate the effect of cell proliferation. The data showed that TM expression was not changed significantly under the low cell density condition, whereas TM level was reduced when cell density is over confluent. We also observed that TM expression level was enhanced by adding VEGF to stimulate BAECs proliferation. Furthermore, RT-PCR and Western blot experiment showed that the decrease of TM expression level was due to the downregulation of TM gene transcription, and independent of internalization and degradation of TM. In addition, similar results were observed in HaCaT cells, a human keratinocyte cell line. TM expression in HaCaT cells was also elevated with cell proliferation. These results suggested that TM expression might be increased when cells proliferated, but decreased after cells reached confluence. Taken together, we concluded that TM expression may be regulated by cell proliferation and cell density.

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