人類凝血酶調節素(Thrombomodulin；簡稱TM)是血管內皮細胞表面的一種醣蛋白，它是身體中重要的抗凝血分子，主要是藉著結合凝血酶而限制凝血酶的促凝血活性及生理活性，科學家們利用包括生化分析及基因改造老鼠等方法探討凝血酶調節素的生理功能，發現凝血酶調節素也扮演抑制血塊溶解，抗發炎反應，抑制腫瘤細胞增生，維持正常的懷孕過程及胚胎發育等重要角色。
　　本論文選用RNA干擾技術(RNA interfenerce，RNAi)探討凝血酶調節素在角質細胞的功能。RNAi是一種具有序列專一性的小片段雙股RNA，可抑制特定基因進行後轉錄表現(post-transcriptional expression)，是近年來研究基因功能的利器。我們設計不同的人類凝血酶調節素的小片段干擾RNA，構築在具有哺乳動物第三型RNA polymerase U6啟動子的載體上，利用U6 啟動子來轉譯小片段干擾RNA(siRNA)，再將siRNA與可以表現凝血酶調節素的質體同時轉染到人類黑色素瘤A2058細胞，發現可以顯著降低凝血酶調節素的蛋白表現。進一步將siRNA的質體轉染到人類上皮細胞HaCaT細胞中，siRNA 亦可以降低HaCaT 細胞內源性的凝血酶調節素，同時細胞表面上的凝血酶調節素活化Protein C的活性也降低。此外，抑制凝血酶調節素表現造成細胞生長與移動能力均增快，顯示凝血酶調節素具有調節HaCaT細胞生長與移動的功能。
　　在另一方面，我們嘗試藉由腺相關病毒(adeno-associated viral，AAV) 傳送siRNA的基因到其他的哺乳類細胞；構築表現加強型綠色螢光蛋白(EGFP)基因以作為標記的siRNA重組腺相關病毒質體，在293細胞中同時轉染此具有EGFP的siRNA重組腺病毒質體，與具有AAV Rep及Cap基因表現的質體，及第三個含腺病毒helper基因的質體，以產生重組病毒，所產出的病毒已經可以成功的感染人類子宮頸上皮細胞HeLa 細胞，使其表現綠色的螢光，將進一步應用於探討凝血酶調節素的功能。

　　Thrombomodulin (TM) is a glycoprotein that was originally identified on vascular endothelium and is well-characterized as a natural endothelial anticoagulant factor. Studies by several investigators have elucidated multiple aspects of TM functions in fibrinolysis, inflammation, proliferation, and in embryogenesis. Functional evaluation of TM in the epithelium cell line HaCaT cells was performed by using RNA interference (RNAi) in this study. RNAi is a sequence-specific post-transcriptional gene silencing mechanism mediated by double-stranded RNA (dsRNA) molecules. It has become a popular research tool in annotating gene function. We used the vector coding a hairpin siRNA against TM gene under the control of mammalian polymerase III U6 promoter. The initial analysis of TM siRNA efficiency was performed in TM negative A2058 cells co-transfected with siRNA and a TM-expressing construct. The best candidate of siRNA that could effectively suppress TM expression was selected and used in the study on the function of HaCaT cells. The endogenous TM level was markedly reduced by siRNA. Protein C activation assay revealed that siRNA diminished TM expression and reduced the TM activity on the cell surface. Moreover, knockdown of TM expression increased the HaCaT cell proliferation and migration, but not in mock cells with control plasmid. These results indicated that siRNA-mediated gene silencing of endogenous TM transcripts has the potential applications for studying TM functions in the epithelium keratinocytes.
　　In another way, we used a commercially adenoassociated viral (AAV) vector for siRNA delivery into mammalian cells. The recombinant AAV vector expresses the enhanced green fluorescent protein (EGFP) under the control of CMV promoter and a U6 promoter-driven hairpin siRNA against TM gene. Recombinant AAV infectious viral particles were produced successfully in 293 cells by cotransfecting three plasmids containing pAAV-EGFP vector genes, adenovirus helper genes, and a vector genome pAAV-RC (replication and capsid). Significantly high level of infection efficiency was achieved by examining the EGFP expression in HeLa cells after infection. We will further apply the infectious virus to deliver siRNA into other TM-expressed cells for studying the function of TM.

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