臺灣博碩士論文加值系統

根據血清流行病學及病理學的研究，肺炎披衣菌會促進動脈硬化與血栓的形成。正常血管內皮在維持血液恆定 (hemostasis) 扮演重要角色，它提供血管non-thrombogenic surface，使血液流動順暢。而當披衣菌感染內皮細胞，對內皮細胞維持正常功能的能力造成影響時，可能和造成動脈硬化重要病理機轉轉有關。由我們的實驗結果發現，肺炎披衣菌感染人類血管內皮細胞會活化內皮細胞促凝活性，抑制內皮細胞中持續表現的兩個抗凝因子血栓調理素 (thrombomodulin, TM) 及組織因子路徑抑制劑 (tissue factor pathway inhibitor, TFPI) 的表現，使內皮細胞維持血液恆定的能力發生變化。內皮細胞的促凝活性在披衣菌感染後逐步上升，18小時達最高值。細胞溶解液中TM抗原量、細胞表面TM的活性及TFPI量在披衣菌感染後48小時皆有明顯下降。我們同時證明，披衣菌感染進入內皮細胞後所新合成的物質可能是造成血液恆定改變的主因。
血栓調理素 (TM) 是血管內皮細胞表面的醣蛋白，與凝血酶 (thrombin) 結合會活化protein C，使凝血路徑中活化的第五、八因子失去活性，達抗凝固功能。受披衣菌感染的內皮細胞TM抗原量及 mRNA在感染後均下降。TM啟動子活性在披衣菌感染後24小時明顯受到抑制。進一步TM啟動子截短分析後，我們發現TM基因上游-69~-33含PyPu box的區域是TM基因轉錄活性必需序列，同時，也是披衣菌感染造成TM下降的影響序列。以site-directed mutagenesis對PyPu box上的GGAA序列作單點突變分析TM啟動子活性發現，單一點突變使TM啟動子活性下降，而且在披衣菌感染時較不受抑制。由以上結果我們認為，披衣菌感染使內皮細胞促凝活性上升，TM及TFPI表現下降，使內皮細胞血液恆定異常，血管處於傾向血栓形成的環境中，進而促使或加速動脈硬化的發生。

Recent seroepidemiological and pathological evidence suggested that infection of Chlamydia pneumoniae (C pneumoniae) might contribute to the instability of atherosclerotic plaque and thrombosis. Normal endothelium plays an important role in maintaining the homostasis by providing a non-thrombogenic surface. Therefore, infection of Chlamydia in endothelial cell may play the key role in the pathogenesis process. In this study, we demonstrated that the hemostatsis of human umbilical vein endothelial cells was altered by C pneumoniae infection. The induction of procoagulant activity peaked at 18 hours post infection and suppression of tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) expression were clearly observed in C pneumoniae infection at 48 hours post infection. We also demonstrated that the new synthesis components of C pneumoniae during infection were involved in the induction of pro-thrombogenic effect in endothelial cells.
Thrombomodulin (TM) is an endothelial surface glycoprotein that alters the procoagulant activity of thrombin and acts as a cofactor of thrombin-catalyzed activation of protein C. Expression of both TM antigen and TM mRNA in endothelial cells decreased in C pneumoniae infection. The TM promoter, which was highly active in endothelial cells, was significantly inhibited by C pneumoniae. Studies with deletion mutants of TM promoter revealed the -69/-33 region containing PyPu box mediated both specific high basal activity and was responsible for the inhibitory effect of C pneumoniae infection. By site-directed mutagenesis of GGAA sequence in PyPu box, TM promoter showed lower basal activity and was less inhibited under C pneumoniae infection. In conclusion, our data supported C pneumoniae infection not only enhanced procoagulant activity, it may also contribute to local thombo-hemostasis dysfunction by inhibiting antithombolic activity through downregulation of TM promoter activity. These events may contribute to the pathogenesis of atherosclerotic process and its sequels.

目 錄
中文摘要 I
英文摘要 II
致謝 IV
目錄 V
表目錄 VII
圖目錄 VIII
縮寫檢索表 X
儀器及藥品 XI
緒論 1
材料與方法
一、細胞培養方法
(一)人類臍帶靜脈血管內皮細胞(Human Umbilical Vein Endothelial Cells; HUVEC)的培養
(二)人類微血管內皮細胞株( Human Microvascular Endothelial Cells; HMEC-1 )的培養
(三)人類子宮頸癌上皮細胞( Human Cervix Epithelioid Carcinoma; Hela 299 )、人類肝細胞( Hep2 )的培養

二、Chlamydia pneumoniae ( TW-183 ) 的培養12
(一)高力價chlamydia pneumoniae的培養
(二)Chlamydia pneumoniae感染─披衣菌力價測定
(三)Chlamydia inclusion body染色─披衣菌力價測定
(四)Chlamydia pneumoniae感染人類內皮細胞

三、凝血功能分析 17
(一)促凝活性( Procoagulant Activity )分析
(二)血栓調理素( Thrombomodulin )抗原量分析
(三)血栓調理素( Thrombomodulin )活性分析
(四)組織因子路徑抑制劑( Tissue factor pathway inhibitor )量分析
四、披衣菌感染後TM mRNA表現分析21
(一)萃取細胞RNA
(二)DNase處理
(三)反轉錄反應( Reverse Transcription )
(四)聚合酶連鎖反應( Polymerase Chain Reaction)
五、啟動子截短及突變-質體的構築 25
(一)構築啟動子截短及突變質體之聚合酶連鎖反應
(二)純化聚合酶連鎖反應產物
(三)質體的接合( Ligation )及轉形( Transformation ) 反應
(四)小量質體的萃取及限制酶作用
六、暫時性表現共同轉染分析( Transient Expression Cotransfection Assay )30
(一)大量質體的萃取
(二)質體的轉染
(三)報告基因的活性分析
七、EMSA分析35
結果 40
討論 48
參考文獻 56
圖 62
表 87

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林嘉群 人類凝血脢調節素啟動者區域之基因突變對其轉錄功能的影響，成功大學醫學院生物化學研究所碩士論文