Abstract
肝素(Heparin)屬於GAGs(glycosaminoglycans)其中之一,其作用常用於心臟脈管手術(cardiopulmonary bypass) 之抗凝劑，以防止血管栓塞,如果肝素使用過量，臨床上則利用硫酸魚精蛋白(protamine sulphate)逆轉肝素的抗凝血作用。目前發現許多種蛋白質或peptide會肝素,為了更進一步探討肝素與peptide的交互作用對二級結構的影響與對凝血機制的作用,我們設計以(i,i+3和i,i+4)的離胺酸(lysine)所間隔的peptide "K34"和與肝素作用並比較硫酸魚精蛋白與肝素作用。 藉由以CD (CircularDichroism)觀察這兩條peptide與肝素作用關係,隨著肝素逐漸加入K34，K34的結構從無序結構(random coil)變成a螺旋結構(a-helix)，與肝素形成的鹽橋(salt-bridge)對於K34的a螺旋結構有重要的影響。就抗凝血實驗而言，K34單獨存在下不會影響外因性凝血路徑(extrinsic pathway)，而且能夠逆轉肝素的抗凝血作用，反觀硫酸魚精蛋白雖然有較強逆轉肝素所造成的抗凝血作用，不過硫酸魚精蛋白單獨存在也會有抗凝血的作用。在HiTrap肝素的實驗中發現K34的作用比硫酸魚精蛋白還要弱，推測在內因性凝血路徑(intrinsic pathway)中看不到K34有逆肝素抗凝血作用，可能是因為K34與肝素的作用力較弱。本實驗看到K34如我們所預期能夠逆轉肝素的抗凝血作用，而且可以避免過量使用之下出現像硫酸魚精蛋白的抗凝血作用，至於逆轉肝素在內因性凝血路徑的作用，則需要再設計與肝素作用較強的peptide。Proliferating cell nuclear antigen (PCNA), an auxiliary factorof DNA polymerase delta, is required in eukaryotic DNAreplication and repair. Previous studies by series deletionhave shown that the rat PCNA promoter possesses the serumresponsiveness and UV inducibility in the region of nucleotidebetween -70 and +125 which contains one AP-1 site and one ATF/CRE site. Furthermore, it was found that the AP-1 mutationpartially loses the serum responsiveness and UV inducibility.In this thesis,we try to characterize the role of AP-1 siteand proximal ATF/CRE site in rat PCNA promoter in the aspectsof serum responsiveness and UV inducibility. Using megaprimingPCR method, we constructed the site-specific ATF/CRE mutatedpromoter and the AP-1/ATF double mutated promoter. In comparisonwith the AP-1 mutant promoter, the ATF/CRE mutant promoterpartially loses the serum responsiveness but represents moreUV inducibility even than the wild type. Furthermore, when theAP-1/ATF double mutated promoter was transfected stably intoCHO.K1 cells, the serum responsiveness and UV inducibilitydecreased remarkably. Results of this thesis suggest that theproximal AP-1 site and ATF/CRE site are both necessary for basalactivity and play crucial roles in serum stimulation of rat PCNApromoter. On the other hand, the ATF/CRE site may be a negativeregulator in UV induction of rat PCNA promoter. It remainsfor further investigation.