謝學真臺灣大學：化學工程學研究所謝宗佑Hsieh, Chung-YuChung-YuHsieh2007-11-262018-06-282007-11-262018-06-282007http://ntur.lib.ntu.edu.tw//handle/246246/52187During atherogenesis, inflammation and coagulation play an important role. Thrombomodulin (TM), in vascular endothelial cells, has anti-coagulation and anti-inflammation properties, it can also regulate cell migration and angiogenesis. Transcription factor, KLF2, has been shown to participate in the regulation of the expression of eNOS, TM and other anti-inflammation genes that can regulate the physiological function of endothelial cells (ECs). ECs are constantly under the influence of flow-induced shear stress. Therefore, shear stress is an important regulator of EC functions. In this study, we focused on the signaling pathway of TM and KLF2 under the shear stress stimulation and the regulatory mechanism of TM protein stability. Shear stress elevated TM promoter activity in ECs after 6 hours under both high (25 dyn/cm2) and low (5 dyn/cm2) shear stress conditions, but high shear had more eminent induction (12 folds vs. unsheared control) than low shear (6 folds vs. control). The mRNA level of TM was increased more than 2 folds in comparison with unsheared control and so did the mRNA level of KLF2 (more than 100-fold increase). As for TM protein level, there was a significant difference between high shear (60% increase) and low shear (23% increase) conditions, but no difference in KLF2 protein level was observed under different shear conditions. These results suggest that shear stress increases the protein stability of TM and the effect is proportional to the magnitude of shear stress. We further explored the regulatory mechanism of TM stability. To simulate the condition of shear-induced NO release, ECs were treated with NOC18 (an NO donor), and we found that TM protein level increased 0.8 folds after 3 hours but no significant increase was observed after 5 hours. It is likely that NO may not be a major factor that regulates TM stability or there are too many down-stream signal molecules activated by NO to observe significant effect of NO on TM. Subsequently, ECs were treated with NAC to mimic the effect of antioxidants which were present in ECs exposed to high shear stress. The results showed that the TM protein level was almost totally down-regulated after 5 hours of shear treatment, but the soluble form of TM in the medium was increased. It suggests that NAC (or other antioxidants) may activate specific protease(s) to cleave TM into the solube form that can promote cell migration or angiogenesis. To explore the effect of phosphorylation/dephosphorylation on the stability of TM, tyrosine kinase inhibitor (PP2) and PTP inhibitor (Na3VO4) were used. We found that TM protein level was increased about 2.2 folds in the presence of PP2, and decreased to 0.2 fold in the presence of Na3VO4 in ECs stimulated by VEGF for 5 hours. Based on these findings, we proposed that the phosphorylation of C-terminal tyrosine residue (Y534) of TM may regulate the TM protein stability. In order to verify this assumption, Y534D (superactive) and Y534A (dominant negative) TM mutants were transfected into HUVECs. We found that Y534D was more unstable in the presence of Na3VO4 but Y534A was not affected by PP2 or Na3VO4. Therefore the phosphorylation of Y534 may destabilize TM protein. Among various PTPs, we found that PTEN was involved in the regulatory mechanism of TM. PTEN silencing down-regulated TM protein level but had no influence on Y534A mutant, and this mechanism was found to be independent of PI3K/Akt pathway. In summary, our data suggest that both high and low shear stresses up-regulate the TM promoter activity and KLF2 mRNA, but only high shear elevates the level of TM mRNA. Besides, shear stress dose-dependently increases the TM protein stability. Further studies indicate that NO may not play a significant role in the regulatory mechanism. As for NAC, it can increase the soluble form of TM, and this phenomenon is due to the cleavage of TM by some unknown protease(s). The key point in the regulation of TM stability seems to be the phosphorylation of Y534. Our results suggest that the phosphorylation of Y534, may be mediated by Src kinase family, decreases the stability of TM, but the activation of PTP such as PTEN enhances the TM protein stability.目錄 誌謝 I 中文摘要 III Abstract V 目錄 VII 圖目錄 XI 表目錄 XV 縮寫與符號說明 XVII 中英名詞對照 XXI 1. 緒論 1 1.1. 動脈粥狀硬化（Atherosclerosis） 1 1.2. 研究動機與目的 7 2. 文獻回顧 9 2.1. 血管內皮細胞與剪力 9 2.1.1. 血管內皮細胞 9 2.1.2. 剪力對血管內皮細胞之影響 13 2.1.3. 內皮細胞對於剪力之感測器 18 2.2. 凝血調節酶（Thrombomodulin, TM） 19 2.2.1. TM之結構與生理功能 19 2.2.2. TM對發炎機制之調控 25 2.2.3. TM對凝血機制之調控 26 2.2.4. TM對細胞附著之調控 28 2.2.5. TM對血管新生及細胞遷移之調控 29 2.2.6. TM表現量之調控 31 2.3. PTEN（Phosphatase and tensin homolog deleted on chromosome 10） 33 2.3.1. PTEN之結構 33 2.3.2. PTEN在生理功能上扮演的角色 36 2.4. Kruppel Like Factor 2(KLF2)之結構與生理功能 42 2.5. 一氧化氮（NO）對於內皮細胞之調控 45 3. 實驗藥品、儀器及方法 49 3.1. 實驗材料 49 3.1.1. 細胞培養及流動實驗所用材料 49 3.1.2. 實驗耗材 51 3.1.3. 細胞轉染所使用之材料 51 3.1.4. 西方墨點轉印法所用之材料 54 3.1.5. 同步定量聚合酶連鎖反應（Real-time quantitative PCR） 55 3.1.6. 啟動子活性測定法（Promoter Activity Assay） 56 3.2. 實驗儀器 57 3.3. 實驗原理與方法 59 3.3.1. 初級人類臍帶靜脈內細胞培養 59 3.3.2. 人類臍帶靜脈內皮細胞繼代培養於玻片 60 3.3.3. 人類臍帶靜脈內皮細胞繼代培養於培養皿 60 3.3.4. 牛動脈內皮細胞繼代陪養 61 3.3.5. 流動室之設計 61 3.3.6. 流動實驗之設計與流程 66 3.3.7. 全細胞之蛋白質（total cell lysate）的抽取 68 3.3.8. 蛋白質含量測定 68 3.3.9. 細胞內特定蛋白質含量測定Western Blot 69 3.3.10. 細胞內全RNA（total RNA）的收取 70 3.3.11. 細胞內特定mRNA含量測定：Real-Time quantitiative PCR 70 3.3.12. 以電穿孔（Electroporation）方式進行細胞轉染 72 3.3.13. 以Lipofectamine2000方式進行細胞轉染 73 3.3.14. 啟動子活性測定 73 3.3.15. β-Galactosidase assay..................................................................... 74 3.3.16. Luciferase assay.............................................................................. 74 4. 結果與討論............................................................................................................ 75 4.1. 不同剪力大小對於TM 及KLF2 之影響..................................................... 75 4.1.1. 不同剪力大小對TM 啟動子活性之調控.................................... 75 4.1.2. 不同剪力大小對TM 及KLF2 mRNA 之影響............................ 78 4.1.3. 不同剪力大小對TM 及KLF2 蛋白質之影響............................. 81 4.2. TM 蛋白質穩定性之量測.............................................................................. 84 4.3. 一氧化氮（NO）對於TM 穩定性之影響.................................................. 86 4.4. 細胞內氧化還原態對於TM 穩定性之影響................................................ 90 4.5. Src kinase family 與PTP 對TM 穩定性之調控........................................... 95 4.5.1. Src kinase family 對TM 穩定性之調控...................................... 95 4.5.2. 蛋白質酪胺酸去磷酸酶（PTP）對TM 穩定性之調控............. 98 4.6. TM 蛋白質穩定性受Y534 調控................................................................. 101 4.7. PTEN 對於調控內皮細胞TM 穩定性之影響............................................ 105 4.8. PI3K/Akt 路徑對TM 穩定性之影響........................................................... 109 4.9. Y534 決定TM 之蛋白質穩定性..................................................................111 4.10. 綜合討論.....................................................................................................114 5. 結論.......................................................................................................................119 5.1. 結論...............................................................................................................119 5.2. 未來研究方向.............................................................................................. 122 參考文獻...................................................................................................................... 1233636400 bytesapplication/pdfen-US剪力凝血酶調節素蛋白質穩定性去磷酸酶內皮細胞shear stressthrombomodulinprotein stabilityphosphataseendothelial cellsthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/52187/1/ntu-96-R94524008-1.pdf