https://scholars.lib.ntu.edu.tw/handle/123456789/63168
DC 欄位 | 值 | 語言 |
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dc.contributor | 謝學真 | zh-TW |
dc.contributor | Hsieh, Hsyue-Jen | en |
dc.contributor | 臺灣大學:化學工程學研究所 | zh-TW |
dc.contributor.author | 張弘勳 | zh-TW |
dc.contributor.author | Chang, Hung-Hsiun | en |
dc.creator | 張弘勳 | zh-TW |
dc.creator | Chang, Hung-Hsiun | en |
dc.date | 2008 | en |
dc.date.accessioned | 2010-06-30T06:09:37Z | - |
dc.date.accessioned | 2018-06-28T17:11:57Z | - |
dc.date.available | 2010-06-30T06:09:37Z | - |
dc.date.available | 2018-06-28T17:11:57Z | - |
dc.date.issued | 2008 | - |
dc.identifier.other | U0001-2907200821442400 | en |
dc.identifier.uri | http://ntur.lib.ntu.edu.tw//handle/246246/186910 | - |
dc.description.abstract | 動脈粥狀硬化致病過程中,發炎反應(inflammation)與凝血反應扮演十分重要的角色。而在血管內皮細胞中的凝血酶調節素(thrombomodulin;簡稱TM)具有抗凝血及抗發炎的功能,近年來亦被發現具有調控細胞遷移與內皮細胞血管新生的效果。由於血管中血液脈動產生的壓力變化會對內皮細胞產生拉伸應力,進而調控內皮細胞的生理功能。本研究使用人類臍帶靜脈內皮細胞(HUVECs)與牛大主動脈內皮細胞(BAECs),探討拉伸應力刺激誘發細胞中TM的訊息傳遞路徑;另外也對TM蛋白質穩定性之調控機制進行研究。內皮細胞受到靜態拉伸應力刺激時,TM的mRNA量有顯著增加,在一小時內增加到控制組的3.2倍,但在六小時後又降至與控制組無異。顯示在拉伸應力的作用下,TM mRNA的轉錄效率在一小時內很快的上昇。但此效果屬於暫態誘發 (transient induction),並無法持續地刺激TM mRNA的轉錄。而在蛋白質層面上,TM蛋白質量亦因為受到拉伸應力的作用而增加達2.2倍以上,且可以至少持續六小時。另外在對細胞做螢光染色後,發現拉伸應力可以促使TM與細胞骨架actin有共位(colocolization),使TM在細胞中呈現類似stress fiber的特殊分佈。於TM蛋白質穩定性的探討,實驗使用短效型及長效型NO donor(分別為SNAP及NOC18)模擬eNOS受拉伸應力活化而釋放NO的效果,並且轉染在C端帶有FLAG作為標記的TM質體觀察TM的降解情形。結果發現NO在短時間內可以抑制TM蛋白質的降解,而在長時間下NO對TM穩定性的影響不大。著探討TM胞內區域磷酸化/去磷酸化對TM蛋白質穩定性的影響,實驗以VEGF刺激細胞內的蛋白激酶及以protein tyrosine phosphatase (PTP) inhibitor(Na3VO4),發現抑制PTP會增加TM的降解而VEGF則沒有明顯的作用,為了證實Y534位置的磷酸化影響TM的穩定性,本研究利用無法磷酸化的Y534A TM變異種來進行實驗。而當胞內區域的Y534位置突變成alanine則幾乎不產生TM的降解片段。顯示TM胞內區域Y534位置經常地受到去磷酸酶的作用,一但抑制了去磷酸酶TM立即遭到降解,而此降解的機制可能是經由泛素化的標記,進而被proteasome降解,實驗顯示TM胞內區域的threonine550以及tyrosine534位置若被磷酸化會造成泛素化的機會增加。探討PTP對TM的作用方式之中,以免疫沉澱法沉澱出與TM有association的蛋白質,發現其中有PTEN的存在,顯示TM 於一般狀態下即會與PTEN有association,但此作用並不受細胞內的NO與ROS影響。amp;#32508;合以上的實驗結果歸納拉伸應力對TM的調控以及影響TM穩定性的可能機制。當內皮細胞受到拉伸應力作用時,在短時間內會增加TM mRNA的表現,並增加TM蛋白質的穩定性,增加細胞內一氧化氮量可以抑制TM的降解。當PTEN對TM胞內區域進行去磷酸化會增加TM穩定性,使其不易受到降解,反之,當胞內區域的的Y534位置磷酸化增加將會受到泛素化,最終被proteasome降解。另外當細胞受到拉伸應力的作用將會增加TM與actin的共位(colocalization),使TM在細胞中呈現類似stress fiber的特殊分布情形。 | zh-TW |
dc.description.abstract | During 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. Vascular Endothelial cells (ECs) are exposed to stretch caused by the blood pressure. Therefore, stretch is an important regulator of EC functions. In this study, human umbilical vein endothelial cells (HUVECs) and bovine aortic endothelial cells (BAECs) were utilized to investigate the signaling pathway of stretch-induced TM and the regulatory mechanism of TM protein stability.hen ECs were exposed to stretch (16% strain) for 1 hour, the mRNA level of TM was increased more than 3.2 folds in comparison with static control, but decreased to the same level as that of static control after 6 hours. These results suggest that stretch transiently induces the mRNA level of TM. As for TM protein level, stretch also induced the protein level of TM up to 2.2 folds, and this effect sustained at least for 6 hours. On the other hand, Double immunofluorescence staining for TM and actin showed that the colocalization between TM and actin was induced by stretch, which led to the special distribution of TM in ECs. We further transfected ECs with TM plasmid containing C-terminal FLAG tag to explore the regulatory mechanism of TM stability. To simulate the condition of stretch-induced nitric oxide (NO) release, ECs were treated with short term and long term NO donor (SNAP and NOC18, respectively), and we found that only short term NO donor protected TM protein from degradation.. To explore the effect of phosphorylation/dephosphorylation on the stability of TM, VEGF and protein tyrosine phosphatase (PTP) inhibitor (Na3VO4) were used. We found that inhibition of PTP activity by Na3VO4 increased the degradation of TM, whereas the use of VEGF to stimulate kinase activity had no effect on the TM stability. These results suggest that the phosphorylation of C-terminal tyrosine residue (Y534) of TM may regulate the TM protein stability. To verify this assumption, Y534A mutant of TM plasmid was transfected into BAECs. We found that in cells transfected with Y534A there was no degradation fragment of TM. Therefore, it is likely that the C-terminal Y534 residue of TM is constantly dephosphorylated by PTP and the dephosphorylation of Y534 residue may stabilize TM protein. Futhermore, our data showed that ubiquitination may play a role in the degradation of TM.Then we used immunoprecipitation to find out that PTEN was associated with TM under normal condition. NO and H2O2 had slight effect on the association between TM and PTEN.n summary, our data suggest that stretch transiently up-regulates the mRNA level of TM. Stretch also upregulates the protein level of TM, and the effect sustains at least for 6 hours. Besides, stretch increases the colocalization between TM and actin and leads to the special distribution of TM in ECs. Further studies indicate that NO may protect TM from degradation. PTEN may dephosphorylate TM Y534 residue and thus stabilize TM protein. On the contrary, the increase in Y534 phosphorylation may lead to the degradation of TM through ubiquitin proteasome system. | en |
dc.description.tableofcontents | 誌謝 I文摘要 IIIbstract V錄 VII目錄 IX目錄 XI寫與符號說明 XIII英名詞對照 XVII. 緒論 1.1. 動脈粥狀硬化(Atherosclerosis) 1.2. 研究動機與目的 7. 文獻回顧 9.1. 血管內皮細胞與拉伸應力 9.1.1. 血管內皮細胞 9.1.2. 拉伸應力對於內皮細胞的影響 13.2. 凝血酶調節素(Thrombomodulin, TM) 15.2.1. TM之結構與生理功能 15.2.2. TM對發炎機制與凝血機制之調控 20.2.3. TM對細胞附著、血管新生及細胞遷移之調控 23.2.4. TM表現量之調控 25.3. PTEN(Phosphatase and tensin homolog deleted on chromosome 10) 28.3.1. PTEN之結構 28.3.2. PTEN在生理功能上扮演的角色 31.4. 一氧化氮(NO)對於內皮細胞之調控 36.5. 泛素化 41. 實驗藥品、儀器及方法 43.1. 實驗材料 43.1.1. 細胞培養所用材料 43.1.2. 實驗耗材 45.1.3. 細胞轉染所使用之材料 45.1.4. 西方墨點轉印法所用之材料 47.1.5. 免疫螢光染色法所用之材料 48.1.6. 同步定量聚合酶連鎖反應(Real-time Quantitative PCR) 48.2. 實驗儀器 49.3. 實驗原理與方法 51.3.1. 初級人類臍帶靜脈內細胞培養 51.3.2. 人類臍帶靜脈內皮細胞繼代培養於PDMS 52.3.3. 人類臍帶靜脈內皮細胞繼代培養於培養皿 52.3.4. 牛動脈內皮細胞繼代培養 53.3.5. 細胞拉伸實驗之設計 54.3.6. 全溶胞產物(total cell lysate)之蛋白質的抽取 56.3.7. 蛋白質含量測定 56.3.8. 免疫沉澱法(Immunoprecipitation) 57.3.9. 細胞內特定蛋白質含量測定(Western Blot) 57.3.10. 細胞內全RNA(total RNA)的收取 59.3.11. 細胞內特定mRNA含量測定:Real-Time quantitiative PCR 59.3.12. 免疫螢光染色法(Immunofluorescence) 61. 結果與討論 63.1. 拉伸應力對於TM之影響 63.1.1. 拉伸應力對TM mRNA之影響 63.1.2. 拉伸應力對TM 蛋白質之影響 65.1.3. 拉伸應力對TM 蛋白質分佈之影響 68.2. 一氧化氮(NO)對於TM穩定性之影響 70.3. 蛋白質酪胺酸去磷酸酶(PTP)對TM穩定性之調控 74.4. 泛素化對於TM穩定性之影響 77.5. PTEN直接作用於TM之胞內區域 79.6. 綜合討論 82. 結論 87.1. 結論 87.2. 未來研究方向 89考文獻 91 | en |
dc.format.extent | 4722239 bytes | - |
dc.format.mimetype | application/pdf | - |
dc.language | zh-TW | en |
dc.language.iso | en_US | - |
dc.subject | 凝血酶調節素 | zh-TW |
dc.subject | 拉伸應力 | zh-TW |
dc.subject | endothelial cell | en |
dc.subject | thrombomodulin | en |
dc.subject | stretch | en |
dc.title | 拉伸應力對內皮細胞中凝血酶調節素之調控 | zh-TW |
dc.title | Regulation of Thrombomodulin in Endothelial Cells by Stretch | en |
dc.type | thesis | en |
dc.identifier.uri.fulltext | http://ntur.lib.ntu.edu.tw/bitstream/246246/186910/1/ntu-97-R95524075-1.pdf | - |
item.fulltext | with fulltext | - |
item.cerifentitytype | Publications | - |
item.openairetype | thesis | - |
item.languageiso639-1 | en_US | - |
item.openairecristype | http://purl.org/coar/resource_type/c_46ec | - |
item.grantfulltext | open | - |
顯示於: | 化學工程學系 |
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ntu-97-R95524075-1.pdf | 23.53 kB | Adobe PDF | 檢視/開啟 |
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