DC 欄位 | 值 | 語言 |
dc.contributor | 謝學真 | zh-TW |
dc.contributor | Hsieh, Hsyeu-Jen | en |
dc.contributor | 臺灣大學:化學工程學研究所 | zh-TW |
dc.contributor.author | 劉芳亘 | zh-TW |
dc.contributor.author | Liu, Fang-Hsuan | en |
dc.creator | 劉芳亘 | zh-TW |
dc.creator | Liu, Fang-Hsuan | en |
dc.date | 2009 | en |
dc.date.accessioned | 2010-06-30T06:29:41Z | - |
dc.date.accessioned | 2018-06-28T17:17:13Z | - |
dc.date.available | 2010-06-30T06:29:41Z | - |
dc.date.available | 2018-06-28T17:17:13Z | - |
dc.date.issued | 2009 | - |
dc.identifier.other | U0001-1108200914013900 | en |
dc.identifier.uri | http://ntur.lib.ntu.edu.tw//handle/246246/187011 | - |
dc.description.abstract | 粥狀動脈硬化(atherosclerosis)為一種慢性的發炎疾病,發炎反應(inflammation)與凝血反應在致病過程中扮演極重要的角色。凝血?調節素(thrombomodulin, TM)為血管內皮細胞中高度醣基化的穿膜蛋白,其主要生理功能為抗凝血、抗發炎反應。而在目前的文獻中亦顯示,凝血?調節素與血管新生、細胞增生與遷移有關。血管中血液脈動產生的壓力變化,對內皮細胞造成拉伸應力的刺激,進而調控內皮細胞的生理功能。本研究使用牛大主動脈內皮細胞(BAECs),探討經拉伸應力誘導之TM表現量上升對內皮細胞遷移的影響;另外也對TM蛋白質穩定性之調控機制進行研究。 實驗將以C端標記flag之TM轉染至BAECs,分別在含低血清(2% FBS)及含高血清(10% FBS)細胞培養液的培養條件下進行7小時的刮傷癒合實驗,觀察TM在細胞內的表現量對刮傷癒合效率的影響。實驗結果發現,在含低血清培養液之培養條件下,相較於轉染empty vector的細胞,轉染TM-flag之細胞其刮傷癒合的程度增加約10%;然而在含高血清培養液培養之下的細胞,兩者之刮傷癒合效果並無顯著差異。類似的實驗,也在BAECs中以TM knock-down的方法進行,在含低血清培養液的培養條件下,經TM knock-down的細胞與控制組相比,7小時的刮傷癒合現象沒有顯著的差異;但是在含高血清之培養條件下,TM knock-down組經過7小時刮傷癒合之後,其癒合程度與控制組相比下降約10%。 內皮細胞經過7小時的拉伸應力(stretch) (16% strain)之後,細胞內TM的蛋白質表現量相較於控制組有2.5~3.5倍上升;但是細胞遷移的現象,無論細胞層的刮痕與應力方向垂直或平行,刮傷癒合的現象比控制組皆下降約20%。然而相同的stretch條件之下,經過15個小時後,細胞內TM蛋白質的表現量卻下降為控制組之0.7倍。表示stretch所刺激的TM表現,並非細胞遷移現象的主要參與者。 在TM穩定性的實驗探討中,以TNF-a處理BAECs經過6小時後,細胞內TM蛋白質的表現量相較於控制組下降為0.6倍。轉染以C端標記flag之TM以及胞內區域有突變之TM-Y/A、TM-T/A、TM-S/A至BAECs並經6小時TNF-a處理之後,發現TM-Y/A以及TM-S/A不受TNF-a影響,表示TM胞內區域之Tyr、Ser去磷酸化現象為維持其蛋白質穩定之關鍵。 MG132預處理內皮細胞1小時再以TNF-a處理6小時後,有效地降低TM受TNF-a誘導之降解現象;但經過anti-ubiquitin抗體進行免疫沉澱的實驗結果,卻沒有明顯的泛素化現象,因此,TNF-a對TM蛋白質穩定性的影響可能並非主要透過泛素化的途徑。 合上述實驗結果,本研究認為,細胞內TM的表現量上升具有促進細胞刮傷癒合的效果,但stretch對TM的調控可能在於對其穩定性的影響,因此無法彰顯促進細胞刮傷癒合的能力。而在TM穩定性的方面,TNF-a會降低TM蛋白質的穩定性使其發生降解,但其胞內區域之Tyr以及Ser突變可導致TM不受TNF-a誘導而被降解,表示Tyr與Ser的去磷酸化對TM的穩定性有調控的作用;而TM受TNF-a誘導之降解過程,泛素化的現象並非主要的調控機制。 | zh-TW |
dc.description.abstract | Atherosclerosis is a chronic inflammatory disease. Inflammation and coagulation play an important role in this disease. Thrombomodulin (TM) is a highly glycosylated transmembrane protein in vascular endothelial cells, and has anti-inflammation and anti-coagulation properties. In recent study, it can also regulate angiogenesis, cell proliferation and migration. Vascular endothelial cells (ECs) are exposed to stretch generated by the blood pressure. Therefore, stretch is an important factor in regulation of EC functions. In this study, bovine aortic endothelial cells (BAECs) were utilized to investigate the influence of stretch-induced TM expression on cell migration and the regulatory mechanism of TNF-a-mediated TM protein stabilityo investigate the effect of TM expression in endothelial cell wound healing ability, BAECs were transfected with TM plasmid containing C-terminal FLAG tag (TM-flag) and the wound healing assay was carried out for 7 hours with low serum (2% FBS) or high serum (10% FBS)-containing culture medium. The results showed that, cultured with low serum-containing culture medium, the degree of healing of BAECs transfected with TM-flag was facilitated about 10% compared with that transfected with empty vector. But when cells cultured with high serum-containing culture medium, there was no significant difference between cells transfected with empty-vector and TM-flag.e also used TM knock-down experiment to investigate its effect on the wound healing. Under low serum culture condition, there was no significant difference in wound healing between TM knock-down cells and scramble control. But when cells cultured with high serum-containing culture medium, the degree of healing of TM knock-down cells was about 10% less than scramble control in a 7-hr wound healing assay.hen endothelial cells were exposed to stretch with 16% strain for 7 hours, TM protein expression was up-regulated by 2.5 to 3.5-fold compared with static control. But weather the scar was perpendicular or parallel to the stretch direction, wound healing ability of BAECs after stretch stimulation for 7 hours was about 20% less than the static control. However, when the same stretch force stimulated cells for 15 hours, TM protein expression was suppressed to about 0.7-fold of static control. The results suggested that stretch-induced TM expression did not substantially regulate endothelial cell migration.o study TM protein stability, we added 10 ng/ml TNF-a to cultured BAECs for 6 hours. The results showed that the TM protein expression was down-regulated to 0.6-fold in comparison with control. When BAECs were transfected with TM-flag, TM-Y/A, TM-T/A or TM-S/A mutants and treated TNF-a for 6 hours, the results suggested that the de-phosphorylation of C-terminal Tyr (Y534) and Ser (S550) residues of TM may be related to TM stability and protect it against TNF-a-induced TM down-regulation. To invesgate the mechanism of TNF-a-induced TM down-regulation, we pre-treared BAECs with 10 uM MG132 for 1 hour followed by 6 hours TNF-a treatment. We found that MG132 protected TM from TNF-a-induced down-regulation, but in the immumoprecipitation experiment, no significant ubiquitination occurred suggesting that the down-regulation of TM by TNF-a is not mainly through ubiquitinaiton pathway.n summary, increased TM expression facilitated endothelial cell wound healing ability. Stretch-induced TM expression was not involved in cell migration. In TM protein stability investigation, its stability was down-regulated by TNF-a. The mutant of C-terminal Tyr (Y534) and Ser (S550) residues of TM increased its stability and protected it from TNF-a-induced down-regulation. | en |
dc.description.tableofcontents | 誌謝.......................................................I文摘要.................................................IIIbstract...................................................V錄.....................................................VII目錄....................................................XI目錄..................................................XIII寫與符號說明............................................XV英名詞對照.............................................XIX. 緒論.............................................1.1. 動脈粥狀硬化(Atherosclerosis)........................1.2. 研究動機與目的.......................................9. 文獻回顧........................................11.1. 血管內皮細胞與拉伸應力 ..............................11.1.1. 血管內皮細胞....................................11.1.2. 拉伸應力對於內皮細胞的影響......................15.2. 凝血酶調節素(Thrombomodulin, TM)之結構與生理功能.......................................................19.3. 凝血酶調節素(Thrombomodulin, TM)之調控機制........26.3.1. TM對凝血機制之調控..............................26.3.2. TM對發炎機制之調控..............................29.3.3. TM對細胞附著之調控..............................31.3.4. TM對細胞增生、血管新生及細胞遷移之調控..........32.3.5. TM表現量之調控..................................34.4. 內皮細胞遷移(Migration).............................36.5. 泛素化..............................................38. 實驗藥品、儀器及方法............................41.1. 實驗材料............................................41.1.1. 細胞培養所用材料................................41.1.2. 實驗耗材........................................43.1.3. 細胞轉染所使用之材料............................44.1.4. 西方墨點轉印法所用之材料........................45.1.5. 收取細胞內總RNA(total RNA)所使用之材料..........46.1.6. 免疫螢光染色法所用之材料........................46.2. 實驗儀器............................................47.3. 實驗原理與方法......................................49.3.1. 牛主動脈內皮細胞培養............................49.3.2. Eahy926細胞培養.................................50.3.3. 內皮細胞繼代培養於PDMS..........................51.3.4. 細胞刮傷癒合分析(Wound Healing Assay)...........52.3.5. 細胞增生實驗(WST-1 Proliferation Assay).........52.3.6. 細胞拉伸實驗之設計..............................53.3.7. 總溶胞產物(total cell lysate)之蛋白質的抽取.....55.3.8. 蛋白質含量測定..................................55.3.9. 免疫沉澱法(Immunoprecipitation).................56.3.10. 細胞內特定蛋白質含量測定(Western Blot)..........57.3.11. 免疫螢光染色法(Immunofluorescence)..............58. 結果與討論......................................59.1. TM表現量對刮傷癒合(Wound Healing)的影響.............59.1.1. 細胞轉染TM之影響................................59.1.2. TM靜默(Konck-Down)之影響........................65.2. 拉伸應力(Stretch)對內皮細胞之影響...................70.2.1. 拉伸應力對細胞癒合之影響........................70.2.2. 拉伸應力在刮傷癒合實驗中對TM表現量的影響........76.3. 細胞遷移中TM受拉伸應力影響之位置分布................80.3.1. TM與actin之共位(Colocalization).................80.3.2. 細胞遷移過程TM與actin分布變化...................82.3.3. 細胞遷移過程受Stretch影響之TM與actin分布變化....84.4. Cytokine對TM磷酸化及穩定性之調控....................88.4.1. Cytokine對TM穩定性之影響........................88.4.2. TM不同位置之胺基酸殘基去磷酸化受Cytokine之影響.......................................................90.4.3. Cytokine調控TM降解可能之機制....................92.5. 綜合討論............................................96. 結論...........................................105.1. 結論...............................................105.2. 未來研究方向.......................................108考文獻................................................109 | en |
dc.format.extent | 11382303 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 | 細胞遷移 | zh-TW |
dc.subject | 細胞激素 | zh-TW |
dc.subject | thrombomodulin | en |
dc.subject | stretch | en |
dc.subject | migration | en |
dc.subject | TNF-{alpha} | en |
dc.title | 拉伸應力誘發之凝血酶調節素(TM)對內皮細胞遷移之影響以及細胞激素對TM穩定性之調控 | zh-TW |
dc.title | Influence of Stretch-induced Thrombomodulin (TM) Expression on Endothelial Cell Migration and Regulation of TM Stability by Cytokine | en |
dc.type | thesis | en |
dc.identifier.uri.fulltext | http://ntur.lib.ntu.edu.tw/bitstream/246246/187011/1/ntu-98-R96524021-1.pdf | - |
item.openairecristype | http://purl.org/coar/resource_type/c_46ec | - |
item.openairetype | thesis | - |
item.languageiso639-1 | en_US | - |
item.grantfulltext | open | - |
item.cerifentitytype | Publications | - |
item.fulltext | with fulltext | - |
顯示於: | 化學工程學系
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