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研究生:吳恩綾
研究生(外文):En Ling Wu
論文名稱:20-羥基二十烷四烯酸抑制血管平滑肌細胞移行之機制:蛋白激酶A之角色
論文名稱(外文):Inhibitory Effects of 20-Hydroxyeicosatetraenoic Acid on Vascular Smooth Muscle Cell Migration: Role of Protein Kinase A
指導教授:馬蘊華
指導教授(外文):Y. H. Ma
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
論文頁數:80
中文關鍵詞:20-羥基二十烷四烯酸移行血管平滑肌細胞蛋白激酶A肌動蛋白
外文關鍵詞:20-HETEMigrationVSMCPKAActin
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20-羥基二十烷四烯酸(20-hydroxyeicosatetraenoic acid; 20-HETE)是花生四烯酸(arachidonic acid)經由細胞色素P450代謝後產生之長鏈不飽和脂肪酸,具有調控血管平滑肌功能之角色。本論文在於探討20-HETE對於抑制血管細胞之移行及其相關機制。研究中利用wound-healing與Transwell assay方式分析細胞移行,發現20-HETE可抑制platelet-derived growth factor (PDGF)引起之血管平滑肌細胞移行,但卻不影響人類內皮細胞移行。以20-HETE (10 nM)與血管平滑肌細胞,R22D細胞,共同培養6小時,可使PDGF引起之細胞移行減少30%,而此作用可被adenylyl cyclase抑制劑SQ22536及protein kinase A (PKA)抑制劑H89所完全逆轉;而20-HETE破壞PDGF引起之F-actin重組排列的現象亦可被SQ22536所逆轉。雖然8-bromo cAMP可有效抑制R22D細胞移行,並隨濃度增加其抑制效果愈好,然而20-HETE並不會改變細胞內cAMP之量,且focal adhesion kinase磷酸化情形也未有顯著差異。雖然如此,20-HETE可顯著增加R22D細胞之PKA活性。以上結果顯示20-HETE具有抑制血管平滑肌細胞移行的作用,而cAMP/PKA訊號路徑可能參與20-HETE抑制細胞移行之調控。
20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonate metabolite, is a potent regulator of vascular function. Since VSMC migration is often associated with vascular wall remodeling in pathological status, we further asked whether 20-HETE inhibits migration of R22D cells. With both wound-healing assay and Transwell assay, we demonstrated that 20-HETE significantly attenuated migration induced by platelet-derived growth factor (PDGF) in a concentration-dependent manner in R22D cells. However, serum-induced migration of endothelial cells was not affected by up to 1 M of 20-HETE. Incubation of 20-HETE (10 nM) for 6 hr attenuated PDGF-induced Transwell migration by approximately 30%, which was reversed by SQ22536, an adenylyl cyclase inhibitor, or H89, a protein kinase A (PKA) inhibitor, suggesting involvement of cAMP/PKA pathway in the inhibitory effects of 20-HETE. Incubation of 8-bromo cAMP for 6 hr also attenuated PDGF-induced cell migration in a concentration-dependent manner. However 20-HETE did not alter levels of cAMP or phosphorylated focal adhesion kinase in R22D cells. Nevertheless, 20-HETE significantly enhanced the PKA activity. In addition, pretreatment of 20-HETE partially disrupted F-actin reorganization induced by PDGF, which was reversed by SQ22536. These results suggest that 20-HETE may inhibit PDGF-induced VSMC migration via sensitization of the cAMP/PKA pathway and consequently interfere F-actin reorganization.
目錄(Contents)
指導教授推薦書.....................................
口試委員審定書.....................................
授權書...........................................iii
致謝.............................................iv
中文摘要.........................................v
英文摘要.........................................vi
目錄.............................................vii
目錄.............................................vii
縮寫表............................................x
第一章 緒論(Introduction).....................................................1
1.1 20-HETE之生物活性......................................................2
1.2 細胞移行之角色...............................................................6
1.3 細胞移行之過程...............................................................8
1.4 引起細胞移行之機制......................................................11
1.5 cAMP/PKA影響細胞移行之作用.................................14
1.6 脂肪酸與細胞移行之關係..............................................16
第二章 研究目的(Specific Aims)..........................................19
第三章 材料與方法(Materials and Methods).......................20
3.1 材料……………………………………………………..20
3.2 血管平滑肌細胞培養(VSM Cell Culture)......................21
3.3 人類臍靜脈內皮細胞的收集..........................................21
3.4 內皮細胞培養(Endothelial Cell Culture)........................22
3.5 Wound-healing細胞移行分析
(Wound-healing Cell Migration Assay) ...........................23
3.6 Transwell 細胞移行分析
(Transwell Cell Migration Assay) ....................................24
3.7 環單磷酸腺甘測量(cAMP assay)...................................25
3.8 蛋白激酶活性分析(PKA assay)......................................25
3.9 螢光染色(fluorescence staining) ....................................27
3.10 西方墨漬法(Western blot) ..............................................28
3.11 統計方法..........................................................................28
第四章 實驗結果(Results)....................................................29
4.1 20-HETE抑制PDGF引起之血管平滑細胞於受傷後之移行...........................................................................................29
4.2 20-HETE抑制PDGF引起之血管平滑細胞穿膜移行...30
4.3 TGF-不影響PDGF引起之細胞移行............................31
4.4 抑制cAMP/PKA反轉20-HETE抑制細胞移行之作用31
4.5 20-HETE不影響細胞內cAMP之量...............................32
4.6 20-HETE增加PKA活性.................................................33
4.7 20-HETE不影響PKA催化子單位之活性....................34
4.8 20-HETE干擾PDGF引起之F-actin重新排列..............34
4.9 20-HETE不影響FAK之磷酸化.....................................35
第五章 討論(Discussion).......................................................37
第六章 結論(Conclusion)......................................................45參考文獻(Reference)................................................................46 Figure Legends and Figures......................................................57
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