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研究生:陳姿吟
研究生(外文):Chih-Ying Chen
論文名稱:利用表現內皮素A型及B型受體細胞以比較受體特性並用於鑑定調節血壓的中草藥
論文名稱(外文):Characterization of ETAR/ETBR and identification of blood pressure modulating herbal medicines by the ETAR/ETBR expressing cells
指導教授:歐樂君
指導教授(外文):Lo-Chun Au
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:65
中文關鍵詞:內皮素受體中草藥血壓
外文關鍵詞:endothelin receptorChinese herbal medicineblood pressure
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內皮素-1 (endothelin-1,ET-1)為一強力血管收縮物質。在高血壓之動物模式已證實其血液中ET-1濃度有上升的現象,臨床研究也指出患有高血壓之病患,其血液中ET-1濃度較正常人高。ET-1必須先與細胞表面的內皮素受體結合才能產生多種生物功能,內皮素受體主要有兩種:A型(ETAR)及B型(ETBR),ETAR主要分部於血管平滑肌細胞,受刺激時會造成血管收縮導致血壓上升;ETBR主要存在於內皮細胞,受刺激時會產生一氧化氮及前列腺素,使血管放鬆血壓下降。由於某些疾病(如高血壓、心血管疾病、敗血症、惡性腫瘤等)發生及惡化與血液中ET-1濃度升高有關,因此臨床上認為內皮素受體拮抗劑可作為高血壓等疾病之治療藥物。中草藥P1於本草綱目記載有降血壓功效,而我們實驗室之前與阮琪昌博士合作,發現P1含rETAR之拮抗劑,若給予高果糖飲食所引起高血壓之大鼠飲用P1水萃液,可降低大鼠之血壓,故在此想探討P1可否作為人類ETAR之拮抗劑。我們利用外送可表現重組質體到CHO-K1細胞以分別表現hETAR及hETBR,並選殖穩定之轉殖株(稱為CHO-ETAR及CHO-ETBR),利用受體結合實驗可得到ETAR之Kd為1533pM,Bmax為233.78 fmol/2×105 cells;ETBR之Kd為482pM,Bmax為122.93 fmol/2×105 cells,顯示ET-1對hETBR之親合力大於hETAR。而ETAR拮抗劑(BQ610)以及ETBR拮抗劑(BQ788)可分別抑制ET-1和ETAR及ETBR結合。以ET-1刺激可使CHO-ETAR細胞內鈣離子濃度上升,並使CHO-ETBR細胞一氧化氮合成酶磷酸化,得知表現於CHO-K1細胞上之內皮素受體是具有功能的。以這兩株細胞為材料,發現P1可直接作用於受體上並抑制ET-1對內皮素受體之結合,P1對ETAR有67%之抑制效果,對ETBR有90%之抑制效果,且可連帶影響由ET-1刺激所引起之CHO-ETAR細胞內鈣離子濃度上升。經由不同比例酒精沉澱濃縮P1中之有效成分,發現F3.3.5x(在3.5倍酒精中不沉澱)對ETAR抑制效果較佳(抑制83%),但對ETBR效果較弱(抑制37%);而F2 2x(在2倍酒精中沉澱)對ETBR抑制效果較佳(抑制92%),但對ETAR效果較弱(抑制48%),由此可知P1中含有兩種不同之有效成分。以人類平滑肌及內皮細胞做實驗,P1及F3 3.5x分別對ETAR有67%及>96%之抑制效果;而P1及F2 2x對ETBR有79%及87%之抑制效果。另外我們發現P1可以抑制由LPS刺激內皮細胞所引起之一氧化氮合成酶磷酸化現象。
Endothelin-1 (ET-1) is a powerful vasoconstrictor that contributes to blood pressure elevation. In different animal models and patients with hypertension, ET-1 is overexpressed in the plasma. The biological effects of ETs are mediated by two receptors:endothelin receptor type A (ETAR) and type B (ETBR). In the vasculature, ETAR is expressed predominantly on the cell membrane of vascular smooth muscle cells and mediates vasoconstrictor effects of ET-1. ETBR is expressed on endothelial cells. Its activation by ET-1 results in vasodilatation through generation of prostacyclin and nitric oxide (NO). Elevation of ET-1 level was found in patients of hypertension, cardiovascular disease, sepsis, and tumor. The endothelin receptor antagonist might be useful for therapeutic purpose. According to the codex P1, a Chinese herbal medicine, has been reported to treat hypertension and reduce the blood pressure. In the previous study, our lab and Dr. Juan found that P1 is rETAR antagonist and reduces the blood pressure in high fructose diet-induced hypertension in rats, and therefore I investigated whether P1 can act as a human ETAR antagonist too. cDNAs of hETAR and hETBR were cloned and inserted into pIRESneo vector. CHO-K1 cells were transfected with the recombinant vectors. The stable clones of CHO-K1 expressing the hETAR or hETBR were designated CHO-ETAR or CHO-ETBR, respectively. The [125I]-ET-1 binding assay was conducted Kd is 1533pM and and Bmax is 233.78 fmol/2×105 cells for CHO-ETAR. Kd is 482pM and Bmax is 122.93 fmol/2×105 cells for CHO-ETBR. The affinity of ET-1 for ETBR seems stronger than ETAR. [125I]-ET-1 binding was inhibited by ETAR specific antagonist BQ610 and the ETBR antagonist BQ788, respectively. ET-1 stimulates intracellular Ca2+ mobilization in CHO-ETAR and eNOS phosphorylation in CHO-ETBR. Binding assay and postreceptor signaling revealed that they are functionally active. We found that [125I]-ET-1 binding was inhibited by P1 water extract. Effective components of P1 directly interact with endothelin receptors and cause 67% inhibition for CHO-ETAR and 90% inhibition for CHO-ETBR. ET-1 stimulated intracellular Ca2+ mobilization was also inhibited by P1 in CHO-ETAR. I enriched the effective components in P1 by precipitating with different volumes of ethanol. We found that there are two effective components in P1. The component enriched in F3 3.5x (the supernatant portion with 3.5V ethanol) is more effective for ETAR (83% inhibition), but less effective for ETBR (37% inhibition). The another component enriched in F2 2x (the precipitate portion with 2V ethanol) is more effective for ETBR (92% inhibition), but less effective for ETAR (48% inhibition). On the other hand, a 67% and a >96% inhibition were achieved by P1 and F3 3.5x respectively when human primary smooth muscle cells were tested. P1 and F2 2x caused 79% and 87% inhibition for human primary endothelial cells. Moreover, we found that LPS stimulated eNOS phosphorylation in endothelial cell can inhibited by P1.
論文電子檔著作權授權書......................i
論文審定同意書..............................ii
誌謝........................................iii
中文摘要....................................iv
英文摘要....................................v
目錄........................................vi
圖目錄......................................vii
表目錄......................................viii
緒論 ...................................1
材料與方法..................................6
結果 ...................................18
討論 ...................................24
圖 ...................................28
表 ...................................43
參考文獻 ...................................49
附錄 ...................................53
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