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系統識別號 U0026-0812200913595244
論文名稱(中文) 血小板對腎上腺素刺激弱反應的中國族群研究
論文名稱(英文) Impaired responsiveness of platelets to epinephrine in Chinese population
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 95
學期 2
出版年 96
研究生(中文) 林技賢
研究生(英文) Jihshyan Lin
電子信箱 t3693407@mail.ncku.edu.tw
學號 t3693407
學位類別 碩士
語文別 中文
論文頁數 59頁
口試委員 口試委員-陳志弘
指導教授-林尊湄
召集委員-陳彩雲
口試委員-邢福柳
中文關鍵字 血小板  腎上腺素  腎上腺素接受器 
英文關鍵字 epinephrine  α2A adrenergic receptor  platelet 
學科別分類
中文摘要 血小板的活化,在血液凝固及血栓形成過程中,扮演著關鍵的角色,而腎上腺素則是在血小板的活化過程中,是一種重要刺激血小板的弱性活化劑,而目前對於腎上腺素與血小板的作用只有少數零星的報導,如果以傳統評估血小板的方法發現,大約6%白種人及16%日本人,其血小板對於腎上腺素的刺激是呈現不反應的情形,雖然個體中對於腎上腺素刺激血小板的反應之變異性很大,但卻是與家族遺傳有關,而其真正的分子調控機轉,目前仍然並不清楚。本研究的主要目的,是想探討血小板對於腎上腺素的刺激,在中國人出現不反應的比例以及其特性,我們以50名健康男性為受檢者進行血小板凝集試驗、流體細胞儀和PFA-100檢測腎上腺素刺激血小板活化情形,發現在血小板凝集試驗的實驗中,可以明顯呈現血小板對於腎上腺素的刺激強和弱反應兩個族群,分別有24名(48%)及26名(52%)。若用1 μM的腎上腺刺激血小板後,再以流體細胞儀分析GPIIbIIIa表現,則強反應者和弱反應者的結果分別是27.1±11.0%, 和 9.9±5.4% (p=0.003)。 而以PFA-100的檢測結果,弱反應者在膠原蛋白/腎上腺素的 closure time比強反應延長(149.6±28.1 vs 114.6±23.3 sec, p=0.03),這些檢測結果比較這兩群,都可發現存在明顯的差距。因為腎上腺素和α2A adrenoceptor (ADRA2A)的交互作用,在正常止血功能和血栓的形成是必需的步驟,已經有研究報告指出個體之間ADRA2A在血小板上的表現的差異,會影響著血小板凝集反應且與家族遺傳有關。因為位在第十對染色體上之ADRA2A基因存在多種基因多型性,本研究中,我們以SSP-PCR的方法分析了其中三個常見的多型性位置,結果顯示啟動子-2210AA 和 3端非轉譯區1780GG或 2372AA基因型,可能與腎上腺素刺激血小板呈弱反應有關聯性。但進一步在啟動子功能性實驗分析中,我們將-2210多型性分別構築在含有luciferase表現基因架構上,卻發現TT多型性有較高的表現活性,因此我們認為-2210 T>A的變異, 可能只是個體對腎上腺素刺激血小板呈弱反應的連結標而已。希望未來能以功能性實驗分析ADRA2A基因的真正變異,去探討在這種生理現象臨床上的表徵和進一步闡明其中的分子機制。
英文摘要 Platelet activation plays a central role in hemostasis and thrombosis. Epinephrine (EP) is known as a weak, but important, agonist for platelet activation. Variability in responsiveness of human platelets towards EP has been documented sporadically in the literature. It has been reported that the responsiveness of platelets to EP was markedly impaired in 6% Caucasian and 16% in Japanese. There is familial clustering of inter-individual variations in the EP-induced platelet aggregation, the molecular basis of which, however, has not been fully understood. The purposes of this study are to screen and characterize the abnormality in healthy Chinese volunteers. A total number of 50 healthy males were recruited to investigate the responsiveness of platelets to EP stimulation by aggregometry, flow cytometry and using epinephrine/collagen cartridge in platelet function analyzer-100 (PFA-100) system. Based on the platelet aggregation, two distinct groups of subjects of EP good and impaired responders were observed in 24 (48.0%) and 26 (52.0%) subjects, respectively. In comparison to the results of flow cytometic analysis after platelet challenged with 1 μM EP, GPIIbIIIa expression of EP good and impaired responders were 27.1±11.0%, and 9.9±5.4% (p=0.003), respectively. Platelet functions were assessed using PFA-100 system, a longer collagen/epinephrine cartridge closure time was demonstrated in impaired EP responders (149.5±28.1 vs 114.6±23.3 sec, p=0.03).
The α2A adrenoceptor (ADRA2A)/EP interaction is an essential step in physiologic hemostasis and thrombus formation. Previous studies demonstrated inter-individual variations in the levels of ADRA2A expression on platelets and EP-induced platelet aggregation, with familial clustering. The gene encoding ADRA2A on chromosome 10 is polymorphic and contains no introns. Three common polymorphisms of ADRA2A gene were analyzed by specific sequence primer polymerase chain reaction. The results demonstrated that promoter -2210AA and 3’UTR 1780GG or 2372AA genotypes of ADRA2A were associated with impaired EP responders. Functional analysis of the polymorphisms of ADRA2A gene were conducted by luciferase reporter gene assays revealed significantly higher promoter activity of the TT allelic variant at this single nucleotide polymorphism site. These observations suggest that the ADRA2A promoter polymorphism-2210T>A might only a linkage marker for responsiveness of platelets to EP. In the future, functional analysis of the alleles of ADRA2A gene is needed to elucidate their clinical significance and to further clarify the underlying molecular pathogenesis.
論文目次 中文摘要 II
英文摘要 IV
致謝 VI
目錄 VII
圖目錄 IX
表目錄 X
附錄圖目錄 XI
縮寫檢索表 XII
儀器及藥品 XIII
緒論 1
研究動機與實驗設計 9
材料與方法 11
一、 檢體收集 11
二、 血小板凝集試驗 (Platelet aggregation studies) 11
三、 流體細胞儀測血小板活性(Detection platelet 11
activation by flow cytometr)
四、 BRL44408抑制腎上腺素活化血小板試驗 11
五、 血小板功能分析儀 (封閉時間)分析PFA-100(Closure time)
analysis 11
六、 西方點漬法(Western blotting) 11
七、 基因多型性分析SSP-PCR (Sequence-Specific Primer-
Polymerase Chain Reaction) 14
八、 基因表現的細胞實驗(Cell-based in vitro gene
expression) 16
ADRA2A(-2210A/T)質體的構築 16
純化PCR產物 18
質體的接合( Ligation )及轉形( Transformation ) 反
應 18
小量質體的萃取及限制酶作用 20
質體的轉染(Transient Expression Assay) 21
Site-direct mutagenesis 23
質體的轉染(Transient Expression Assay)24
Dual leucifease 活性分析 25
結果 26
討論 30
參考文獻 37
圖 43
表 52
附錄圖 55
自述 58
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