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研究生:許杏如
研究生(外文):Hsu hsin-ju
論文名稱:人類血小板型血栓素受體的選殖,表現及定性研究
論文名稱(外文):Cloning, expression and characterization of the human platelet type thromboxane receptor
指導教授:簡偉明
指導教授(外文):Kan wai-ming
學位類別:碩士
校院名稱:國立成功大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:100
中文關鍵詞:血栓素血小板亞型血栓素受體磷酸鈣轉染法
外文關鍵詞:Thromboxane A2platelet-type TXA2 receptorcalcium phosphate transfection method
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血栓素為血小板活化及血管平滑肌收縮的強效刺激劑。 血栓素的作用主要是透過血栓素受體。 血栓素受體普遍存在不同的動物種類、不同組織及不同的細胞上。 血栓素受體是G蛋白連結的受體(G-protein coupled receptors) ,具有七個穿膜結構。 目前知道血栓素受體有二個亞型,即血小板亞型 (platelet type) 和內皮細胞亞型(endothelial cell type),二者的主要差異在C端上的序列不同。而後學者發現在血小板上同時有血小板亞型和內皮細胞亞型血栓素受體的存在。為了更徹底的研究人類血小板亞型血栓素受體 (hpTXR) 的作用,所以建構一個表現hpTXR的哺乳動物細胞株成為當務之急。
我們從人類的胎盤基因組庫(human placenta cDNA library)利用聚合連鎖反應(polmerase chain reaction)複製後,得到hpTXR的DNA片段。將hpTXR的DNA片段利用內限制切割位點選殖到哺乳類的表現載體-pcDNA3,根據DNA定序的結果證實hpTXR的DNA片段是正確的。將 pcDNA3- hpTXR 質體以磷酸鈣轉染法(calcium phosphate transfection method) 送入HEK-293細胞株中表現,再以geneticin (G418) 進行細胞株的篩選,成功送入質體的細胞株對G418有抵抗力,在篩選下可以存活。接著以血栓素受體催動劑-U46619刺激篩選後的細胞,測量細胞內鈣離子的變化,藉此測試細胞上血栓素受體的功能是否正常。由實驗結果得知,U46619刺激下確實使細胞內鈣離子增加,而沒有表現hpTXR的原細胞株鈣離子則沒有明顯的增加。以血栓素受體拮抗劑-IPA進一步確定細胞內鈣離子增加確實是透過血栓素受體所造成。之後我們從多株細胞中挑選五個單株細胞進行培養,並觀察U46619刺激下細胞內鈣離子的變化,發現其中有一株細胞的鈣離子增加較其他細胞株高,顯示此細胞株可能表現有較多的血栓素受體。利用西方墨點法,以辨識受體第三細胞內結構及C端的抗體,顯示在轉染後的細胞上確實有人類血小板亞型血栓素受體的表現,而原細胞株上則沒有血栓素受體。另一方面,為了製備效果較好的人類血小板亞型血栓素受體抗體,將hpTXR的DNA片段利用pMAL-c2原核細胞表現載體進行選殖,將質體送入E. coli.中表現MBP(maltose binding protein)-hpTXR融合蛋白。由西方墨點法的結果顯示,我們不論以的MBP抗體或是辨識受體第三細胞內結構及C端的抗體,都可以看到一個80kDa染色帶,顯示MBP-hpTXR融合蛋白能正確的在E. coli.中表現。往後本實驗室可以利用此融合蛋白製備人類血小板亞型血栓素受體抗體。建立此一表現血小板亞型血栓素受體的細胞株,將可提供日後研究血栓素受體更豐富的工具;而MBP-hpTXR融合蛋白的產生,使得往後製備人類血小板亞型血栓素受體特異性抗體更加方便。

Thromboxane A2 (TXA2) is a potent stimulator for platelet aggregation and vascular smooth muscle contraction. TXA2 actions are mediated via thromboxane receptors (TXRs) which are widely distributed in different species, tissues and cell lines. Thromboxane receptors are G-protein coupled receptors which have seven transmembrane domains. Two isoforms of TXRs, platelet and endothelial types, have been found and differ mainly in the C-terminal receptor sequence. However, recently both isoforms was found to exist in platelet. In order to clarify the function of human platelet type thromboxane receptor (hpTXR), we constructed a mammalian cell line which expresses only hpTXR for pharmacological study.
To obtain hpTXR DNA sequence, we amplified hpTXR DNA fragment from human placenta cDNA library by PCR. The PCR fragment was cloned into pT7Blue-3 cloning vector. Moreover, the insert was subcloned from the cloning vector to the mammalian expression vector (pcDNA3). According to the DNA sequencing results, the hpTXR DNA sequence was found to be correct. pcDNA3-hpTXR plasmid was transfected in HEK-293 cells by calcium phosphate transfection method, and selected with geneticin (G418). The successfully transfected cells is resistant to G418 and survive. The selected cells were stimulated by TXR agonist-U46619, and the change of intracellular Ca2+ was measured. The function of hpTXR in HEK-293 was characterized by measuring of the change of intracellular Ca2+. When the transfected HEK-293 cells were stimulated by U46619, it resulted in an intracellular Ca2+ increase, while the untransfected cells were not. The increase in Ca2+ was mediated via TXR was confirmed with specific TXR antagonist-IPA. Furthermore, clones expressing hpTXR were isolated from transfected cells and the change of intracellular Ca2+ were measured. One of the clones showed higher Ca2+ increase, which suggested that the clone might express higher density of thromboxane receptors. The expression of human platelet type TXR in transfected HEK-293 cells were further confirmed by Western blotting, using antibodies recognized TXR third intracellular loop and C-terminus. In untransfected cells no expression was detected. For generation of high affinity hpTXR antibodies, hpTXR DNA was cloned into pMAL-c2 expression vector, and then transformed in E. coli. to express MBP-hpTXR fusion protein. A 80kDa protein could be detected by anti-MBP or anti-L3 and anti-C antibodies. These results revealed that MBP-hpTXR fusion protein is expressed in E. coli. successfully. In summary, the construction of hpTXR HEK-293 cell line provide us a useful tools for the studies of thromboxane receptor. The MBP-hpTXR fusion protein may be for useful anti-hpTXR antibodies generation.

致謝
中文摘要
英文摘要
符號及縮寫
壹. 緒論 1
貳. 實驗設計 12
參. 實驗材料及方法 14
肆. 實驗結果 55
伍. 實驗討論 62
陸. 結論 71
參考文獻 72
圖表 78
自述 100

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