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研究生:鄭雅柔
研究生(外文):Ya-Jou Cheng
論文名稱:巨噬細胞中Pyk-2在內質網調控脂多醣產生自由基之角色
論文名稱(外文):The role of Pyk-2 in endoplasmic reticulum regulated the productionof free radicals stimulated by lipopolysaccharide on macrophages.
指導教授:陳有任
指導教授(外文):Yu-Jen Chen
學位類別:碩士
校院名稱:元培科學技術學院
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:43
中文關鍵詞:內質網鈣離子蓄池
外文關鍵詞:Proline-rich tyrosine kinase-2(Pyk-2)ThapsigarginEndoplasmic reticulum Ca2+ store
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細胞中內質網鈣離子蓄池(endoplasmic reticulun Ca2+ pool)是細胞維持細胞內鈣離子濃度平衡的重要胞器,其中的鈣離子在訊息傳遞及生理作用中都扮演著重要的角色。而proline-rich tyrosine kinase-2(Pyk-2)是focal adhesion kinase(FAK)家族中的一員,它是ㄧ個Ca2+-dependent的non-receptor tyrosine kinase,可藉由tyrosine磷酸化而被活化,Pyk-2完全活化需要在具功能性的特定位置被磷酸化,包含了自體磷酸位Tyr-402和catalytic domain Tyr-580。本研究主要利用thapsigargin(TG),一內質網鈣離子幫浦(Ca2+-ATPase)抑制劑,來探討老鼠巨噬細胞株(J774)中內質網鈣離子蓄池被抑制之後,對巨噬細胞誘導之一氧化氮(nitric oxide,NO)產生、iNOS表現及細胞存活率的影響,進而探討Pyk2與相關訊息p38、src在其中所扮演之角色。另外使用兩種Ca2+ ionophore:A23187、ionomycin及另一個可逆性的內質網幫浦抑制劑:DBHQ來與TG比較。細胞先TG處理15分鐘,再以lipopolysaccharide (LPS)刺激,結果發現,在自由基產生方面,用TG和DBHQ前處理,再以lipopolysaccharide(LPS)刺激,能夠減少NO及H2O2的產生,並且iNOS表現量也明顯下降。而Pyk-2的表現,以TG和DBHQ前處理,能減少total Pyk-2的表現量,但活化狀態Pyk-2之磷酸化 (Tyr-402、Tyr-580)卻沒有明顯變化。而且磷酸化之p38與src亦沒有明顯改變。綜合以上結果,我們認為以TG或DBHQ內質網幫浦抑制劑後,再以LPS刺激,自由基的產量的確明顯減少,指出內質網鈣離子蓄池可調控部份訊息,雖然Pyk-2表現量有受到抑制,但活化型態卻沒明顯改變,而其他與Pyk2有關之訊息傳遞蛋白:p38與src亦沒有明顯改變。因此研究結果指出,於巨噬細胞中內質網鈣離子蓄池,可調節細胞的自由基產生,但Pyk-2並沒有明顯參與在產生自由基之訊息傳遞中。
The endoplasmic reticulum(ER)、a major intracellular reservoir of Ca2+、is an important organelle to maintain intracellular calcium balance. The intracellular free calcium plays a vital role in regulating the cellular physiological and biochemical functions. Proline-rich tyrosine kinase-2 (Pyk-2)、a member of the focal adhesion kinase (FAK) subfamily、is a calcium- dependent non-receptor tyrosine kinase. Pyk-2 can be activated by tyrosine phosphorylation. Full Pyk-2 activation requires phosphorylation at functionally distinct sites、including autophosphorylation site Tyr-402 and catalytic domain Tyr-580. This study used thapsigargin (TG)、an ER Ca2+–ATPase inhibitor、to explore the effects of the inhibition of ER Ca2+-ATPase on nitric oxide (NO) and H2O2 production、iNOS expression and cell viability in lipopolysaccharide- stimulated J774 macrophage、and then to investigated the role of Pyk-2、src and p-38 involving in this mechanism. Besides、we used Ca2+ ionophore A23187、ionomycin and DBHQ、another ER Ca2+–ATPase inhibitor、to compare the effects induced by TG. The production of NO and H2O2 were decreased when cells treated with TG for 15 min and then stimulated by LPS. The expression of iNOS also significantly decreased in these treatments. The expression of total Pyk-2 decreased when cells treated with TG for 15 min and then stimulated by LPS. However、the activated forms of phosphorylated Pyk-2 (Tyr-402 and Tyr-580) had remained unchanges in these treatments. Neither p38 nor src had any significant changes in their phosphate state in these treatments. Following the above results、we thought that macrophages treated with TG or DBHQ inhibiting ER Ca2+– ATPase and then stimulated by LPS、the production of free radicals significantly reduced、that indicated ER Ca2+ pool regulated some signal processes. The expression of Pyk-2 obviously reduced、but activated forms of Pyk-2、p38 and Src had no significantly change. Thus、ER Ca2+ pool in macrophages could regulate the production of free radicals、Pyk-2 obviously did not involve in these signal processes.
誌謝……………………………………………………………………………Ⅰ
中文摘要 ……………………………………………………………………Ⅱ
英文摘要 …………………………………………………………………Ⅲ
目錄 ………………………………………………………………………Ⅳ
圖目錄 ……………………………………………………………………Ⅵ
表目錄 ……………………………………………………………………Ⅶ
第一章 緒論 ……………………………………………………………… 1
第二章 研究材料與方法 ………………………………………… ………7
2.1細胞培養
2.1.1小鼠巨噬細胞株的培養 ………………………………… 7
2.2細胞毒性測試 ……………………………………………………7
2.3自由基之測定
2.3.1一氧化氮(NO)的測定…………………………………………8
2.4過氧化氫(H2O2)的測定………………………………………………8
2.5細胞蛋白質之萃取及分析
2.5.1全細胞(Whole cell)蛋白質之萃取 ………………………9
2.5.2磷酸化(Phosphorylation form)蛋白質之萃取……………9
2.5.3蛋白質濃度測定 ……………………………………………10
2.6 SDS-聚丙烯醯胺版膠電泳法(SDS-PAGE) ………………………11
2.7 西方轉漬法 ………………………………………………………13
2.8 統計分析 …………………………………………………………………14
2.9 實驗藥品 ……………………………………………………………14
第三章 研究結果
3.1 TG、DBHQ、A23187及Ionomycin對巨噬細胞誘發NO之影響…15
3.2 TG對巨噬細胞誘發H2O2之影響 …………………………………16
3.3 TG、DBHQ、A23187及Ionomycin對巨噬細胞誘發iNOS表現
之影響 ……………………………………………………………17
3.4 TG、DBHQ、A23187及Ionomycin對巨噬細胞誘發Pyk-2表現
之影響 ……………………………………………………………18
3.5 TG、DBHQ、A23187及Ionomycin對巨噬細胞誘發Pyk-2之磷
酸化(Tyr-402、Tyr-580)表現之影響 …………………………18
3.6 TG、DBHQ、A23187及Ionomycin對巨噬細胞誘發src之磷酸
化(p-src)表現之影響 ……………………………………………19
3.7 TG、DBHQ、A23187及Ionomycin對巨噬細胞誘發p-38之磷酸
化表現之影響 ……………………………………………………19
3.8 TG、DBHQ、A23187及Ionomycin對巨噬細胞誘發HSP-70及
HSP-90表現之影響 ………………………………………………20
第四章 討論與結論 …………………………………………………………21
第五章 圖表與圖表說明 ……………………………………………………25
參考文獻 ……………………………………………………………………38
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