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標題: H9c2大鼠心臟細胞Gelsolin影響細胞形態和caveolin訊號路徑抗雙氧水誘導的氧化壓力
Gelsolin Affects Cell Morphology and Caveolin Signaling Against Hydrogen Peroxide-induced Oxidative Stress in H9c2 Rat Cardiomyoblasts
作者: 何舒婷
He, Shu-Ting
關鍵字: 大鼠心臟細胞株H9c2;H9c2 Rat Cardiac cells;gelsolin (GSN);caveolins (cavs);氧化壓力;肌動蛋白絲;自由基;細胞骨架;SOD活性;Gelsolin (GSN);Caveolins (cavs);Oxidative stress;Actin filaments;ROS;Anti-oxidants;HO-1;Cu-SOD;catalase;stress fiber
出版社: 生命科學系所
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The Journal of biological chemistry 278, 1248-1258.
Gelsolin (GSN)是一個廣泛分佈,具有鍵結肌動蛋白與調控肌動蛋白絲的組裝與拆卸。GSN也具有促凋亡和抗凋亡的功能;蛋白分子的 N端具有促凋亡的特性,但全長的GSN會與PIP2形成複合物則有抗凋亡的特性。但是到目前為止對於GSN在心臟細胞中所扮演的功能與分子調控機轉還不清楚。另外, caveolins (cavs)在心臟疾病上扮演保護的角色;能增加抗氧化能力與減少氧化傷害。本研究利用過量表現GSN (GSN op)與緘默GSN (si-GSN)的H9c2細胞株,藉由外加雙氧水使細胞面臨氧化壓力的模式下,探討GSN是否會影響cavs在H9c2細胞株內的功能。首先,反轉錄聚合酶連鎖反應確認pcDNA6-GSN有轉染送入到H9c2細胞內(GSN op);西方轉漬墨點法也顯示GSN蛋白表現有增加的情形。螢光光譜儀和倒立式顯微鏡檢測細胞內鈣離子濃度和細胞形態,結果發現過量表現GSN會增加胞內鈣離子濃度和使細胞形態變得較短小。隨後MTT assay檢測細胞增長時間,再利用免疫螢光顯微鏡術檢測細胞內肌動蛋白絲的分子組裝組裝與拆卸的變化,再以流式細胞儀檢測細胞週期。結果發現:過量表現GSN會減緩細胞生長,且細胞分布在S期和G2期的比例上升,還有增加細胞內肌動蛋白絲去聚合反應。因此,GSN在H9c2細胞內過度表現可能經由影響肌動蛋白絲動態組裝與卸載的機轉因而影響細胞週期與生長。另外,過量表現GSN也會增加cavs的表現;相反地,緘默GSN則抑制cavs的表現。進一步探討過量表現GSN增加細胞內cavs對細胞抗氧化壓力的影響;因此,檢測細胞內自由基與SOD活性變化,結果發現在雙氧水誘導氧化壓力增加的正常與GSN過量表現的H9c2細胞,過量表現GSN能減緩雙氧水減少cavs蛋白表現和SOD活性,以及細胞內自由基的產生,終而增加細胞抗凋亡的能力。綜合以上結果,過量GSN表現可能增加細胞內切割和加帽的能力,增加肌動蛋白絲的降解,使降低細胞分裂的能力,進而延長細胞生長時間。另外,也影響了細胞內cavs 傳導路徑,進而促使細胞內抗氧化能力增加,達到保護的效果。由於cavs傳導路徑在心臟保護上可能扮演很重要的角色,所以未來經由影響細胞骨架重組來調控cavs訊號路徑可作為研究心臟保護的新方向。

Gelsolin (GSN) is a Ca2+-dependent actin-regulatory protein that can sever actin filaments and cap the quickly growing ends of filaments, and thus promoting actin disassembly. Accordingly, GSN plays a role in the organization of the cytoskeleton assembly/disassembly, cell motility, cell growth, and apoptosis. The biological function of GSN and its interaction with scafold proteins-caveolin in cardiac cells is not clear thus far. Caveolin (cav) is known to play an important role in myocardial protection signaling for controling intracellular SOD activity and modulating oxidative stress in cardic cells. It is of interest to define the role of GSN and to establiah relationship between the stress fiber formation by cytoskeleton and the expression of Cavs in cardic cells. In this study, GSN over-expressed (GSN op) and GSN silenced (si-GSN) H9c2 rat cardiomyoblasts in a simulated condition with oxidative stress by hydrogen peroxide were used to explore the potential mechanism of GSN in cardiac protection aganist oxidative stress. A pcDNA6-GSN containing full-length human GSN transfected into H9c2 cells caused increases in intracellular calcium concentration and alteration of cell morphology and actin filament-associated structure. GSN over-expressing cells changed morphology to a “shorter and broader” form, while GSN silencing cells displayed a less change in cell shape as compared with wild-type cells. MTT assay and flow cytometry indicated that GSN over-expression slowed down cell proliferation and increased cell populations sorted in S and G2 phases. These results suggested that GSN over-expression may enhance the ability of capping and severing F-actin such as to increase dissembly of actin filaments, and to slow down cell division and cell growth. In addition, GSN was found to modulate the expression of Cavs and their cellular functions in protecting cardic cells from oxidative stress induced by H2O2. The experiments designed to investigate the impact of an increase intracellular Cavs on anti-oxidative effects in H9c2 cells showed that GSN over-expressing cells increased their anti-oxidative capacity in H2O2-induced oxidative stress by preventing the down-regulation of Cavs, and by attenuating the H2O2-increased intracellular ROS levels, and by elevating mRNA expression for associated anti-oxidants such as HO-1, Cu-SOD, and catalase, and by increasing anti-oxidative SOD activity. Taken together, the results found in this study suggested that GSN might induce remodeling of actin filaments and that linked to mediate the Cav signaling for anti-oxidative effects in cardiac cells.
其他識別: U0005-2108201319465700
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