2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647995摘要：我們長期來都在探討腹膜透析(peritoneal dialysis, PD)病人的腹膜硬化症(peritoneal fibrosis, PF)發生機轉、治療與處置。我們已發表: 非生理性之腹膜透析液會造成腹腔內的: 氧化壓力、慢性發炎反應，及引起腹膜硬化症。過去三年，我們在國科會計畫[97-2314-B-002-053-MY3, paper submitted]支持下，發現: PD 病人發生腹腔內出血(hemoperitoneum)，會誘發氧化壓力、慢性發炎反應，及引起腹膜硬化症。這部分目前正在投稿審查程序中。本計畫的第一年的研究主題是: 腹膜表面細胞衰老過程中，heme oxygenase-1(HO-1)的角色，這個部分又可以分成兩段: 先建立之細胞研究模式，再配合刺激自由基反應，研究HO-1 與細胞衰老過程，接著再knock-down HO-1 基因表現，觀察對腹膜表面細胞衰老過程的影響。在第二年的研究主題中: 我們測試如果增強HO-1 的基因表現，觀察對腹膜表面細胞衰老過程又有什麼影響。HO-1 的訊息傳遞與作用機轉產物中，會有ferritin 的表現，因此，我們也探討: ferritin 的heavy-chain 在腹膜硬化症的發生機轉中所扮演的角色。這部分同時也和我們之前的腹腔內出血(hemoperitoneum)研究結合，包括不同細胞狀態下的調控與基因表現。第二年的另一個研究重點(預計會橫跨入第三年，因為機轉目前為之但可能很複雜)，也就是: senescence 與老化相關標記(markers)，如: p16-INK4a, p66shc, Sirt1,FoxO, Klotho, Werner syndrome protein WNR, and mTOR signaling 等。另外，接續之前細胞吞噬(autophagy)的機轉觀察，也與PF 發生有關，會納入研究中。第三年後半的主題其實最難，但卻是統合這六年來的一個重要問題: 有關自然療程(即:無感染，只是PD 藥水治療，加上clinical or subclinical 的腹腔內出血，這些都是病人臨床上會經驗到的歷程)中，腹膜表面細胞的衰老過程，和腹膜硬化症的發生究竟有無關聯。結論: 本計畫的轉譯(translation)價值在於: 找出的機轉與臨床上可用之生物標記，可以提供PF 發病早期的細胞與組織變化，及做為臨床上藥物治療的重要研究平台。更可能做為分析PF 預後的重要參考。<br> Abstract: We had studied for years focusing on the management of peritoneal fibrosis. Wehad identified that the un-physiological peritoneal dialysis (PD) solutions may induceoxidative stress, chronic inflammatory reaction within the peritoneal cavity. We alsohad documented that hemoperitoneum occurs not infrequently in PD patients, and mayinduce peritoneal fibrosis through oxidative stress and ferritin reaction(97-2314-B-002-053-MY3, paper submitted).In this proposal we will investigate the role of heme oxygenase-1 (HO) andferritin heavy chain in cellular senescence of cultured human omentum-derivedperitoneal mesothelial cells. In the first year, we checked the hypothesis that duringcellular senescence free radical may accumulate and therefore HO-1 may also beinduced. Induction of the enzyme is protective for the cell from rapid progression ofsenescence. We will characterize the expression pattern of HO-1 alteration during thesenescence course of cultured human peritoneal mesothelial cells. To study their rolein deterring cellular senescence, we will use RNA interference to knock downexpression of each gene. Studies have supported that aging is associated with theconsequence of free radical damage by various endogenous reactive oxygen species.In the second year project, we will study further the free radical induction of HO-1expression, which may further cause generation of ferritin heavy chain. We also aim tostudy the alteration of senescence- and aging-related markers, including populationdoubling number, p16-INK4a, p66shc, Sirt1, FoxO, Klotho, Werner syndrome proteinWNR, and mTOR signaling will be examined. Telomere length, autophagy markerLC3, oxidative stress marker 8-hydroxydeoxyguanosine and Fe (II) to Fe (III) ratiowill be measured as well. To further support our hypothesis, in the third year, we alsoattempt to over-express HO-1 into the cultured cells by introducing an expressionvector carrying HO-1 full-length cDNA, and analyze the senescence phenotypes. Totest the hypothesis that ferritin heavy chain is a down stream effector of HO-1, we willintroduce ferritin heavy chain expression vector into the cells that bear HO-1interference RNA. If HO-1 knock-down cells can have then a slower pace ofsenescence when they are forced to express ferritin heavy chain, it will support thehypothesis. Through this 3-year project, together with the previous 3-year NSC project(97-2314-B-002-053-MY3, paper submitted), we can measure directly what reallyhappen in sub-clinical PD patients, and answer one important but unanswered question:“Why uneventful PD patients will get PF at the end of long-term PD therapy?”In summary, Our findings can be a big jump by making clear the HO-1 andferritin heavy chain serving as protectors of cellular senescence. And our proposal willprovide value as an important basis for future translational research and give a betterview in this uncharted territory.我們長期來都在探討腹膜透析(peritoneal dialysis, PD)病人的腹膜硬化症 (peritoneal fibrosis, PF)發生機轉、治療與處置。我們已發表: 非生理性之腹膜透析 液會造成腹腔內的: 氧化壓力、慢性發炎反應，及引起腹膜硬化症。過去三年， 我們在國科會計畫[97-2314-B-002-053-MY3, paper submitted]支持下，發現: PD 病 人發生腹腔內出血(hemoperitoneum)，會誘發氧化壓力、慢性發炎反應，及引起 腹膜硬化症。這部分目前正在投稿審查程序中。 本計畫的第一年的研究主題是: 腹膜表面細胞衰老過程中，heme oxygenase-1 (HO-1)的角色，這個部分又可以分成兩段: 先建立之細胞研究模式，再配合刺激 自由基反應，研究HO-1 與細胞衰老過程，接著再knock-down HO-1 基因表現， 觀察對腹膜表面細胞衰老過程的影響。在第二年的研究主題中: 我們測試如果增 強HO-1 的基因表現，觀察對腹膜表面細胞衰老過程又有什麼影響。HO-1 的訊息 傳遞與作用機轉產物中，會有ferritin 的表現，因此，我們也探討: ferritin 的 heavy-chain 在腹膜硬化症的發生機轉中所扮演的角色。這部分同時也和我們之前 的腹腔內出血(hemoperitoneum)研究結合，包括不同細胞狀態下的調控與基因表 現。第二年的另一個研究重點(預計會橫跨入第三年，因為機轉目前為之但可能很 複雜)，也就是: senescence 與老化相關標記(markers)，如: p16-INK4a, p66shc, Sirt1, FoxO, Klotho, Werner syndrome protein WNR, and mTOR signaling 等。另外，接續 之前細胞吞噬(autophagy)的機轉觀察，也與PF 發生有關，會納入研究中。第三 年後半的主題其實最難，但卻是統合這六年來的一個重要問題: 有關自然療程(即: 無感染，只是PD 藥水治療，加上clinical or subclinical 的腹腔內出血，這些都是 病人臨床上會經驗到的歷程)中，腹膜表面細胞的衰老過程，和腹膜硬化症的發生 究竟有無關聯。 結論: 本計畫的轉譯(translation)價值在於: 找出的機轉與臨床上可用之生物 標記，可以提供PF 發病早期的細胞與組織變化，及做為臨床上藥物治療的重要 研究平台。更可能做為分析PF 預後的重要參考。We had studied for years focusing on the management of peritoneal fibrosis. We had identified that the un-physiological peritoneal dialysis (PD) solutions may induce oxidative stress, chronic inflammatory reaction within the peritoneal cavity. We also had documented that hemoperitoneum occurs not infrequently in PD patients, and may induce peritoneal fibrosis through oxidative stress and ferritin reaction (97-2314-B-002-053-MY3, paper submitted). In this proposal we will investigate the role of heme oxygenase-1 (HO) and ferritin heavy chain in cellular senescence of cultured human omentum-derived peritoneal mesothelial cells. In the first year, we checked the hypothesis that during cellular senescence free radical may accumulate and therefore HO-1 may also be induced. Induction of the enzyme is protective for the cell from rapid progression of senescence. We will characterize the expression pattern of HO-1 alteration during the senescence course of cultured human peritoneal mesothelial cells. To study their role in deterring cellular senescence, we will use RNA interference to knock down expression of each gene. Studies have supported that aging is associated with the consequence of free radical damage by various endogenous reactive oxygen species. In the second year project, we will study further the free radical induction of HO-1 expression, which may further cause generation of ferritin heavy chain. We also aim to study the alteration of senescence- and aging-related markers, including population doubling number, p16-INK4a, p66shc, Sirt1, FoxO, Klotho, Werner syndrome protein WNR, and mTOR signaling will be examined. Telomere length, autophagy marker LC3, oxidative stress marker 8-hydroxydeoxyguanosine and Fe (II) to Fe (III) ratio will be measured as well. To further support our hypothesis, in the third year, we also attempt to over-express HO-1 into the cultured cells by introducing an expression vector carrying HO-1 full-length cDNA, and analyze the senescence phenotypes. To test the hypothesis that ferritin heavy chain is a down stream effector of HO-1, we will introduce ferritin heavy chain expression vector into the cells that bear HO-1 interference RNA. If HO-1 knock-down cells can have then a slower pace of senescence when they are forced to express ferritin heavy chain, it will support the hypothesis. Through this 3-year project, together with the previous 3-year NSC project (97-2314-B-002-053-MY3, paper submitted), we can measure directly what really happen in sub-clinical PD patients, and answer one important but unanswered question: “Why uneventful PD patients will get PF at the end of long-term PD therapy?” In summary, Our findings can be a big jump by making clear the HO-1 and ferritin heavy chain serving as protectors of cellular senescence. And our proposal will provide value as an important basis for future translational research and give a better view in this uncharted territory.