乙醯基半胱氨酸避免 傳統腹膜透析液導致的 人類腹膜間皮細胞氧化性壓力

N-acetylcysteine prevents conventional peritoneal dialysate fluid - induced oxidative stress in human peritoneal mesothelial cells

蕭惠旭(Hui-Hsu Hsiao)

指導教授 ： 陳香文

高雄醫學大學/醫學院/醫學研究所/碩士(2008年)

摘要

灣的透析病人之發生率與盛行率，是分別高居世界第一及二位。連續性可攜帶式自動腹膜透析（Continuous ambulatory peritoneal dialysis；CAPD）是末期腎臟衰竭病患治療方式之ㄧ。臨床發現，目前使用的傳統腹膜透析溶液 (peritoneal dialysate fluids；PDF)，易有生物相容性不佳的問題而產生人類腹膜間皮細胞(human peritoneal mesothelial cells; HPMCs)損傷及腹膜纖維化（peritoneal fibrosis）的合併症。本實驗研究目的，是利用以1.5% dextrose為基質的腹膜透析液，來探討腹膜透析液對人類腹膜間皮細胞的毒性作用。及抗氧化劑可能的效應。
HPMCs是取自腹膜透析病人已使用的腹膜透析液。使用組織免疫染色法確立細胞形態。利用MTT assay（3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl Tetrazolium Bromide）來測試細胞存活率。選擇具有抗氧化作用的藥劑:薑黃素（curcumin；CUR）和乙醯基半胱氨酸（N-acetylcysteine；NAC）進行探討。利用H2DCFDA和dHE染劑偵測氧自由基（reactive oxygen species；ROS）。西方墨點法來偵測熱休克蛋白72（heat shock protein72；HSP72）、熱休克蛋白27（heat shock protein 27；HSP27）的變化。利用微盤分析儀（ELISA Reader），進行抗氧化酵素-過氧化氫酶(catalase)和超氧化歧化酶 (superoxide dismutase; SOD) 的活性偵測，及麩胱甘肽(glutathione) 的偵測。
結果顯示當HPMC暴露到傳統腹膜透析液中，細胞存活率會隨著暴露時間的增加而降低。預先加入NAC培養的HPMC，則會增加其細胞存活率。加入CUR培養的HPMC，並沒有顯著的差異。利用流式細胞儀進行氧化性脅迫(oxidative stress)分析。結果顯示，暴露到透析液中的細胞，綠色螢光(DCF)和紅色螢光(EB)有明顯的增加。而事先加入NAC培養，則可減少ROS的累積，且降低細胞死亡率。從西方墨點法的結果顯示暴露到傳統腹膜透析液後，將導致HSP72有增加情形。NAC可降低因腹膜透析液刺激，導致腹膜細胞HSP72的表現。而各組間，抗氧化酵素-過氧化氫酶和超氧化歧化酶的活性偵測，並無明顯差異。腹膜透析液暴露後，將導致HPMCs內還原態麩胱甘肽 (GSH)的下降，NAC的處置有助於改善此情況。
傳統腹膜透析液的暴露，將導致HPMC內氧自由基的堆積，及GSH的下降。而NAC的前處置或共同培養，可藉由維持GSH的產生，而降低HPMC中ROS堆積。降低腹膜透析液刺激導致的氧化性傷害。其中HSP72的表現可以當作HPMC受到氧化脅迫的指標。

關鍵字

腹膜透析液 ； 人類腹膜間皮細胞 ； 乙醯基半胱胺酸 ； 氧化性壓力 ； 氧自由基

並列摘要

Introduction and aims: Chronic peritoneal dialysis is one of the major therapies for patients with end stage renal disorders. Dialysates with bio-incompatibility may lead to peritoneal mesothelial cell injury, fibrosis and subsequent poor dialysis efficacy. The aim of this study investigates the toxic effects of conventional glucose-based dialysates in primary human peritoneal mesothelial cells (HPMCs) and the possible roles of amtioxidants in HPMC during conventional dialysate exposure.

Methods：HPMCs were collected from peritoneal effluent of patients receiving peritoneal dialysis (PD). Conventional 1.5% dextrose peritoneal dialysate was utilized. HPMCs were exposed to conventional peritoneal dialysate with 1.5% dextrose for different time course and then the survival rate of HPMCs was evaluated by MTT assay. Curcumin（CUR）and N-acetylcysteine (NAC) were utilized as antioxidants. ROS accumulation, such as intracellular hydrogen peroxide, and superoxide anion in HPMCs were detected with flow cytometry by using intracellular probes, H2DCFDA and dHE, respectively,. Protein expression of HSP72 and HSP27 were detected by Western blots analysis. Antioxidant enzymes, such as catalase and superoxide dimutase (SOD), and glutathione were detected by ELSA reader.

Result：Present study shows that conventional 1.5% dextrose dialysate exposure resulted in significant decrease of cell survival in a time dependent manner. NAC treatment contributes to decrease the conventional dialysate-induced cell death. However, curcumin can not offer the protective effects. Moreover, HSP72 is induced in HPMCs after dialysate exposure. NAC treatment decreases HSP72 induction. The intensity of DCF green fluorescence and the EB red fluorescence are increased, and shifting to right showed in histogram after conventional dialysate exposure. The accumulation of intracellular hydrogen peroxide and superoxide anion are in an exposure time-dependent manner. However, NAC treatment contributes to decrease dialysate induced-ROS accumulation. Activity of SOD and catalase is not different in every group. However, the reduced glutathione is significant decrease after dialysate exposure and NAC treatment contributes to preserve the expression of reduced glutathione in HPMCs.

Conclusion: ROS accumulation during convention peritoneal dialysate exposure is an important cause of leading cell damage. NAC treatment contributes to protect the HPMCs from conventional dialysates-induced cellular damage by preserving the reduced glutathione to decrease ROS accumulation and oxidative stresses. HSP72 could be used as a stress marker.

並列關鍵字

HPMCs ； ROS ； HSP72

參考文獻

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