高濃度葡萄糖引發人類腹膜間皮細胞凋亡機制中粒線體功能與形態的改變

Functional and Morphological Change of Mitochondria in High Glucose Induced Human Peritoneal Methothelial Cells Apoptosis

楊得政(Te-Cheng Yang)

指導教授 ： 洪冠予

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.00733

摘要

研究背景與目的
膜腹透析採用連續性的透析方式，利用人體本身的腹膜作為透析膜，移除尿毒症病患體內的代謝廢物與水分。然而，長期使用的腹膜透析液會傷害腹膜，造成人類腹膜間皮細胞(human peritoneal mesothelial cells, HPMCs)凋亡、細胞外間質堆積，最後引起腹膜纖維化，讓腹膜喪失脫水功能，無法繼續進行腹膜透析治療。因此，腹膜的功能健全與否，關係著膜腹透析治療能否有效成功的進行。目前使用的腹膜透析液，具有高糖、高滲透壓、及低酸鹼值等生物相容性不佳的問題。過去的研究指出，高糖會引起HPMCs凋亡，但是分子路徑和調控機制目前仍未釐清。不過，糖尿病的相關研究發現，高糖引起活性氧自由基(reactive oxygen species, ROS)激增，正是引起組織細胞傷害的主因；同時，近來的研究顯示，粒線體的分裂參與多種不同訊號引起的細胞凋亡。本研究的目的是為了探討高糖引起HPMCs凋亡的路徑中，ROS、粒線體功能和形態改變，這幾項因素之間的相互關係。希望作為研發藥物治療及調整腹膜透析液的理論基礎，有助於降低對HPMCs的傷害，維持健康的腹膜以利透析治療的進行。

研究方法
我們利用HPMC細胞培養，經過不同濃度的葡萄糖處理24小時，先檢測細胞存活率，再評量高糖對於HPMCs凋亡的影響。接著以西方墨點法，評估cytochrome c 釋出、caspase-9和caspase-3的活化，以及PARP-1 cleavage，以探討HPMCs凋亡路徑。另一方面，測量高糖引發ROS的增加，並分析脂質和DNA的氧化傷害；同時以JC-1染劑檢測高糖對粒線體膜電位的影響。下一步，比較有無先以抗氧化劑NAC處理HPMCs，對高糖所引發的各種效應，有什麼不同?最後，以電子顯微鏡和Mito-Tracker染色在共軛焦顯微鏡下，觀測高糖引發粒線體形態的改變。

研究結果
研究結果發現，高糖降低HPMCs的存活率同時引起細胞凋亡；進一步發現，粒線體釋出cytochrome c，caspase 9和caspase 3的活化及PARP-1 cleavage等，都因高濃度葡萄糖顯著地表現；顯示高糖引發HPMCs凋亡，是經由粒線體所調控的內在路徑。同時，高糖造成粒線體內ROS過量產生，引起脂質和DNA氧化傷害，降低粒線體膜電位。若先以NAC處理HPMCs，原本高糖誘發ROS的過量產生會降低，減少了它對脂質和DNA的氧化傷害，進而改善粒線體膜電位失衡以及細胞凋亡。利用共軛焦顯微鏡觀發現，在高糖的環境下，原本呈現長管狀的粒線體會變短和變小，甚至出現斷裂的現象；高倍電子顯微鏡下更發現，粒線體斷裂成啞鈴狀或扁盤狀或；同時，原本由內膜向內皺褶形成的粒線體嵴變得鈍平糢糊，甚至出現內膜結構崩解的現象。

討論與展望
我們的研究發現，在高糖引發HPMCs細胞凋亡的路徑中，最立即也最直接的效應是ROS增加，而粒線體功能與形態的變化則扮演著樞紐的角色。ROS的過量產生，造成粒線體脂質和DNA氧化傷害，粒線體功能失衡，膜電位降低，打開mPT pore，釋放凋亡蛋白；同時，高糖也造成粒線體分裂，持續增加粒線體ROS的生成。兩者形成惡性循環，最後導致內膜結構崩解，粒線體發生永久性的斷裂。希望更進一步對於高糖引發HPMCs凋亡分子路徑和調控機制的釐清，有助於維持病患腹膜的功能，以利腹膜透析治療的長期進行。

關鍵字

腹膜透析 ； 高濃度葡萄糖 ； 人類腹膜間皮細胞 ； 細胞凋亡 ； 活氧性自由基 ； 粒線體斷裂

並列摘要

Background and Purpose
Peritoneal dialysis (PD) is a continuous mode of dialysis, using the patient's peritoneum as a dialysis membrane across which fluid and metabolic waste products are exchanged from the blood. However, long-term use of PD solution impact a great harm to peritoneum, resulting in human peritoneal mesothelial cells (HPMCs) apoptosis, ECM accumulation, peritoneal fibrosis and ultimately peritoneal function failure. Current conventional PD solution has many characters of bioincompatibility, such as high glucose,high osmotic pressure, and low pH value. It has been pointed out that high glucose is the major reason to cause HPMCs apoptosis. But its pathways and regulatory mechanism haven't yet been completely clarified. Identification of mechanisms of high glucose induced HPMCs apoptosis could be useful in clinical applications to maintain the function of peritoneum for peritoneal dialysis.

Methods
HPMCs were cultured in media containing 5 (as control), 84, 138, 236 mM glucose for 24 hours. Survival rate of HPMCs was evaluated and HPMCs apoptosis was analyzed by Annexin-V/PI stain and flow cytometry. Cytochrome c release, caspase activation and PARP-1 cleavage were evaluated by Western blot. In addition, ROS generation and ensuing lipid peroxidation and oxidative damage of DNA were measured. Mitochondrial membrane potential (MMP) change was monitored by JC-1 dye. Furthermore, the antioxidant NAC is used to pretreat HPMCs before exposure to high glucose to determine whether HG-induced apoptosis of HPMCs could be contributed to ROS overproduction. Finally, mitochondrial morphology change was observed under TEM and confocal microscopy after staining by MitoTracker Red.

Results
After exposure to high glucose, the viability of HPMCs decreased and there is a significant increase in apoptosis of HPMCs. The results of Western blot assay on cell lysate showed cytochrome c release from mitochondria increased and subsequent caspase activation cascades and PARP-1 cleavage could be also demonstrated in HG condition. In addition, HG induced decreasing of MMP, assayed by JC-1. NAC exhibited attenuating effect on HG-induced ROS production, MMP decrease and HPMCs apoptosis simultaneously. Finally, under confocal microscopy and TEM, high glucose was found to cause mitochondria shortening and fragmentation.

Discussion and Prospective
In this study, we examined the correlations among high glucose, ROS, MMP, and morphological change of mitochondria, and clarified whether and how they participate in HG-induced HPMCs apoptosis. One of the initial consequences of HG is an increased generation of ROS, which is a pervasive source to injure mitochondria by causing lipid peroxidation and mtDNA damage. ROS-mediated reducing of MMP and subsequent opening of the mitochondrial permeability transition pore (mPT pore) cause cytochrome c release, and trigger the intrinsic pathway of apoptosis. In addition, we found that mitochondria undergo fragmentation concomitantly after exposure to high glucose. It may perturb structural organization of ETC connectivity, and is causally associated with ROS overpruduction and HPMCs apoptosis. Further understanding the mechanism by which HG induced HPMCs apoptosis is vital before a therapeutic intervention can be developed to maintain peritoneal membrane function for the PD patients.

並列關鍵字

peritoneal dialysis ； high glucose ； human peritoneal mesothelial cells apoptosis ； reactive oxygen species ； mitochondria fission

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