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標題: | 探討維他命D調控粒線體分裂與粒線體自噬對於心肌缺血再灌流所引發細胞凋亡的影響 To study the effects of vitamin D on ischemia/reperfusion-induced cardiac injury via mitochondrial fission and mitophagy |
作者: | Yi-Chieh Lee 李奕潔 |
指導教授: | 陳玉怜(Yuh-Lien Chen) |
關鍵字: | 心肌缺血再灌流,粒線體自噬,粒線體分裂,維他命D3,細胞凋亡,粒線體失活, Cardiac ischemia/reperfusion,Mitophagy,Mitochondrial fission,Vitamin D3,Apoptosis,Mitochondrial dysfunction, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 近年來心血管疾病成為已開發國家主要死亡原因之一,其中又以心肌梗塞(myocardial infraction,MI) 擁有極高的發病率以及致死率。早期再灌流是治療心肌梗塞最好的方式,但心臟的缺血再灌流(ischemia/reperfusion,IR) 會造成過量的氧化壓力、粒線體失能和細胞凋亡等途徑,導致術後心肌細胞損傷更加嚴重進而使心臟恢復不良。粒線體的動態平衡 (粒線體分裂和粒線體自噬) 在心臟缺血再灌流中扮演著重要的角色,透過創新的藥物研發或小分子干擾進一步減緩再灌流所造成的傷害更是現今急需解決的問題。文獻指出,維他命D3會去調節心血管的功能,但更詳細的生理調節途徑以及對粒線體動態平衡的影響尚未明瞭。在細胞模式的部分,我們利用缺氧再灌氧(hypoxia/reoxygenation,H/R) 的環境誘導H9c2心肌母細胞,模擬動物缺血再灌流的氧化壓力傷害。H9c2心肌母細胞在H/R的環境下,存活率明顯降低。使用TUNEL染色以及觀察細胞凋亡相關蛋白cleaved caspase 3的活化,結果顯示H/R會誘導細胞凋亡。此外,在H/R的情況下,Mff (mitochondrial fission factor) 會調控粒線體分裂相關蛋白p-Drp1 (dynamin related protein 1),由細胞質轉移到粒線體上並促進粒線體的分裂現象。接著利用西方墨點法觀察粒線體自噬相關蛋白LC3BII/I和BNIP3 (BCL2 Interacting Protein 3) 的活化,吖啶橙螢光染色檢測自噬溶小體 (autolysosome) 的多寡以及透過穿透式電子顯微鏡觀察超微結構,顯示在H/R的情況下,粒線體分裂以及粒線體自噬作用皆有上升的現象,而加入維他命D3後會減緩以上的結果。在動物模式 (I/R) 部分,藉由結紮心臟左前降支冠狀動脈(left anterior descending coronary artery,LAD) 造成心肌缺血30分鐘後,再灌流3小時取其心臟組織做觀察。結果顯示在I/R的情況下,無論是細胞凋亡、粒線體分裂或是粒線體自噬皆會有上升的現象,加入維他命D3後也會有減緩傷害的效果。綜合以上的實驗結果,心臟缺血再灌流的情形下,粒線體分裂以及過量的粒線體自噬會促進細胞死亡,而維他命D3可以透過這條途徑減緩細胞凋亡的現象。 Myocardial infarction is a leading cause of morbidity and mortality worldwide. Early reperfusion is the best strategy for the rescue of ischemic myocardium. However, myocardial reperfusion leads to the excessive production of reactive oxygen species, the activation of apoptotic pathways, and the induction of mitochondrial dysfunction, all of which contribute to postischemic cardiomyocyte death and exacerbate cardiac injury. Mitochondrial dynamics (mitochondrial fission and mitophagy) are crucial to the control of cell survival in cardiac I/R injury. Novel pharmacological or molecular interventions mitigating reperfusion injury, adjunctive to current reperfusion therapies, are in great need. The vitamin D3 regulates the cardiovascular function, but its physiological contribution in the reperfused heart, particularly its influence on mitochondrial homeostasis, is unknown. To mimic myocardial I/R injury, we established a hypoxia/reoxygenation (H/R) model in H9C2 cells in vitro. H/R treatment significantly decreased the cell viability of H9c2 cells by MTT assay. H/R condition also increased apoptosis by TUNEL assay and caspase 3 activation. In addition, H/R treatment increased mitochondrial fission demonstrated by the increased expression of dynamin related protein 1 (p-Drp1) and mitochondrial fission factor (mff) as well as mitochondrial translocation of Drp1. H/R condition triggered excessive mitophagy, as demonstrated by the increase of the expression of LC3BII/I and BNIP3 (BCL2 Interacting Protein 3) by western blot, the number of autolysosomes by acridine orange staining and ultrastructure by transmission electron microscopy. In contrast, vitamin D3 reversed these effects. In I/R mouse model, mice were subjected to 30 minutes of ligating the left anterior descending coronary artery, followed by 3 hours of reperfusion (I/R group). The I/R group showed higher apoptotic cells, mitochondrial fission and mitophagy. Vitamin D3 treatment showed decreasing trends in apoptosis, mitochondrial fission and mitophagy. Based on these findings, the I/R could increase mitochondrial fission and excessive mitophagy to induce cell death while vitamin D3 treatment can attenuate the cell apoptosis via these pathways. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70458 |
DOI: | 10.6342/NTU201802975 |
全文授權: | 有償授權 |
顯示於系所單位: | 解剖學暨細胞生物學科所 |
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