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標題: | 缺血-再灌流引起之心肌細胞內鋅離子釋放導致細胞凋亡機制之研究 Intracellular zinc release contributes to ischemia reperfusion-induced myocyte apoptosis |
作者: | Chien-Liang Lin 林建良 |
指導教授: | 陳瑞芬 |
關鍵字: | 細胞凋亡,缺血再灌流傷害,粒線體,鋅離子,心肌細胞, apoptosis,I/R injury,mitochondria,Zn2+,cardiomyocyte, |
出版年 : | 2012 |
學位: | 博士 |
摘要: | Part I:
氧化壓力 (oxidative stress) 和硝化壓力 (nitrosative stress) 都被認為參與了心肌缺血再灌流 (I/R) 所造成的傷害。本研究乃探討是否再灌流時引起之細胞內鋅離子的釋放所造成GSK-3B活化而導致心臟之傷害,而若螯合鋅離子的釋放則能保護心肌細胞。使用時間推移共軛焦顯微鏡 (time-lapse confocal microscopy) 與高親和力超氧化物 (O2-•) 和鋅離子 (Zn2+) 探針對心肌細胞作研究,我們這個研究是首先證明,缺血再灌流 (I/R)、活性氧 (reactive oxygen species) 與活性氮 (reactive oxygen species) 皆會造成顯著地細胞內O2-•增加,進而導致在細胞質和線粒體的Zn2+釋放出來。將心肌細胞置身於一種細胞穿透力強且高親和力之細胞內Zn2+螯合劑,TPEN,可以大大地中和掉Zn2+釋放後所造成的影響 (至少持續24小時),進而明顯地保護細胞,免於受到缺血再灌流、活性氧、活性氮,或鋅離子/鉀離子所誘導之心肌細胞凋亡。在成鼠的心臟實驗,TPEN及baicalein (一種強效的flavonoid) 可明顯保護I/R所造成的傷害。黃酮 (Flavonoids) 和U0126 (一種MEK1/2的抑製劑)在很大程度上抑制心肌細胞凋亡和TPEN敏感性之缺血再灌流或補充細胞內的鋅離子量會持續的誘導細胞外信號調節激酶1和2(ERK1/2) 磷酸化、肝醣合成酶激酶GSK-3B上的P-Ser9去磷酸化和P-Tyr216、GSK-3B和p53在細胞核積累及易位。沉默GSK-3B或p53基因表現是有心臟保護的效果的,顯示出ERK-GSK-3B-P53信號路徑的活化參與鋅離子敏感性的細胞死亡。此外,ERK依賴性的NOXA-髓細胞白血病-1 (MCL-1) 的途徑也參與其中,當沉默NOXA基因表現,會有心臟的保護,而U0126會明顯減少NOXA的表現和MCL-1降解。這樣的結果指出,細胞內鋅離子所造成GSK-3B的活化與心臟之I/R injury有關。TPEN總是相較於其他抑制劑對心臟的保護有更好的效果。因此,在再灌注開始時之急性上游鋅離子螯合劑的使用及天然產品的使用,如: 類黃酮,可能有益於心臟缺血再灌流損傷的治療。 Part II: 咖啡酸(CA)是一種天然酚類化合物,且大量存在藥用植物中。CA的具有多種生物效應,如抗細菌和抗腫瘤的生長。也有報導指出,在小鼠模式中,CA會引起前胃和腎腫瘤。在這裡,我們用兩種人類肺癌細胞株A549和H1299,釐清CA在癌細胞增殖中所扮演的角色。利用生長實驗顯示,CA可適度地促進肺癌細胞的增殖。此外,在處理抗癌藥物紫杉醇(PTX)前,先給予CA,可抑制PTX對癌細胞的毒殺效果。西方墨點法分析表明,CA可促進NF-kB的下游標的-促生存蛋白Survivin和Bcl-2的表現。這和CA誘導的NF-kBp65的核易位的觀察是一致的。我們的研究表明,CA是通過NF-kB信號路徑,而對於PTX治療的肺癌細胞有促細胞增值的作用。這可以提供癌症在化療阻抗性的有進一步的了解。 Part I: Oxidative stress and nitrosative stress are both suggested to be involved in cardiac ischemia-reperfusion (I/R) injury. This study investigate whether reperfusion-induced intracellular Zn2+ (Zn2+i) release activates GSK-3B, resulting in cardiac I/R-injury, and whether acute chelation of the Zn2+i release salvages the live myocardium. Using time-lapse confocal microscopy of cardiomyocytes and high affinity O2-• and Zn2+ probes, the present study is the first to show that I/R, ROS, and RNS all cause a marked increase in the [O2-•]i, resulting in cytosolic and mitochondrial Zn2+ release. Exposure to a cell-penetrating, high affinity Zn2+i chelator, TPEN, largely abolished the Zn2+i release and markedly protected myocytes from I/R-, ROS-, RNS-, or Zn2+/K+ (Zn2+i supplementation)-induced myocyte apoptosis for at least 24 hr after TPEN removal. In adult rat hearts, TPEN or baicalein (a potent flavonoid) again markedly inhibited cardiac I/R injury. Flavonoids and U0126 (a MEK1/2 inhibitor) largely inhibited the myocyte apoptosis and the TPEN-sensitive I/R- or Zn2+i supplement-induced persistent ERK1/2 phosphorylation, dephosphorylation of p-Ser9 on GSK-3B, and the translocation into and accumulation of p-Tyr216 GSK-3B and p53 in, the nucleus. Silencing of GSK-3B or p53 expression was cardioprotective, indicating that activation of the ERK-GSK-3B-p53 signaling pathway is involved in Zn2+i-sensitive myocyte death. Moreover, the ERK-dependent Noxa-Mcl-1 pathway is also involved, since silencing of Noxa expression was cardioprotective and U0126 abolished both the increase in Noxa expression and Mcl-1 degradation. Our results demonstrated that Zn2+i -sensitive activation of GSK-3B is involved in cardiac I/R injury. TPEN always provided more cardioprotection than the other tested inhibitors. Thus, acute upstream Zn2+i chelation at the start of reperfusion and the use of natural products, i.e. flavonoids, may be beneficial in the treatment of cardiac I/R injury. Part II: Caffeic acid (CA), a natural phenolic compound, is abundant in medicinal plants. CA possesses multiple biological effects such as anti-bacterial and anti-cancer growth. CA was also reported to induce fore stomach and kidney tumors in mouse model. Here we used two human lung cancer cell lines, A549 and H1299, to clarify the role of CA in cancer cell proliferation. The growth assay showed that CA promoted the proliferation of the lung cancer cells moderately. Furthermore, pre-treatment of CA rescues the proliferation inhibition induced by sub-IC50 dose of paclitaxel (PTX), an anticancer drug. Western blot showed that CA up-regulated the pro-survival proteins survivin and Bcl-2, the down-stream targets of NF-kB. This is consistent with the observation that CA induced nuclear translocation of NF-kB p65. Our study suggested that the pro-survival effect of CA on PTX-treated lung cancer cells was mediated through NF-kB signaling pathway. This may provide mechanistic insights into chemoresistance of cancer cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65374 |
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