臺灣博碩士論文加值系統

心臟在缺血狀態下心肌會產生凋亡，缺血後所產生的氧化壓力以及鈣離子超載是缺血誘導心肌凋亡的主要原因，我們假設KMUP-1可以減少缺血後所產生的氧化壓力以及鈣離子超載，而具有心臟保護作用。本論文利用低氧模式並且去除血清以及葡萄糖以模擬體內心肌缺氧之生理環境，經由去氧核醣核酸裂片(DNA fragmentation)的產生以及Hoechst 33342 陽性反應得知，心肌細胞在此低氧模式下會產生細胞凋亡。由本實驗結果發現：KMUP-1可以減少低氧模式所造成的細胞凋亡，而且在正常氧壓下，KMUP-1對H9c2並沒有明顯毒性；KMUP-1能減少一些誘導細胞產生凋亡的因子，例如：DNA fragmentation的產生、活性氧化物(ROS)的生成、以及Bcl-2/Bax蛋白質比值的下降；KMUP-1會增加eNOS表現並且促進一氧化氮(NO)的生成，KMUP-1也會活化sGC進而增加PKG表現，已知NO對心肌缺血現象有許多的益處，例如抑制Ca2+內流以及減少心臟細胞的耗氧量；KMUP-1可以增加低氧環境下磷酸化ERK表現量，並且減少磷酸化JNK與p38的表現量；此外，KMUP-1也可以防止脫脂酸磷脂醯膽鹼(LPC)引起的H9c2細胞毒性，並抑制LPC所引起的ROS生成、細胞內鈣離子上升、ERK磷酸化減少，以及p38磷酸化增加之作用。由以上結果得知KMUP-1對於低氧模式以及LPC模式所誘導的細胞凋亡現象有改善之效果，其心臟保護作用機轉是KMUP-1可能經由 MAPK訊息傳導路徑、NO-cGMP-PKG路徑，並且減少細胞內ROS的產生以及Ca2+超載之作用，而防止心肌細胞因缺氧或LPC所因起的細胞損傷。

Cardiac myocytes undergo apoptosis under condition of ischemia. Myocardial oxidative stress and Ca2+ overload induced by ischemia may be involved in the development and progression of myocardial apoptosis. We hypothesized that KMUP-1 could prevent the ischemia-induced reactive oxygen species production and Ca2+ overload, leading to decreased calcium-responsive signaling in myocardial dysfunction. We showed that serum/glucose deprivation and hypoxia, components of ischemia in vivo, resulted in apoptosis of H9c2 cardiomyoblasts. H9c2 cells apoptosis is evidenced by an increase in DNA ladder and Hoechst 33342 positive. In this model of simulated ischemia, represented by serum/glucose deprivation and hypoxia, KMUP-1 (0.1, 1, and 10 μM) could protect H9c2 cells against simulated ischemia-induced apoptosis. KMUP-1 also inhibited several of the molecular events of apoptosis that follow simulated ischemia, such as the DNA fragmentation, the production of ROS, and the ratio of Bcl-2 to Bax. Furthermore, KMUP-1 could modulate eNOS and enhances NO production. KMUP-1 also could stimulate soluble guanylate cyclase (sGC) and activate PKG. NO has been shown to exert a number of actions that would be expected to be beneficial during myocardial ischemia, including inhibition of Ca2+ inflow and a decrease in myocardiac calculated oxygen consumption. Moreover, our results suggested that KMUP-1 can not only increase the expression of ERK, but can also decrease the expression of JNK and p38. In addition, pretreatment with KMUP-1 could protect against LPC-induced cytotoxicity in H9c2 cells and the drug can also inhibit LPC-stimulated ROS production, intracellular calcium elevation, the decrease of ERK phosphorylation, and the increase of p38 phosphorylation. In conclusion, these results demonstrate that KMUP-1 can increase the survival rate of H9c2 cells in the hypoxia and LPC models. The cardioprotective effect of KMUP-1 may via MAPK and NO-cGMP-PKG pathway, decrease intracellular ROS production, and Ca2+ overload to prevent myocardial ischemic injury.

1. 中文摘要 --------------------------------- 1
2. 英文摘要 --------------------------------- 2
3. 縮寫表 ----------------------------------- 4
4. 緒論 ------------------------------------- 5
5. 研究材料 --------------------------------- 14
6. 研究方法 --------------------------------- 24
7. 研究結果 --------------------------------- 38
8. 討論 ------------------------------------- 45
9. 結論和未來展望 --------------------------- 51
10. 參考文獻 --------------------------------- 53
11. 附圖 ------------------------------------- 59

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