許多研究顯示一氧化碳 (carbon monoxide, CO) 與一氧化氮 (nitric oxide, NO) 為具有類似生物功能的氣體分子。本實驗探討一氧化氮和一氧化碳對 naïve rat pheochromocytoma (PC12) 細胞存活之相關性。
西方墨點法分析結果得知 PC12 細胞中，三種一氧化氮合成酶之蛋白質表現量低，而原血紅素氧化酶的蛋白質表現量高，尤其 heme oxygenase-1 (HO-1) 蛋白質表現量更高。投予 PC12 細胞一氧化氮清除劑 (carboxy-2-phenyl-4,4,5,5,-tetramethyl-imidazoline- 1-oxy-1-3-oxide, carboxy-PTIO，2 μmol) 或原血紅素氧化酶拮抗劑 (zinc protoporphyrinIX, ZnPP，25 nmol) 24 小時，藉由 WST-1 (4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) 測試分析粒線體中去氫酶的活性，進而反映出細胞逐漸死亡。另一方面，一氧化氮供給劑 (amino-3-morpholinyl-1,2,3- oxadiazolium chloride, SIN-1，1 μmol) 或 (S-Nitroso-N-acetylpenicill- amine, SNAP，1 μmol) 或一氧化碳前驅物 (hematin，500 nmol) 會增加 PC12 細胞的存活率。同時投予 SIN-1 (1 μmol) 和 carboxy-PTIO (2 μmol) 或 SIN-1 (1 μmol) 和 ZnPP (25 nmol) 各 24 小時，SIN-1 可以削減 carboxy-PTIO 或 ZnPP 對細胞造成的死亡。然而 nNOS 拮抗劑 (Nω-propyl-L-arginine, NPLA，100 pmol)、iNOS 拮抗劑 (S-methylisothiourea, SMT，10 nmol)、eNOS 拮抗劑 (N5-1-Iminoethyl-L-ornithine dihydrochlorid, L-NIO，4 nmol) 和一氧化氮前驅物 (L-arginine, L-Arg，2 μmol) 對 PC12 細胞存活之影響無顯著差異，且 L-Arg 和 carboxy-PTIO 同時投予，也不能改變 carboxy-PTIO 對細胞所造成的死亡。
綜合以上實驗結果得到結論是：內生性一氧化氮和一氧化碳貢獻於 naïve PC12 細胞中維持其生存。

Recent studies suggest that carbon monoxide (CO) is another gas molecule that has similar biological actions as nitric oxide (NO). The purpose of this study is to investigate the relationship between NO and CO in the survival of naïve rat pheochromocytoma PC12 cells.
Western blot analysis revealed that all three isoforms of nitric oxide synthase (NOS) exhibited low expression and two isoforms of heme oxygenase (HO), especially HO-1, exhibited higher expression in PC12 cells under basal condition. Exposure of PC12 cells for 24 h to the NO scavenger, carboxy-2-phenyl-4,4,5,5,- tetramethylimidazoline-1-oxy-1-3-oxide (carboxy-PTIO, 2 μmol) or HO inhibitor, zinc protoporphyrinIX (ZnPP, 25 nmol) resulted in a progressive reduction in mitochondria dehydrogenase activity reflected cell viability as determined by the WST-1 (4-[3-(4-lodophenyl)- 2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay. On the other hand, incubation with NO donors, amino-3-morpholinyl- 1,2,3-oxadiazolium chloride (SIN-1, 1 μmol) or S-Nitroso-N-acetyl- penicillamine (SNAP, 1 μmol), or the CO precursor, hematin (500 nmol), resulted in an elevation in cell viability. The progressive reduction in cell viability induced by carboxy-PTIO (2 μmol) or ZnPP (25 nmol) was significantly blunted by co-treatment with SIN-1 (1 μmol). However, incubation with the NO precursor, L-arginine (L-Arg, 2 μmol), or the selective inhibitors for nNOS, iNOS or eNOS, Nω-propyl-L-arginine (NPLA, 100 pmol), S-methylisothiourea (SMT, 10 nmol) or N5-1-Iminoethyl-L-ornithine dihydrochloride (L-NIO, 4 nmol) did not significantly alter cell viability. Co-treatment with carboxy-PTIO (2 μmol) and L-Arg (2 μmol) was also ineffective.
These results suggest that NO or CO contributes to the survival of naïve PC12 cells.

中文摘要………………………………………………………………..1

英文摘要………………………………………………………………..2

緒 論………………………………………………………………..3

研究目的……………………………………………………………….15

材料和方法…………………………………………………………….16

實驗結果………………………………………………………………..33

討 論………………………………………………………………..39

結 語………………………………………………………………..50

參考文獻………………………………………………………………..51

附 圖………………………………………………………………..59

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