香煙萃取物誘發人類單核球細胞蛋白質羰基化機制之探討

The mechanism of cigarette smoke extract induces protein carbonylation in THP-1 cells

蘇磁慧(Tzu-Hui Su)

指導教授 ： 王祖興

中山醫學大學/醫學科技學院/生物醫學科學學系碩士班/碩士(2008年)

https://doi.org/10.6834/CSMU.2008.00037

摘要

香煙中含有許多氧化物、自由基及金屬離子，會對細胞造成氧化傷害，其中蛋白質羰基化作用為一典型之氧化傷害，會使蛋白質失去正常的結構及功能，因此偵測蛋白質羰基化的程度，可視為細胞受到氧化傷害的指標。本研究，利用香煙萃取物 (cigarette smoke extract, CSE) 處理人類單核球細胞 (THP-1)，以圓點轉漬法 (dot blot) 結合 2,4-dinitrophenyl hydrazine 技術，分析蛋白質羰基化表現的程度，探討香煙萃取物造成蛋白質羰基化之特性及機制。以 20% 的 CSE 處理 THP-1 細胞不同時間 (0.5 ~ 24 小時)，測量細胞蛋白質羰基化表現的程度，結果發現在 0.5 小時蛋白質羰基化表現程度最高，增加倍數分別為 9.0 ± 5.1 倍，處理 1、3、6、24 小時後，增加倍數隨之遞減，分別增加 5.7 ± 2.7、4.3 ± 1.0、3.3 ± 0.8 及 1.9 ± 0.5 倍。為進一步探討銅鐵離子及氧化壓力在 CSE造成蛋白質羰基化的角色，分別利用不同性質的銅鐵金屬離子螯合劑 (1,10-phenanthroline (PHE)、Neocuproline (NC)、N-acetyl-L-cysteine (NAC)、D-Penicillamine (D-Pen)、EDTA) 與抗氧化劑 (vitamin C、trolox、S-allyl-L-cysteine (SAC)、α-tocopherol) 前處理 THP-1 細胞後，再與 CSE 一起處理，結果發現，D-Pen 與 NAC 具有顯著的降低蛋白質羰基化的表現 (P < 0.005)，其它金屬螯合劑及抗氧化劑則無明顯降低，推測 CSE 誘發蛋白質羰基化表現可能原因與細胞中銅離子釋放有密切關係，此外分離細胞中的細胞質與細胞核 (cell fractionation) 結合圓點轉漬法分析CSE 造成 THP-1 細胞誘發蛋白質羰基化表現在細胞內的分佈情形，發現羰基化發生在細胞核明顯高於細胞質蛋白質；西方墨點法分析更顯示，分子量在大於 100 kDa (100~211 kDa) 的細胞核蛋白質的羰基化表現最強烈。綜合上述結果推測，CSE 造成 THP-1 細胞蛋白質羰基化表現，可能與細胞內銅鐵離子及氧化壓力增加有密切關係，且細胞核可能是發生蛋白質羰基化主要的場所。

關鍵字

香煙萃取物；蛋白質羰基化；金屬螯合劑；抗氧化劑；細胞分離；圓點轉漬法

並列摘要

Cigarette consists of oxidants, free radicals, and metal ions that cause cellular oxidative damage. Carbonylation is one kind of cellular oxidative damage that makes proteins lose their correct conformation and functions. Therefore, the detection of carbonylation can be an indicator of the oxidative damage of proteins. In this study, we treated human monocyte (THP-1) with cigarette smoke extract (CSE) and then analyzed the carbonylation of the cells using dot blot with 2, 4-dinitrophenyl hydrazine. Cigarette smoking extract (20%) induced protein carbonylation over a wide range of time course in THP-1 cells was analyzed by immuno-dot blot assay. Maximum induction of protein carbonylation was observed at 0.5 h after cigarette smoke extract (CSE) exposure. The induction fold was 9.0 ± 5.1. The protein carbonylation levels showed a transitory decrease over 0.5 hour of treatment with CSE. The induction folds of protein carbonylation induced by 20% CSE were 5.7 ± 2.7, 4.3 ± 1.0, 3.3 ± 0.8, and 1.9 ± 0.5 at 1h, 3h, 6h, and 24h, respectively. We further characterized the metal-ion-mediated oxidative stress by testing whether metal ion chelators (10-phenanthroline (PHE), neocuproline (NC), N-acetyl-L-cysteine (NAC), D-penicillamine (D-Pen), and EDTA) or antioxidants (vitamin C (vit C), trolox, S-allyl-L-cysteine (SAC), and α-tocopherol) could block CSE-induced protein carbonylation in THP-1 cells. The results showed that D-Pen and NAC could significantly inhibit CSE-induced protein carbonylation in THP-1 cells, and the inhibition percentages were 82.7% and 78.6%, respectively. To determine the distribution and protein profiles in CSE-induced protein carbonylation in THP-1 cells, carbonylated proteins were analyzed by immuno-dot blot assay and Western blot combined with a cell fractionation system. The results showed that the CSE-induced protein carbonylation was much higher in nuclear subfraction than that in cytosolic one. The proteins with molecular weight of 100 kDa, 110-121, and 121-211 in nuclear subfraction exhibited high protein carbonylation signals.In conclusion, our findings suggest that copper-ion-mediated oxidant stress and the nuclear subfraction play an important role in CSE-induced protein carbonylation. To identify specific carbonylated proteins induced by CSE and its relationship with smoking-related diseases need further investigations.

並列關鍵字

cigarette smoke extract (CSE)；protein carbonylation；metal ion chelators；antioxidants；cell fractionation；dot blot

參考文獻

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