使用荧光探针二苯基-1-苯甲基膦估计活细胞的脂质过氧化。,Free Radical Biology and Medicine

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使用荧光探针二苯基-1-苯甲基膦估计活细胞的脂质过氧化。
Free Radical Biology and Medicine ( IF 7.1 ) Pub Date : 2001 Jul 15
Takahashi, M, Shibata, M, Niki, E

与苯氢过氧化物化学计量反应生成荧光产物DPPP氧化物的二苯基-1-苯甲酰基膦（DPPP）用作活细胞中脂质过氧化的荧光探针。DPPP已成功整合到U937细胞中。通过荧光显微镜确认了DPPP掺入细胞膜。通过监测细胞荧光强度的增加来检查DPPP与氢过氧化物的反应。发现脂溶性氢过氧化物例如亚油酸氢过氧甲酯优选与DPPP反应，而过氧化氢不与位于膜中的DPPP反应。观察到荧光强度的增加和施加到细胞上的亚油酸氢过氧甲酯的量之间的线性相关性。DPPP对细胞增殖影响不大，细胞活力或细胞形态至少3 d。DPPP的荧光产物DPPP氧化物在活细胞膜中至少稳定2 d。用马来酸二乙酯（DEM）或2,2'-偶氮双（2-ami基丙烷）二盐酸盐（AAPH）处理或以低血清含量培养后，测量DPPP标记的细胞的荧光。这些试剂和培养条件引起荧光的剂量和/或时间依赖性增加。维生素E的添加有效地抑制了荧光的增加。当用NO，H（2）O（2），AAPH和DEM处理DPPP标记的细胞和DCFH-DA标记的细胞以比较膜和胞质溶胶中过氧化物的形成时，观察到过氧化物形成的独特模式。

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Estimation of lipid peroxidation of live cells using a fluorescent probe, diphenyl-1-pyrenylphosphine.

Diphenyl-1-pyrenylphosphine (DPPP), which reacts with lipid hydroperoxides stoichiometrically to yield fluorescent product DPPP oxide, was used as a fluorescent probe for lipid peroxidation in live cells. DPPP was successfully incorporated into U937 cells. Incorporation of DPPP into the cell membrane was confirmed by fluorescence microscopy. Reaction of DPPP with hydroperoxides was examined by monitoring increase in fluorescence intensity of the cell. It was found that lipid-soluble hydroperoxides such as methyl linoleate hydroperoxide preferably react with DPPP, whereas hydrogen peroxide did not react with DPPP located in the membrane. Linear correlation between increase in fluorescence intensity and the amount of methyl linoleate hydroperoxide applied to the cell was observed. DPPP gave little effect on cell proliferation, cell viability or cell morphology for at least 3 d. DPPP oxide, fluorescent product of DPPP, was quite stable in the membrane of living cells for at least 2 d. Fluorescence of DPPP-labeled cells was measured after treating with diethylmaleate (DEM), or 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), or culturing with low serum content. These reagents and culture condition induced dose- and/or time-dependent increase in fluorescence. Addition of vitamin E effectively suppressed increase in fluorescence. When DPPP-labeled cells and DCFH-DA-labeled cells were treated with NO, H(2)O(2), AAPH, and DEM to compare the formation of hydoperoxides in the membrane and cytosol, distinct patterns of peroxide formation were observed. These results indicate that fluorescent probe DPPP is eligible for estimation of lipid peroxidation proceeding in the membrane of live cells, and use of this probe is especially advantageous in long-term peroxidation of the cell.

更新日期：2017-01-31

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