Bilski P, Kukielczak B M, Chignell C F
Technology Planning & Management Corp, Research Triangle Park, NC, USA.
Photochem Photobiol. 1998 Nov;68(5):675-8.
In vivo, keratinocyte skin cells are exposed to photooxidative processes, some of which can be mediated by singlet molecular oxygen (1O2), a species that is very difficult to detect spectrally in cells. We photosensitized 1O2 in cultured HaCaT keratinocytes stained with rose bengal (RB) that localizes exclusively inside the keratinocyte hydrophobic regions, as evidenced by strongly red-shifted absorbance and intense fluorescence. We used keratinocytes grown in a monolayer on a plastic coverslip and in suspension. The phosphorescence spectrum (1200-1350 nm) from 1O2 was strongest when the coverslip containing RB-stained keratinocytes was irradiated in air. The spectral intensity decreased when the coverslip was immersed in D2O during irradiation and was almost completely quenched when it was irradiated while immersed in water. Water not only shortens the 1O2 lifetime but also reabsorbs part of the 1O2 phosphorescence, processes that do not occur when 1O2 is produced in a keratinocyte layer exposed to air. Because the RB was inside keratinocytes, singlet oxygen must also be produced inside the keratinocytes. However, the sensitivity to the extracellular environment suggests that most of the detectable 1O2 phosphorescence originates from those 1O2 molecules that escaped from the cell through its membrane into D2O or into the air, where 1O2 has longer lifetimes. Our results confirm directly that 1O2 is indeed photosensitized in living cells by RB. They also suggest that keratinocyte monolayers may be a good cell model to examine in vitro the production of 1O2 by other photosensitizers of environmental and photomedical interest.
在体内，角质形成细胞会暴露于光氧化过程中，其中一些过程可能由单线态分子氧（1O2）介导，而单线态分子氧是一种在细胞中很难通过光谱检测到的物质。我们用孟加拉玫瑰红（RB）对培养的HaCaT角质形成细胞进行光敏化处理，RB专门定位于角质形成细胞的疏水区域内，这一点可通过强烈的红移吸收和强烈的荧光得到证明。我们使用了在塑料盖玻片上单层生长以及悬浮生长的角质形成细胞。当含有RB染色角质形成细胞的盖玻片在空气中受到照射时，1O2的磷光光谱（1200 - 1350 nm）最强。当盖玻片在照射期间浸入重水（D2O）中时，光谱强度会降低，而当它浸入水中进行照射时，磷光几乎完全淬灭。水不仅会缩短1O2的寿命，还会重新吸收部分1O2的磷光，而在暴露于空气的角质形成细胞层中产生1O2时，这些过程不会发生。由于RB在角质形成细胞内部，单线态氧也必定在角质形成细胞内部产生。然而，对细胞外环境的敏感性表明，大多数可检测到的1O2磷光源自那些通过细胞膜从细胞逸出进入D2O或进入空气的1O2分子，在D2O或空气中1O2具有更长的寿命。我们的结果直接证实了1O2确实在活细胞中被RB光敏化。它们还表明，角质形成细胞单层可能是一个很好的细胞模型，用于在体外研究其他具有环境和光医学意义的光敏剂产生1O2的情况。
