Notingher I, Verrier S, Haque S, Polak J M, Hench L L
Imperial College of Science, Technology and Medicine, Department of Materials, South Kensington Campus, London SW7 2BP, United Kingdom.
Biopolymers. 2003;72(4):230-40. doi: 10.1002/bip.10378.
The noninvasive analysis of living cells grown on 3-dimensional scaffold materials is a key point in tissue engineering. In this work we show the capability of Raman spectroscopy for use as a noninvasive method to distinguish cells at different stages of the cell cycle and living cells from dead cells. The spectral differences between cells in different stages of the cell cycle are characterized mainly by variations in DNA vibrations at 782, 788, and 1095 cm(-1). The Raman spectrum of dead human lung derived (A549 line) cells indicates the breakdown of both phosphodiester bonds and DNA bases. The most sensitive peak for identifying dead cells is the 788 cm(-1) peak corresponding to DNA Obond;Pbond;O backbone stretching. The magnitude of this peak is reduced by 80% in the spectrum of dead cells. Changes in protein peaks suggest significant conformational changes; for example, the magnitude of the 1231 cm(-1) peak assigned to random coils is reduced by 63% for dead cells. The sharp peak of phenylalanine at 1005 cm(-1) drops to half, indicating a decrease of stable proteins associated with cell death. The differences in the 1190-1385 cm(-1) spectral region also suggest a decrease in the amount of nucleic acids and proteins. Using curve fitting, we quantify these spectral differences that can be used as markers of cell death.
对生长在三维支架材料上的活细胞进行无创分析是组织工程中的一个关键点。在这项工作中，我们展示了拉曼光谱作为一种无创方法区分细胞周期不同阶段的细胞以及活细胞与死细胞的能力。细胞周期不同阶段的细胞之间的光谱差异主要由782、788和1095 cm⁻¹处DNA振动的变化来表征。人源肺（A549系）死细胞的拉曼光谱表明磷酸二酯键和DNA碱基均发生了断裂。用于识别死细胞的最敏感峰是对应于DNA O键；P键；O主链拉伸的788 cm⁻¹峰。在死细胞光谱中，该峰的强度降低了80%。蛋白质峰的变化表明存在显著的构象变化；例如，归属于无规卷曲的1231 cm⁻¹峰的强度在死细胞中降低了63%。苯丙氨酸在1005 cm⁻¹处的尖锐峰降至一半，表明与细胞死亡相关的稳定蛋白质减少。1190 - 1385 cm⁻¹光谱区域的差异也表明核酸和蛋白质的量减少。通过曲线拟合，我们对这些可作为细胞死亡标志物的光谱差异进行了量化。
