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用荧光探剂DPH分别标记药物敏感的肺腺癌细胞A549和抗顺铂药物的肺腺癌细胞A549/DDP, 对其膜脂物理状态的变化研究结果表明, 对顺铂药物敏感的A549的DPH各向异性值(P)为0.162, 而抗顺铂药物的A549/DDP者为0.194, 统计分析表明二者具有明显的差异. 当用可探测细胞膜脂双层不同层次的荧光探剂2-AS, 7-AS和12-AS进一步测定不同层次的膜脂质分子的流动性变化时, 结果表明: 分别反映膜表层和中层的2-AS和7-AS的各向异性值, 对敏感性的A549细胞分别为0.134和0.144, 具抗药性的A549/DDP细胞则分别为0.171和0.178. 而反映膜深层脂质分子变化的12-AS的各向异性值二者却无显著差异. 这提示, 两种细胞膜脂流动性的变化主要反映在膜的表层和中层. 同时, 用MC540荧光探剂标记两种细胞膜在FCM (flow cytometry)上测定反映膜脂质分子头部堆积程度差异的二维散点图及频数分布直方图的结果分析也表明, A549/DDP细胞膜脂质分子的有序性增加, 即流动性降低. 用气相色谱测量两株细胞膜脂肪酸的不饱和度, A549/DDP细胞膜脂肪酸的不饱和度明显低于A549细胞, 进一步肯定了上述结果. 结果提示, 在药物长期作用下, 膜脂物理状态的变化亦可能是肿瘤细胞具有抗药性的原因之一.
The fluorescent probe DPH was used to label drug-sensitive lung adenocarcinoma cell line A549 and anti-cisplatin drug lung adenocarcinoma cell line A549/DDP, respectively. The results of their changes in the membrane lipid state showed that DPH was sensitive to cisplatin-sensitive A549 cells. The anisotropy value (P) was 0.162, while the anti-cisplatin drug A549/DDP was 0.194. The statistical analysis showed that there was a significant difference between the two. When using detectable cell membrane lipid bilayers of different levels of fluorescent probe 2-AS , 7-AS and 12-AS further determined changes in the mobility of membrane lipid molecules at different levels. The results showed that the anisotropy values of 2-AS and 7-AS, respectively, reflecting the surface and middle layers of the membrane, were A549 cells were 0.134 and 0.144, respectively, and drug-resistant A549/DDP cells were 0.171 and 0.178, respectively. There was no significant difference in the anisotropy of 12-AS, which reflects changes in membrane deep lipid molecules. This suggests that The changes in the lipid fluidity of the two cell membranes are mainly reflected in the surface layer and the middle layer of the membrane. Meanwhile, the two cell membranes labeled with the fluorescent probe of MC540 were used to determine the two-dimensional dispersion reflecting the difference in the accumulation degree of the membrane lipid molecules on FCM (flow cytometry). Analysis of results of histogram and frequency distribution histogram The results showed that the order of lipid molecules in A549/DDP cells increased, that is, the fluidity was decreased. The unsaturation of fatty acids in cell membranes was measured by gas chromatography. The unsaturation of A549/DDP cell membrane fatty acids was significantly lower than that of A549 cells. The above results suggest that, under the long-term effects of drugs, changes in the physical state of membrane lipids may also be one of the reasons for the resistance of tumor cells.