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目的:通过建立一种新型的荧光薄层层析-分光光度法,精确而直接测定细胞内葡萄糖神经酰胺合酶GCS的活性,以评价其对耐药性肿瘤细胞的作用。方法:加入不同剂量的含荧光的NBD-C6-Ceramide,运用荧光薄层层析-分光光度法测定不同反应时间下人乳腺癌细胞MCF-7-AdrR中,葡萄糖神经酰胺(glucosylxeramide,GlcCer)和神经酰胺(ceramide,Cer)含量,计算两者的比值大小反映GCS酶活性情况,放射性酶学测定作为对照。Western-blot及免疫荧光分析MCF-7-AdrR及其转化细胞(MCF-7-adrR/GCS及MCF-7-adrR/asGCS)中GCS与P-糖蛋白(P-gp)的蛋白表达关系。结果:研究表明5μmol/L NBD-Cer是细胞内神经酰胺糖基化作用的最佳浓度,糖基化作用随时间延长而增强,此方法精确测定MCF-7-AdrR各细胞及其它耐药型细胞中的GCS酶活性,MCF-7-adrR与MCF-7-adrR/GCS较药物敏感组MCF-7的糖基化作用(GC/Cer)明显增强(P均<0.01),而在MCF-7-adrR/asGCS中糖基化作用急剧下降(P<0.01)。放射性酶学测定结果与之相符。Western-blot及免疫荧光显示GCS表达与耐药标志物P-gp表达在MCF-7-AdrR各细胞中呈明显相关。结论:不同于传统的同位素酶学测定,这种新型荧光薄层层析-分光光度法,是测定细胞内神经酰胺糖基化作用的一种简单易行、重复性好的实验方法,为进一步应用于耐药性肿瘤的体内实验奠定基础。
OBJECTIVE: To establish a new fluorescence thin-layer chromatography-spectrophotometry to accurately and directly determine the activity of intracellular glucosylceramide synthase GCS to evaluate its effect on drug-resistant tumor cells. Methods: Different concentrations of NBD-C6-Ceramide with different dosages were added. The fluorescence intensity of glucosylceramide (GlcCer) and the expression of GlcCer in MCF-7-AdrR cells were detected by fluorescence thin layer chromatography-spectrophotometry Ceramide (Cer) content, calculate the ratio between the two reflect the GCS enzyme activity, radioactivity determination as a control. The protein expression of GCS and P-glycoprotein (P-gp) in MCF-7-AdrR and its transformed cells (MCF-7-adrR / GCS and MCF-7-adrR / asGCS) was analyzed by Western- Results: The study showed that 5μmol / L NBD-Cer is the best concentration of intracellular ceramide glycosylation, glycosylation increases with time, this method accurate determination of MCF-7-AdrR cells and other drug-resistant GCS activity, MCF-7-adrR and MCF-7-adrR / GCS in MCF-7 cells significantly increased compared with MCF-7 cells (all P <0.01) Glycosylation abruptly decreased in 7-adrR / asGCS (P <0.01). Radiometric determination of results consistent with. Western-blot and immunofluorescence showed that the expression of GCS was correlated with the expression of P-gp, a marker of drug resistance, in all MCF-7-AdrR cells. CONCLUSION: Unlike traditional isotope enzyme assays, this new fluorescence thin-layer chromatography-spectrophotometry is a simple and reproducible experimental method for the determination of intracellular ceramide glycosylation. In vivo experiments for drug-resistant tumors to lay the foundation.