新型荧光薄层层析-分光光度计测定细胞内GCS酶活性
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国家自然科学基金资助项目(30770666)


A new approach to evaluate cellular enzyme activity of glucosylceramide synthase based on fluorescent thin-layer chromatography and spectrophometer
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    摘要:

    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5 μmol/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-

    Abstract:

    Objective:To establish a new approach to evaluate cellular enzyme activity of glucosylceramide synthase(GCS) accurately by fluorescent thin-layer chromatography and spectrophometer,and decide its role in drug-resistant cancer cells. Methods:Using fluorescent NBD C6-ceramide,GlcCer/Cer and ceramide glycosylation of drug-resistant breast cancer cell(MCF-7-AdrR)was measured under different concentrations of NBD C6-Ceramide and increasing treatment time. Radioenzymatic assay were performed on these cells as control method. Protein expressions of GCS and P-gp were assessed by Western-blot analysis and immuno-fluorescence staining. Results:These data indicated that 5 μmol/L of NBD-C6-Ceramide was the optimal concentration,and GCS activity increased with time delay. This method has been used to accurately evaluate the levels of GCS enzyme in drug-resistant cells,which is consistent with radioenzymatic assay. Ceramide glycosylation in MCF-7-adrR and MCF-7-adrR/GCS cells was dramatically increased compared with that in MCF-7 cells(P < 0.01),while the level of glycosylation in MCF-7-AdrR/asGCS was much lower than MCF-7-adrR(P < 0.01). Conclusion:The cell-based fluorescent method is direct,reproducible for assessing ceramide glycosylation. It was applicable to validate GCS activity in drug-resistant cancer cells and would further help to measure GCS activity in vivo.

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殷冬梅,贾 辛,杨 博,祝 佳,沈 勤.新型荧光薄层层析-分光光度计测定细胞内GCS酶活性[J].南京医科大学学报(自然科学版),2011,(5):715-719

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  • 收稿日期:2010-11-16
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