荧光测定法体外快速筛选抗肿瘤活性物质的研究

Development of fluorometric assay for screening compounds with antitumor activity in vitro

目的:建立一种基于FDA荧光信号的体外快速筛选抗肿瘤活性物质的方法。方法:通过筛选合适的FDA浓度,建立了荧光测定法,并对该方法进行了线性范围和精密度的考察以及可靠性验证。应用本方法对两种药用植物的抗肿瘤活性部位进行了初筛。结果:该方法在68~1.5×10~4个/孔的范围内,荧光信号强度与细胞数之间具有良好的线性关系(R^2=0.996 7),板内精密度为10.9%。应用本方法检测得到盐酸阿霉素抑制MCF-7细胞增殖的IC_(50)值为3.04μg/mL,说明本方法用于抗肿瘤活性物质筛选是准确可靠的。经过本方法的筛选,获得了沙煲暗罗茎乙酸乙酯部位、沙煲暗罗茎石油醚部位和海南染木树石油醚部位抑制MCF-7细胞增殖的IC_(50)值分别为134.1μg/mL、78.46μg/mL和86.87μg/mL。结论:本方法快速、灵敏、细胞线性范围宽,可用于天然产物抗肿瘤活性物质的体外快速高通量筛选。

Objective：To develop a FDA fluoresesce siginal-based method for quick screening of antitumor compounds in vitro. Method：A fluorometric assay was established through selecting the appropriate FDA concentration. The liner range,accuracy,and reliability of this method were evaluated. Preliminary study on the antitumor activity of the extracts of two medicinal plants was done using the fluorometric assay. Results：The linearity of this method for cells stained with FDA was validated（R^2=0.996 7）in the range of 68 ~1.5 ×10~4 per well,and the in-plate precision was 10.9%. The IC_（50） value obtained for MCF-7 cells on doxorubicin hydrochloride was 3.04 μg/mL by this method,thus the fluorometric assay was reliable. Screening with this fluorometric assay,the acetic ether fraction and petroleum ether fraction from the stem of Polyalthia oblique,the petroleum ether fraction from the leaves of Saprosma hainanense showed antitumor activity,the IC_（50） values were 134.1,78.46 and 86.87 μg/mL,respectively. Conclusion：The fluorometric assay provided high sensitivity and wide linear range,can be used in screening of antitumor compounds from natural products in vitro.