刃天青还原法检测细粒棘球绦虫原头蚴总数

Detection of amount of protoscolices of Echinococcus granulosus with resazurin reduction method

目的建立刃天青还原法体外检测细粒棘球绦虫存活原头蚴总数的实验方法。方法根据刃天青在活细胞线粒体酶作用下被还原成强荧光物质的原理,将体外培养的原头蚴,用2mg/mL刃天青染色10h后,用荧光分光光度计(激发光波长在560nm,发射长590nm)测定荧光值,并利用激光扫描共聚焦显微镜形态观察,通过统计学方法建立活原头蚴总数与对应的荧光值之间的数学模型,用于存活原头蚴总数的检测。结果激光扫描共聚焦显微镜观察发现,死亡的原头蚴经刃天青染色没有荧光,活的原头蚴经刃天青染色发出红色荧光。活原头蚴总数与对应的荧光值呈线性关系,建立直线方程y=0.019x+6.453,在检验中将测得的荧光值(y)带入该公式即可获得活原头蚴总数,此数学模型相关系数r=0.994,P<0.01,说明每个浓度下活原头蚴总数与每个浓度对应的荧光值之间的相关系数r极显著。结论本研究建立了刃天青还原法体外快速检测细粒棘球绦虫存活原头蚴总数的实验方法,该方法可应用于抗包虫病药物体外筛选,具有避免人为误差、降低人力消耗等优点。

We aimed to establish a new experimental method to detect the total number of living protoscolices of Echinococ- cus granulosus based on the reduction characteristic of resazurin that the resazurin could emit strong fluorescence when reduced by mitochondrial enzyme. The fluorescence values of the protoseolices stained with resazurin was determined by fluorescence spectrophotometer （excitation wavelength at 560 nm, emission wavelength 590 nm）, and the morphology were observed by la- ser scanning confocal microscope. A mathematical model was established based on the relationship between the number of living protoscolices and the corresponding fluorescence values through the statistical method. Results of the laser scanning confocal microscope showed that the living protoscolices stained by resazurin emitted red fluorescence while the death one emitted little fluorescence. According to the relationship between the measured fluorescence values and corresponding protoscolices number, a linear equation y=0. 019x＋6. 453 was established, and the total numbers of living protoscolices could be obtained by putting the fluorescence value （y） into the formula. The correlation coefficient of the mathematical model was r=0. 994 and P〈0.01, which showed that the correlation coefficient r between living protoscolices number in each concentration and corresponding flu- orescence values of each concentration was very significant. This study established a rapidly experimental method, which based on the reduction characteristic of resazurin, to detect the total number of living protoscolices of Echinococcus granulosus in vitro. This method avoids artificial errors and reduce the hu- man consumption, and could be applied to screening anti-echi- nococcosis drugs in large scale in vitro.