重组酵母测评系统检测环境雌激素的影响因素探讨

STUDY ON INFLUENCING FACTORS OF THE RECOMBINANT YEAST ESTROGEN SYSTEM IN ENVIRONMENTAL ESTROGENS DETECTION

目的 :探讨重组酵母测评系统中影响 β -半乳糖苷酶活性的因素。 方法 :以乙烯雌酚为受试物 ,研究在检测环境雌激素的重组酵母测评系统中 ,振荡和非振荡培养、酵母扩增时间、受试物作用时间和裂解方式等对酶活性的影响。结果 :在非振荡培养条件下 ,可检出乙烯雌酚的雌激素活性 ,且与振荡培养条件下的雌激素活性差异无统计学意义(P >0 0 5 )。非振荡培养时 ,在 18～ 30h内 ,随酵母扩增时间的延长 ,酶活性逐渐增加 ,30h后 ,酶活性则随扩增时间的延长而逐渐降低 ;各剂量组的乙烯雌酚作用时间以 30h时酶活性最高 ,若将作用时间延长至 4 8h ,酶活性则明显下降 (P<0 0 1) ;在不同裂解方式下 ,各剂量组乙烯雌酚的酶活性大小顺序为 :液氮裂解 >Y -PER裂解 >彗星实验裂解液裂解 >SDS +氯仿 (CHCl3 )裂解 >超声破碎。结论 :重组酵母测评系统检测环境雌激素的优化组合为 :恒温培养箱中酵母扩增 2 4～ 30h ,受试物作用 30h ,Y -PER或液氮裂解酵母细胞。

Objective:To explore influencing factors of the recombinant yeast estrogen system on β-galactosidase activity.Methods:Diethylstilbestrol was used as tested substance.The effects of vibrating and non-vibrating incubation, yeast multiplying time, exposure time and lysis methods on β-galactosidase activity were studied.Results:By the means of non-vibrating incubation, the estrogen-like activity of Diethylstilbestrol could be detected at all dose tested and there were no significantly statistical differences compared with vibrating incubation(P>0.05).During the multiplying time of 18～30h,β-galactosidase activity was increased with the increasing of the time. On the contrary, β-galactosidase activity was gradually decreased after 30h. For exposure time, at the point of 30h,β-galactosidase activity was the highest, and the β-galactosidase activity significantly decreased when the exposure time was prolonged to 48h(P<0.01). Lysis methods had a great impact on the β-galactosidase activity. In this experiment, the lysis effect of the five methods from high to low was:liquid nitrogen >Y-PER> comet assay lysis solution> sodium lauryl sulfate+ chloroform>ultrasonic breakage.Conclusion:The optimal experimental conditions for the recombinant yeast estrogen system in environmental estrogens detection were that the yeast cells were multiplied under non-vibrating from 24h to 30h, the exposure time of tested substances was 30h, and yeast cells were broken by liquid nitrogen or Y-PER.