【作者】 张永芳； 夏宗勤； 胡雅儿；

【Author】 ZHANG Yong-fang,XIA Zong-qin,HU Ya-er (Shanghai Jaotong Univweisity School of Medicine,Shanghai 200025,China)

【机构】 上海交通大学医学院；

【摘要】 目的:研究知母活性成分(ZMS)提高毒蕈碱样乙酰胆碱2型受体(M2受体)mRNA基因表达的机制。方法:以转基因的CHOm2细胞系为对象,在培养基中加放线菌素D抑制mRNA合成,不同时间取样用实时荧光定量RT-PCR方法测定M2受体mRNA的含量,根据其指数曲线的下降速率,算出其mRNA的半衰期,观察ZMS对半衰期的影响,并在此基础上,用环己酰亚胺(cycloheximide,CYC)抑制蛋白质的新生时,观察ZMS调节M2受体mRNA半衰期的影响。结果:无CYC时,ZMS使M2受体mRNA半衰期显著延长,与对照组相比,分别为4.75h±0.54h和2.13h±0.23h,P<0.05。在ZMS或溶媒作用前4h先加CYC,ZMS的作用消失,两组相比半衰期分别为3.06h±0.23h和3.00h±0.20h,P>0.05。但是如果让ZMS或溶媒先作用24h再加CYC,则不能消除ZMS的作用,两组的半衰期分别是5.43h±1.13h和2.46h±0.09h。结论:ZMS提高M2受体mRNA的稳定性需要有新的蛋白质合成。更多还原

【Abstract】 Aim The aim of this work is to study the elevation mechanism of ZMS on muscarinic M2 receptor mRNA expression.Methods Actinomycin D was addied to cultured CHOm2 cells to stop the de novo synthesis of M2 receptor mRNA and samples were taken at various times to determine the time course of mRNA of M2 receptor with real-time quantitative RT-PCR.Half-life of M2 receptor mRNA and the effect of ZMS on the half-life was obtained from the slope of the exponential curves.Cycloheximide was added at 4h prior to and 24h after the addition of ZMS to examine the effect of de novo protein synthesis on the action of ZMS.Results The half-life of m2 mRNA was prolonged by ZMS treatment without cycloheximide(4.75h±0.54h and 2.13h±0.23h for ZMS and vehicle treated groups,respectively,P<0.05).When cycloheximide was added to the culture medium 4h prior to the addition of ZMS,the effect of ZMS in prolonging the half-life of m2 mRNA disappeared(3.06h±0.23h and 3.00h±0.20h for cells with and without ZMS,respectively).However, when the ZMS was added to the medium 24h prior to the addition of cycloheximide,the action of ZMS was not abolished by cycloheximide(half-life was 5.43h±1.13h and 2.46h±0.09h for cells with and without ZMS,respectively).Conclusion These data suggest that de novo protein synthesis was required for the increase in M2 mRNA stability induced by ZMS.更多还原