上海交通大学学报（医学版）

›› 2009, Vol. 29 ›› Issue (11): 1324-.

• 论著（基础研究） • 上一篇    下一篇

新蛋白质合成介导知母活性成分对M2受体稳定性的调节作用

张永芳, 夏宗勤, 胡雅儿   

  1. 上海交通大学 基础医学院细胞调控研究室, 上海 200025
  • 出版日期:2009-11-25 发布日期:2009-11-24
  • 通讯作者: 胡雅儿, 电子信箱: yaerhu@shsmu.edu.cn。
  • 作者简介:张永芳（1976—）, 女, 讲师, 博士；电子信箱: zhangyongfang1@yahoo.com。
  • 基金资助:

    上海市卫生局基金（054038）

ZMS regulation of M2 muscarinic receptor stability mediated by de novo synthesis of protein

ZHANG Yong-fang, XIA Zong-qin, HU Ya-er   

  1. Research Laboratory of Cell Regulation, Basic Medical College, Shanghai Jiaotong University, Shanghai 200025, China
  • Online:2009-11-25 Published:2009-11-24
  • Supported by:

    Shanghai Municipal Health Bureau Foundation, 054038

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摘要：

目的 探讨知母活性成分ZMS提高CHOm2细胞毒蕈碱样乙酰胆碱2型受体 (M2受体) mRNA表达的机制。方法 体外培养至80%～90%融合的CHOm2细胞分为ZMS 1组（单纯添加1×10-5 mol/L ZMS作用24 h）、ZMS 2组（添加1×10-5 mol/L ZMS作用24 h后加入1 μg/mL环己酰亚胺作用12 h）、ZMS 3组（加入1 μg/mL 环己酰亚胺预处理4 h后加入1×10-5 mol/L ZMS作用24 h），以上各组分别设立对照组（以等体积DMSO替代ZMS，其他处理与相应ZMS组相同）。各组细胞培养基中均加入放线菌素D抑制mRNA合成，于不同时间点收集CHOm2细胞，Real-time PCR检测M2受体mRNA相对表达量并计算半衰期。结果 与相应对照组比较，ZMS 1组和ZMS 2组CHOm2细胞M2受体mRNA的半衰期明显延长，分别为（4.75±0.54）h vs（2.13±0.23）h和（5.43±1.13）h vs (2.46±0.09) h（均P<0.05）；添加1 μg/mL 环己酰亚胺预处理的ZMS 3组CHOm2细胞M2受体mRNA半衰期的(3.06±0.23)h与其相应对照组的(3.00±0.20)h比较，差异无统计学意义（P>0.05）。结论 ZMS提高M2受体mRNA的稳定性需要有新蛋白质合成的参与。

关键词: 知母活性成分, CHOm2细胞, 毒蕈碱样乙酰胆碱2型受体mRNA, Real-time PCR, 蛋白质合成抑制剂

Abstract:

Objective To explore the mechanism of ZMS regulation of M2 muscarinic receptor mRNA expression. Methods In vitro cultured CHOm2 cells were divided into ZMS 1 group (treatment with 1×10-5 mol/L ZMS for 24 h), ZMS 2 group (treatment with 1×10-5 mol/L ZMS for 24 h and 1 μg/mL cycloheximide for 12 h) and ZMS 3 group (treatment with 1 μg/mL cycloheximide for 4 h and 1×10-5 mol/L ZMS for 24 h), and their corresponding control groups were also established (substitution of ZMS by DMSO). Actinomycin D was added to cultured CHOm2 cells of each group to inhibit the synthesis of mRNA. CHOm2 cell samples were taken at different time points, the relative expression of M2 receptor mRNA was detected by Real-time PCR, and half life of M2 receptor mRNA was calculated. Results Compared with corresponding control groups, the half life of M2 receptor mRNA of CHOm2 cells in ZMS 1 group and ZMS 2 group was significantly prolonged [(4.75h±0.54) h vs (2.13±0.23) h, P<0.05; (5.43±1.13) h vs (2.46±0.09) h, P<0.05].There was no significant difference in half life of M2 receptor mRNA of CHOm2 cells between ZMS 3 group and its corresponding control [(3.06±0.23) h vs (3.00±0.20) h, P>0.05]. Conclusion De novo protein synthesis is required for the enhancement of M2 receptor mRNA stability regulated by ZMS.

Key words: ZMS, CHOm2 cells, muscarinic M2 receptor mRNA, Real-time PCR, protein synthesis inhibitor