siRNA抑制大鼠心肌细胞MagT1基因表达的研究

The study of gene expression of siRNA inhibited magT1 in rat cadiocyte

目的 Mg2+是活细胞中含量最丰富的二价阳离子之一,是多种酶的辅助因子,还是维持大分子有活性的构象,脂类和磷酸肌醇类第二信使的调控,多种离子通道和转运体的调控所必需的在哺乳动物细胞生命活动中起着重要作用。临床观察表明,心肌细胞内游离Mg2+浓度过低时会导致心律失常等现象。为了深入研究MagT1基因在心肌细胞中的功能,本研究试图确定沉默效果好的MagT1 siRNA,为后续实验奠定基础。方法体外分离大鼠心肌细胞,并检验心肌细胞的纯度;委托上海吉玛生物技术公司设计4对siRNA,与转染试剂混合并转染进大鼠心肌细胞,使用RT-PCR方法检测siRNA沉默MagT1基因的效果。结果经过RT-PCR的检测,发现4对siRNA中只有1对siRNA沉默效果最好,序列为5'-GAUUGGACUUGUUGUGUUATT-3';5'-UAACACAACAAGUCCAAUCTT-3'。结论找到了沉默效果好的siRNA,为使用RNA干扰方法深入研究MagT1的功能奠定了基础。

Objective Mg2＋is one of the most abundant cations in living cells which is cofactor of a variety of enzymes and maintained the conformation of the macromolecules activity ,regulated lipids and phosphoinositide second messenger.Mg2＋plays im-portant role in mammal′s life activity which is necessary to the regulation of many kinds of ion channels and transporters .Clinical ob-servation showed that arrhythmia phenomenon appeared when the concentration of Mg 2＋which in myocardial cells is low.In order to study the function of MagT 1 gene in myocardial cells deeply ,this study tried to determined the MagT 1 siRNA which with good silence effect,and to lay the foundation for follow -up study.Methods Isolated rat myocardial cells in vitro and checked the purity of myo-cardial cells ,entrusted Shanghai Jima Biotech Corp to designed four pairs of siRNA and mixed then dyed into rat cadiocyte ,tested the effect of siRNA silenced MagT 1 gene by the method of RT -PCR.Results Only one pairs of siRNA silence effect were found best though the test of RT -PCR,the sequence were 5′-GAUUGGACUUGUUGUGUUATT-3′;5′-UAACACAACAAGUCCAAUCTT-3′.Conclusion The siRNA that have better silence effect was found which laid the foundation for study the function of MagT 1 deep-ly by the method of RNA interference.