人周围血单核细胞载脂蛋白E基因表达的竞争性逆转录-聚合酶链反应定量分析

A Competitive Reverse Transcription-Polymerase Chain Reaction to Study Quantification of Apolipoprotein E Gene Expression in Human Monocyte-derived Macrophages

为建立人周围血单核细胞载脂蛋白E基因表达检测方法 ,研究载脂蛋白E基因与儿童健康的关系 ,我们抽取 2 6例健康儿童外周静脉血 ,分离血单核细胞 ,抽提RNA ,采用逆转录—聚合酶链式反应检测载脂蛋白E基因表达 ,并以正常人cDNA作定量标准物 ,待测样品与定量标准物共扩增 ,计算出待测样本的个体的mRNA量。研究发现载脂蛋白E基因能在健康儿童周围血单核细胞表达 ,健康儿童载脂蛋白E基因表达量为 0 .37± 0 .15mol/molmRNA。表明采用竞争性逆转录—聚合酶链反应方法来检测人周围血单核细胞载脂蛋白E基因表达的分析方法快速、简便、灵敏、实用、可靠 ,且能准确定量 ,值得推广应用。

Aim To study the relationship between the expression status of apolipoprotein E gene and children health,developed a new method for the quantification of apolipoprotein E mRNA in human monocyte-derived macrophages. Methods 10 mL venous blood was obtained from 26 health children.The mononuclear cells were isolated.Total RNA from the monocytes was extracted.Apolipoprotein E gene express were determined by reverse transcription-polymerase chain reaction. After synthesis of the internal apolipoprotein E DNA standard,we amplified internal standard DNA and cDNA of samples studied. Quantification of apolipoprotein E gene express was calculated. Results Apolipoprotein E mRNA was 0.37±0.15 mol/mol GAPDH mRNA in health children. Conclusions The competitive reverse transcription- polymerase chain reaction was a rapid,simple,sensitive method to study expression of apolipoprotein E gene. This method might be a valuable tool to assess apolipoprotein E gene expression in human disease.