定量RT-PCR测定冠心病患者外周血Siglec-1(CD169)的基因表达水平

Real-time quantitative RT-PCR in measuring Siglec-1 gene expression levels in peripheral blood mononuclear cells of patients with coronary heart disease

目的:建立检测人Siglec-1(sialic acid-binding immunoglobulin-like lectins,唾液酸结合的免疫球蛋白样凝集素1,CD169)mRNA含量的实时荧光定量RT-PCR的方法,并用来测定冠心病患者外周血单个核细胞(PBMCs)中Siglec-1的基因表达水平,探讨Siglec-1基因表达水平与冠状动脉粥样硬化发生发展的关系。方法:基于SYBR Green荧光标记技术,建立实时荧光相对定量RT-PCR方法,在ABI PRISM 7000荧光定量PCR仪上测定了57例冠状动脉粥样硬化性心脏病患者和38例健康对照者外周血中Siglec-1 mRNA的含量。生化常规测定所有入选者血脂水平。结果:冠心病组Siglec-1 mR-NA的平均拷贝数显著高于健康对照组,为健康对照组的3.23(1.28～8.11)倍(P<0.01);其中急性心肌梗死组(AMI)、稳定型心绞痛组(SA)和不稳定型心绞痛组(UA)分别为对照组的3.32(1.38～7.97)、2.56(0.88～7.42)、3.35(1.25～8.96)倍,而三组之间差异无统计学意义;冠心病血脂正常组和血脂异常组的Siglec-1 mRNA平均拷贝数无显著差异。结论:成功建立了人Siglec-1基因表达含量的实时荧光定量RT-PCR检测方法;冠心病患者Siglec-1 mRNA的含量显著高于健康对照组,且其含量高低与血脂无关;Siglec-1mRNA含量在冠心病患者外周血单核细胞上表达显著升高,说明冠心病患者外周血单核细胞已经发生巨噬细胞化,单核巨噬细胞介导的免疫炎症反应在冠心病发生发展过程中起重要作用。

Objective： To establish a real-time quantitative reverse transcription polymerase chain reaction （RT-PCR） method for examining the expression of sialic acid-binding immunoglobulin-like lectin-one （Siglec-1）gene in the peripheral blood mononuclear cells （PBMCs） in patients with coronary heart disease （CHD）,so as to explore the relationship between Siglec-1 mRNA expression and the development/progression of CHD. Methods： Eased on the fluorescent SYBR Green method, a real-time quantitative RT-PCR was set up. The expression of Siglec-1 gene in the PBMCs of 57 patients with CHD and 38 healthy controls were measured by ABI PRISM 7000 Sequence Detection Systems. The blood lipid levels were determined in all the subjects by routine biochemical examination. Results： The mean Siglec-1 mRNA copy was significantly higher in the CHD group than that in the healthy control group （mean 3. 23 folds, ranging 1. 28-8. 11 folds, P〈0. 01 ）. The expression in the acute myocardial infarction group,stable angina and unstable angina group were 3.32（1.38-7.97） ,2.56（0.88-7.42） ,and 3.35（1.25- 8.96） folds that of the control group, respectively. No significant difference was observed in the normalized Siglec-1 mRNA copy number between CHD group with normal level of serum lipids and abnormal level of serum lipids. Conclusion： A real time quantitative RT-PCR method for detecting the expression of Siglec-1 gene in PBMCs has been successfully established. The expression of Siglec-1 is dramatically increased in PBMCs in CHD patients.