定量RT-PCR测定冠心病患者外周血中Toll样受体4的基因表达水平

Measurement of the Toll like receptor 4 gene expression levels in peripheral blood of the atherosclerotic by real-time quantitative RT-PCR

目的建立检测人Toll样受体4(Toll like receptor 4,TLR4)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TLR4的基因表达水平,探讨TLR4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TaqMan荧光探针技术,构建克隆载体pGEM-T-TLR4作为定量模板,建立实时荧光定量RT-PCR方法.在GeneAmp 5700型检测仪上测定了50例冠状动脉粥样硬化性心脏病患者和40名健康对照者外周血中TLR4 mRNA的含量.结果TLR4 mRNA在50例冠心病患者的表达范围为7.7×105～2.3×107拷贝/μg RNA;40名健康对照者的表达范围为9.8×104～5.5×106拷贝/μg RNA,冠心病组TLR4 mRNA的平均拷贝数显著高于健康对照组[(5.3±3.8)×106vs(1.8±1.4)×106;P＜0.01].而冠心病血脂正常组和血脂异常组的TLR4 mRNA平均拷贝数无明显差别[(5.1±4.7)×106 vs(5.4±3.0)×106;P＞0.05].结论成功建立了人TLR4基因表达含量的荧光定量检测方法;冠心病患者TLR4 mRNA的含量显著高于健康对照组,且其含量高低与血脂无关,TLR4及其介导的天然免疫应答与动脉粥样硬化的发生发展存在一定的相关性.

Objective To establish a real-time quantitative reverse transcription-pelymerase chain reaction（RT-PCR） method for detecting the expression of TLR4 gene in peripheral blood mononuclear cells（PBMCs） and explore the relationship between TLR4 mRNA expression and coronary arteriosclerosis disease（CAD）. Methods Based on fluorescent TaqMan methodology, a real-time quan- titative RT-PCR was set up. In this method, a cloning vector pGEM-T-TLR4 was constructed as a standard plasmid. The specific expression of TLR4 in 50 patients with CAD and 40 healthy controls were measured by using GeneAmp 5700 Sequence Detection Systems. Results The TLR4 mRNA copy number ranged from 7.7 ×10^5 - 2.3 ×10^7 copy/μgRNA in 50 CAD patients and 9.8 ×10^4 - 5.5 ×10^6 copy/μgRNA in 40 healthy controls. The mean TLR4 mRNA copy number in CAD group was significantly higher than that in controls（（5.3 ± 3.8） ×10^6 vs（1.8 ± 1.4） ×10^6 ; P 〈 0.01）. But no significant difference was found in the mean TLR4 mRNA copy number between CAD group with normal level of plasma lipids and the CAD group with abnormal level of plasma lipids（ （5.1 ± 4.7） ×10^6 vs （5.4 ± 3. 0） ×10^6 ; P 〉 0. 05 ）. Conclusion A real-time quantitative RT-PCR method for detecting the expression of TLR4 gene in PBMCs has been successfully established. The expression level of TLR4 is increased in atherosclerotie. And the mean TLR4 mRNA copy number has no correlation with the level of plasma lipids. TLR4 and the innate immune response mediated by it are associated with arteriosclerosis.