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

Objective To establish a real-time quantitative reverse transcription-polymerase 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 quantitative 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;P0.01). 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;P0.05). 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 atherosclerotic. 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.