Establishment Of The Nested-Real-Time-PCR For Detection Of Human Transcription Factor FOXP3mRNA Expression And Its Clinical Application In Dignosis Of Rheumatoid Arthritis

ObjectiveTo develop a Nested-Real-Time-PCR method for the detection of F0XP3mRNA in human peripheral blood mononuclear cells(PBMC), study the correlation of F0XP3mRNA with CD4~+CD25~+ Regulatory T cell, ratio of CD4 and CD8, Th1 and Th2, syndrome types of Traditional Chinese Medicine(TCM).MethodsRNA was isolated from PBMC and reversed transcription into cDNA as template for PCR. Design outer and inner primers by FOXP3 gene sequence. Detect F0XP3mRNA gene expression level in Nested-Real-Time-PCR using Taqman probe, detect number of CD4~+CD25~+ Regulatory T cell using flow cytometry and detect IFN- γ and IL-4 in plasma using Enzyme-Linked Immune Sorbent Assay (ELISA)at the same time.ResultsThe production of PCR was proved as specific FOXP3 segment by sequence assay. Total mean Ct(Cycle Threshold) in 6 tests was 14.66. The mean CV (coefficient of variation) was 5.81%. The standard curve showed a fine linear relationship between Ct and template concentration, and the correlation coefficient was 0.999. The sensitivity was 100 times higher than normal real-time PCR by reach 10° copy. FOXP3mRNA expression, number of CD4~+CD25~+ Regulatory T cell, ratio of CD4 and CD8(CD4/CD8), ratio of IFN-γ and IL-4(IFN-γ/IL-4) were detected in 31 Rheumatoid Arthritis(RA) patients and 10 health person. FOXP3mRNA expression, number of CD4~+CD25~+ Regulatory T cell, CD4/CD8 of RA patients were higher than health peason(P<0.01). The FOXP3mRNA expression and the number of CD4~+ CD25~+ Regulatory T cell were positive correlation, P=0.004. The 31 patients belongs...