关键词: 实时荧光定量PCR

Abstract:

Objective To assess the preferential expressions of peripheral blood T cell receptor beta chain variable region （TRBV） subfamilies in patients with psoriasis vulgaris（PV）, and to estimate their role in the pathogenesis of psoriasis. Methods Thirty-three upstream primers were designed to target the human functional TRBV genes, downstream primers to target the common T cell receptor beta constant （TRBC） gene, with T cell receptor alpha constant （TRAC） gene as the internal reference. Total RNA was extracted from the peripheral blood T cells of 10 health human controls and 10 patients with PV, and transcribed into cDNA. Then, TRBV genes were amplified by real-time fluorescence quantitative PCR （RFQ-PCR） and the fluorescence intensity of each samples was detected. The expression levels of TRBV genes in the control group were used to calculate the cut-off values （mean expression levels of TRBV subfamilies in the 10 normal controls + 3 standard deviations）. When the expression level of a TRBV subfamily from patients with PV was equal to or higher than the cut-off value, it was considered as the preferentially expressed TRBV subfamily. Results The threshold cycle （Ct） value varied from 21 to 24 for TRAC gene. The difference in the Ct value between TRBV subfamily genes and TRAC gene in patients with PV was 2.98 for TRBV2 gene, 3.24 for TRBV5-7 gene, 2.52 for TRBV6-6/6-9 gene, 2.04 for TRBV 12 gene, 3.56 for TRBV 24 gene, and 4.12 for TRBV 29 gene, and the expression levels of these subfamily genes were significantly higher than those in the normal controls （all P < 0.05）. According to the above standard, TRBV6-6/6-9, TRBV12 and TRBV29 were considered to be preferentially expressed subfamilies. Conclusions There is a preferential expression of TRBV gene subfamilies in peripheral blood of patients with psoriasis vulgaris, which may play a vital role in the abnormal T cell-mediated immune responses in psoriasis.

Key words: Real-time fluorescence quantitative polymerase chain reaction