Expression And Detection Level Of Significance Of IL-21RmRNA In The Peripheral Blood Mononuclear Cells In Patients With Rheumatoid Arthritis

Objective To establish a Nested-Real-Time-PCR method for the detection of IL-21RmRNA in human peripheral blood mononuclear cells(PBMC),(1)detect IL-21RmRNA in patients with Rheumatoid Arthritis and explore clinical significance; (2)explore the role of IL-21RmRNA in RA's bone destruction,further reveal the pathogenesis of RA;(3) explore the relevance of IL-21RmRNA with IL-21(interleukin-21)in RA patients' serum;(4) explore IL-21RmRNA in the detection of the expression level and its significance in RA patients with different TCM syndrome types.Method Design outer primers by span intron,design inner primers,and apply Nested-Real-Time-PCR.To increase specific template,we decrease concentration of primers and enzyme and cycle times in the first PCR amplification,then detect Real-Time fluorescent quantity in the second PCR amplification.Theβ-actin gene was used as internal reference, and the results were presented as the ratios of IL-21RmRNA toβ-actinmRNA.At the same time,detect the level of IL-21 in RA's serum by a competitive enzyme-linked immunosorbent assay(ELISE) and number of CD4~+CD25~+ Regulatory T cell by using flow cytometry.Results To confirme IL-21RmRNA for the specific purpose of the fragments by the results of the sequencing analysis of PCR fragments.The value between Ct and the initial template concentration has a good linear relationship;the minimum concentration is up to 10~1 copies.The level of IL-21 in RA patients' serum is detected by ELISA,the correlation coefficient is 0.9984.To compare 60 cases of patients with RA with 30 case of healthy control grope,the expression of IL-21RmRNA and the level of IL-21 in patients with RA increase,the statistical analysis has significant differences(P＜0.01);According to joint function in patients with RA,the expression ofⅠlevel andⅡlevel IL-21RmRNA and the level ofⅠlevel andⅡlevel IL-21 have no significant differences(P＞0.05).To compare withⅢ,Ⅰlevel,Ⅱlevel different significantly(P＜0.01);To compare with non-active,the expression of IL-21RmRNA and IL-21in active patients is higher;After treatment,the expression of IL-21RmRNA and IL-21 is lower obviously,The difference is significant (P＜0.01).TCM syndrome compared each other were no significant differences.The expression level of IL-21RmRNA in human peripheral blood mononuclear cells(PBMC),the content of IL-21 in RA serum and the number of peripheral blood CD4~+CD25~+T lymphocytes do Pearson correlation analysis.The expression of IL-21RmRNA and the levels of IL-21 in RA serum correlate significantly,the both is not related to the number of peripheral blood CD4~+CD25~+T lymphocytes.Conclusions The expression of IL-21RmRNA and IL-21 in RA patients is significantly increased,the level of IL-21RmRNA and IL-21 is associated with the severity of RA,the detection of PBMC IL-21RmRNA and serum IL-21 in RA patients may contribute to the understanding of RA patients Immune function and help guide the treatment of RA.Nested-Real-Time-PCR for the detection of PBMC IL-21RmRNA and ELISA for serum IL-21 in RA patients are simple,repeatable,high specific and sensitive methods and can be used as means of testing used in clinical.