类风湿关节炎患者外周血单个核细胞IL-21R mRNA的检测及其临床意义

Detection and clinical significance of IL-21R mRNA in peripheral blood mononuclear cells of patients with rheumatoid arthritis

目的建立检测类风湿关节炎(RA)患者外周血单个核细胞(PBMC)IL-21R mRNA的巢式实时荧光定量PCR的方法,研究IL-21R mRNA在RA PBMC中的表达水平并探讨其临床意义。方法根据IL-21R mRNA序列,跨内含子设计外引物,采用巢式实时荧光定量PCR方法。首次扩增以增加原始模板浓度,并以减少引物、酶的浓度及循环次数的方法保持扩增片段的特异性,再以首次扩增产物为模板,进行二次PCR扩增作实时荧光定量检测;以β-actin作内参,以二者比值作为IL-21R mR-NA表达水平的指标。结果60例RA患者与30例健康人对照相比,RA患者组IL-21R mRNA表达上调(P<0.01);将RA患者关节功能分级作IL-21R mRNA表达水平比较,Ⅰ级、Ⅱ级相比无显箸差异(P(0.05);Ⅰ级、Ⅱ级分别与Ⅲ级以上相比,差异显著(P(0.01);RA活动期IL-21R mRNA水平较非活动期增高,治疗后IL-21R mRNA的表达明显下降,治疗前后比较有显著差异(P<0.01)。结论IL-21R mRNA在RA患者PBMC中高表达,其表达水平对RA的诊断和治疗具有重要的参考价值。本文建立的检测方法重复性好,敏感且特异,可作为一种常规检测手段应用于临床。

Objective To establish a nested-real-time-PCR for the detection of IL-21R mRNA in peripheral blood mononuclear cells （PBMC） of patients with rheumatoid arthritis, and explore its clinical significance. Methods The outer primers of span intron and inner primers were designed for nested-real-time-PCR. To increase specific template, the concentration of primers and enzyme, cycle times were decreased in the first amplification, and the real-time fluorescent quantity assay was performed in the second amplification. The β- actin gene was used as internal reference, and the results were presented as the ratios of IL-21R mRNA to β-actin mRNA. Results IL- 21R mRNA was detected in 60 patients with RA and 30 healthy controls. The expression of IL-21R mRNA gene in the patients with RA was increased, and the statistical analysis has significant differences （ P 〈 0. O1 ）. According to the grades of joint function in RA pa- tients, the expression of IL-21R mRNA has no significant differences between the grade I and grade II （P 〉 0.05）, but a significant difference was found in the patients of grade III compared with those of grade I and II （P 〈0. O1 ）. The expression levels of IL-21R mRNA in active patients was higher than those in non-active patients. After treatment, the expression of IL-21R mRNA was lower obvi- ously, and the difference was significant （P 〈 0.01 ）. Conclusions Nested-real-time-PCR is a simple, repeatable, high specific and sensitive method for the detection of IL-21R mRNA in human PBMC. The expression level of IL-21R mRNA may be valuable for diag- nosis of RA and analysis of therapeutic effect.