系统性红斑狼疮患者T淋巴细胞CD70基因表达及其启动子区域甲基化状态的研究

Expressions of CD70 mRNA and protein and methylation status of CD70 gene promoter in T cells frompatients with systemic lupus erythematosus

目的探讨系统性红斑狼疮（SLE）患者T淋巴细胞CD70mRNA和蛋白的表达及CD70基因启动子DNA甲基化的状态。方法分离15例活动期SLE患者、15例非活动期SLE患者和15例健康对照外周血CD4＋与CD8＋细胞，用实时定量逆转录PCR（RT-PCR）方法检测CD4＋和CD8＋细胞CD70mRNA转录水平，流式细胞仪检测CD4＋CD70＋细胞和CD8＋CD70＋细胞百分率，亚硫酸氢钠基因测序法检测CD4＋细胞和CD8＋细胞CD70基因启动子区域甲基化水平。组间比较采用单因素方差分析，组间两两比较采用SNK-q检验。结果①活动期、非活动期SLE患者CD4’细胞CD70mRNA转录水平分别为0．82±0．12和0．73±0．11，明显高于健康对照组（O．45±0．09），F=53．017，P〈0．01，活动期SLE患者CD4细胞的CD70mRNA转录水平显著高于非活动期SLE患者（P〈0．05）。活动期、非活动期SLE患者外周血CD4＋CD70＋细胞百分率分别为80．30％±11．04％和66．80％±3．98％，明显高于健康对照组（12．48％±3．45％），F=311．517，P〈0．01，活动期SLE患者CD4＋CD70＋细胞百分率显著高于非活动期SLE患者（P值〈0．05）。SLE患者外周血CD70＋CD4＋细胞百分率与SLE疾病活动度呈显著正相关（r=0．792，P=0．000）。活动期、非活动期SLE患者组的CD4＋细胞CD70基因启动子序列-60～－300bp区域平均甲基化水平分别为0．32±0．05和0．36±0．05，明显低于健康对照组（0．62±0．05），F=152．64，P〈0．01，活动期平均甲基化水平明显低于非活动期SLE患者组（P〈0．05）。结论CD4＋细胞CD70基因启动子区域处于低甲基化状态，这种低甲基化状态可能是CD70过度表达的直接原因。

Objective To detect the expressions of CD70 mRNA and protein and to determine the methylation status of CD70 gene promoter in T cells from patients with systemic lupus erythematosus （SLE）. Methods Peripheral blood CD4＋ and CD8＋ T cells were isolated from 15 patients with active SLE, 15 patients with inactive SLE and 15 healthy control subjects. Real-time quantitative reverse transcription-PCR was carried out to quantify the mRNA expression of CD70, flow cytometry to determine the frequency of CD4＋CD70＋and CD8＋ CD7＋ T cells, and bisulfite sequencing to evaluate the methylation status of CD70 gene promoter in CD4＋ and CD8＋ T cells. Differences in these parameters among these groups were analyzed by one-factor analysis of variance and SNK-q test. Results Compared with the healthy controls, the patients with active SLE and inactive SLE showed a significant increase in CD70 mRNA expression in CD4＋ T cells （0.82 ± 0.12 and 0.73 ± 0.11 vs. 0.45 ±0.09, F = 53.017, P 〈 0.01 ） and in the frequency of CD70＋CD4＋ T cells （80.30% ：1：11.04% and 66.80%± 3.98% vs. 12.48% ±3.45%, F = 311.517, P 〈 0.01）. Also, the expression of CD70 mRNA in CD4＋T cells and the frequency of CD70＋CD4＋ T cells were significantly higher in patients with active SLE than in patients with inactive SLE （both P 〈 0.05）. There was a positive correlation between the frequency of peripheral CD70＋CD4＋ T ceils and disease activity in SLE in these patients （r = 0.792, P 〈 0.01）. The average methylation index of the region between -600 bp and -300 bp of CD70 gene promoter in CD4＋ T cells was 0.32 ± 0.05 and 0.36 ± 0.05 respectively in the patients with active and inactive SLE, significantly lower than that in the healthy controls （0.62 ：t： 0.05, F = 152.64, P 〈 0.01 ）, and the patients with active SLE showed a significantly lower methylation index than those with inactive SLE （P 〈 0.05）. Conclusions The CD70 gene promoter in CD4＋T cells is significantly hypomethylated in natients with SLE. which may directly lead to the overexoression of CD70.