免疫相关性血细胞减少症患者CD4^＋T淋巴细胞中CD70表达水平及其启动子甲基化水平研究

Hypomethylation and overexpression of CD70 in CD4^＋ T cells of the patients with immuno-related pancytopenia

目的研究免疫相关性血细胞减少症（IRP）患者外周血CD4^＋T淋巴细胞CD70mRNA和蛋白的表达水平及CD70启动子甲基化水平，探讨CD70在IRP发病中的作用。方法选取35例IRP患者（初治组16例，恢复组19例）及15名正常对照，应用密度梯度离心法分离其外周血单个核细胞，免疫磁珠分选CD4^＋T淋巴细胞，应用实时定量PCR（RT-PCR）检测CD70mRNA相对表达水平，采用流式细胞术检测CD4^＋CD70^＋/CD4^＋细胞比例，采用甲基化特异性PCR（MSP）法检测CD4^＋T淋巴细胞CD70基因启动子区域甲基化水平。结果IRP患者外周血CD4^＋CD70^＋／CD4^＋细胞比例初治组[（7．46±1．51）％]高于恢复组[（5．95±1．34）％]和正常对照组[（1．83±0．60）％]，恢复组高于正常对照组（P值均〈0．05）；IRP初治组、恢复组及正常对照组CD70 mRNA的表达水分别为2.314（0．200～6．084）、1．021（0．135～3．434）及0．353（0．008～2．258），两两比较差异均有统计学意义（P〈0．05）。IRP患者CD70启动子甲基化水平低于正常对照组（P〈0．05）。IRP患者CD70表达率与CD5^＋B淋巴细胞比例呈正相关（r=0．533，P〈0．01）。结论CD4^＋T淋巴细胞CD70启动子区域低甲基化水平导致CD70的过表达可引起其免疫紊乱，可能在IRP的发病中发挥重要作用。

Objective To study the expression of CD70 and the methylation level of CD70 promoter in immuno-related pancytopenia （IRP） patients, and explore the role of CD70 in the pathogenesis of IRE Methods Thirty- five IRP patients and fifteen healthy donors were enrolled in this study. Peripheral blood mononuclear cells were isolated from venous blood by density gradient centrifugation, and CD4^＋ T cells were isolated by immunomagnetic beads. The mRNA level and the percentage of CD70 of CD4^＋ T cells were measured by real-time quantitative polymerase chain reaction （RT-PCR） and flow cytometry respectively. Methylation-Specific PCR （MSP） was performed to determine the methylation level of CD70 promoter. Results The percentage of CD70 expression on CD4^＋ T cells of untreated IRP patients [ （7.46± 1.51 ）% ] was significantly higher than that of recovered ones [ （5.95±1.34）% ] and normal controls [ （1.83 ±0.60）% ], and that of recovered IRP patients was significantly higher than of normal controls （P〈0.05）. The relative expressions of CD70 mRNA in CD4^＋ T cells were 2.314 （0.200-6.084）, 1.021 （0.135-3.434）, 0.353 （0.008-2.258） in three groups respectively. The differences among untreated IRP patients, recovered IRP patients and normal controls were significant （P〈0.05）. The CD70 promoter methylation level in CD4^＋ T cells of all IRP patients was significantly lower than that of normal controls（P〈0.05）. The expression of CD70 positively correlated to the ratio of CD5^＋ B cells （r=0.533, P〈0.01）. Conclusions The overexpression of CD70 may lead to immunologic disarrangement of patients, which may play important role in the pathogenesis of IRP.