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目的:建立高分辨率溶解技术定量检测系统性红斑狼疮患者血细胞中CD70启动子甲基化的方法,探讨CD70甲基化程度与系统性红斑狼疮发病的相关性。方法:319例系统性红斑狼疮患者及323例健康体检者外周血标本,抽提DNA后进行甲基化修饰。以CpGenome Universal Methylated DNA作为100%甲基化的样本,以100%非甲基化的健康体检者外周血DNA作为稀释剂,分别制成100%,80%,50%,30%,10%,0系列浓度的标准曲线;采用高分辨率溶解技术定量检测CD70甲基化水平。结果:100%,80%,50%,30%,10%,0甲基化标准品的高分辨率溶解曲线从右向左依次排列,待测样本在标准曲线上的位置即代表了其甲基化程度。高分辨率溶解技术重复性好,可检测低至10%甲基化水平。CD70甲基化水平与系统性红斑狼疮发病显著相关(P=0.00047)。结论:建立高分辨率溶解技术定量检测CD70甲基化程度的方法,CD70启动子低甲基化有望成为评估系统性红斑狼疮患病的分子指标。
OBJECTIVE: To establish a method for the quantitative detection of CD70 promoter methylation in blood cells of patients with systemic lupus erythematosus by high resolution lysis technique and to explore the correlation between the degree of CD70 methylation and the incidence of systemic lupus erythematosus. Methods: Peripheral blood samples were obtained from 319 patients with systemic lupus erythematosus and 323 healthy controls. DNA was extracted and methylated. Using 100% methylated CpGenome Universal Methylated DNA as a 100%, 100%, 80%, 50%, 30%, 10%, 100% 0 series of concentration of the standard curve; using high-resolution lysis technique for quantitative detection of CD70 methylation levels. Results: The high resolution curves of 100%, 80%, 50%, 30%, 10% and 0 methylated standards were arranged from right to left. The position of the sample to be tested on the standard curve represents its A Degree of High-resolution lysis technology reproducibility, can detect as little as 10% methylation level. The level of CD70 methylation was significantly associated with the onset of systemic lupus erythematosus (P = 0.00047). Conclusion: The establishment of a high-resolution lysis method to quantitatively detect the degree of CD70 methylation, CD70 promoter hypomethylation is expected to be a molecular index to evaluate the prevalence of systemic lupus erythematosus.