基于差异基因表达谱的慢性乙型肝炎病证结合研究

Study of Chronic Hepatitis B Based on Differential Gene Expression Profile by Method of Disease Integrated with Traditional Chinese Medical Syndrome

【目的】通过检测分析慢性乙型肝炎(简称乙肝)典型证候的差异基因表达谱,探讨乙肝中医证候分型与基因表达之间的关联。【方法】采集乙肝患者和健康者的外周血样本,提取白细胞中的总RNA;运用基因芯片技术检测基因表达,并对其表达谱进行比较分析;使用实时定量RT-PCR验证部分基因表达。【结果】乙肝患者与健康者之间以及乙肝肝郁脾虚证、湿热蕴结证和肝肾阴虚证3个证候的患者之间的基因表达谱均存在明显差异。对3个证候组相对于健康组的差异表达基因进行差异基因、聚类、功能(gene ontology,GO)分析和信号传导通路(Pathway)分析发现,肝郁脾虚证单独调变的基因主要与细胞动力学过程等有关;湿热蕴结证单独调变的基因主要与脂质储存的正向调节等有关;肝肾阴虚证单独调变的基因主要与氮氧化合物合成酶调节物活性等有关。采用实时定量RT-PCR验证部分基因的表达,结果与基因芯片检测结果趋势基本一致。【结论】乙肝肝郁脾虚证、湿热蕴结证和肝肾阴虚证患者白细胞具有特征性的基因表达谱和显著性差异表达基因,提示乙肝中医临床辨证分型具有分子生物学物质基础。

Objective To investigate the differential gene expression in chronic hepatitis B （CHB） patients with typical syndromes of traditional Chinese medicine （TCM） syndromes, and to explore the relationship between TCM syndromes and gene expression. Methods Peripheral blood samples were collected from CHB patients and healthy volunteers before treatment. After total RNA of leukoeytes was isolated, the gene expression profiles were detected by microarray. The expression levels of partial genes were tested by real-time quantitative reverse transeription-polymerase chain reaction （RT-PCR） . Results Mieroarray analysis results showed that there were significant differences of gene expression between CHB patients and healthy volunteers, and among CHB patients with the syndrome of liver-qi stagnation and spleen deficiency, syndrome of damp-heat accumulation, and syndrome of liver-kidney yin deficiency. The results of gene ontology （GO） and signal pathway analysis between the healthy control and CHB patients with various syndromes showed that the specially-regulated genes of CHB patients with liver-qi stagnation and spleen deficiency were mainly related to cytokinetic process, those in patients with dampness-heat accumulation were mainly related to the positive regulation of lipid storage, and those of patients with liver-kidney yin deficiency were mainly related to the activities of nitric oxide synthase regulator. The real time RT-PCR for partial genes presented the similar results with those of gene microarray. Conclusion There are specific expression profiles of differential genes and significant differential genes in CHB patients with the syndrome of liver-qi stagnation and spleen deficiency, syndrome of damp-heat accumulation, and syndrome of liver-kidney yin deficiency, which may be the molecular foundation for the classification of TCM syndromes of CHB patients.