mRNA差异显示技术对环境诱发系统性红斑狼疮基因表达谱改变的研究

mRNA differential display for environment-induced changes in gene expression profiles of systemic lupus erythematosus

目的 比较环境因素诱发系统性红斑狼疮患者（SLE）与健康人外周血单个核细胞基因表达水平的差异.方法 选择5例无家族发病案例的SLE患者与5例健康人外周血单个核细胞,提取总RNA进行扩增、Cy3染色后,与Agilcnt 4X44K人全基因组芯片杂交,筛选差异表达基因进行基因实体（GO）分析和信息通路（pathway）分析.以qRT-PCR验证芯片结果.结果 5例SLE患者共同有2435个相对于健康人的差异基因表达（判断值=2.0）.GO分析结果表明,生物过程中差异表达基因占32.08%（512/1596）,其他比例较高的有细胞过程、生物调控、细胞代谢等;分子功能中差异表达基因占34.09%（544/1596）,其较高比例依次为结合、催化活性、传感活性和转录调节活性;细胞组分方面差异表达基因33.83%（540/1596）,主要涉及细胞与细胞部分两方面,其次为细胞器.Pathway分析显示免疫系统信号及B细胞受体通道中各有12个基因的表达发生了差异性改变,而T细胞受体、表皮生长因子受体1、IL-12介导的信号等通道均有10个基因表达明显差异.分析发现SLE患者在炎性反应与自身免疫相关基因方面有45个基因发生明显改变,其中11个上调,34个下调;细胞外基质和黏附分子方面有5个相关基因改变其中5个上调,1个下凋.qRT-PCR结果与芯片结果比较差异无统计学意义（P〉0.05）.结论 5例无家族发病案例的SLE患者与健康人外周血单个核细胞基因表达水平存在明显的差异.

Objective To compare the gene expression profiles of peripheral blood mononuclear （PBMN） cells between patients with environment-induced systemic lupus erythematosus （SLE） and healthy individuals. Methods PBMN cells were separated from five patients with non-familial SLE and 5 healthy individuals,and total RNAs extracted,which were then amplified,Cy3 stained,hybridized with Agilcnt 4X44K human whole-genome microarray,and screened for differentially expressed genes to be subjected to GO analysis and pathway analysis. qRT-PCR was used to verify the results of microarray. Results Five patients with SLE were found to have 2435 differential gene expressions as compared with healthy people （Cut off=2.0）. GO analysis showed 32.08% （512/1596） of the differential gene expressions accounted for biological processes,while higher percentages of such expressions also accounted for cellular processes,biological control,cell metabolism,etc. 34.09% （544/1596） of differential gene expressions affected molecular functions which included,based on a descending frequency,the binding,catalysis,transduction and transcriptional regulation activity. 33.83% （540/1596） of differential gene expressions affected cellular component,mainly involving the whole or parts of a cell,and organelles as well. Pathway analysis showed 12 gene expression differences each controlling the immune system signals and B-cell receptor channel,and 10 each controlling T-cell receptors,epidermal growth factor receptors 1,and IL-12-mediated channel signals.SLE patients were found to have 45 differentially expressed genes that affected autoimmune-related inflammatory response genes （with 11 up-regulated and 34 down-regulated）,and another 5 that affected extracellular matrix and adhesion molecules （with 4 up-regulated and 1 down-regulated）. qRT-PCR results did not differ significantly from those of microarray （P〉0.05）. Conclusion Remarkable differences may exist in expression of PBMN cells between 5 cases of non-familial SLE and healthy individuals.