重症继发性肺结核患者基因表达谱的分析和验证

Gene expression profiling and verification for severe secondary pulmonary tuberculosis patients

目的 分析和验证重症继发性肺结核患者基因表达谱.方法 收集解放军三○九医院结核病研究所2012年5月至2013年10月住院患者中符合入选标准的继发性肺结核患者103例,根据病情严重程度分为重症组（57例）和轻症组（46例）,并以同期进行健康体检且年龄相匹配的45名健康人为对照组.选取重症组4例、轻症组3例和对照组5名用Affymetrix基因表达谱芯片进行全基因组检测和分析,对重症组比对轻症组筛选的差异基因做聚类分析和生物信息学分析;对以上各组剩余的53例、43例和40名用实时荧光定量聚合酶链反应（RT-PCR）验证芯片结果,其中验证代表基因原癌基因（JUN）表达水平是重症组、轻症组各20例,对照组8名;用方差分析和非参数检验统计方法判断组间的统计学差异.结果 芯片结果显示重症组比对轻症组差异基因为406条,下调基因264条,上调基因142条,差异基因以表达下调为主;聚类分析显示同组样本表达谱有相似性,提示芯片试验有一定重复性,结果可靠;基因本体论注释显示差异基因主要参与了生物学过程,包括免疫反应、信号转导、DNA依赖的转录调节、炎症反应、抗原处理与提呈、趋化作用等;信号通路分析显示差异基因参与了22条炎症和免疫反应相关通路,主要有B细胞受体信号通路、抗原处理与提呈、Toll样受体信号通路、丝裂原活化蛋白激酶通路、转化生长因子-β通路等;RT-PCR验证JUN吸光度值在组间比较的统计学结果为重症组比轻症组P ＜0.001,重症组表达下调,与表达谱芯片结果相符.结论 重症继发性肺结核与轻症继发性肺结核患者相比对有大量差异表达基因,其肺结构严重损害与基因水平差异有关.

Objective To explore the gene expression profiles of severe secondary pulmonary tuberculosis patients.Methods From May 2012 to October 2013,a total of 103 eligible patients with secondary pulmonary tuberculosis were recruited from Institution of Tuberculosis Research of PLA Hospital No.309.They were divided into severe secondary pulmonary tuberculosis （severe group） （n =57） and mild secondary pulmonary tuberculosis （mild group） （n =46） by the severity of disease.At the same time age-matched healthy controls （n =45） were selected from healthy subjects undergoing physical examination.Whole genome expression profiling was performed with Affymetrix Gene expression chips for 4 cases in severe group,3 in mild group and 5 in healthy group.Cluster and bioinformatics analysies were performed on differentially expressed genes in severe versus mild group.The remainders of three groups were 53,43 and 40 cases respectively used for verify the results of gene chip by real-time fluorescence quantitative PCR （RT-PCR）.And 20 cases in severe group,20 in mild group and 8 in control group were used to verify the expression level of jun oncogene （JUN） on behalf of differential expressed genes.Analysis of variance and nun-parametric tests were used for statistic difference analysis among three groups.Results There were 406 differentially expressed genes for severe and mild groups.There were 264 down-regulated gene and 142 upregulated ones.The down-regulated genes were predominant.Cluster analysis show the similarity of gene expression profile in the same group.The result confirmed that the gene chip experiments were both repeatable and reliable.According to gene ontology,the differentially expressed genes were mainly involved in such biological processes as immune response,signal transduction,regulation of transcription （DNA-dependent）,inflammatory response,antigen processing and presentation and chemotaxis,etc.Pathway analysis showed differentially expressed genes were involved in 22 pathways of immune response and inflammation.The major pathways included B cell receptor signaling,antigen processing and presentation,Toll-like receptor signaling,MAPK signaling and transforming growth factor-beta （TGF-β） signaling.Realtime fluorescence quantitative PCR （RT-PCR） analysis showed that the statistics of optical density for JUN was P ＜0.001 in severe versus mild group.It was down-regulated in severe group.And the expression of JUN was conformed with the result of gene expression chip.Conclusions The patients of severe group have a larger number of differential expressed genes versus those of mild group.And severe lung tissue damage in severe group may be correlated with differences in gene expression.