基于数字基因表达谱(DGE)分析异烟肼诱导家蚕脂肪体损伤的差异表达基因  

An Analysis on Differentially Expressed Genes in Bombyx mori Fat Body with Injury Induced by Isoniazid Based on Digital Gene Expression Library

作　　者：王海英[1] 魏广兵[1] 刘影霞[1] 杜贝贝[1] 岳玲[1] 黄涛[1] 王立鹏[1] 徐世清[1] 司马杨虎[1] 

机构地区：[1]苏州大学基础医学与生物科学学院,江苏苏州215123

出　　处：《蚕业科学》2016年第3期404-414,共11页ACTA SERICOLOGICA SINICA

基　　金：现代农业产业技术体系建设专项(No.CARS-22)

摘　　要：以无脊椎动物家蚕为实验动物,采用高通量测序技术分析人结核病治疗药物异烟肼(INH)诱导蚕体脂肪体损伤状态下的相关基因信息。供试5龄第3天家蚕幼虫分别按照1、2 mg/头的剂量经口给予INH后8 h,解剖获取家蚕脂肪体组织并提取总RNA,利用RNA-Seq测序技术分别构建INH诱导组和CK对照组家蚕脂肪体的数字基因表达谱(digital gene expression,DGE)文库。比对2个DGE文库的差异表达基因,并进行GO功能分析和KEGG通路分析,结果显示:INH影响了家蚕脂肪体中功能基因的表达,其中下调表达的基因数目明显较多,这些差异表达基因的GO分类和所在的KEGG通路,均集中在核酸损伤、细胞外信号转导、细胞凋亡与氧化应激过程、蛋白质代谢、药物代谢过程和免疫毒性等几大通路中。选择4个分别与肝损伤、药物代谢、线粒体能量代谢有关的差异表达基因进行qRT-PCR检测,结果与DGE文库的表达一致。另外,家蚕脂肪体经HE和DAPI染色后亦可观察到INH对细胞的损伤;测定不同浓度INH及作用时间下家蚕脂肪体中与肝损伤有关的谷丙转氨酶(ALT)和谷草转氨酶(AST)的活性显著提高,但2种酶的变化趋势不一样。推测INH造成机体组织损伤可能与其和机体内的大分子物质结合,从而影响到这些大分子发挥参与多种代谢通路调节机体正常生理活动的作用有关。In this paper,using silkworm（ Bombyx mori） as experimental animal,we studied the differential gene expression in injured fat body induced by isoniazid（ INH） which is prescribed for as antituberculotics through high-throughput sequencing technique. At 8 h after oral administration of INH to day 3 silkworm larvae of the 5th instar at dosage of 1 and2 mg per larva,total RNA was extracted from fat body,and digital gene expression（ DGE） libraries of fat body from INH treatment group and from blank control group（ CK） were constructed using RNA-Seq sequencing technology. Comparative analysis on the differentially expressed genes between two DGE libraries indicated that INH treatment affected the expression of functional genes in fat body,especially the number of down-regulated genes was significantly higher.GO annotation and KEGG pathway analysis showed that these differentially expressed genes were enriched in nucleotide damage,extracellular signal transduction, cell apoptosis and oxidative stress,protein metabolism,drug metabolism and immunotoxicity etc. We randomly selected 4 differentially expressed genes involved in hepatic injury,drug metabolism and mitochondrial energy metabolism to perform qRT-PCR detection,and the results were consistent with that in DGE library. Observation of fat body after HE and DAPI staining also verified that INH could induce cell injury. Activities of alanine aminotransferase（ ALT） and aspartate aminotransferase（ AST）,which are related to liver damage,in fat body of silkworm at different time points after treatment with INH of different concentrations were determined,and the results showed that they were all significantly increased and had different variation tendency. It is speculated that the binding of INH to relevant macromolecules affected various metabolic pathways with involvement of these macromolecules,and resulted in tissue injury.

关 键 词：异烟肼 家蚕 脂肪体 肝损伤 RNA-Seq测序 差异表达基因 GO功能 KEGG分析 

分 类 号：R965[医药卫生—药理学]