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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。
Invertebrate silkworm (Bombyx mori) was used as experimental animal, and high-throughput sequencing was used to analyze the related gene information of human tuberculosis fatal injury induced by isoniazid (INH). The 5th instar larvae of 3rd instar larvae were orally administered with 1, 2 mg / head of INH 8 h after oral administration, and adipose tissue of silkworm (Bombyx mori) was obtained and total RNA was extracted. RNAi-Seq sequencing was used to construct INH induction group and CK, a digital gene expression (DGE) library of silkworm fat body. The results showed that INH affected the expression of functional genes in the fat body of silkworm (Bombyx mori), and the number of differentially expressed genes in the two DGE libraries was significantly higher than that in the other two DGE libraries. The number of differentially expressed genes The GO classification and the KEGG pathway are all focused on pathways such as nucleic acid damage, extracellular signal transduction, apoptosis and oxidative stress, protein metabolism, drug metabolism and immunotoxicity. Four differential expression genes related to hepatic injury, drug metabolism and mitochondrial energy metabolism were selected for qRT-PCR. The results were consistent with those of DGE library. In addition, the injury of INH to cells could also be observed after HE and DAPI staining. The levels of ALT and AST in the fat body of silkworm (Bombyx mori) were measured at different concentrations of INH and time. The activity of the two enzymes was significantly increased, but the trend of the two enzymes was not the same. It is hypothesized that INH causes damage of body tissue and its binding with macromolecules in the body, thus affecting the role of these macromolecules in regulating the normal physiological activities of the body through a variety of metabolic pathways.