基因表达谱分析非酒精性脂肪性肝病中谷胱甘肽转移酶的作用  

Role of glutathione transferase in nonalcoholic fatty liver disease:An analysis based on gene expression profile

作　　者：沈婷婷[1] 朱哿瑞 王帆 孙鑫 黄恺 彭渊[1] 陶艳艳[1] 刘成海[1,2] SHEN Tingting;ZHU Gerui;WANG Fan;SUN Xin;HUANG Kai;PENG Yuan;TAO Yanyan;LIU Chenghai(Institute of Liver Diseases,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Shanghai Key Laboratory of Traditional Chinese Clinical Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学附属曙光医院·肝病研究所,上海201203 [2]上海市中医临床重点实验室,上海201203

出　　处：《临床肝胆病杂志》2023年第1期89-96,共8页Journal of Clinical Hepatology

基　　金：上海市中医药事业发展三年行动计划(ZY[2018-2020]-CCCX-5001);上海市科学技术委员会科技计划项目(20Z21900100);上海市临床重点专科建设项目(shslczdzk01201)。

摘　　要：目的用二代转录组测序(RNA-seq)技术,结合差异基因GO分析、KEGG富集分析,探讨谷胱甘肽转移酶(GST)在高脂饮食诱导的小鼠非酒精性脂肪性肝病(NAFLD)中的作用。方法14只雄性C57BL/6J小鼠随机抽样分为对照组(n=6)和模型组(n=8)。对照组小鼠喂养普通饲料;模型组喂养高脂饲料,连续7周,构建NAFLD模型。试剂盒检测血清ALT、AST活性及TG水平、苏木精-伊红(HE)和油红染色观察肝组织病理和脂滴沉积情况;提取肝组织RNA进行高通量转录组测序,将基因表达量差异倍数≥2.0且P<0.05定义为差异基因,筛选对照组与模型组肝组织差异基因,应用GO、KEGG数据库进行功能分析,并采用qRT-PCR验证差异基因表达。符合正态分布的计量资料两组比较采用独立样本t检验。结果对照组与模型组小鼠体质量、血清ALT、AST差异无统计学意义(P值均>0.05)。与对照组相比,模型组血清TG水平明显高于对照组[(2.02±0.50)mmol/L vs(1.00±0.29)mmol/L,t=-4.45,P=0.001]。HE染色提示:模型组可见弥漫性脂肪变性和气球样变。油红染色显示:模型组肝细胞胞浆内橘红色脂滴明显增多,且肝细胞脂肪变分级明显高于对照组(1.88±0.64 vs 1.00±0.00,t=-3.86,P=0.006)。转录组测序提示两组差异基因1367个,其中上调基因数608个、下调基因数759个;且两组中GST基因差异表达17个。选择差异倍数最明显的前10个GST基因进行验证。与对照组相比,GSTa2、GSTa3、GSTa4、GSTm1、GSTm2、GSTm3、GSTm4、GSTp1、GSTo1表达下调,GSTk1表达上调。实验结果与测序结果一致。结论GST通过参与类固醇代谢过程、脂肪酸代谢过程、胆固醇代谢过程等多个生物学过程影响脂质代谢,与NAFLD发病密切相关。Objective To investigate the role of glutathione transferase in nonalcoholic fatty liver disease(NAFLD)induced by high-fat diet using the RNA-Seq technique in combination with gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses of differentially expressed genes.Methods A total of 14 male C57BL/6J mice were divided into control group with 6 mice and model group with 8 mice by random sampling.The mice in the control group were fed with normal diet,and those in the model group were fed with high-fat diet for 7 consecutive weeks to establish a model of NAFLD.Kits were used to measure the activities of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)and the level of triglyceride(TG),and HE staining and oil red staining were used to observe liver pathology and deposition of lipid droplets.Liver tissue RNA was extracted for RNA-Seq,and genes with a fold change of≥2.0 and a P value of<0.05 were defined as differentially expressed genes;after differentially expressed genes were screened out between the control group and the model group,GO and KEGG enrichment analyses were performed,and qRT-PCR was used to validate the expression of the differentially expressed genes.The independent samples t-test was used for comparison of normally distributed continuous data between two groups.Results There were no significant differences between the two groups in body weight and the serum levels of ALT and AST(all P>0.05).Compared with the control group,the model group had a significantly higher serum level of TG(2.02±0.50 mmol/L vs 1.00±0.29 mmol/L,t=-4.45,P=0.001).HE staining showed diffuse steatosis and ballooning degeneration in the model group,and oil red staining showed that the model group had a significant increase in orange-red lipid droplets in the cytoplasm of hepatocytes and a significantly higher grade of hepatocyte steatosis than the control group(1.88±0.64 vs 1.00±0.00,t=-3.86,P=0.006).RNA-seq results showed a total of 1367 differentially expressed genes betwee

关 键 词：非酒精性脂肪性肝病 谷胱甘肽转移酶 基因表达谱 

分 类 号：R575.5[医药卫生—消化系统]