自身免疫性肝炎小鼠模型差异表达基因的筛查及柴胡皂苷D对部分差异表达基因表达的影响  

Screening for differentially expressed genes in mice with autoimmune hepatitis and effect of saikosaponin-d on the expression of several differentially expressed genes

作　　者：陈浩 郝健亨 李振城 徐慧超 高艳 苗宇船 郝慧琴 刘杨 CHEN Hao;HAO Jianheng;LI Zhencheng(School of Basic Medical Sciences, Shanxi University of Chinese Medicine, Jinzhong, Shanxi 030619, China)

机构地区：[1]山西中医药大学基础医学院,山西晋中030619

出　　处：《临床肝胆病杂志》2020年第4期840-846,共7页Journal of Clinical Hepatology

基　　金：国家自然科学基金项目(81470190);基于炎症反应的重大疾病创新药物山西省重点实验室开放课题基金资助项目(SXIDL-2018-07);山西省卫生健康委科研课题计划(2018002);山西中医药大学“肝脏炎性疾病中西医结合基础研究创新团队”资助项目(2018TD-016)。

摘　　要：目的对自身免疫性肝炎(AIH)小鼠模型的差异表达基因进行基因芯片筛查,并观察柴胡皂苷D(SS-d)对部分差异表达基因表达的影响,探讨AIH的发病机制及SS-d对该病的治疗作用机制。方法健康、雌性SPF级C57BL/6小鼠40只[体质量(20±2)g],分为基因芯片组(n=8)和SS-d治疗组(n=32)。基因芯片组小鼠又分为正常对照组(n=4)和模型组(n=4),正常对照组常规饲养,模型组小鼠按15 mg/kg剂量给予尾静脉注射刀豆蛋白A(Con A),12 h后处死并提取肝组织,按Agilent芯片说明书进行差异表达基因的筛选,采用荧光定量PCR技术对部分差异表达基因进行验证。SS-d治疗组小鼠随机分为正常组(常规饲养)、模型组(按15 mg/kg剂量给予尾静脉注射Con A)、SS-d低剂量组和SS-d高剂量组(分别按2.5 mg/kg和5.0 mg/kg剂量给予腹腔注射SS-d预处理,8 h后按15 mg/kg剂量给予尾静脉注射Con A)(每组8只)。12 h后收集各组小鼠静脉血,ELISA法检测血清ALT、AST。无菌条件下提取小鼠肝组织,部分多聚甲醛固定后切片,并进行HE染色;部分肝组织用于提取RNA,采用荧光定量PCR技术检测部分差异表达基因(IFNγ、IL-4、IL-5、IL-13和IL-17)的mRNA水平变化。多组间比较采用单因素方差分析,进一步两两比较采用LSD-t法检验;两组间比较采用t检验。结果基因芯片组小鼠共筛查差异表达基因11512个,其中上调5189个,下调6323个,显著富集于138条信号通路;荧光定量PCR验证结果显示,与正常对照组比较,模型组小鼠IFNγmRNA和IL-17 mRNA的表达升高(P值均<0.01),而IL-4 mRNA、IL-5 mRNA和IL-13 mRNA的表达下调(P值均<0.01),与芯片筛查结果一致。在SS-d治疗组中,与正常对照组比较,模型组小鼠血清ALT、AST水平均升高(P值均<0.01);肝组织内可见大量淋巴细胞浸润;IFNγmRNA和IL-17 mRNA的表达水平明显升高(P值均<0.05),而IL-4 mRNA、IL-5 mRNA和IL-13 mRNA的表达水平明显降低(P值均<0.05)。与模型组比较,SS-d�Objective To investigate the differentially expressed genes in autoimmune hepatitis(AIH)mice by microarray screening,the effect of saikosaponin-d(SS-d)on several differentially expressed genes,the pathogenesis of AIH,and the therapeutic mechanism of SS-d.Methods A total of 40 healthy female specific pathogen-free C57BL/6 mice(with a body weight of 20±2 g)were divided into gene chip group with 8 mice and SS-d treatment group with 32 mice.The mice in the gene chip group were further divided into normal control group and model group,with 4 mice in each group;the mice in the normal control group were given normal feeding,and those in the model group were given injection of concanavalin A(Con A)via the caudal vein at a dose of 15 mg/kg;the mice were sacrificed 12 hours later to collect liver tissue samples,the differentially expressed genes were screened out according to the instructions of Agilent chip,and some differentially expressed genes were verified by quantitative real-time PCR.The mice in the SS-d treatment group were randomly divided into normal control group(routine feeding),model group(injection of Con A via the caudal vein at a dose of 15 mg/kg),and low-and high-dose SS-d groups(pretreated with intraperitoneal injection of SS-d at doses of 2.5 mg/kg and 5.0 mg/kg,respectively,followed by the injection of Con A via the caudal vein at a dose of 15 mg/kg 8 hours later),with 8 mice in each group.Venous blood samples were collected 12 hours later,and ELISA was used to measure serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST).Liver tissue samples were collected under sterile conditions;some samples were fixed by paraformaldehyde to prepare sections,and HE staining was performed;some liver tissue samples were used to extract RNA,and quantitative real-time PCR was used to measure the changes in the mRNA expression of several differentially expressed genes(interferonγ[IFNγ],interleukin-4[IL-4],interleukin-5[IL-5],interleukin-13[IL-13],and interleukin-17[IL-17]).A one-way analysis of varianc

关 键 词：肝炎 自身免疫性 柴胡皂苷-D 基因表达 小鼠 近交C57BL 

分 类 号：R575[医药卫生—消化系统]