严重型脊髓性肌萎缩症小鼠肝脏代谢紊乱的分子病理机制  

Molecular pathological mechanism of liver metabolic disorder in mice with severe spinal muscular atrophy

作　　者：刘力赫 朱明芮 王一凡[1] 万波 姜智[1] LIU Lihe;ZHU Mingrui;WANG Yifan;WAN Bo;JIANG Zhi(Suzhou Medical College of Soochow University,Suzhou 215000,China)

机构地区：[1]苏州大学苏州医学院,江苏苏州215000

出　　处：《南方医科大学学报》2023年第5期852-858,共7页Journal of Southern Medical University

基　　金：苏州大学“大学生创新创业训练计划”(202010285009/202010285045Z)。

摘　　要：目的 探讨严重型(Ⅰ型)脊髓性肌萎缩症(SMA)小鼠肝脏代谢紊乱的分子病理机制。方法 观察Ⅰ型SMA和同窝对照小鼠出生后喝奶状况和体质量变化;验证腹腔注射20%的葡萄糖溶液能否延长Ⅰ型SMA的生存时间;利用生物信息学方法分析Ⅰ型SMA和同窝对照小鼠肝脏组织转录组RNA测序数据,寻找差异表达基因及其相关信号通路;实时定量PCR、蛋白免疫印迹验证肝脏组织中脂代谢、糖代谢等相关基因表达变化;亚硫酸盐测序法定量分析新生小鼠肝脏组织Fasn基因启动子区CpG岛甲基化水平;测试DNA甲基转移酶抑制剂对Ⅰ型SMA小鼠原代肝细胞脂代谢基因表达的影响。结果 Ⅰ型SMA小鼠能正常喝奶,出生后第2天开始体质量低于同窝正常对照小鼠;每隔12 h腹腔注射葡萄糖溶液能使Ⅰ型SMA小鼠生存时间由9±1.3 d延长至11±1.5 d(P<0.05);分析肝脏组织RNA测序数据结合定量PCR验证,结果显示Ⅰ型SMA小鼠发病过程中肝脏内PPARα正调控的代谢相关基因下调表达;Ⅰ型SMA小鼠肝脏组织中Fasn基因启动子区域甲基化程度(76.44%)高于对照小鼠(58.67%);培养基中添加DNA甲基转移酶抑制剂5-AzaC,SMA小鼠原代培养肝细胞中Fasn等脂代谢相关基因表达增加1倍以上(P<0.01)。结论 严重的SMA小鼠发生肝脏代谢紊乱,肝组织中PPARα调控的脂代谢、糖代谢等相关基因持续甲基化而表达下调,加速SMA病程发展。Objective To explore the molecular pathological mechanism of liver metabolic disorder in severe spinal muscular atrophy(SMA).Methods The transgenic mice with type I SMA(Smn-/-SMN20tg/2tg)and littermate control mice(Smn+/-SMN20tg/2tg)were observed for milk suckling behavior and body weight changes after birth.The mice with type I SMA mice were given an intraperitoneal injection of 20%glucose solution or saline(15μL/12 h),and their survival time was recorded.GO enrichment analysis was performed using the RNA-Seq data of the liver of type I SMA and littermate control mice,and the results were verified using quantitative real-time PCR.Bisulfite sequencing was performed to examine CpG island methylation level in Fasn gene promoter region in the liver of the neonatal mice.Results The neonatal mice with type I SMA showed normal milk suckling behavior but had lower body weight than the littermate control mice on the second day after birth.Intraperitoneal injection of glucose solution every 12 h significantly improved the median survival time of type I SMA mice from 9±1.3 to 11±1.5 days(P<0.05).Analysis of the RNA-Seq data of the liver showed that the expression of the target genes of PPARαrelated to lipid metabolism and mitochondrialβoxidation were down-regulated in the liver of type I SMA mice.Type I SMA mice had higher methylation level of the Fasn promoter region in the liver than the littermate control mice(76.44%vs 58.67%).In primary cultures of hepatocytes from type I SMA mice,treatment with 5-AzaC significantly up-regulated the expressions of the genes related to lipid metabolism by over 1 fold(P<0.01).Conclusion Type I SMA mice have liver metabolic disorder,and the down-regulation of the target genes of PPARαrelated to lipid and glucose metabolism due to persistent DNA methylation contributes to the progression of SMA.

关 键 词：脊髓型肌萎缩症 小鼠 肝脏代谢 分子病理机制 

分 类 号：R744.8[医药卫生—神经病学与精神病学]