【作者】 张静；

【导师】 程昕然；

【作者基本信息】 电子科技大学 ， 儿科学， 2024， 硕士

【摘要】 目的探讨miR-27a在肝脏及肝细胞在脂肪代谢中的作用,并初步探讨其发病机制。方法首先,使用高脂饲料喂养小鼠16w造模肥胖及脂肪肝模型,通过分析比较高脂组与低脂组肝脏病理、肝酶、脂质、miR-27a及INSR及下游相关信号分子;然后对小鼠进行尾静脉注射miR-27a沉默慢病毒,通过体内实验研究miR-27a沉默后小鼠肝脏INSR及其下游信号分子变化,肝酶、脂质变化;接着从体外实验研究,予以高脂干预AML-12细胞48小时,使用油红O染色及脂质检测分析是否造模成功;其次使用miR-27a过表达或沉默慢病毒转染细胞并高脂干预,对不同组细胞检测油红O染色、脂质、肝酶测定、分子检测等;最后,使用双荧光素酶实验检测miR-27a与INSR之间的相关性。结果(1)成功构建AML-12脂肪肝细胞及动物脂肪肝模型。(2)体内实验研究结果显示高脂干预后,动物肝脏脂质增加,肝细胞损伤肝酶增加,q PCR测定miR-27a增加、INSR及其下游信号分子PIK3R3、PPARα等降低。miR-27a沉默后可引起INSR、PIK3R3、PPARα分子表达上升,可改善小鼠脂肪肝病理结构,降低肝脏脂质及肝酶,缓解肝细胞损伤。(3)体外实验结果显示高脂干预后,AML-12脂肪肝细胞TC、TG含量增加,而FFA不变,高脂干预后肝细胞存活率下降,肝细胞损伤肝酶上升。高脂干预后肝细胞miR-27a升高,INSR、PIK3R3、PPARα降低,miR-27a沉默后,肝细胞INSR及其下游信号分子表达上升,而miR-27a过表达后,结果反之。miR-27a过表达的AML-12脂肪肝细胞与miR-27a沉默的AML-12脂肪肝细胞TC、TG、FFA均较高脂组升高,其中LV-O+FA组TC、FFA升高明显,LV-I+FA组TG升高明显,并且后者与前者相比,TC、FFA下降,而TG升高。miR-27a沉默后肝细胞存活率上升,并且miR-27a沉默及过表达的肝细胞肝酶均有下降趋势。(4)双荧光素酶实验报告提示INSR为miR-27a下游靶基因。结论在MAFLD中,miR-27a可影响肝细胞脂质代谢,并且miR-27a可靶向抑制INSR,降低下游信号分子PPARα表达,影响MAFLD脂代谢。更多还原

【Abstract】 Objective: To explore the role of miR-27 a in liver and liver cell lipid metabolism,and preliminary explore its pathogenesis.Method:Firstly,a 16 w model of obesity and fatty liver was established in mice fed with high-fat diet.The liver pathology,liver enzymes,lipids,miR-27 a,INSR,and downstream related signaling molecules were analyzed and compared between the high-fat and low-fat groups;Then,mice were subjected to tail vein injection of miR-27 a silencing lentivirus,and in vivo experiments were conducted to study the changes in liver INSR and downstream signaling molecules,as well as changes in liver enzymes and lipids after miR-27 a silencing;Subsequently,through in vitro experimental research,high-fat intervention was administered to AML-12 cells for 48 hours,and oil red O staining and lipid detection were used to analyze whether the modeling was successful;Secondly,miR-27 a overexpression or silencing of lentivirus transfected cells and high-fat intervention were used to detect oil red O staining,lipid and liver enzyme assays,molecular detection,etc.on different groups of cells;Finally,the correlation between miR-27 a and INSR was detected using dual luciferase assay..Result:(1)Successfully constructed AML-12 fatty liver cells and animal fatty liver models.(2)In vivo experimental studies have shown that after high-fat intervention,animal liver lipids increased,liver enzymes damaged by liver cells increased,miR-27 a increased by q PCR,INSR and its downstream signaling molecules PIK3R3,PPARα decreased,etc.Silencing miR-27 a can cause an increase in the expression of INSR,PIK3R3,and PPARα molecules,improve the pathological structure of mouse fatty liver,reduce liver lipids and enzymes,and alleviate liver cell damage.(3)The results of in vitro experiments showed that after high-fat intervention,the content of TC and TG in AML-12 adipose liver cells increased,while FFA remained unchanged.After high-fat intervention,the survival rate of liver cells decreased,and liver enzyme damage increased,meanwhile,miR-27 a in liver cells increased,while INSR,PIK3R3,and PPARα decreased.After miR-27 a was silenced,the expression of INSR and its downstream signaling molecules in liver cells increased,while overexpression of miR-27 a resulted in the opposite.AML-12 adipocytes overexpressing miR-27 a and AML-12 adipocytes silencing miR-27 a showed higher levels of TC,TG,and FFA compared to the high lipid group.Among them,TC and FFA increased significantly in the LV-O+FA group,while TG increased significantly in the LV-I+FA group.Compared with the former,TC and FFA decreased while TG increased.After miR-27 a silencing,the survival rate of liver cells increased,and both miR-27 a silencing and overexpression of liver enzymes in liver cells showed a decreasing trend.(4)The dual luciferase assay report suggests that INSR is a downstream target gene of miR-27 a.Conclusion : In MAFLD,miR-27 a can affect liver cell lipid metabolism,and miR-27 a can target the inhibition of INSR,reduce downstream signaling molecule PPARα expression,and affect MAFLD lipid metabolism.更多还原