11β-羟基类固醇脱氢酶诱发脂质蓄积的实验研究

Overexpression of 11β-hydroxysteroid dehydrogenase type1 in C57BL/6 mice into fatty liver but not obesity

目的:探讨I型11β-羟基类固醇脱氢酶(11β-HSD1)对脂质蓄积的影响及机制。方法:构建I型11β-羟基类固醇脱氢酶腺病毒(Ad-11β-HSD1),并在293A细胞中进行扩增和纯化;C57BL/6小鼠按随机数字表法分组,实验组10只,通过尾静脉注射Ad-11β-HSD1,对照组10只。2周后采集小鼠血浆,收集肝脏组织。油红O染色观察肝脏组织脂质蓄积情况;酶法测定血脂以及肝脏中脂质含量;利用实时荧光定量聚合酶链式反应技术分析和蛋白免疫印迹,分析脂代谢相关基因和蛋白表达情况;分析AKT/GSK胰岛素信号通路的改变。结果:与对照组相比,2周后实验组肝脏脂质蓄积,脂滴富集明显,脂质定量显著增加[(59.61±3.21)vs.(80.47±5.1)μg/mg,P<0.05];实验组脂肪酸合成通路主调控因子SREBP1及其合成通路相关蛋白FAS、SCD1和ACC1的表达水平均不同程度升高(P<0.05),而基因表达水平无差异。胰岛素信号通路受损,p-AKT,p-GSK水平降低(P<0.05)。血浆中TG和TC水平无差异。结论:11β-HSD1可能通过损伤胰岛素信号通路,并激活脂肪酸合成通路而促进小鼠脂肪肝形成。

Objective： To investigate the effect and mechanism of 11β-hydroxysteroid dehydrogenase type 1（ 11β-HSD1） on lipid accumulation. Methods： 11β-HSD1 adenovirus（ Ad-11βHSD1） was constructed,amplified and purified in 293A cells,and Ad-11βHSD1 was injected into the C57 BL/6 mice via the tail vein. Two weeks after adenovirus infection,plasma and liver tissues were collected. Oil red O staining was used to observe the accumulation of lipids in the liver tissues. Serum lipids and lipids in the liver were determined by enzymatic method. Lipogenesis and insulin signaling pathway were analyzed by Western blot,the former also was analyzed by Quantitative Real-time PCR（ Q-PCR）. Results： Two weeks after overexpression of 11β-HSD1 in the mouse liver,oil red O staining confirmed lipid accumulation and lipid droplets enrichment.The lipid quantification result further confirmed the above result（ 59. 61 ± 3. 2） μg/mg pro in control group;（ 80. 47 ± 5. 1） μg/mg pro in experimental group（ P〈 0. 05）. The main regulatory factor Sterol Regulatory Element Binding Protein-1（ SREBP1） of the fatty acid synthesis pathway and its downstream regulatory genes FAS,SCD1 and ACC1 were all increased significantly（ P〈 0. 05）,but no difference was found in gene levels.Insulin signaling pathway was impaired,and the ratio of p-AKT/AKT and p-GSK/GSK were reduced largely（ P〈 0. 05）. There was no difference in plasma triglyceride and cholesterol levels. Conclusion： After 2 weeks of 11β-HSD1 overexpression,the mice developed into fatty liver,which is tightly related to the damage of insulin signaling pathway and the activation of fatty acid synthesis pathway.