聚集素对肝脂肪变性细胞的作用及机制  

Effect and mechanism of clusterin on hepatic steatosis cells

作　　者：苏静 胡以平 SU Jing;HU Yiping(Department of Cell Biology,Naval Medical University,Shanghai 200082,China)

机构地区：[1]海军军医大学细胞生物学教研室,上海200082

出　　处：《新乡医学院学报》2023年第1期1-10,共10页Journal of Xinxiang Medical University

基　　金：国家重点研发计划项目(编号:2018YFA0107500);上海市自然科学基金资助项目(编号:21ZR1477400)。

摘　　要：目的探讨聚集素(CLU)对脂肪变性Hep1-6细胞中脂沉积、脂代谢、糖代谢及氧化应激的影响及作用机制。方法将Hep1-6细胞随机分为对照组、CLU组、短发夹(Sh)组和绿色荧光蛋白(GFP)组,对照组细胞不给予任何处理,CLU组细胞加入过表达CLU慢病毒上清,Sh组细胞加入敲减CLU慢病毒上清,GFP组细胞加入对照病毒上清。分别采用蛋白免疫印迹法和实时荧光定量聚合酶链式反应法检测细胞中CLU蛋白和mRNA表达来验证稳定过表达和敲减CLU细胞株的成功建立。待对照组、CLU组、Sh组和GFP组细胞培养至密度约80%时,加入游离脂肪酸工作液1 mmol诱导24 h构建脂肪变性细胞模型。细胞模型建立成功后,Sh组细胞随机分为Sh-CLU低剂量组和Sh-CLU高剂量组,Sh-CLU低剂量组每毫升培养基中加入400 ng CLU,Sh-CLU高剂量组每毫升培养基中加入800 ng CLU,继续培养24 h。另取Hep1-6细胞以每孔4×10^(5)个接种于12孔培养板,细胞贴壁后,加入含有体积分数1%牛血清白蛋白(BSA)的达尔伯克改良伊格尔无血清培养基培养24 h(BSA组)。油红染色观察对照组、BSA组、CLU组、Sh组和GFP组细胞中脂滴沉积情况,并采用酶标仪检测细胞中脂滴沉积量。采用实时荧光定量聚合酶链式反应法检测对照组、CLU组、Sh组、Sh-CLU低剂量组和Sh-CLU高剂量组细胞中脂肪酸合成、脂肪酸氧化、脂滴形成、脂转运、脂解、氧化应激和糖代谢相关基因mRNA表达水平。结果与对照组相比,CLU组细胞中CLU蛋白和mRNA表达水平均显著升高,Sh组细胞中CLU蛋白和mRNA表达水平均明显降低。与对照组相比,CLU组细胞中脂滴沉积显著减少,Sh组细胞中呈现更多的红色脂滴沉积。与对照组相比,CLU组、Sh-CLU低剂量组和Sh-CLU高剂量组细胞中脂肪酸合成相关基因、脂肪酸氧化相关基因、脂解相关基因、氧化应激相关基因、脂转运和糖代谢相关基因mRNA表达水平总体显著升高,脂�Objective To explore the effect and mechanism of clusterin(CLU)on lipid accumulation,lipid metabolism,glucose metabolism and oxidative stress in steatosis cells of Hep1-6.Methods Hep1-6 cells were randomly divided into control group,CLU group,short hairpin(Sh)group and green fluorescent protein(GFP)group.The cells in control group were not treated with any treatment;the cells in CLU group were added with CLU overexpression lentivirus supernatant;the cells in Sh group were added with CLU knockdown lentivirus supernatant;the cells in GFP group were added with control virus supernatant.The expressions of CLU protein and mRNA were detected by Western blot and real-time fluorescence quantitative polymerase chain reaction method respectively to verify the successful establishment of stable overexpression and knockdown CLU cell lines.When the cell density in the control group,CLU group,Sh group and GFP group was about 80%,1 mmol of free fatty acid working solution was added for inducing 24 h to construct the steatosis cell model.After the cell model was established,the cells in the Sh group were randomly divided into Sh-CLU low-dose group and Sh-CLU high-dose group.The Sh-CLU low-dose group was added 400 ng CLU permilliliter of medium,the cells in the Sh-CLU high-dose group was added 800 ng CLU permilliliter of medium,and the cells in the above two groups were cultured for 24 h.Another Hep1-6 cells were taken and inoculated in 12-well culture plate with 4×10^(5) cells per well.After the cells attached to the wall,Dulbecco′s modification of Eagle′s serum-free medium containing 1%bovine serum albumin(BSA)was added for culturing 24 h(BSA group).The lipid droplet deposition in cells in the control group,BSA group,CLU group,Sh group and GFP group was observed by oil red staining,and the amount of lipid droplet deposition in cells in the above five groups was detected by enzyme-labeled instrument.The expressions of fatty acid synthesis,fatty acid oxidation,lipid droplet formation,lipid transport,lipolysis,oxidative stres

关 键 词：脂肪变性 聚集素 游离脂肪酸 

分 类 号：R575.5[医药卫生—消化系统]