摘要
目的观察其受抑制后对二硫键A氧化还原酶样蛋白(disulfide-bond A oxidoreductase-like protein,Dsb A-L)和高分子量(high molecular weight,HMW)脂联素表达的影响.方法利用短发夹RNA(short hairpin RNA,shRNA)构建慢病毒载体,沉默3T3-L1细胞的叉头状转录因子O1(fork head box transcription factor O1,FoxO1)基因,建立携带sh RNAFoxO1的慢病毒载体,用qPCR和Western blot法从基因和蛋白水平筛选出最佳干扰效果的sh RNA-FoxO1慢病毒载体.在后续实验中利用此FoxO1-sh RNA慢病毒载体沉默3T3-L1 FoxO1基因,通过Western blot检测Dsb A-L、HMW脂联素的表达.结果在3T3-L1细胞中筛选出sh RNA-1组对FoxO1基因的表达的沉默效果最佳,抑制率达到60%以上(与对照组相比,基因相对表达量1.04±0.04 v s 0.37±0.05,P<0.001);在后续实验选用sh RNA-1干预3T3-L1细胞,用Western blot检测,并与对照组比较,结果FoxO1蛋白表达显著减少(与GAPD H光密度比值分别是1.02±0.08和0.38±0.08,P<0.001),而Dsb A-L和HMW表达明显增加(Dsb A-L与GAPDH光密度比值分别是0.28±0.06和0.53±0.07,P=0.009;HMW脂联素与GAPDH光密度比值分别是0.05±0.02和0.11±0.03,P=0.043).结论在3T3-L1细胞中,沉默FoxO1基因后可促进Dsb A-L和HMW脂联素的表达.
AIM To observe the effect of fork head box transcription factor Ol（FoxO1） gene silencing on the expression of disulfide-bond A oxidoreductase-like protein（DsbA-L） and high molecular weight（HMW） adiponectin.METHODS Lentiviral vector carrying short hairpin RNAs（shRNAs） targeting the FoxO1 gene was constructed,and the shRNA with the highest inhibition of FoxO1 expression（shRNA-FoxO1）in 3T3-L1 fat cells was selected by real-time quantitative PCR and Western blot and used for subsequent experiments.The expression of DsbA-L and HMW adiponectin in 3T3-L1 fat cells was determined by Western blot after infection with lentiviral vector carrying shRNAFoxO1.RESULTS The lentiviral vector carrying the shRNAFoxO1 had the most significant effect on the expression of FoxO1 in 3T3-L1 cells.The inhibition rate reached over 60%,and the relative expression levels of FoxO1 gene between the shRNA-FoxO1 and control groups were 0.37±0.05 and 1.04±0.04,respectively（P0.001）.Western blot analysis showed that compared with the control group,the expression of FoxO1 was significantly inhibited（1.02±0.08 vs 0.38±0.08,P0.001）,but the expression of DsbA-L and HMW adiponectin was significantly increased（0.28±0.06 vs 0.53± 0.07,P= 0.009;0.05±0.02 vs 0.11±0.03,P=0.043） in the shRNA-FoxO1 group.CONCLUSION In 3T3-L1 cells,silencing FoxO1 gene promotes the expression of DsbA-L and HMW adiponectin.
出处
《世界华人消化杂志》
CAS
2017年第1期56-63,共8页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No.81360596
广西自然科学基金重点资助项目
No.2014GXNSFDA118029
广西八桂学者建设专项经费基金资助项目~~
