摘要
目的应用RNA干扰(RNAi)技术,构建针对3T3-L1脂肪细胞型脂肪酸结合蛋白(A-FABP)的microRNA表达载体,研究沉默A-FABP基因的表达对脂肪细胞合成甘油三酯及脂联素分泌的影响。方法构建靶向A-FABP基因的microRNA表达载体转染3T3-L1脂肪细胞后,RT-PCR及Western blot法检测A FABP mRNA及蛋白表达。建立A-FABP基因沉默细胞模型,0.5 mmol/L游离脂肪酸和成熟脂肪细胞共孵育24 h,分为对照组、脂肪酸组、RNAi组、RNAi+脂肪酸组。ELISA法分别检测甘油三酯及脂联素水平,RT PCR法检测脂肪细胞A-FABP及脂联素mRNA的表达。结果 microRNA表达载体转染脂肪细胞后,能显著抑制A-FABP mRNA和蛋白表达。随着脂肪酸浓度的升高,脂肪细胞甘油三酯合成明显增加,脂联素分泌明显减少(P<0.05)。与对照组和脂肪酸组比较,RNAi组和RNAi+脂肪酸组甘油三酯水平明显降低,脂联素水平明显升高,脂联素mRNA表达明显上调(P<0.05)。结论阻断A-FABP基因有可能成为防治动脉粥样硬化及糖尿病的新靶点。
Objective To study the effect of 3T3-L1 adipocyte fatty acid-binding protein（A-FABP） gene on secretion of adipocyte-synthesized triglyceride and adiponectin by constructing its microRNA expression vector using RNAi technology.Methods A microRNA expression vector for A-FABP gene was constructed and transfected into 3T3-L1 adipocytes.Expression of A-FABP mRNA and protein was detected by RT-PCR and Western blot,respectively.A model of silent A-FABP gene was established.Free fatty acid（FFA） cells（0.5 mmol/L） and mature adpocytes were incubated for 24 h and divided into control group,FFA group,RNAi group,and RNAi＋FFA group.Triglyceride and adiponectin levels were measured by ELISA.Expression of A-FABP and adiponectin mRNA was detected by RT-PCR.Results The microRNA expression vector after transfected into 3T3-L1 adipocytes could significantly inhibit the expression of A-FABP mRNA and protein.The synthesis of triglyceride increased significantly while the secretion of adiponectin decreased significantly with the increasing fatty acid concentration in adipocytes（P〈0.05）.The triglyceride level was significantly lower while the adiponectin secretion level and the adiponectin mRNA expression level were significantly higher in RNAi and RNAi＋ groups than in control and fatty acid groups（P〈0.05）.Conclusion Silent A-FABP gene may become a new target for preventing and treating atherosclerosis and diabetes mellitus.
出处
《中华老年心脑血管病杂志》
CAS
北大核心
2012年第5期464-467,共4页
Chinese Journal of Geriatric Heart,Brain and Vessel Diseases
基金
衡阳市科学技术发展计划项目(2009KJ64)
