RNA干扰技术沉默脂肪细胞型脂肪酸结合蛋白基因对脂肪细胞合成甘油三酯及分泌内脂素的影响

Effect of Adipocyte Fatty Acid Binding Protein Silencing on Triglyceride Production and Visfatin Secretion in 3T3-L1 Adipocyte

目的观察RNA干扰技术沉默脂肪细胞型脂肪酸结合蛋白基因后,脂肪细胞甘油三酯合成及内脂素分泌的变化。方法将3T3-L1前脂肪细胞采用体外诱导分化为脂肪细胞,将脂肪细胞与0～1 mmol/L不同浓度脂肪酸共孵育后,测定其甘油三酯及内脂素的浓度。构建针对脂肪细胞型脂肪酸结合蛋白基因的微小RNA质粒表达载体,将上述载体转染3T3-L1脂肪细胞,用逆转录聚合酶链反应和Western blot检测脂肪细胞型脂肪酸结合蛋白基因及蛋白的表达,将0.5 mmol/L脂肪酸与沉默前后的脂肪细胞共孵育24 h,用逆转录聚合酶链反应检测脂肪细胞型脂肪酸结合蛋白基因沉默前后脂肪细胞内脂素mRNA表达的变化,并测定其甘油三酯及内脂素浓度的变化。结果随着脂肪酸浓度的增高,与其共孵育的脂肪细胞内甘油三酯浓度也随着增加(P<0.05),其分泌的内脂素浓度也随着增加(P<0.05);构建脂肪细胞型脂肪酸结合蛋白微小RNA质粒载体,转染脂肪细胞后,能显著抑制脂肪细胞内脂肪细胞型脂肪酸结合蛋白的mRNA及蛋白表达水平(P<0.05);沉默脂肪细胞型脂肪酸结合蛋白基因后脂肪细胞内甘油三酯浓度明显低于沉默前(P<0.05),其分泌的内脂素浓度也明显低于沉默前(P<0.05)。结论经RNA干扰技术沉默脂肪细胞内脂肪细胞型脂肪酸结合蛋白基因能明显降低脂肪细胞甘油三酯的合成及内脂素的分泌。

Aim To explore the influence of adipocyte＇s triglyceride anabolism and visfatin secretion after silence adipocyte fatty acid binding protein（A-FABP） genes by RNA interference. Methods 3T3-L1 preadipocytes were cultured in vitro and were induced to differentiate into adipocytes,then the adipocytes were cultured with concentration from 0 to 1 mmol/L fatty acid,adipocyte＇s triglyceride production and visfatin secretion were measured.The micro RNA（miRNA） expression vector plasmid about A-FABP genes was constructed and then transfected with the 3T3-L1 mature adipocytes.A-FABP mRNA expression and A-FABP protein expression were measured by reverse transcription polymerase chain reaction（RT-PCR） and Western blot.After and before slience A-FABP,the adipocytes were cultured with concentration of 0.5 mmol/L fatty acid,visfatin mRNA expression in adipocyte were measured by RT-PCR,adipocyte＇s triglyceride anabolism and visfatin secretion were detected. Results With the concentration of fatty acid risen in adipocytes,the concentration of triglyceride in adipocyte were raised（P0.05）,the concentration of visfatin in adipocyte were raised too（P0.05）.Constructing the miRNA expression vector plasmid about A-FABP genes,then transfecting the 3T3-L1 mature adipocytes,the expression of mRNA and protein about A-FABP genes were obviously restrained（P0.05）.After silence AFABP genes,the concentration of adipocyte＇s triglyceride was obviously lower than before（P0.05）,the concentration of visfatin secretion was obviously lower than before（P0.05）. Conclusion After silence A-FABP genes by RNA interference,the concentration of adipocyte＇s triglyceride anabolism and visfatin secretion were lower than before.