脂肪酸结合蛋白4在Hcy致泡沫细胞胆固醇聚积中的作用

The Effect of FABP4 on Homocysteine Induced Cholesterol Accumulation in the Foam Cells

目的探讨脂肪酸结合蛋白4(FABP4)在同型半胱氨酸(Hcy)致泡沫细胞胆固醇聚积中的作用和意义。方法体外复制THP-1单核细胞/泡沫细胞模型,油红O染色验证模型复制是否成功,给予100μmol/L Hcy处理细胞24 h后,实时定量PCR和Western blot检测泡沫细胞中FABP4 mRNA和蛋白的表达;体外构建FABP4过表达载体,双酶切法鉴定;转染FABP4至泡沫细胞,荧光倒置显微镜观察转染效率;胆固醇检测试剂盒检测细胞内胆固醇聚积情况。结果油红O染色显示泡沫细胞质内充满了大量红染脂质颗粒,胞核呈蓝色,且100μmol/L Hcy处理后细胞内胆固醇含量升高了2.4倍(P<0.01);实时定量PCR结果显示100μmol/L Hcy处理后泡沫细胞中FABP4 mRNA表达上调(P<0.01);Western blot检测结果显示FABP4的蛋白表达与其mRNA表达变化一致(P<0.01)。FABP4过表达载体转染细胞后,可见大量绿色荧光蛋白表达;重组质粒组泡沫细胞中FABP4的表达水平明显升高(P<0.01),细胞内胆固醇含量升高1.9倍,差异具有显著性(P<0.01)。结论 FABP4表达上调可能是Hcy致泡沫细胞胆固醇聚积的重要机制之一。

Aim To explore the effect and significance of fatty acid binding protein 4（ FABP4） on the formation of foam cells and the accumulation of cholesterol induced by homocysteine. Methods The macrophage-derived foam cell model of THP-1 monocytic cell line was duplicated and oil red O staining was used to validate whether the model was successfully established in vitro,and foam cells were treated with 100 μmol / L homocysteine. The protein and mRNA expressions of FABP4 in the foam cells were detected by Western blot and real time-polymerase chain reaction（ RT-PCR）.The FABP4 recombinant plasmid was constructed and then transfected to foam cells which had been identified by restriction enzyme digestion and fluorescent microscope respectively. Results Cytoplasm was filled with large quantities of red lipid droplets and the nuclear was stained to blue,and the content of total cholesterol treated with 100 μmol / L Hcy were significantly increased to 2.4-fold. The results of RT-PCR and Western blot exposed an up-expressed FABP4 mRNA and protein in 100 μmol / L Hcy group（ P0.01）. GFP protein expressed at a high level after the FABP4 recombinant plasmid was transfected into foam cells and the expression of FABP4 was also greatly elevated,the content of TC was significantlyincreased to 1.9-fold,and the difference was significant compared to the control group（ P0.05 or P0.01）. Conclusion The up-regulation of FABP4 might be one of the important mechanisms for Hcy which mediated the accumulation of cholesterol in foam cells.