中国生物工程杂志

Aim:To construct the short hairpin small interfering RNA(shRNA) eukaryotic expression vector specific to mouse lectin like oxidized low density lipoprotein receptor 1(LOX-1) gene and to observe its silencing effect on LOX-1 in RAW264.7 cells. Methods:(1)The pLOX-1-shRNA expression vector was constructed by gene recombination, Then transfected into the cultured RAW264.7 cells. At 48 h after Transfection, the expression of LOX-1 mRNA in RAW264.7 cells were determined by semi-quantitative RT-PCR, the expression of LOX-1 proteins examined by Western blot.(2) Oil Red O Dyeing experiment was used to show the cellular lipid droplets in lipid-loaded cells. The method of cholesterol oxidase analysis was performed to determine the content of cellular cholesterol.The ability of uptake Dil-ox-LDL in RAW264.7 cells was assayed by fluorescence microscopy. Results: pLOX-1-shRNA expression vector was successfully constructed. Transfection of pLOX-1-shRNA expression vector into RAW264.7 cells down regulaled the expression level of LOX-1 gene, as compared with the control group,transfection of the RAW264.7 cells with LOX-1-shRNA led to a remarkable reduction of the number macrophages transformed into foam cell,and could suppress the uptake of ox-LDL.Conclusion:The pLOX-1-shRNA expression vector can inhibit the expression of LOX-1 in RAW264.7 cells and the transformation of the macrophages into foam ,which may he beneficial in searching new gene therapy of atherosclerosis.