A Study in Construction of Short Hairpin Small Interfering RNA Expression Vector Target Lectin Like Oxidized Low Density Lipoprotein Receptor-1 Gene and Its Effect on Foam Cells
- VernacularTitle:LOX-1特异性小发夹RNA表达载体的构建及其对巨噬细胞源性泡沫细胞形成的影响
- Author:
Hui-Yu YANG
;
Yun-Fei BIAN
;
Zhi-Ming YANG
;
Na-Na ZHANG
;
Chuan-Shi XIAO
;
- Publication Type:Journal Article
- Keywords:
Lectin like oxidized low density lipoprotein receptor-1 Short hairpin siRNA RNA interference Foam cells
- From:
China Biotechnology
2006;0(10):-
- CountryChina
- Language:Chinese
-
Abstract:
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.
