Expression And Primary Function Study Of Human Metabolic Related Protein And The Action Of Astragalus Polysaccharides On Cholesterol Efflux In THP-1 Macrophage-derived Foam Cell

Part IExpression and primary function study of human metabolic related proteinOBJECTIVE: To clone and express human metabolism related protein(MRP) gene, and study the function of MRP gene. METHODS: Full length of MRP gene was cloned into prokaryotic expression vector pPROEX HTa by Using RT-PCR method, and eukaryotic expression vector pEGFP—C3. MRP protein was highly effectively expressed in E.col ROSSET(DE3 )strain and the fused protein was purified, with which the New Zealand rabbit was immunized to generate polyclonal antibody against MRP protein .Laser confocal microscopy was employed to reveal the localization of MRP in HEK293 cell line. MRP mRNA and protein expression profile was demonstrated in various tissues by RT-PCR and Western Blot. Using immunofluorescence immunochemistry and laser confocal scan microscopy, localization of MRP in pancreas cells was also demonstrated. MRP mRNA and protein expression in primary human adipocytes treated with various concentrations of glucose for different time periods was analysised with RT-PCR and Western Blot. RESULTS: Polyclonal antibody with higher specificity and higher potency was prepared successfully. The antibody was tested by Western Blot, and the results showed a specific 15kd band when the antibody was diluted with 1:1000. MRP was located in cytoplasm and nuclear of HEK293 cell under laser confocal microscope. MRP was widely expressed in tissues and particularly high level in brain, muscle, adipose. Immunofluorescence immunochemistry and laser cofocal microscope showed that MRP located in pancreas cells.MRP expression responding to glucose was time-dependent in human adipocytes. As time increased, the gene expression levels of MRP first increased, then decreased after eight hours. Conclusion: A novel gene (MRP gene) which widely expressed in human tissues was cloned. MRP expression might be adjusted by glucose. part IIThe Action of Astragalus polysaccharides on Cholesterol Efflux in THP-1 Macrophage-derived Foam CellOBJECTIVE: To study the action of astragalus polysaccharides (Aps) on apoptosis , proliferation and cholesterol efflux in THP-1 macrophage-derived foam cell.METHODS: After exposure of the cultured THP-1 macrophage-derived foam cell to Aps at different dose, apoptosis of THP-1 macrophage-derived foam cell were determined by caspase3 , Annexin V/PI and Hoechst stain; proliferation were determined by XTT, cholesterol efflux and ABCA1 protein level were determined by liquid scintillator and flow cytometer , respectively.RESULTS: Apoptosis of THP-1 macrophage-derived foam cell was induced by Aps in dose-dependent manners. Cell proliferation was inhibited by Aps. Aps increased cholesterol efflux in THP-1 macrophage-derived foam cell with dose dependent pattern and flow cytometer showed that exposure of the cultured THP-1 macrophage-derived foam cell to Aps at different dose, resulted in increase in the expression of ABCA1 protein in THP-1 macrophage-derived foam cell with dose-dependent pattern.Conclusion: Aps may induce cell apoptosis and inbihits proliferation of THP-1 macrophage-derived foam cell; the effect that Aps increase cholesterol efflux might be related to it's activity of up regulating ABCA1.