The Establishment Of Foam Cell Model And The Study On Mechanisms Of PPARs In Atherosclerosis

Objective To establish the in vitro model of THP1 macrophages derived foam cells,to observe the target gene expression of THP1 cells and the effect of PPARs agonist on lipid metabolism of THP1 macrophages and to explore the mechanism of subtypes of PPARs family involved in the development of atherosclerosis.Method 1.THP1 suspension cultured cells were induced with 5ng/ml PMA for 24hours,adherent cells were collected after observed under the electron microscopy.Serum was isolated from the patients with hyperlipidemia and added into the THP1 cells culture medium in concentration of 10%and go on incubated for 12 hours,intracellular lipid accumulation were observed under microscopy after fixed and stained by oil red O.2.THP1 cells and HUT78 cells were singlely or co-cultured,induced by PMA and/or bezafibrate,six wells for each group,cells were collected after incubated for 24 hours.MTT assay was used to evaluate the proliferation of the cells by messurement of absorbance(OD value).3.THP1 cells were induced by PMA in concentration of 5ng/ml for 24 hours,and then exposured to bezafibrate in concentration of 0.1.1.10.50?mol/L for 12 hours,respectively,and to compare with the groups of single THP1 cells and of treated by PMA or 10?mol/L bezafibrate.Then cells were collected for the isolation of RNA,and rt-PCR were used to analysized the levels of mRNA,including PPAR??PPAR??PPAR??ANGPTL3?ANGPTL4?ABCA1?ABCGI1?LPL.The effects of bezafibrate in different concentration on activating PPARs and the subtype of PPARs on lipid metabolism were observed.Results 1.After inducing by PMA at 5ng/ml for 24 hours,the isolated monocytes were successfully transferred to macrophages.When co-incubating with 10%human serum of the patients with hyperlipidemia for 12 hours,a great deal of lipid droplets were observed in the cultured cells under the microscope by oil red O staining.2.MTT assay revealed that,the OD value was increased in the group that of THP1 and HUT78 co-cultured compared with HUT78 cells cultured alone.The growth rate of THP1 after induced by PMA was inhibited compared with the un-induced group.THP1 cells were inhibited after added in bezafibrate compared with cultured alone.The growth rate of co-cultured group was inhibited in the PMA induced group than non-induced group.The differences were all statistically significant(P<0.05).There's no statistically difference between the groups of HUT78 with and without.3.The PPARs mRNA expression of THP1 macrophages showed a dose-dependent increase when cells were incubated with differrent concentrations of bezafibrate.When bezafibrate concentration was 10?mol/L,the expression of PPAR? mRNA was significant higher than other groups(P<0.05).The highest expression of PPAR? and PPAR? were observed for bezafibrate at the concentration of 50?mol/L compared with other groups(P<0.05).4.By Spearman corralation analysis,PPAR??PPAR??PPAR? were all positively related with ABCA1 and ABCG1 mRNA expression(P<0.05).PPAR??PPAR? were positively related with ANGPTL3 and LPL(P<0.05).ANGPTL3 was positively related with LPL(P<0.05).Conclusion A foam cell model was successfully established by the use of PMA and human serum from cases with hyperlipidemia and provide a new access for the study of atherosclerosis mechanism in vitro.2.When treated with PMA,THP1 cells have the characteristics of human macrophages and do not increase itself and no longer stimulate the T cells(HUT78)proliferation.Bezafibrate inhibite the growth of THP1 cells.3.All subtypes of PPARs can be activated by bezafibrate in dose dependent manner.The same patten was worked in mRNA expression of ANGPTL3?ANGPTL4?ABCA1?ABCG1 and LPL.And it was related to the activation of PPARs pathway.