Study On The Mechanism Of Leptin On The Expression Of ACAT-1 In The Mononclear Cells Differentiation Into Foam Cells

Artherosclerosis,the chronic inflammation of the artery,is related with various risk factors.One of the important pathochanges in the early phase was the migration of the mononuclear cells to the endothelial underlayer when damages happened to the endothelial cells of the artery, followed by the intake of a mass of lipids and differentiation to the macrophages and foam cells which was the major cause of the lipid stripe formation.The cholesterin esters,as the depot format of chlesterin,comprise of principal ingredient of lipids in the foam cells,was transformed by the catalysis of acyl coenzyme A-cholesterol acyltransferase(ACAT).When the cholesterin reached a certain concentration in cells,it was esterificated by ACAT so as to avoid the damages to the cells.While the activity of ACAT increased abnormally at pathological conditions and too much lipid was taken in by the macrophas and transformed to the chlesterin esters,which played a key point in the self-differentiation to the foam cells.Two subtypes of ACAT have been found,the ACAT-1 and ACAT-2,the former was the major isomerase in the macrophages.A lot of researches have discovered that the activity of ACAT-1 upregulated obviously in the formation of foam cells,but the mechanism was not completely known,needing the further expreiments.Leptin,a kind of hormone secreted by adipocytes,functioned as a central regulator to the energy intaking and expenditure,adipopexis,et al. The leptin could also played as a peripheral regulator to the immunocytes, the pancrease B cells,adipocytes and muscular cells.More and more researches haved conformed that the hypedeptinemia was a risk factor to the artherosclerosis.The leptin performed its function by combining to the conrespoding receptors OB-R,which included 5 isoform in humanbeing,among them only OB-Rb could regulate the energy metabolism via the signaling pathway.JAK-STAT was considered as the major pathway for the leptin signaling pathway.Besides the pathway abov,the leptin could also affected several target protein by activating Ras protein,among which the Raf kinase played an important role and the reciprocal signal way was called Ras-Raf-MEK-MAPK signal pathway.Under the hyperleptinemia,the synthesis of cholesterin esters increased and was considered to be results of the upregulation of ACAT-1 in the differentiation from mononuclear cells to the foam cells.But what a pity that whether there are a time dependant or dose dependant pattern are still unknown and no related papers about the signaling pathway in the effects of leptin on the ACAT-1.So in this doctorial dissertation,cell culture assay and molecular biology were applied to conform:1.Expression and activity of ACAT-1 in the mononuclear cells differentiation to the foam cell.2.Effects of leptin on expression and activity of ACAT-1 in the differentiation of mononuclear cells to the foam cell and whether the regulation effects show a time dependant or dose dependant pattern.3.Signaling pathway in the leptin effects on the expression of ACAT-1 in the differentiation from mononuclear cells to the foam cells. (-/-)Part 1 Cholesterol acyitransferase-1 expression in the the mononuclear cells differentiation to the foam cellsObjectiveTo explore cholesterol acyltransferase-1 expression in the differentiation from the mononuclear cells to the foam cells.Methods(1)Mononuclear cell line was resuscited and cultured as the subject of the research.(2)The mononuclear cells line THP-1 were cocultured with phorbol myristate acetate for 1-4 days after resuscitation and the morphology was recorded under the reverse microscope.Flow cytometry was applied to detected CD14 expression,RT-PCR assay,western bloting assay and liquid phase scintillation counting were employed to examine the expression of ACAT-1 mRNA,protein and the activity.(3)The macropahges were cocultured with oxidized low density lipoprotein for 1-4days followed the stain by red oil.RT-PCR assay, western bloting assay and liquid phase scintillation counting were employed to examine the expression of ACAT-1 mRNA,protein and the activity.Result(1)the mononuclear cell line THP-1 presented a suspension growth pattern after resuscitation,and showed a significant changes after 24 hours induction by PMA including the adhensive growth pattern, ovalisation shap,obvious enlarged volume,puffing of the cytolymph, increased cellular organelle and the pseudopod formation at the cellular membrane.The mononuclear cells line THP-1 expressed the CD14 with a 90%positive rate after induced by PMA.(2)the macrophages presented an enlarged volume,adhensive growth pattern and the puffed cytolymph.Some red stained substance could be found in the cytolymph,indicating that the the macrophages have differentiated into foam calls.(3)Compared with the mononuclear cell goup,expression of ACAT-1 mRNA and protein,the ACAT-1 activity in macrophages obviously increased(P＜0.05),showing a time dependant pattern,and reached a highest expression at day 3.(4)Compared with the macrophages,expression of ACAT-1 mRNA and protein,the ACAT-1 activity in foam cells increased,showing a time dependant pattern,and reached a highest expression at day 2,but has no siginificant difference compared with control group.(P＞0.05)Conclusion:(1)The mononuclear cells line THP-1 could differentiated to macrophage and foam cells after induction by PMA and ox-LDL.(2)The ACAT-1 gene expression increased during the mononuclear cells differentiation to the foam cells,especially from the mononuclear cells to the mocrophages. Part 2 Effects of Leptin on the ACAT-1 expression in the mononuclear cells differentiation to foam cellsObjectiveTo explore the effects of leptin on the ACAT-1 expression in the mononuclear cells differentiationMethods1.The mononuclear cells line THP-1 was induced resuscitation and cultured;2.The different concentration gradients of leptin were added to the medium and the cytotoxity was recorded to find the optimal intervention concentration for the further experiment;3.The mononuclear cells were cocultured with the PMA and 1mmol/L,2.5mmol/L,5mmol/L,10mmol/L和15mmol/L leptin and 2.5mmol/L anti-leptin antibody for 1-4 days,RT-PCR assay,western bloting assay and liquid phase scintillation counting were employed to examine the expression of ACAT-1 mRNA,protein and the activity in different groups,the time and dose dependant pattern in the regulation, and the blockage effects of anti-leptin antibody on the expression and activity of ACAT-1.4.The macrophages were cocultured with the OX-LDL and 1mmol/L,2.5mmol/L,5mmol/L,10mmol/L和15mmol/L leptin and 2.5mmol/L anti-leptin antibody for 1-4 days,RT-PCR assay,western bloting assay and liquid phase scintillation counting were employed to examine the expression of ACAT-1 mRNA,protein and the activity in different groups,the time and dose dependant pattern in the regulation, and the blockage effects of anti-leptin antibody on the expression and activity of ACAT-1.Result1.Cells in the groups within the 15mmol/L leptin didn't presented obvious changes in the morphology,and cell survival rate in the 20mmol/L leptin group showed less than 50%.2.Mononuclear cells derived macrophages in all groups showed mRNA and protein expression,activity of ACAT-1.Compared the control group,the ACAT-1 expression increased in leptin groups(P＜0.05), showing a time and dose dependant pattern,with the highest expression in 10 mmol/L group at day 3,about 2 time more than the control group. The upregulation effects of leptin could be blockaged totally by the anti-leptin antibody group.3.Macrophages derived foam cells in all groups showed mRNA and protein expression,activity of ACAT-1.Compared the control group,the ACAT-1 expression increased in leptin groups(P＜0.05),showing a time and dose dependant pattern,with the highest expression in 10 mmol/L group at day 2,about 1.5 time more than the control group.The upregulation effects of leptin could be blockaged totally by the anti-leptin antibody group.Conclusion1.Leptin could upregulate the expression of ACAT-1 during the mononuclear cells differentiation into the foam cells;2.The upregulation effects of leptin's on the ACAT-1 expression presented a time and dose dependant pattern. Part 3 Mechanism of leptin on the expression of ACAT-1 in the macropgahe differentiation into foam cellsObjectiveTo explore the mechanism of leptin effects on the expression of ACAT-1 in the mononclear cells differentiation into foam cellsMethod1.The mononuclear cells line THP-1 was resuscitated followed by induction to the macrophage and foam cells;2.Expression of leptin receptor OB-Rb was detected in the cells after induction.3.On the basic backgroud of interventions to the mononuclear cells by PMA and leptin,RAF inhibitor,JAK inhibitor,SATA3 inhibitor and MAPK inhibitor were,separately,added to the medium for 72 hours.RT-PCR assay,western bloting assay and liquid phase scintillation counting were employed to examine the expression of ACAT-1 mRNA,protein and the activity.3.On the basic backgroud of interventions to the macrophage by OX-LDL and leptinRAF inhibitor,JAK inhibitor,SATA3 inhibitor and MAPK inhibitor were,separately,added to the medium for 48 hours.RT-PCR assay,western bloting assay and liquid phase scintillation counting were employed to examine the expression of ACAT-1 mRNA,protein and the activity.Result1.The expression of leptin receptor OB-RB increased obviously during the differentiation from the mononuclear cells to the macrophages (P＜0.05),and further increase could be found in the macrophages differentiation into the foam cells(P＜0.05).2.The upregulation effects of leptin on the ACAT-1 expression could retarded most by JAK inhibitor,SATA3 inhibitor and partially by the RAF inhibitor and MAPK inhibitor during the mononuclear cells differentiation into the macrophages.3.The upregulation effects of leptin on the ACAT-1 expression could retarded most by JAK inhibitor,SATA3 inhibitor and partially by the RAF inhibitor and MAPK inhibitor during the macrophages differentiation into the foam cells.ConclusionTwo signaling pathways could be invovled in the leptin upregulaton effects on the ACAT-1 expression,including the OB-RB-JAK-SATA3 pathway and OB-RB--Raf--MAPK pathway,in which the former played a more important role.