Study On The Effects Of Oxidized LDL On Expression Of Toll-Like Receptor-4 In Cultured Human Monocyte-drived Macrophages

Chapter â…  Increased Expression of Toll-Like Receptor-4 in Human Monocyte/Macrophage-drived Foam CellsObjective To explore the mechanisms of increased expression of toll-like receptor 4 ( TLR-4 ) during the formation of foam cells.Methods Low density lipoprotein ( LDL ) were prepared by density gradient ultracentrifugation. LDL was oxidized by incubation with CuCl₂. The THP-1 human monocytic leukemia cell line ( THP-1 ) was chosen in our study and cultured in RPMI 1640. The differentiation of THP-1 cells into macrophages ( MP ) was induced by using myristate acetate ( PMA ) for 48 h.The MP cells were then incubated with oxidized LDL (ox-LDL) to generate foam cells ( FC ).The mRNA and the protein mass levels of TLR-4 were determined in all three groups respectively. The mRNA and the protein mass levels were detected by Immunocytochemistry, Western Blotting and Reverse Transcription Polymerase Chain Reaction ( RT-PCR ).Results The TLR-4 mRNA and the protein mass level were significantly increased not only during the differenciation of monocytes into macrophages ( p <0.05 ) but also during the formation of lipid-laden foam cells (p <0.05 ).Conclusions The upregulation of TLR-4 gene expression during the differentiation of monocytes into macrophages and the differentiation of macrophages into foam cells could increase TLR-4 protein synthesis dramatically, which may be closely related to ability of foams' inflammation reaction in atherosclerosis. Chapter â…¡ Effects of Oxidized LDL on Expression of Toll-Like Receptor-4 in Cultured Human Monocyte-drivedMacrophagesObjective To explore the effects of LDL oxidizing on the expression of toll-like receptor 4 (TLR-4 ) in human monocyte-drived macrophage.Methods Low density lipoprotein (LDL) were prepared by density gradient ultracentrifugationt. LDL was oxidized by incubation with CuCl₂. The THP-1 human monocytic leukemia cell line (THP-1) was chosen in our study and cultured in RPMI 1640. The differentiation of THP-1 cells into macrophages (MP) was induced by using myristate acetate (PMA) for 48 ho The macrophage cells were then incubated with oxidized LDL (ox-LDL ) or LDL. The protein mass and the mRNA levels of TLR-4 were determined in all groups respectively.The protein and mRNA levels were detected by Immunocytochemistry, Western Blotting and Reverse Transcription Polymerase Chain Reaction (RT-PCR).Results The TLR-4 protein mass and mRNA level were significantly increased by ox-LDL (p <0.01 ).In contrast, LDL effectes neither the expression of TLR-4 protein nor of TLR-4 mRNA significantly.Conclusions The upregulation of TLR-4 gene expression in human monocyte-drived macrophages by ox-LDL could increase TLR-4 protein synthesis dramatically, but LDL has not the effect; LDL oxidizing may be one of triggers for inflammation in atherosclerosis. Chapter â…¢ Effect of Interferon-gamma on the Expression of Toll-Like Receptor-4 in Human Monocyte/Macrophage-drived Foam CellsObjective To observe the effect of recombinant human Interferon-gamma (IFN-r) on the expression of human toll-like receptor 4 (TLR-4) during the formation of foam cells.Methods Low density lipoprotein (LDL) were prepared by density gradient ultracentrifugationo LDL was oxidized by incubation with CuCl₂. The THP-1 human monocytic leukemia cell line ( THP-1 ) was chosen in our study and cultured in RPMI 1640o The differentiation of THP-1 cells into macrophage cells ( MPs ) was induced by using myristate acetate ( PMA ) for 48 hours a The macrophage cells were then incubated with oxidized LDL ( ox-LDL ) to generate foam cells ( FCs ). In this process, recombinant human IFN-r was given or not. The mRNA and the protein mass levels of TLR-4 were determined respectively . The mRNA and the protein mass levels were detected by Immunocytochemistry , Western Blotting and Reverse Transcription Polymerase Chain Reaction ( RT-PCR ).Results The TLR-4 mRNA and the protein mass level were significantly increased during the formation of lipid-laden foam cells (p <0.05). INF-r enhanced the TLR-4 mRNA and the protein mass level in this process.Conclusions The upregulation of human TLR-4 gene expression during the differentiation of monocytes into foam cells could increase the TLR-4 protein synthesis dramatically, IFN-r enhanced the expression further. This may be closely related to ability of foam cells inflammation reaction in atherosclerosis.