RESUMO
Lipoma preferred partner (LPP) has been identified as a protein which is highly selective for smooth muscle progenitor cells (SMPCs) and regulates differentiation and migration of SMPCs, but mechanisms of LPP expression are not elucidated clearly. The aim of the present study was to discuss the mechanisms by which LPP expression is regulated in the differentiation and migration of SMPCs induced by TGF-β1. It was found that TGF-β1 could significantly increase the expression of LPP, smooth muscle α-actin, smooth muscle myosin heavy chain (SM-MHC), and smoothelin in SMPCs. Moreover, inactivation of Rho kinase (ROK) with ROK inhibitors significantly inhibited LPP mRNA expression in TGF-β1-treated SMPCs and mouse aortic smooth muscle cells (MAoSMCs). At the same time, LPP silencing with short interfering RNA significantly decreased SMPCs migration. In conclusion, LPP appears to be a ROK-dependant SMPCs differentiation marker that plays a role in regulating SMPCs migration.
RESUMO
Lipoma preferred partner (LPP) has been identified as a protein which is highly selective for smooth muscle progenitor cells (SMPCs) and regulates differentiation and migration of SMPCs, but mechanisms of LPP expression are not elucidated clearly. The aim of the present study was to discuss the mechanisms by which LPP expression is regulated in the differentiation and migration of SMPCs induced by TGF-β1. It was found that TGF-β1 could significantly increase the expression of LPP, smooth muscle α-actin, smooth muscle myosin heavy chain (SM-MHC), and smoothelin in SMPCs. Moreover, inactivation of Rho kinase (ROK) with ROK inhibitors significantly inhibited LPP mRNA expression in TGF-β1-treated SMPCs and mouse aortic smooth muscle cells (MAoSMCs). At the same time, LPP silencing with short interfering RNA significantly decreased SMPCs migration. In conclusion, LPP appears to be a ROK-dependant SMPCs differentiation marker that plays a role in regulating SMPCs migration.
Assuntos
Animais , Camundongos , Medula Óssea , Metabolismo , Fisiologia , Diferenciação Celular , Genética , Movimento Celular , Genética , Células Cultivadas , Proteínas do Citoesqueleto , Genética , Metabolismo , Proteínas com Domínio LIM , Genética , Metabolismo , Camundongos Endogâmicos C57BL , Músculo Liso , Metabolismo , Fisiologia , Células-Tronco , Metabolismo , Fisiologia , Fator de Crescimento Transformador beta1 , Genética , Metabolismo , Quinases Associadas a rho , Genética , MetabolismoRESUMO
To construct the eukaryotic expression plasmid of human PRX3 and measure its expression in the HEK-293FT cells, the full-length coding region of human PRX3 was cloned by PCR and inserted into the eukaryotic expression vector pcDNA4-Xpress (A). HEK-293FT cells were transiently transfected with the recombinant plasmid. Western blot and immuofluorescence were used to detect the expression of the fusion protein. In the experiment, restriction analysis identified the construction of the recombinant plasmid and the inserted sequence was identical with that published on GenBank. Western blot and immunofluorescence confirmed the expression of the recombinant protein in transfected HEK-293FT cells. It was concluded that the eukaryotic expression plasmid of human PRX3 was constructed successfully and the recombinant could be expressed efficiently in HEK-293FT cells, which provides a sound basis for the further study on human PRX3.
Assuntos
Linhagem Celular Transformada , Clonagem Molecular , Estruturas Embrionárias , Células Eucarióticas/metabolismo , Expressão Gênica , Vetores Genéticos , Rim/citologia , Rim/metabolismo , Peroxidases/biossíntese , Peroxidases/genética , Plasmídeos/genética , TransfecçãoRESUMO
To construct the eukaryotic expression plasmid of human PRX3 and measure its expression in the HEK-293FT cells, the full-length coding region of human PRX3 was cloned by PCR and inserted into the eukaryotic expression vector pcDNA4-Xpress (A). HEK-293FT cells were transiently transfected with the recombinant plasmid. Western blot and immuofluorescence were used to detect the expression of the fusion protein. In the experiment, restriction analysis identified the construction of the recombinant plasmid and the inserted sequence was identical with that published on GenBank. Western blot and immunofluorescence confirmed the expression of the recombinant protein in transfected HEK-293FT cells. It was concluded that the eukaryotic expression plasmid of human PRX3 was constructed successfully and the recombinant could be expressed efficiently in HEK-293FT cells, which provides a sound basis for the further study on human PRX3.
Assuntos
Humanos , Linhagem Celular Transformada , Clonagem Molecular , Embrião de Mamíferos , Células Eucarióticas , Metabolismo , Expressão Gênica , Vetores Genéticos , Rim , Biologia Celular , Metabolismo , Peroxidases , Genética , Peroxirredoxina III , Peroxirredoxinas , Plasmídeos , Genética , TransfecçãoRESUMO
AIM: To study the effect of ginsenosides on lipopolysaccharide-induced expression of tissue factor (TF) and plasminogen activator inhibitor type-1 (PAI-1) in vascular endothelial cells (EC), and to investigate the mechanism of ginsenosides in the healthy protection and treatment of cardiovascular diseases. METHODS: Human umbilical vein endothelial cells (HUVEC) were cultured by trypsin digestion method. PAI-1 was measured in the conditioned medium of HUVEC by a specific enzyme-linked immunosorbent assay (ELISA), whereas TF activity was measured in the lysates of these cells by a single step clotting assay. Specific mRNA expressions were determined by Northern blotting. RESULTS: Treatment of HUVEC with LPS resulted in a significant increase in PAI-1 antigen and TF activity. Ginsenosides inhibited this LPS-induced upregulation of PAI-1 protein and TF activity in HUVEC. These effects were also confirmed on the level of specific PAI-1 and TF mRNA expression by Northern blotting. CONCLUSION: Ginsenosides counteract activated endothelial cells by inhibiting LPS-induced PAI-1 and TF expression in these cells. This ability of ginsenosides might explain its efficacy in the healthy protection and the treatment of cardiovascular diseases.
RESUMO
AIM: To investigate the effect of homocysteine (HCY) on the induction of macrophage inflammatory protein-1? (MIP-1?) expression in cultured human umbilical vein endothelial cells (HUVECs). METHODS: After exposure of the cultured HUVECs to HCY at increasing concentrations (0.1, 0.5 and 1 mmol/L) for 8 h, the MIP-1? mRNA expression was determined by in situ hybridization using a MIP-1? cDNA probe, and the MIP-1? protein expression was measured by cell enzyme-linked immunosorbent assay (ELISA) using a goat anti-human MIP-1? monoclonal antibody. RESULTS: The in situ hybridization showed that cultured HUVECs were able to express MIP-1? mRNA at a low level that was purplish blue granules in cytoplasm. After exposure to HCY at the concentrations mentioned above, the expression of MIP-1? mRNA was significantly increased in a dose-dependent manner. Analysis of variance showed that there was significant difference between groups ( F= 606.38, P
RESUMO
AIM: To understand whether native and oxidized low density and very low density lipoprotein (n-LDL, n-VLDL, ox-LDL, ox-VLDL) enhance the expression of macrophage inflammatory protein (MIP)1? mRNA in cultured aortic smooth muscle cells (SMCs). METHODS: Native low density and very low density lipoprotein were isolated from normal blood donors by density gradient ultracentrifugation, and were oxidatively modified by adding CuCl 2. After a 24 h-exposure of the cultured SMCs to n-LDL, n-VLDL, ox-LDL and ox-VLDL, respectively, the expression of MIP-1? mRNA was determined by in situ hybridization and RT-PCR. RESULTS: Cultured aortic SMCs expressed MIP-1? mRNA at low level. N-LDL, n-VLDL, ox-LDL and ox-VLDL enhanced the expression of MIP-1? mRNA in SMCs, ox-LDL and ox-VLDL showed stronger effect than n-LDL and n-VLDL, respectively. The effect of ox-VLDL was most striking. There was a significant difference between groups ( P