The Regulation Mechanism Of Cell Cycle Regulative Protein Hcdc14A On HBVECs Damages Induced By High Glucose, High FFA And Hypoxia

Partâ… The influences of different stimuli on the expressions ofHcdc14A and its related cyclins,cell proliferation andapoptosis in HBVECOBJECTIVE:To investigate the influence of high glucose,high FFA and hypoxiaon protein Hcdcl4A,related cyclins,cell proliferation and apoptosis in HBVEC.METHODS:HBVECs were treated with high glucose,high FFA and hypoxia.Theexpression of Hcdcl4A at the mRNA and protein level was detected by RT-PCR andWestern Blot,respectively.At the same time,the expressions of cyclinB,cyclinD,cyclinE and P53 were detected by Western Blot.The cell proliferation ability andsurvival ability of HBVECs which were treated by above conditions were furtherconfirmed by XTT and CCK8.The cell apoptosis was detected by AnnexinV/PI andcaspase3 activity,the cell cycle by flow cytometry,the spatial structure ofcytoskeletal protein by fluorescent probe,and cell ultrastructure by electronmicroscope.RESULTS:1.The expression of mRNA and protein of Hcdcl4A were decreased inHBVECs,as stimulated by high glucose,high FFA and hypoxia in different ways,with the most obvious decrease of Hcdcl4A existing in the combined intervention ofabove stimuli.2.The expression of protein cyclinB,cyclinD and cyclinE weredecreased,and P53 was increased after different stimuli.3.The proliferation abilityand survival ability of HBVECs were reduced after being stimulated by aboveconditions.4.The cell apoptosis was increased and the caspase3 activity was raised.5.The cell proportion of S phase and G2-M phase after above stimulations were lower than that of control.6.The spatial structure of cytoskeletal protein waschanged after above stimulations.7.The apoptosis cell and apoptotic body wereobviously increased after stimulations under electron microscope.CONCLUSIONS:The expression of protein Hcdc 14A was decreased in cells treatedwith high glucose,high FFA and hypoxia in different ways,and related cyclins werealso influenced.The cell cycle was partly blocked,cytoskeletal protein and cellultrastructure were interfered or destroyed,the proliferation ability and survival abilityof HBVECs were reduced,and the cell apoptosis was increased after being treated byabove conditions.Partâ…¡The influence of overexpressing of Hcdcl4A on relatedcyclins,cell proliferation and apoptosisOBJECTIVE:To investigate the influence of overexpressing Hcdc14A on relatedcyclins,cell proliferation and cell apoptosis.METHODS:The full-length cDNA sequence was subcloned into green fluorescencerotein vector pEGFP to study the location of Hcdcl4A in the HEK293 cells and Helaells by confocal microscope.We also tried to detect the expressions of cyclinB,cyclinD,cyclinE and P53 by Western Blot when Hcdcl4A was overexpressed.Theproliferation ability of HBVECs was further confirmed by CCK8,the cell apoptosiswas detected by AnnexinV/PI and caspase3 activity respectively,the cell cycle by flow cytometry,the spatial structure of cytoskeletal protein by fluorescent probe,andcell ultrastructure by electron microscope after Hcdc 14A was overexpressed.RESULTS:1.Hcdcl4A was located on the two poles of spindle apparatus andcellular centrosome.2.Not only the expression of protein Hcdcl4A was strikinglyincreased,but also cyclinB,cyclinD,cyclinE and P53 were all increased afterHcdcl4A was overexpressed.3.The cell proliferation ability was increased afterHcdc 14A was overexpressed.4.The cell apoptosis was not obviously increased,butthe caspase3 activity was obviously raised after Hcdcl4A was overexpressed.5.Thecell proportions of S phase and G2-M phase after Hcdcl 4A was overexpressed wereraised.6.The cytoskeletal protein were obviously depolymerized after Hcdcl 4A wasoverexpressed.7.There were some tumor-like cell found under electron microscopeafter Hcdcl4A was overexpressed.CONCLUSIONS:The overexpression of protein Hcdcl4A could up-regulated theexpression of cyclinB,cyclinD,cyclinE and P53,accelerate cell cycle,promote cellproliferation,induced depolymerization of cytoskeletal protein,and perhaps disturbthe normal apoptosis proceeding.Thus these results suggested that the interactionbetween Hcdcl4A and cellular cyclical proteins after Hcdcl4A was overexpressedmight play a role in cell proliferation and apoptosis. OBJECTIVE:To investigate the influence of knockdown of Hcdcl4A with siRNAon related cyclins,cell proliferation and cell apoptosis.METHODS:To synthesize three pairs siRNA targeting definite sequence ofHcdcl4A and transfect HEK293 cell.After the total RNA and protein was extractedfrom these treated cells,the expressions of RNA and protein of Hcdcl4A weredetected by RT-PCR and Westem Blot respectively to verify the change of Hcdcl 4Ain the HEK293 cells.We also tried to detect the expressions of cyclinB,cyclinD,cyclinE and P53 by Westem Blot after siRNA transfection.The cell proliferationability of HBVECs was confirmed by CCK8,the cell apoptosis was detected bycaspase3 activity,the cell cycle by flow cytometry,the spatial structure ofcytoskeletal protein by fluorescent probe,and cell ultrastructure by electronmicroscope after siRNA transfection.RESULTS:1.Not only the expression of protein Hcdcl4A was strikinglydown-regulated,but also that of cyclinB and cyclinD were all decreased and that ofP53 was increased after siRNA transfection.2.The cell proliferation ability wasdecreased compared with that of control after siRNA transfection.3.The cellapoptosis was obviously increased compared with that of control after siRNAtransfection.4.The cell proportions of S phase and G2-M phase after siRNAtransfection were decreased compared with that of control.5.The cytoskeletalprotein became polymerized and obscure after siRNA transfection.6.The apoptosiscell and apoptotic body were obviously increased after siRNA transfection.CONCLUSIONS:The knockdown of Hcdcl4A could also affect the expression ofcyclinB,cyclinD and P53,block cell cycle,promote polymerization of cytoskeletalprotein,suppress cell proliferation,and induce cell apoptosis.Thus these resultssuggested that knockdown of Hcdcl4A cause functional disorder of cell cycleregulation network and disturb the cell normal physiological function. Partâ…£The influence of G-CSF on Hcdcl4A and its related cyclinsand the mechanism of G-CSF cytoprotective action onHBVECOBJECTIVE:To detect the influence of G-CSF on Hcdcl4A and its related cyclins,and investigate G-CSF cytoprotection mechanism on HBVEC after combinedintervention.METHODS:After HBVECs treated by G-CSF in different ways,the proliferationability and cell apoptosis were confirmed by CCK and caspase3 activity respectively.After different G-CSF pretreatment,the cell proliferation ability and survival abilityof HBVECs stimulated with high glucose,high FFA and hypoxia were examined byXTT and CCK8.After G-CSF pretreatment the apoptosis of HBVECs treated withabove combined stimulation was confirmed with AnnexinV/PI and caspase3activity.We also tried to detect the expressions of Hcdcl4A,cyclinB,cyclinD,cyclinE and P53 by Westem Blot after combined stimulation with G-CSFpretreatment.After combined stimulation with G-CSF pretreatment,the cell cyclewas detected by flow cytometry,the cytoskeletal protein by fluorescent probe,cellultrastructure by electron microscope,oxidative stress related indexes including ROS,NO,eNOS,Ca²⁺and MMP by fluorescent probe,and PI3K/AKT and MAPK signalpassway by immunofluorescence.RESULTS:1.G-CSF(100nM for 72h)displayed obvious contribution on promotingproliferation and suppressing apoptosis.2.G-CSF pretreatment for 12h or 24h hadthe similar effects on raising cell proliferation and decreasing cell apoptosis(P＞0.05).3.Not only the expressions of protein Hcdcl4A,cyclinB and cyclinE were strikinglyincreased,but also that of P53 was decreased after combined stimulation withG-CSF pretreatment.4.After combined stimulation with G-CSF pretreatment, theaflavinehe cell proportions of S phase and G2-M phase were raised,thecytoskeletal protein was obviously depolymerized,and the apoptosis cell andapoptotic body were obviously decreased.5.G-CSF pretreatment could relieve theoxidative stress by elevating NO,eNOS and MMP,degrading ROS and Ca²⁺.6.G-CSF pretreatment could lessen the damage on HBVECs induced with combinedstimulation by refreshing AKT and ERK1/2 and blocking JNK and p38 passways.CONCLUSIONS:G-CSF could up-regulate the expressions of Hcdcl4A,cyclinB,and cyclinE,and down-regulate the expression of P53,regulate cell cycle,promotecell proliferation,suppress cell apoptosis,reduce oxidiative stress in HBVECsinduced by high glucose,high FFA and hypoxia.G-CSF perhaps producedcytoprotection and anti-apoptotic effect through regulating PI3K/Akt and MAPKpathways.