Influence Of Homocysteine On The Expression Of Tissue Factor Human Endothelial Cells And The Intervention Of Genistein

PartⅠInfluence of homocysteine on Endothelial Cells to express tissue factorObjective To investigate the effects of homocysteine on tissue factor(TF) expression in primary cultured human great saphenous vein endothelial cells(GSEC).Methods The endothelial cells were obtained from human and cultured by enzyme digestion method.The morphology of GSEC was studied by phase contramicroscope.Its molecular markers were observed byⅧfactor immunofluorescence.Passage 3～4 cells were used and divided into different groups randomly:(1) Blank control group:treated without intervention;(2)groups of different homocysteine concentrations:GSEC were incubated with different concentrations ofhomocysteine(10,20,50,80,100μmol/L) for 24h.(3) homocysteine groups of different intervention time:GSEC were coincubatedwith 50μmol/L homocysteine for different periods(2,4,8,24,48,72h).After intervention,the culture cells and supernatant were collected.LDH activity in supernatant was detected by LDH test box;TF mRNA was detected by quantitative reverse transcription polymerase chain reaction (RT-PCR);Concentration of TF in supernatant of culture cellswere detected by the method of double mono-cloned antibody ELISA.Results1.Normal cells were flat polygonal and grown well.The border of cells was clear, and cytoplasm was rich.The nucleus were round or oval at the center of cell.With the increase of cell density,cultured cells were "paving stone-like" arrangement.Most cells displayed green fluorescence under fluorescent microscopy,which accords with the characteristics of EC.2.After intervention of homocysteine,cells were rough,which varied the border. The activity of LDH in groups of different homocysteine concentrations were higher than blank control group(p＜0.05);In groups of different intervention time,the activity of LDH gradually increased from 4 hour group,and there was significant difference with blank control group(p＜0.05).These showed that homocysteine has a direct cytotoxic effect.3.After the intervention of homocysteine(10,20,50,80,100μmol/L) for 24 hours, the levels of TF expression increased initially with the concentration raise(p＜0.05),the endothelial cells TF expression have a dose-response relationship,in a dose-dependent manner.4.After cells were coincubated with 50μmol/L homocysteine,the expression of TF increased beginning from 4hour,which raised as the stimulation extension and 72 hour to the highest,in a time-dependent manner(p＜0.05).PartⅡEffects of genistein on homocysteine-induced expression of TFObjective To investigate the effects of genistein on homocysteine induced Endothelial Cells expression of tissue factor.Methods groups:(1) blank control group:treated without intervention;(2) homocysteine groups:cells were incubated with 50μmol/L,homocysteine for 24h;(3) genistein groups of different concentrations:cells were firstly incubated with(5,10,20,40,50mg/L) genistein respectively for 1h,then coincubated with 50μmol/L,homocysteine;(4) genistein groups of different intervention times:cells were firstly coincubated with 40mg/L genistein for 1h,then coincubated with 50μmol/L,homocysteine for different period(2,4,8,12,24,48,72h). MTT reduction assay was used to observe the genistein drug effect on cell proliferation.TF mRNA was detected by quantitative reverse transcription polymerase chain reaction (RT-PCR).The levels of TF in supernatant of culture cells were detected by the method of double mono-cloned antibody ELISA.Results1.After the intervention of different concentrations of genistein for 6,12,24,48,72 h,respectively,compared with the blank control group,there was no significant changes in cell proliferation(P＞0.05).It showed that drug intervention itself had no toxic effects on the cells.2 After intervention with Genistein with different concentration(24h),Both TF antigen concentration and mRNA level induced by homocystein(50μmol/L) were decreased in a dose dependent manner(R=0.994,0.992,respectively,p＜0.05).3 After intervention with Genistein(40mg/L) during different time,both TF antigen concentration and mRNA level induced by homocystein(50μmol/L) were decreased in a time dependent manner(R=0.972,0.982,respectively,p＜0.05).Conclusions1.Homocysteine have a direct cytotoxic effect on the endothelial cells.2.Homocysteine indued the expression of TF in a concentration dependent and time-dependent manner.3.Genistein inhibits the expression of TF in primary cultured human great aphenous vein endothelial cells endothelial cells induced by homocysteinein a concentration and time dependent manner.