Effect Of Homocysteine On The Saphenous Vein Smooth Muscle Cell Growth And The Impact Of Genistein Intervention

Objective: To study the impact of the homocysteine (Hcy) on the proliferation and apoptosis in the primary culture of saphenous vein smooth muscle cells (GSVSMC), to find suitable concentration and time for GSVSMC proliferation.Methods: The saphenous vein smooth muscle cells (GSVSMC) were cultured by tissue explant method. The morphology of GSVSMC was studied by phase contramicroscope. Its molecular markers were observed by actin immunofluorescence assay. 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: GSVSMC were incubated with different concentrations of homocysteine(100μmol/L,200μmol/L,500μmol/L,1000μmol/L)for 24h. (3) homocysteine groups of different intervention time: GSVSMC were coincubated with 500μmol/L homocysteine for different periods(6h,12h,24h,48h).After intervention, we determinated optical absorption by MTT, accounted the rate of cell proliferation .we analysised apoptosis of GSVSMC by flow cytometry AnnexinⅤ/ PI double labeling staining.Results: 1. Normal cell were radioactive and grown well, showing SMC typical "peak" and "valley"-like growth. Most cells displayed green fluorescence under fluorescent microscopy, which accords with the characteristics of SMC. The brown granules can be seen in the cytoplasm byα-actin immunohistochemical staining, which is paralleled with the long axis of the cell .2. After the intervention of homocysteine (100μmol/L,200μmol/L,500μmol/L,1000μmol/L)for 24h, MTT assay shows that the proliferation of GSVSMC is more obvious with the increase of the homocysteine concentration. But there was no significant difference between 500μmol/L group and 1000μmol/L group (P>0.05).Under the same conditions of hcy concentrations, the proliferation of GSVSMC is more obvious with the extension of time.3. Flow cytometry showed that under the same conditions time, GSVSMC apoptosis decreased with the increase of Hcy concentrations (p<0.05), and there was negative correlation between GSVSMC apoptosis and Hcy concentrations; role in the same hcy concentration conditions, GSVSMC apoptosis decreased with the extension of time (P<0.05), and there was negative correlation between GSVSMC apoptosis and intervention time.PartⅡInfluence of homocysteine on the GSVSMC growth and the intervention of genisteinObjective: To investigate the effects of genistein on homocysteine induced GSVSMC growth Methods: groups: (1) blank control group: treated without intervention;(2) homocysteine groups: cells were incubated with 500μmol/L homocysteine for 24h;(3) genistein groups of different concentrations: cells were firstly incubated with 500μmol/L homocysteine respectively for 24h, then coincubated with(5 mg/L,10 mg/L,20 mg/L,40mg/L)genistein for 24h; (4) genistein groups of different intervention times: cells were firstly coincubated with500μmol/L homocysteine respectively for 24h , then coincubated with genistein for different period(12h,24h,48h,72h).After intervention, we determinated optical absorption by MTT, accounted the rate of cell proliferation inhibition.we analysised apoptosis of GSVSMC by flow cytometry AnnexinⅤ/ PI double labeling staining .Results: 1. MTT assay shows that the proliferation inhibition ratio of GSVSMC increased with the concentration of Gen increased, under the condition of the same time; the proliferation inhibition ratio of GSVSMC increased with the time of Gen intervention, under the conditions of same Gen concentration, there was significant difference with the control group (p<0.05). 2. Flow cytometry showed that GSVSMC apoptosis increased with Gen concentration increased under the same conditions of intervention time; GSVSMC apoptosis increased with the time of Gen intervention increased.Conclusions: 1. GSVSMC can promote the proliferation and reduce GSVSMC apoptosis under high concentrations of Hcy, and in a certain range has time and concentration-dependent manner. 2.Gen can reduce GSVSMC proliferation after high concentrations of Hcy intervention, and increase GSVSMC apoptosis, they has time and concentration-dependent manner in a certain range.