EPCs Culture From Peripheral Blood In Vitro And The Study Of Tongxinluo On Adhesion And Proliferation Activity Of EPCs For CHD Patients

Objective:Endothelial progenitor cells (EPCs) are the precursors of endothelial cells (EC). EPCs is not only involved in angiogenesis of human embryos, as well as maintaining the integrity of endothelial cell function and vascular homeostasis.Vascular endothelial cell injury is the central and central link of cardiovascular disease. The reduction and dysfunction of EPCs is considered to be a future predictor of cardiovascular events. The dynamic balance between endothelial damage and repair is the key to maintan the normal function.EPCs are important factors that involved in the process of repair.Tongxinluo is made of Collateral theory of traditional medicine. The study have confirmed that Tongxinluo can significantly improve endothelial function of CHD patients, stabilize plaques, delay the process of atherosclerosis in patients with CHD.At present, Tongxinluo has been widely used in the prevention and treatment.of clinical cardiovascular disease. To esplore the effects of Tongxinluo on the adhesion and proliferation of EPCs from human circulating blood in vitro.In order to provide information for reasonable application of Tongxinluo in treatment of CHD.Methods:This study can be divided into syntrophic two parts.1. Study on the isolation, culture and identification of EPCs from peripheral blood: The mononuclear cells (MNCs) were isolated from 10ml of peripheral blood by Ficoll density gradient centrifugation and cultured in EndoCult Liquid Medium Kit with 10% excellent FBS on 25μg/ml fibronectin-coated culture dishes. The process of cells growth and morphology change were observed under inverted microscope every day. The cells were characterized as Di-LDL uptaking and FITC-I, and further demonst rated by expressing CD34 and CD 133 using flow cytometry.2. The effect of Tong xinluo on activity of EPCs:At the basement of the first step, cells which culture in 7 days were dissociated,resuspensed.EPCs were incubated with with various concentrations of groups for 48hours and with 500ug/ml group for vario-us for various time point.EPCs were viewed with an inverted fluorescent microscope. EPCs proliferation activity were assayed by MTT assay and adhesion activity was performed by replating cells on fibronectin-coated dishes then adherent cells were counted.Results:1.1x106-2x106 MNCs were isolated in one ml peripheral blood by Ficoll density gradient centrifugation when we adjust centrifuge rate to 1500rpm. Trypan blue staining measured the viabilities of MNCs were about 96±2%. In the morning of culture, the most of cells shaped round or oval, mixed with small amounts of fusiform cells. A part of round MNCs attached dishes become fusiform EPCs in the fifth day. About in the seventh day, cell colonies contains several cells emerged. Cells become long-spindle form in the tenth day.After fourteen days, cells begin senesce, disintegration. The detection of endothelial progenitor cell line specific marker VEGFR-2, CD133and vWF are strong positive in seventh day. The function of EPCs for ac-LDL-uptaking and UEA-I binding were both strong positive in seventh day.2. Effect of AMI on the adhesive capacity of EPCs:Tongxinluo improved the adh-esive capacity of isolated EPCs in dose-and time-dependently(P<0.05).The number of adhered cells increased after 24hours(P<0.05),with a peak at 48hours(P<0.05). Tongxinluo had a peak effect at 500μg/ml concentration (P＜0.05).3. Effect of AMI on the proliferative capacity of EPCs:Tongxinluo improved the proliferative capacity of isolated EPCs in dose-and time-dependently(P<0.05).The proliferative capacity of EPCs improved after 24hours(P<0.05),with a peak at 48hou-rs (P<0.05).Tongxinluo had a peak effect at 500μg/ml concentration(P<0.05).Conclusions:1. At 26-37℃, MNCs can be better isolated from peripheral blood when use 1500rpm by Ficoll density gradient centrifugation. It can satisfy the culture of EPCs.2. The identification of phenotype and mensuration of activity for EPCs exhibit the period of prosperity in the seventh day to tenth day after culture in vitro.3. Tong xinluo improves the activity of proliferation and adhesive capacity and In vitro vasculogenesis capacity of EPCs in dose-and time-dependent manner.