The Effect Of Acrylonitrile On The Function Of Endothelial Progenitor Cells

Part Ⅰ:Acrylonitrile chronic modelObjective:To select the appropriate dose to establish acrylonitrile chronic model of rat.Methods:The median lethal dose of acrylonitrile is100mg/kg. According to this, we expose Sprague-Dawley rats to25mg/kg、50mg/k、70mg/kg、100mg/kg randomly via intragastric administration.Results:Found that half of the rats exposed to LD50of acrylonitrile died in two weeks; rats exposed to three-quarters of LD50inactive, malaise, weight loss, then, have died, the dose wes not suitable for chronic exposure; meanwhile, rats exposed to acrylonitrile of25mg/kg or50mg/kg had no significant weight loss or obesity, no deaths.Conclusion:Three groups of Sprague-Dawley rats were exposed to water、25mg/kg (A group)、50mg/kg (B group) randomly via intragastric administration with on working day for3months. Part Ⅱ:Isolation, culture and identification of endothelial progenitor cells from peripheral bloodObjective:The study is to establish the isolation, culture, purification, proliferation method of rat endothelial progenitor cells (EPCs) from peripheral blood in vitro and examine their phenotype.Methods:EPCs were isolated from rats’peripheral blood according to density gradient centrifugation and purified by their characteristic of adhering to plastic surfaces. The EPCs in culture were observed under phase microscopy and examined the expression of surface markers CD133.Results:We obtained many EPCs through the combination of density gradient centrifugation and adherence method. After passage, the cells were further purified. 2×105cells were seeded in25cm2cell culture flask with standard culture medium of10%fetal calf serum, and were passaged after80%～90%confluence. High purity and active proliferation MSCs can maintain their biological characters without differentiation. After two weeks of primary culture, cells were passaged for the first time. Then they were passaged every7days and displayed a rather homogenous population of fibroblast-like cells.Conclusion:We could purify EPCs in vitro by the combination of density gradient centrifugation and adherence method. Part Ⅲ:The study of influential factors of proliferation, migration and vasculogenesis of endothelial progenitor cellsObjective:To investigate the effects of acrylonitrile on proliferation, migration and vasculogenesis of rat endothelial progenitor cells from peripheral blood in vitro.Methods:Rat peripheral blood mononuclear cells (PBMCs) were cultured in vitro. Then cellular growth curve was plotted. EPCs were observed with phase contrast microscope to find morphological changes. Cell proliferation was assessed by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay, and CD133surface antigens of EPCs were analyzed by flow cytometry, migration assay was performed in Transwell chambers, vasculogenesis assay was performed by Matrigel, eNOS were detected by Western blot.Results:(1) Cell differentiation:Acrylonitrile had no obvious effect on cell surface antigens (CD133) expressed by EPCs.(2) Cell proliferation:The stage of latency of passage cells was about48h. Day5to day7after passage, EPCs entered the exponential phase of growth, and then went into plateau phase at day9. OD490values of EPCs treated by acrylonitrile in vivo were lower than control group (p<0.05), while OD490values of B group were lower than A group (p<0.05).(3)Cell migration:The number of cells crossing the filter in C、A and B group was respectively45±2.76、42.5±3.08、42±4.32. The number of cells crossing the filter was not significantly decreased in A and B group (p>0.05).(4)Cell vasculogenesis:The number of vasculogenesis in C、A and B group was respectively34±4.22、24±3.74、19±3.56. The number of vasculogenesis was significantly decreased in A and B group (p<0.05).(5) The expression of eNOS was reduced in A and B group (p<0.05).Conclusion:Acrylonitrile could decrease the proliferative and vasculairzation capacities of EPCs; meanwhile, they did not decrease the migratory capacities of EPCs. Acrylonitrile have no obvious effect on differentiation of EPCs.The effects of acrylonitrile on proliferative and vasculairzation capacities maybe have something to do with eNOS.