Ffect Of Soluble Epoxide Hydrolase Inhibitor On Function Of Endothelial Progenitor Cell

Part Ⅰ Isolation, culture and identification of mice bone marrow-derived vascular endothelial progenitor cellsObjectives Study the methods of isolation, culture and identification of mice bone marrow-derived vascular endothelial progenitor cells.Methods1.The isolation and culture of mice bone marrow-derived vascular endothelial progenitor cells:Using density gradient entrifugation separating single nuclear cells from mouse femoral and tibial bone marrow, move into the cell culture bottles which have been coated by fibronectin according to the5x103/cm2density, culture by EBM-2medium which contains FBS and growth factors, observe the endothelial progenitor cells shape in different time points by confocal microscope.2. Double fluorescence staining of mice bone marrow-derived vascular endothelial progenitor cells:Cells culture for7days, remove the culture medium, add DiI-ac-LDL (10mg/L), avoid light and incubation stick wall cells for4h in37℃, then add FITC-UEA-1(10mg/L), and incubation stick wall cells for1h in37℃. The fluorescence microscope observe cells absorb Dil-AcLDL and combined with FITC-UEA-1double positive cells for Endothelial progenitor cells.3.The identification of mice bone marrow-derived vascular endothelial progenitor cells:After7days culture, count the cell numbers which express CD34,CD133,CD31,F1k-1surface antigen differentiation.Results1.After4days culture, colony formation can be seen by single nuclear cell in microscope, the circular cells in central, round by spindle cells;7days after culture, the cell colony significantly expanded and the circular cells in central transform to the spindle cells; in day8, cable shape transformation can be visible in culture cells; after10days culture, the endothelial progenitor cells can achieve80%-90%fusion, priority to circular and polygonal cells, spindle cells Also visible.2. In microscope, the cells absorbed Dil-AcLDL and combined with FITC-UEA-1double positive cells for more than95%.3. Flow cytometric test results shows:The each cell surface differentiation antigen content for CD34(53.89±0.34)%, CD133(52.79±0.67), CD31(36.67±0.93)%,Flk-1(43.88±0.48)%.Conclusion Through the morphology, double dyeing and flow cytometric test, we have proved the cultured cells is endothelial progenitor cells. Part Ⅱ The influence and mechanism of soluble epoxide hydrolase inhibitors t-AUCB to mice bone marrow-derived vascular endothelial progenitor cellsObjectives Study the influence and mechanism of different concentration t-AUCB/and PPAR γ receptor blockers (GW9662) influence mice bone marrow-derived vascular endothelial progenitor cells proliferation and adhesion, migration, the nest, angiogenesis, and secretion VEGF and HIF1-α after intervention.Methods After7days inoculation, endothelial progenitor cells intervened by different concentration (0,1,10,50,100μmol/L t-AUCB,GW9662+100μmol/L t-AUCB), then take the same number (1×105)cells process the following experiments:1. After digesting, the cells were planted to96orifice plate, the hole added MTT, after24hours, determine the uptake value one by one in the enzyme league immune monitoring, calculate the influence of proliferation.2. The cells were moved into the cell culture bottles which have been coated by fibronectin, incubation1h in37℃, remove not stick wall cells, count adhesion cell number under the inverted microscope. Calculate the influence function of EPCs's adhesion by t-AUCB.3.The cells were add to the upper plate of transwell, the lower strata add migration buffer contains different concentration of t-AUCB,37℃incubation4h, determine the cell number of moving to lower strata, calculate the influence function of EPCs's migration by t-AUCB.4. Together in a culture with2μg/mL Dil-ac-LDL, EPCs were intravenous to mice immediately through the tail vein after the left anterior descending ligation.24h later, the heart be got from mice and cut into small grain, digestive in liquid enzymes. After centrifugal, counts the number of Dil mark positive cells in fluorescence microscopy, calculate the influence function of EPCs's homing by t-AUCB.5. The same Number of cells move into the cell culture bottles which have been coated by matrigel before, incubation24h in37℃, counts the number of angiogenesis under the inverted microscope.6.The cells were digested, extraction protein, and the western blot test the ability of secretion VEGF and HIF1-α function.Results1.From0μmol/L-100μmol/L, along with increased concentration, t-AUCB can obviously increase the EPCs proliferation capacity continue, compared with control group (0μmol/L),1,10,50,100μ mol/Lt-AUCB can significant increase the proliferation capacity in EPCs (P<0.05); PPAR y receptor blockers (GW9662) can inhibit its function (P<0.05).2.From0μmol/L-100μmol/L, along with increased concentration, t-AUCB can obviously increase the EPCs adhesion capacity continue, compared with control group (0μmol/L),1,10,50,100μmol/Lt-AUCB can significant increase the adhesion capacity in EPCs (P<0.05); PPAR y receptor blockers (GW9662) can inhibit its function (P<0.05).3.From0μmol/L-100μmol/L, along with increased concentration, t-AUCB can obviously increase the EPCs homing capacity continue, compared with control group (0μmol/L),1,10,50,100μmol/Lt-AUCB can significant increase the homing capacity in EPCs (P<0.05); PPAR y receptor blockers (GW9662) can inhibit its function (P<0.05).4.From0μmol/L-100μmol/L, along with increased concentration, t-AUCB can obviously increase the EPCs migration capacity continue, compared with control group (0μmol/L),1,10,50,100μmol/Lt-AUCB can significant increase the migration capacity in EPCs (P<0.05); PPAR γ receptor blockers (GW9662) can inhibit its function (P<0.05).5.From0μmol/L-100μmol/L, along with increased concentration, t-AUCB can obviously increase the EPCs angiogenesis capacity continue, compared with control group (0μmol/L),1,10,50,100μ mol/Lt-AUCB can significant increase the angiogenesis capacity in EPCs (P<0.05); PPAR y receptor blockers (GW9662) can inhibit its function (P<0.05).6.From0μmol/L-100μmol/L, along with increased concentration, t-AUCB can obviously increase the EPCs generation and secretion VEGF and HIF1-α capacity continue, compared with control group (0μmol/L),1,10,50,100μmol/Lt-AUCB can significant increase the generation and secretion VEGF and HIF1-α capacity in EPCs (P<0.05); PPAR γ receptor blockers (GW9662) can inhibit its function (P<0.05).Conclusion From0umol/L-100umol/L, along with increased concentration, t-AUCB can obviously increase the EPCs proliferation and adhesion, migration, the homing, angiogenesis, generation and secretion VEGF,HIF-α ability, the PPAR y receptor play a importtent role. Part Ⅲ The influence and mechanism of transplant EPCs intervened by soluble epoxide hydrolase inhibitors t-AUCB to mice myocardial infarctionObjectives Study the influence and mechanism of transplant EPCs intervened by different concentration soluble epoxide hydrolase inhibitors t-AUCB and PPAR y receptor blockers (GW9662) to mice myocardial infarction.Methods1. Through the ligation left anterior descending method to make myocardial infarction model, and record the infarction electrocardiogram.2.1hour after infarction respectively through the tail intravenous200ul endothelial progenitor cells intervened by different concentration (0,1,10,50,100μmol/Lt-AUCB,GW9662+100μmol/L t-AUCB) t-AUCB.3. In24hours,3days,7days,14days and28days after myocardial infarction, to death mice, take out the heart, formalin fixed, determination of myocardial infarction area accounts for total area of the cross-section to the ligation point percentage.4. Do Ⅷand VEGF immunohistochemical stains determination the angiogenesis number of myocardial infarction fringe area.5. Compare the myocardial infarction area ratio and angiogenesis quantity of different concentrations group in the same time point intervened by different concentration of t-AUCB.6. Compare the myocardial infarction area ratio and angiogenesis quantity intervened by same concentration of t-AUCB in different time points.Results1. In making myocardial infarction model, ligation left before descending branch instantly visible the ligation line distal myocardial mobility is abate, left ventricular wall shows pale then become purple. Doing ECG, the line of front wall shows ST segment the arch upward bid up, prove that model making success. 2. At the same time point, the myocardial infarction area percentage in different concentrations transplantation groups shows:after transplantation24hours, there is no significant difference between different concentration intervention group (P>0.05); after3days,7days,14days and28days transplantation, along with increased concentration, the intervention group gradually reduce the ratio,there was significant difference between the intervention group (P<0.05).3. The area percentage of the same concentration of t-AUCB intervention in different time points after myocardial infarction shows:(1) In0μmol/L group:from24hours to14d, with prolonged, heart infarction area increase gradually; from14d to28d, area reduced gradually, and between different time points had significant differences (P<0.05).(2) In1μmol/L group:from24h to3d, the area is reduced gradually than former;3days to14days, the area is increase gradually than former;14days to28days, the area is reduced gradually than former, and between different time points had significant differences (P<0.05).(3) In10,50,100μmol/L group:from24hours to7days, the area gradually narrowed than former;7days to14days, the area is increase gradually than former, but still less than24hours area; from14days to28days, the area is reduced gradually than before, and between different time points had significant differences (P<0.05).(4) In100μmol/L t-AUCB+gw9662group:from24hours to3days, the area increase gradually than former;3days to7days, the area is reduce gradually than former;7days to14days, the area is increases than former; from14days to28days, myocardial infarction area ratio decrease than former, and between different time points had significant differences (P<0.05).4. The angiogenesis quantity of different concentrations group in the same time point intervened by t—AUCB shows, with increasing concentration, the vascular number significantly increased, and t-AUCB concentration has positive correlation between vascular number, GW9662+100umol/L results between0and1mol/L. Each group had significant differences in between (P<0.05).5. The angiogenesis quantity of the same concentration intervention group in different time points intervened by t—AUCB shows, With the intervention prolonged, there is significantly more blood vessels number in the same intervention group;the results of100umol/Ll+GW9662group is between0umol/1and1umol/1. Between each group has significant difference (P<0.05).Conclusion Through intervene EPCs, sEH inhibitors t-AUCB participate in positive control in myocardial infarction repair, prompting myocardial ischemia area vascular reconstruction; t-AUCB positive control and activate the EPCs function is PPAR γ relevant.