| Objective: To observe the effects 17βestradiol (17β-estradiol, 17β-E2) on oxidized low-density lipoprotein (ox-LDL) induced apoptosis of endothelial progenitor cells and its possible mechanism designed to investigate the effects of estrogen suppression atherosclerosis and its possible mechanism reducing the probability of female coronary heart disease. Past estrogen internal study on the impact of endothelial progenitor cells have more emphasis on the promotion of endothelial progenitor cells proliferation and migration and angiogenesis, whereas the internal side of endothelial progenitor cells from apoptosis has not placed enough emphasis. In recent years, several documents were found estrogen also inhibit endothelial cell apoptosis, in this experiment we use oxidized low-density lipoprotein as the induction agent to observe the effects of estradiol on endothelial cell progenitor cells in vitro before apoptosis. ox-LDL takes an important part in atherosclerosis (AS), thus estrogen inhibited apoptosis of endothelial cell progenitor cells by ox-LDL-induced is better able to explain the role of its anti-AS. This study also try to observe the apoptosis mechanisms and means of 17β-estradiol on inhibition endothelial cell progenitor cells, that observe whether 17β-estradiol through its receptor plays a role, whether have relation with the PI3K/Akt pathway .Methods : (1) in peripheral mononuclear cells separation: Human peripheral blood samples 50ml in heparin anticoagulation (heparin 1000U). PBS for dilution by 1:1 at people add on lymphocyte separation medium, 2000R/min centrifugal, drawing white layer between mononuclear cells, 1500R / M centrifugal washing three times, taking equal volume cell suspension mixed with 2g / L Units Trypan blue, the calculation of cell viability (dead cells - the blue, live cells are not colored), the number of viable cells counted for more than 98% can be vaccinated. Finished above in four hours after Blood samples are collected (2) induction of differentiation: fibronectin dissolved with PBS diluted to 1 ng / ml after the package was 24 plate, each hole add 100ul, at 37℃temperature box incubated 2h, before the use drawing-off excess liquid, with PBS washing twice. Hours with M199 culture medium adjust cell density, were inoculated culture flask fibroblasts more than 24 hours adherent, so cultivate 24 hours after admission to non-adherent cells inoculation density of 4 X 106/cm2 on coated fibronectin (FN) of 24 well culture plates (per hole M199 culture medium add 1 ml), each hole add 10% FBS, 1% Green streptomycin (10,000 units / ml), VEGF 50 ng / ml, b-FGF50 1ng / ml, placed in 5% CO2, saturated humidity, 37 oC incubation box cultivate four days, washed out non-adherent cells with phosphate buffer, changing the culture medium to continue to cultivate 7d, further use of PBS away non-adherent cells, adherent cells for experimental use. (3) EPCs Identification: Using immunohistochemical method to detect cell forms of anti-CD34 and VEGFR-2; the use of immunofluorescence assay cell epitope of anti-C133; endothelial progenitor cells using specific antibodies DiI-ac-LDL and FITC-UEA-1 adherent cells on immunofluorescence staining. (4) EPCs apoptosis observation: the use of fluorescein isothiocyanate-labeled Annexin-v, propidium iodide (PI) double staining immunofluorescence method observe EPCs apoptosis. Adherent cells directly to cultivate 7d on coverslip, then washing with PBS twice at 500ul of Binding Buffer added 2ul fluorescein isothiocyanate-labeled Annexin-v, 5ul PI mixing. Will add to the above solution coverslip surface, so that cells have a long coverslip surface evenly covered with dark, room temperature reaction for 10 minutes. Coverslip upside down on the slide will be up in two-color filter fluorescence microscope observation. (5) estrogen internal observation of apoptosis of endothelial progenitor cells: different concentrations of estrogen intervention by ox-LDL-induced apoptosis of EPCs, to observe its dose-dependent and time-dependent manner. (6) mechanism add estrogen receptor antagonist ICI182, 780 and the PI3K inhibitor LY294002 observed after apoptosis.Results: 1. The effect of estrogen on EPCs have inhibition of apoptosis, under 100 nmol /L concentrations of E2 , the apoptosis rate was (8.23±0.6)%, compared with the control group (12.31±0.34)% , the apoptosis rate has significantly The statistical significance (P <0.01). 2. EPCs estrogen suppression of apoptosis in a dose-dependent. At E2 concentrations 10nmol/L-100nmol/L range of concentration with an increase at 24h post-intervention testing, its anti-apoptotic role in gradually. At 10nmol/L, 50nmol/L, 100nmol/L concentrations of E2 were under the apoptosis rate (22.39±0.68)%, (20.42± 2.56)%, (13.93±0.3)%, compared with ox-LDL-induced group (31.43±1.05)% apoptotic rate , there is significant statistical significance. (P<0.01) at 48h after the intervention in apoptosis rate were detected (23.28±0.64)%, (19.25±1.16)%, (14.57±0.45)%, compare to the 24h results there is no significant statistical significance (P>0.05 ). 3. Estrogen receptor antagonist ICI182, 780 with the PI3K inhibitor LY294002 can inhibit the anti-apoptotic role of E2, the apoptosis rate added estrogen receptor antagonist ICI182, 780 group (n = 8) increased to (28.6±1)%, while the apoptosis rate added PI3K inhibitor LY294002 group (n = 8) increased to (25.2±1)%, compared to E2 group (13.9±0.3)% , the apoptosis rate have obvious statistical Study significance (P<0.01).Conclusion: 1. 17β-estradiol has effect of anti-human peripheral blood endothelial progenitor cells from apoptosis. 2. ox-LDL can induce human peripheral blood endothelial progenitor cells from apoptosis, and 17β-estradiol can be a dose-dependent manner against ox-LDL-induced endothelial progenitor cells from apoptosis. 3. Anti-17β-estradiol ox-LDL-induced human peripheral blood endothelial progenitor cells apoptosis has relation with the mechanism of estrogen receptor and PI3K pathways .
|
PDF Full Text Request