| Part One Detection of Axl expression in EPCs from preeclampsia patients and normal patientsObjective To detect the expression of Axl in EPCs from preeclampsia patients and normal patientsMethods1.Separate PBMCs from the cord blood of the two groups,and culture for7 days;2 The expression of Axl m RNA in EPCs from two groups were determined by RT-PCR;3 The expression of Axl protein from two groups were determined byWestern Blotting.Results1.After cultured until the seventh day, CFUs(clony-forming units)can be found consisting of round cells in the central and spindle shaped cells in the peripheral.2.The m RNA(25.77±8.69 vs. 1.55±0.67, P < 0.05) and protein(17.00±6.36 vs. 1.03±0.20, P < 0.05) expression of Axl in EPCs from preeclampsia patients were significantly higher than that of normal pregnancy.Conclusion The study shows increased expression of Axl in Preeclampsia EPCs than in normal EPCs.Part Two The effect of Axl inhibitor on EPCs functionsObjective To investigate the effect of Axl signaling pathway inhibition on the proliferation, differentiation, migration and adhesion capacity of EPCs and the role of Axl in the pathogenesis of preelampsia.Methods1.First,Separate and culture the cord blood EPCs.Then devide them intothree groups:experimental group?negative control group and blank control group. The experimental group is treated with Axl inhibitor(BMS777-607, Bio Vision, USA) which already showed inhibition to Gas6/Axl signal at the concentration of 5.64ng/ml, and the same volume of DMSO(the solution of BMS777-607) was added to the culture medium of the negative control group, and the blank control group was treated with nothing. Then, EPCs from the three groups go through the following cell function assays;2.The migration ability of EPCs was evaluated by transwells with 8.0-mm pore sized PET membranes(Corning, USA);3.Cell proliferation was measured by the CCK-8 assay;4.The cell differentiation assay was performed according to Yan T's previous summary;5.The cell adhesion assay was performed according to Ushiyama S's previous summary with little alteration.Results1.Treatment with Axl inhibitor caused a decrease on EPC proliferation in experimental group compared to both control and negative control group(0.97±0.06 vs. 1.20±0.11, P < 0.01; 0.97±0.06 vs. 1.18±0.05,p<0.05).While the control group and negative control group showed nosignificant difference( 1.20±0.11 vs. 1.18±0.05,P>0.5).2.Histogram showed the average number of spindle-shaped cells decreased in experimental group compared to the control and negative control group(21.80±4.33 vs. 41.12±2.93, P < 0.05;21.80±4.33 vs.37.80±9.58, P < 0.05), while no significant difference between the control group and negative control group(41.12±2.93 vs. 37.80±9.58, P>0.5).The percentage of differentiated cells decreased in the experimental group(12.02±2.18 vs. 17.78±1.81, P < 0.01;12.02±2.18 vs. 16.36±2.03, P< 0.05) compared to both the control group and negative control group,while there is no significant difference between the control group and the negative control group(17.78±1.81 vs. 16.36±2.03,P>0.5).3.The migration ability of EPCs was significantly decreased after inhibition of Axl(experimental group) compared with the negative control group(50.35±15.48 vs. 79.88±5.69, P <0.05), while there was no significant difference between the control group and negative control group(72.40±6.24 vs. 79.88±5.69, P>0.5)and between the control group and experimental group(50.35±15.48 vs72.40±6.24,P>0.5).4.The the number of adhered cells was significantly decreased in the experimental group compared with both the control group and the negative control group(62.90±6.43 vs. 80.73±4.80, P <0.05; 62.90±6.43 vs. 83.20±4.52, P<0.01), but there was no significant difference betweenthe control group and the negative control group(80.73±4.80 vs.83.20±4.52, P>0.5).Conclusion After inhibition of Axl signaling pathway(treatment with Axl inhibitor),the proliferation, differentiation, migration and adhesion capability of EPCs decreased. |
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