| Part oneThe role of miR-126and EPCs in the pathogenesis of preeclampsiaObjectiveTo investigate the expression of miR-126in endothelial progenitor cells and placental tissue from patients with preeclampsia and their relations to placenta perfusion.MethodsTwelve patients with pre-eclampsia and twelve normal late pregnant women were investigated in part one. The numbers and colony formation of EPCs were examined in vitro. The expression of miR-126was determined with RT-PCR in the EPCs and placental tissue respectively. The relationship between EPCs number and expression of miR-126were determined. And microvessel density (MVD) were detected in two groups.Results 1. After7days culture in vitro, round mononuclear cells gradually turned into spindle-shaped cells. Clusters consisting of round cells centrally with multiple spindle-shaped cells sprouting from the central core, which we called colony-forming units (CFU). Identification of EPCs was performed by staining with Dil-acetylated-low-density lipoprotein (DiI-Ac-LDL, red color) and FITC-labeled Ulex europaeus agglutinin-I (FITC-UEA-I, green color). The number of EPCs in preeclampsia group (77±13) was much lower than control group (136±23). CFU in control group (8.7±2.2) was much higher than that in preeclampsia group (4.3±1.3).2. The values of miR-126in EPCs from cord blood in control group and pre-eclampsia group were1.0±0.15and0.55±0.36; The values of miR-126in placenta tissue from control group and pre-eclampsia group were0.88±0.25and0.38±0.22(P<0.05)3. There was positive correlation between the expression of miR-126and number of EPCs in preeclampsia group (r=0.65, P<0.05). There was no correlation between the expression of miR-126and number of EPCs in control group.4. The microvessel density (MVD) in preeclampsia group and control group were54.8±5.72and77.6±7.92(P<0.05). The S/D ratio in preeclampsia group (3.08±0.95) was much higher than control group (2.37±0.63)ConclusionThe study shows that the decrease of number and miR-126level in EPCs from preeclampsia patients may be one of the possible reasons for the incidence of pre-eclampsia. Part twoMicroRNA-126improves biological function of endothelial progenitor cells in vitroObjectiveTo investigate the effect of MicroRNA-126(miR-126) on the proliferation, migration differentiation and colony formation capacity of endothelial progenitor cells (EPCs) from patients with preeclampsia and the role of miR-126in the onset and development of preeclampsia.MethodsThe experiment was divided into three groups:groups of transfection, negative control and blank control which corresponded to groups of miR-126mimic transfection, mimic control transfection and no transfection. Expression of miR-126and target genes were detected by real-time polymerase chain reaction and western blot analysis. Changes of cell proliferation, migration and differentiation capacity were respectively detected by MTT, transwell assay and dfiierentiation assay.Results1. After24h of transfection, transfection efficiency was determined by Cy3-siRNA transfection control. All cells presented strong and extensive cytosolic delivery of Cy3-siRNA. Cy3positive cells composed90%of the total cells. The expressions of miR-126in transfection groups, negative control and blank control were7.5±1.8,3.8±2.2and3.3±1.5;2. The mRNA level of miR-126target gene PIK3R2in transfection groups, negative control and blank control were0.25±0.28,1.34±0.29and1.0±0.32. The mRNA level of PI3K in three groups were0.46±0.22,0.54±0.2and0.85±0.23respectively. The protein level of target genes were the same trend as the mRNA level. Compared with negative control and the transfection group, the difference has importance (P<0.05). There were no significant difference between negative control and blank control group (P>0.05)3. The integral absorbance (A) values of the miR-126mimic transfection group and negative control group were0.78±0.12and0.46±0.07respectively, which was significantly different (P<0.05); the (A) value of blank control group was0.53±0.09, which was not significantly different compared to negative control group (P>0.05).4. The cell migration number of transfection group and negative control group were136.4±2.5and93.2±3.1respectively, with a statistically significant difference (P<0.05); and the number of blank control group was84.2±2.7, which was not significantly different compared with negative control group (P>0.05).5. The number of endothelial-like spindle-shaped cells in transfection group and negative control group were96.1±10.3and53.2±11.3respectively, with a statistically significant difference between them (P<0.05); the number of blank control group was47.4±9.6, which was not significantly different compared with negative group (P>0.05).ConclusionmiR-126is probably involved in the pathogenesis of preeclampsia by regulating proliferation, differentiation, and migration capacity of EPCs. Part threeIntraplacental transfection of miR-126to improve placental perfusion in preeclampsia ratObjectiveTo test the hypothesis that enhanced leval of miR-126expression might improve placental vasculogenesis and pregnancy outcome.Methods1.Placenta ischimia model was established by intraperitoneal injection of L-NAME in pregnant rat;2.21model rat were devided into three groups(control, preeclampsia and treatment group) and treatment group were treated with local injection of agomir-126on placenta;3. In vivo transfection effect of agomir-126was observed through frozen section of placenta under fluorescence microscope;4. Placenta and fetus weight, size were measured at day21of gestation;5. Expression level of miR-126of transfected placenta was detected by real time quantitative PCR;6. Placental microvessel density (MVD) after transfected was detected by immnunohistochemical assay (CD34).Results1. One rat died from anesthesia accident, only one preterm birth of all the21rats; All other rats were alive and went through the whole pregnancy.2. Strong expression of Cy3was observed throughout the placenta under fluorescence microscope. It means ideal effect of transfection in vivo.3. The blood pressure on Day15in treatment group and preeclampsia group were significant higher than control group. On Day20, the blood pressure in preeclampsia group was higher than control group and treatment group. There were no significant difference compared between control group and treatment group.4. Under contrast-enhanced ultrasonography by ultrasounic microbubble, we got the data about the size, diameter and blood perfusion of placenta. First, we used conventional ultrasound to detect the largest section of placenta in three groups, results showed that the sectional area in preeclampsia group was the lowest in three groups. At8sec after microbubbles injection, there signal of placenta was lower in preeclampsia group than control group. The time-density curve showed that the arrival time and time to peak were larger in preeclampsia group than control group and treatment group.5. The mean weight of fetus in control group, preeclampsia group and treatment group were5.26±0.06,4.15±0.08and5.04±0.11. The weight of placenta in control group, preeclampsia group and treatment group were0.60±0.03,0.43±0.02and0.52±0.04respectively, The diameter of placenta in control group, preeclampsia group and treatment group were1.43±.14,1.25±0.18, and1.40±0.11. The weight and size of fetus and placenta in preeclampsia group were smaller than other two groups, meanwhile, the placenta in preeclampsia group were paler than other two groups.6. The values of miR-126in treatment group negative control group and preeclampsia group were respectively4.2±0.31,1.0±0.27,0.693±0.16, with a statistically significant difference between treatment group and preeclampsia group(P<0.05)7. The endothelial cells in the placental tissue were dyed brown (CD34) in the section. In the control group, there were strong dying in the section, however, in the preeclampsia group, there were only light dying in the section. The number of placental microvessels in treatment group and preeclampsia group were respectively69.2±4.15and39.1±5.34, which had significant difference. There was no significant difference between control group and treatment group. Expression of miR-126and protein in placenta can be elevated efficiently through intraplacental transfection of agomir-126. MiR-126promotes placenta vasculogenesis and perfusion in preeclampsia rat. |
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