Study Of Mechanism Of Extravillous Trophoblasts In Uterine Spiral Arteries Remodeling During Pregnancy

Part one Study of expression of MMP-9 and FasL in placenta of preeclampsiaObjective:To investigate the involement of MMP-9 and FasL in the pathological mechanism of preeclampsia and the correlation between them.Methods:The expression of MMP-9 and FasL of placenta tissue in 24 cases of normal term preganancy and 48 cases of preeclampsia women was determined by immunohistological method.Results:Expression of MMP-9 and FasL was mainly located in the cytoplasm and membrane of trophoblasts. Compared with control group, the expression of MMP-9 in the preeclampsia group was significantly lower(P<0.05), while the expression of FasL in the preeclampsia group was significantly higher than those of control group(P< 0.05). There was a significantly negative correlation between the expression of MMP-9 and FasL in placenta tissue(r=-0.700,P<0.05).Conclusions:1. Altered expression of MMP-9 and FasL in trophoblasts might influence the invasion of trophoblasts and artery remodeling, thus contribute to the pathogenesis of preeclampsia.2. Trophoblast invasion might be associated with endothelial apoptosis, both of which were involved in the mechanism of preeclampsia. Part twoStudy of role and mechanism of MMP-9 of extravillous trophoblasts on FasL secretionObjective:To investigate the release of FasL by regulation of MMP-9 secreted by extravillous trophoblasts(TEV-1), MMP-9siRNA was transfected into TEV-1 by lipofectamine.Methods:1. MMP-9 siRNA was constructed and then transfected into TEV-1 cells by lipofectamine.2. After transfection, the level of MMP-9, FasL mRNA were assessed by real-time PCR, and the intracellular or extracellular level of these proteins were measured by ELISA.3. After administration of recombinant MMP-9, the intracellular or extracellular level of FasL was measured by ELISA.Results:1. The cells transfected with MMP-9 siRNA had a remarkable decrease in MMP-9 mRNA level as compared with those transfected with control siRNA(P<0.05), whereas FasL mRNA level between the two groups had no significant changes(P>0.05).2. MMP-9 siRNA caused a significant decrease in the secretion of MMP-9(P<0.05), whereas control siRNA did not alter the secretion. There was a decrease expression of intracellular MMP-9 in MMP-9 siRNA transfected cells when compared with cells transfected with control siRNA(P<0.05).The level of sFasL were significantly reduced in cells transfected with MMP-9 siRNA as compared with that in cells transfected with control siRNA(P<0.05). Increased expression of intracellular FasL was detected in cells transfected with MMP-9 siRNA as compared with that in cells with control siRNA(P<0.05).3. sFasL release was markedly recovered by adding exogenous MMP-9 to MMP-9 siRNA transfected cells(P<0.05). Following recombinant MMP-9 added to control siRNA, a significant increase of sFasL was present with exogenous MMP-9 treatment(P<0.05). Expression of intracellular FasL was recovered to control level after adding exogenous MMP-9 to MMP-9 siRNA treated cells(P<0.05). Exogenous MMP-9 to control siRNA was observed to cause significant decrease of intracellular FasL expression(P<0.05).Conclusions:1. MMP-9 siRNA efficiently and specifically degraded MMP-9 mRNA in TEV-1 cells.2. MMP-9 secreted by TEV-1 cells regulated both secreted and soluble FasL protein.3. Recombinant MMP-9 effected both secreted and soluble FasL protein in TEV-1 cells.Part threeStudy of role and mechanism of MMP-9 of extravillous trophoblasts on endothelial apoptosisObjective:To investigate the involvement of MMP-9 in trophoblasts-induced endothelial apoptosis, MMP-9siRNA was transfected into extravillous trophoblasts (TEV-1) by lipofectamine.Methods:MMP-9siRNA was transfected into extravillous trophoblasts (TEV-1) by lipofectamine. Then trophoblasts transfected with MMP-9siRNA or control siRNA were cocultured with endothelial cells respectively in a transwell system. The apoptosis of endothelial cells were detected by flow cytometry. Besides, EVC-304 cells were cultured under MMP-9 for different period of time to observe whether MMP-9 has direct apoptosis effect on EVC-304 cells.Results:1. The percentage of cells externalizing phosphatidylserine, quantified by flow cytometry, showing that the level of endothelial apoptosis were significantly decreased when cocultured with trophoblasts transfected with MMP-9 siRNA, as compared with that when cocultured with trophoblasts transfected with control siRNA(P<0.05). Yet, the apoptosis level was recovered to control level by adding MMP-9 back to MMP-9 silencing cells(P<0.05). It showed MMP-9 administration(5ng/mL) of TEV-1 cells caused increased endothelial apoptosis(P<0.05).2. We also studied whether MMP-9 alone, in addition to FasL, directly induces endothelial apoptosis. Apoptosis effect was investigated after stimulation of the cultures with MMP-9 (5ng/mL) for 6,12, or 24 hours. No difference was found as compared with control(P>0.05).Conclusions:1. MMP-9 secreted by extravillous trophoblasts induced endothelial apopotosis.2. MMP-9 alone has no direct apoptosis effect on endothelial cells, and MMP-9 secreted by trophoblasts contributes to endothelial apoptosis via extracellular release of soluble FasL.