Study The Mechanism Of Extravillous Trophoblast Apoptosis And The Matricellular Protein Cyr61 Down-regulation Caused By Hypoxia In Preeclampsia

Part oneStudy expression of Cyr61 and CTGF in placentas from preeclamptic womenObjective:To detect the expression of Cyr61 and CTGF in placentas from normal term pregnancy and preeclampsia women, and explore their roles in the pathogenesis of preeclampsia.Methods:The protein and mRNA expression levels of Cyr61 and CTGF of placenta tissue in 16 cases of normal term pregnancy and 34 cases of severe preeclampsia women were determined by immunohistochemistry and real-time fluorescence quantitative PCR, respectively. Results:1. Cyr61 was expressed in syncytiotrophoblast, cytotrophoblast and endothelial cell and mainly located in the cytoplasm of the placental villous syncytiotrophoblast. CTGF was expressed in cytotrophoblast and endothelial cell and mainly located in the cytoplasm of the placental villous cytotrophoblast. Compared with control group, the expression of Cyr61 in the severe preeclampsia group was significantly lower (P<0.01), while CTGF in the severe preeclampsia group was significantly higher than that of control group (P<0.05). 2. Cyr61 mRNA expression in the severe preeclampsia group was significantly lower than normal levels, the difference was statistically significant (P<0.05); CTGF mRNA expression in severe preeclampsia were significantly increased comparing with normal levels, the difference was statistically significant (P<0.01).3. There was a negatively correlation between the expression of Cyr61 and CTGF in placentas from severe preeclampsia (r=-0.34, P<0.01). Conclusions:1. Abnormal expression of Cyr61 and CTGF in trophoblast might lead toVEGF reduce and the failure of ECM formation and remodeling thus resulted in the pathogenesis of preeclampsia.2. The expression of Cyr61 and CTGF in preeclampsia is negatively. Therefore, the addition of exogenous VEGF or Cyr61 could be antagonized the effect of CTGF in the pathogenesis of preeclampsia.Part twoThe cell proliferation and apoptosis of extravillous trophoblast under hypoxiaObjective:To investigate the proliferation and apoptosis of extravillous trophoblast under hypoxic conditions and Cobalt chloride (CoCl2) was used to mimic the effects of hypoxia in villous explants.Methods:1. Methl thiazolyl tetrazolium (MTT) assay was implied to evaluate the cell proliferation function. Cells were treated with 150 or 300μmol/L of cobalt chloride. TEV-1 cells were treated with cobalt chloride for 6h,12h,24h,48h and 72h. Normoxic cell lines were cultured in the same way without the use of cobalt chloride.2. Flow cytometry analysis was used to detect trophoblast apoptosis and the cells were treated with 300μmol/L of cobalt chloride for 6h,12h,24h,48h and 72h.Results:1. Compared with the control, CoCl2 inhibited growth parameters of TEV-1 cells.The cell proliferation was remarkably inhibited at a concentration of 300μmol/L CoCl2 in contrast to 150μmol/L CoCl2(P<0.05). When TEV-1 cells were treated with 300μmol/L CoCl2 for 24h, the cell reached the lowest point of proliferation(P<0.01).2. Annexin-V/PI double staining assay showed that, compared to the cells cultured in normal group, exposure of extravillous trophoblasts to low oxygen concentration(300μmol/L CoCl2), enhanced cell apoptosis in a time-dependent manner, with a significant increase in apoptosis levels after 12h (10.7+/-0.8-fold; P<0.01), reaching a maximum of 24.0+/-3.4-fold at 24h(P<0.05).Conclusions:Cobalt chloride, which is used to mimic the effects of hypoxia in TEV-1 cells, inhibits the cell proliferation and promotes cell apoptosis. As the hypoxic condition extends, TEV-1 cells apoptosis increases and peaks, exposure to hypoxic conditions for 24h.Part threeThe expression of Cyr61 in extravillous trophoblast under hypoxia and its role in the pathogenesis of preeclampsiaObjective:To examine the expression of matricellular protein Cyr61 functional changes involved in adaptation to hypoxia of the TEV-1 cells, using cobalt chloride (CoCl2) as hypoxic mimic and explore Cyr61 activity in the pathological mechanism of preeclampsia. Methods:1. Immunofluorescence microscopy analysis was used to detect the location and expression of Cyr61 in extravillous trophoblast.2. Real-time quantitative PCR was implied to analyze hypoxic regulation of Cyr61 mRNA.3. The protein level of Cyr61 was investigated by Western blot analysisResults:1. The matricellular protein Cyr61 was mainly observed in the cytoplasm and partly in nuclear.2. Expression of Cyr61 in extravillous trophoblast increased upon hypoxic treatment for 6h and 12h. However, as the hypoxic time extended, Cyr61 expression decreased.Conclusions:Although hypoxia transiently induced Cy61 production, as the hypoxic condition extended, the expression of Cyr61 was down-regulated.Part fourPreeclamptic sera induces Cyr61 decrease in extravillous trophoblast and its relation with preeclampsiaObjective:To investigate the expression of Cyr61 in extravillous trophoblast and the cells was treated with normal pregnant serum and PE serum, espectively.Methods:1. The location and expression of Cyr61 in extravillous trophoblast was demonstrated by immunofluorescence microscopy analysis.2. The protein and transcriptional level of Cyr61 in extravillous trophoblast, which was cultivated under PE serum for 24h, was investigated by using real-time quantitative PCR and Western blot analysis, respectively.Results:1. Cyr61 protein was mainly located in the cytoplasm and partly in nuclear.2. In contrast to normal pregnant serum, the Cyr61 mRNA expression level as well as protein decreased in extravillous trophoblast exposed to PE serum (P<0.05).Conclusions:Preeclamptic sera evoked Cyr61 down regulation and Cyr61 deficiency might play roles in the pathogenesis of preeclampsia.