A Dissertation Submitted To Huazhong University Of Science And Technology For The Degree Of Doctor Of Obstetrics And Gynecology In Medicine

PartⅠExpression of osteopontin in placental tissues from healthy pregnant women and the patients with preeclampsiaObjective:To study the expression of OPN (osteopontin) in the placental tissues from healthy pregnant women and the patients with preeclampsia, and to explore the relationship between OPN and preeclampsia.Methods:We obtained 20 cases of placental tissues from healthy pregnant women and 25 cases of those from patients with preeclampsia, including 10 cases of mild preeclampsia,15 cases of severe preeclampsia. First, we observed structure of placental tissues from normal pregnancies and those with preeclampsia after HE (hematoxylin-eosin) staining. Then, we detected OPN expression in the placenta of each group by RT-PCR, Western Blotting and immunohistochemistry.Results:1. We found that in preeclampsia placenta, there were villus swelling, interstitial vacuolization, fibrinoid necrosis, nodules increased in syncytiotrophoblast and a large number of apoptotic cells in villous syncytiotrophoblast, which were rare in the normal placenta. 2. Immunohistochemical results showed OPN in placenta were mainly expressed in the cytoplasm and membrane of placental trophoblast cells and also expressed in placental capillary endothelial cells.3. Western blotting results displayed that relative value of OPN expression in normal late pregnancy group, mild preeclampsia group and severe preeclampsia group was 1.65±0.11, 1.19±0.15 and 0.61±0.10, respectively.4. RT-PCR results revealed that relative value of OPN mRNA expression in normal late pregnancy group, mild preeclampsia group and severe preeclampsia group was 1.01±0.08, 0.71±0.07 and 0.39±0.06, respectively.These results suggested that compared with healthy late pregnancy group, OPN expression was significantly decreased in the placental tissues from the patients with preeclampsia, and its expression was negatively related to the severity of preeclampsia.Conclusion:Low expression of OPN in placenta from preeclampsia patients suggested it would play an important role in the pathogenesis of preeclampsia and be able to reflect the severity of preeclampsia. PartⅡEffect of hypoxia on the expression of Osteopontin in trophoblast cellsObjective:To investigate the effect of hypoxia on OPN expression in human placental choriocarcinoma trophoblast cell line, BeWo, and to explore the role of OPN in the pathogenesis of preeclampsia.Methods:Expression of OPN in BeWo cells was measured by real-time PCR, immunohistochemistry and Western boltting under different oxygen concentrations.Results:1. The morphology of BeWo cells was obviously changed in hypoxic condition.2. Immunohistochemical results revealed that OPN was expressed in each group of BeWo cells, which were treated on different oxygen concentration and processing time. OPN was mainly localized in the cytoplasm and membrane of BeWo cells. Moreover, there was no obvious changes of OPN expression after 24 hours hypoxic treatment and its expression significantly decreased after 48 hours hypoxic treatment.3. The results were consistent with above by Western blotting, but we also found the expression of OPN was not affected in Hela cells, in which OPN was expressed, on the same hypoxic conditions.4. Real-time PCR results suggested that OPN was expressed in a concentration and time-dependent manner in BeWo cells, which means OPN expression was significantly decreased with reducing oxygen concentration and extending hypoxia time.Conclusion:OPN was expressed in a concentration and time-dependent manner in BeWo cells after hypoxic treatment, which was the same as decreased OPN expression in preeclampsia placenta with the worsening illness. So we reckoned that decreased expression of OPN was closely related to the invasiveness of trophoblast cells caused by hypoxia. PartⅢThe effect of hypoxic on the invasiveness of trophoblastic cellsObjective:To study the relationship between hypoxic treatment and invasiveness of BeWo cells and to analyze the relationship between expression of OPN and invasiveness of trophoblast cells in the patients with preeclampsia.Methods:We simulated preeclampsia process by using an air distributing device (QT-MTX-3), and observed the effects of hypoxic treatment on BeWo invasiveness by using transwell system.Results:BeWo cells could invase to lower compartment of the transwell system in each group. Comprared to normaxia group, the infiltration index of BeWo cell in 8% O2 group and in 2% O2 group was 0.2988 and 0.0917, respectively. It is suggested that the lower oxygen concentration could decrease the number of BeWo cells which infiltrated into lower compartment.Conclusion:Hypoxia inhibited the invasion of BeWo cells, which means its invasiveness was conditioned by lower oxygen concentration.