| Section OneThe different expression of PPAR y in peripheral blood and umbilical cord blood plasma, amniotic fluid between full-term pregnancy with or without gestational diabetes mellitusObjectives:The aim of this study was to investigate the expression of PPAR y in vitro by analyzing the expression of GDM pregnant women and normal pregnancy’s peripheral blood and umbilical cord blood plasma, amniotic fluid.Methods:Collect peripheral blood and umbilical cord blood plasma, amniotic fluid during selective cesarean section surgery at38-41weeks in Obstetrics and Gynecology hospital affiliated to fudan university in October2012to November2012. The detection method was enzyme-linked immunosorbent assay (ELISA).Results:The expression of PPAR y in GDM peripheral blood was obviously higher than normal pregnant women (p<0.001). The expressions of PPAR y in the GDM umbilical cord blood plasma and amniotic fluid have no significant diffrences with normal pregnant women.Conclusion:This is the first study to find the expression of nuclear transcription factor PPARy protein in the extracellular fluid. We hypothesized that PPAR y may release to the extracellular through certain mechanism, and the expression of PPAR y protein in GDM peripheral blood plasma reactively increased in order to maintain steady state of PPAR y protein in the fetus and amniotic fluid of pregnant women. But the influence of PPAR y protein on maternal-fetal interface cell is needed for further research.Section TwoThe expression of PPAR y in GDM pregnant’placenta, amniotic and chorionic membrane Objectives:The aim of this study was to investigate the possible role of PPAR y in lipid metabolism pathway and lay the experimental foundation for the next cell in vitro experiment by analyzing the expression of PPAR y in GDM pregnant’ placenta, amniotic membrane and chorionic membrane.Methods:Collect placenta, amniotic membrane and chorionic membrane during selective cesarean section surgery at38-41weeks in Obstetrics and Gynecology hospital affiliated to fudan university in October2012to November2012. The detection method was immunohistochemical method.Results:In GDM pregnant women’s placenta tissues, PPAR y was immunolocalized on the nucleus of syncytiotrophoblastic cells, but not present in the cytotrophoblastic cells. In GDM pregnant women’s amniotic membrane tissue, PPAR y was immunolocalized in the amniotic membrane on the nuclei of the simple cuboidal epithelium cells and in the cortex layer of fibroblasts fibroblasts on the nuclei of the fibroblasts. It was also expressed in the unformed stroma. In GDM pregnant women’s chorionic tissues, PPAR y was immunolocalized in the network layer on the membrane of cells and on the nuclei of the fibroblasts.Conclusion:In the placenta, the expression of PPAR y protein was localized on the nucleus and cytoplasm in the placenta syncytiotrophoblast. Visibly, PPAR y protein localized on the nucleus secreted into the extracellular potential through certain mechanism. It also supports the expression of PPAR y protein in cord blood plasma and amniotic fluid in clinical experiment. In the amnion, chorion layer, PPAR γ protein was localized on the nucleus of the amniotic epithelial cells, amorphous matrix and the cell membrane of chorionic reticular layer, it further supports the secretion function of membranes. PPAR y protein may be secreted into the amniotic fluid through the membranes. PPAR y protein may play a role by exocrine secretion.Section ThreeEffects of PPAR γ on cellular glucolipid metabolismObjectives:The aim of this study was to establish the co-culture system in vitro consisting of trophoblastic cell and endometrial stromal cells. The system simulates the maternal-fetal interface interaction between cells. We analyse the excreted molecular of the trophoblastic cell and endometrial stromal cells and the glucolipid metabolism ability in two kinds of cells by changing the concentration in co-culture system of PPAR y protein. Further to understand whether PPAR y protein play the role of maternal-fetal interaction or the regulation influences the glucolipid metabolism of cells.Methods:The cell co-culture system includes human trophoblast cell lines (Be Wo) and endometrial stromal cells (ESC). We change the concentration of free PPAR y protein in the cell culture environment. Setting up three points of the PPAR y protein’s concentration such as0.25ng/μL,5ng/μL,10ng/μL. The expression of cellular glucose transporter1(GLUT1), Stearic acid CoA to saturated enzyme (SCD) was detected by RT-PCR. The expression of syncytin in BeWo cells was detected by RT-PCR. The expression of vascular endothelial growth factor (VEGF) in ESC cells was detected by RT-PCR. Lipid storage capacity of BeWo and ESC cells in the co-culture system was detected by Oil red O staining.Results:1. The expression of cellular glucose transporter1(GLUT1), Stearic acid CoA to saturated enzyme (SCD) in BeWo cells:In the separate training system, RT-PCR detection found that as the PPAR γ concentration higher, BeWo cells GLUT1mRNA and SCD mRNA expressions have no significant differences. In co-culture system, the expression of GLUT1mRNA increase significantly when the PPAR y concentration is5ng/μL and10ng/μL. SCD mRNA expression rises as the PPAR y density higher, but without significant differences. Glut1mRNA, SCD mRNA expression in the co-culture system was increased compapared that in the separate training system. Only when the PPAR y protein concentration is1Ong/m, SCD mRNA expression rate in the co-culture system increased with significant difference than that in the separate culture of system relative expression (P<0.05).2. The expression of cellular glucose transporter1(GLUT1), Stearic acid CoA to saturated enzyme (SCD) in ESC:The expressions of GLUT1mRNA and SCD mRNA in ESC have no significant differences in the co-culture system and the separate training system.3. Lipid storage capacity of BeWo cells:In co-culture system:With the increase of protein concentration, oil red O staining founded that fat content in BeWo cytoplasm increased. When the PPAR y protein concentration was10ng/ml, fat content in BeWo cytoplasm increased obviously.4. Lipid storage capacity of ESC:In co-culture system, with the increase of protein concentration, oil red O staining founded that fat content in ESC cytoplasm increased. When the PPAR y protein concentration was10ng/ml, fat content in ESC cytoplasm increased obviously.5. The expression of syncytin in BeWo cells:In the separate culture system, the syncytin mRNA expression increases with the increasing of protein concentration without statistical differences. In the co-culture system, the relative expression of Syncytin mRNA has no tendency.6. The expression of VEGF in ESC cells:The expressions of VEGF mRNA in ESC have no significant differences in the co-culture system and the separate training system.Conclusion:Study on transport and metabolism of glucose and lipid in the BeWo cells and ESC cell, we found only in the co-culture system, when the PPAR y protein concentration is5ng/ml and lOng/ml, BeWo cells expressing Glutl mRNA relative expression rate relative to the blank control group increased significantly, and the co-culture system relative separate training system Glutl mRNA, SCD mRNA relative expression rate of increase, SCD mRNA relative expression rate increased significantly when the PPAR y protein concentration is lOng/ml. There may be PPAR y protein play in regulating glucose metabolism in cells depends on interactions between BeWo cells and ESC cells. Oil red O staining indicated that intracellular lipid droplets in BeWo cell and ESC cell accumulated increasing with increasing on centrations of PPAR y protein. It suggests that PPAR y protein can improve the fat metabolism and may improve peripheral hyperlipidemia cycle condi.The syncytin secretion of BeWo cell increased trapezoidally in cultured alone system, and syncytia formation of BeWo cells were increased under light microscope, it showed that PPAR y protein could promote the synthesis of trophoblastic cells, but the accumulation has not yet reached the qualitative change. Although there was no statistical difference, but the synthesis of BeWo cells is still in good condition in the effects of PPAR y protein. While in the co-culture system with the increase of PPAR y protein concentration, BeWo cells express syncytin without no difference in mRNA levels. It showed that BeWo cells have abnormal synthesis in the effect of ESC cells. VEGF mRNA expression rate in ESC cells has no change with the increase of PPAR y protein concentration, indicating that PPAR y in extracellular fluid may has no obvious influence on the receptivity.To sum up, PPAR y protein promotes the expression of cell glucose transporter1(GULT1) and cellular lipid metabolism (SCD) of trophoblast in co-culture system, and promote the uptake of liposomes in the co-cluture, but no obvious influence on the two kinds of synthesis of protein molecules Syncytin and receptivity molecule VEGF. It suggests that PPAR y protein in extracellular fluid may effect of the lipid transport function of maternal-fetal interface cell and improve the lipid uptake. Combined with the results of clinical trials of the first part, PPAR y in maternal peripheral blood of GDM increased may be compensatory.In order to maintain the fetus steady state by increasing the uptake and transport of glucose and lipid on maternal-fetal interface cells. |
PDF Full Text Request