Expression Of Cytokines And Growth Factors During Human Embryo Implantation And Regulation Of IL-1β And HCG

Human embryo implantation is a series of cellular or molecular biology events from the fertilized egg to embryo implantation and a very complicated physiological process,including free blastocyst position,adhesion,invasive and placentation. Judging from the current animal and human research data,embryo implantation ix regulated by many genes,involving hundreds of moleculars,which form cascade network systems,and these moleculars may vicarious function each other.As invasive state uterus and receive state fetus,the cytokines and Growth factors secreted from them are very crucial for the reconstruction of matrix and the control of invasion.It is also research base on regulating embryo implantation.But studies on these molecular of the maternal-fetal interface confined in a single factor.Moreover,the effect of exogenous hormones and cytokines on the regulation of embryo implantation and the interaction of cytokines is not yet over.Useing Luminex system examine two types of maternal-fetal cell culture supernatant of leptin,TGF-β1,IL-6,bFGF,IL-1ra,VEGF, EGF,protein expression.IL-1βis a necessary factor implantation of the embryo,maybe the first initiation factor of fetal-maternal dialogue,and IL-1βas an important factor regulating,with the corresponding high-affinity receptor binding followed by the second wave of cytokine cascade waterfall effect,can also affect the body in many cytokine secretion. IL-1βright now and the regulatory role of the majority is against MMPs and TIMPs. How to play its maternal-fetal interface HCG is the earliest embryo one of the elements,Moreover,it shows that the fetus was one of the signs.In the process of implantation through paracrine mode role of the local impact of pregnancy.HCG is found in the new angiogenesis important VEGF cytokine stimulation was significant, HCG and makes a significant increase of MMP-9,TIMP-1 is not affected,thereby contributing to the further trophoblast invasion.HCG currently on the right embryo implantation of the majority of the use of choriocarcinoma cell lines to explore,using primary cultured trophoblast cells and decidual cells during early pregnancy its study of the role should be more valuable,But rarely reported in the literature.Therefore, respectively,and IL-1βHCG as a regulator,Study them to the above two categories of maternal-fetal cell secretion of the above eight cytokines and growth factors,to explore cytokines and growth factors on trophoblast invasion,embryo implantation how to regulate it,Find regulation of embryo implantation of cytokines and growth factors network molecular mechanism to embryo implantation and pregnancy establish more theoretical basis.PartⅠDifference of human cytokines and growth factors in extravillous cytotrophoblasts and decidualized stromal cellsOBJECTIVETo investigate the difference of cytokines and growth factors expressed in early pregnant decidua and trophoblast and their relationship with implantation.METHODSCollect the villi and deciduas of Women Early Pregnancy which from 6-10weeks legal voluntary interruption pregenncy were mechanically isolated and cultured in vitro.5×105 EVCT and DSC were placed in 24-well plate and culture with serum-free medium.Isolated cells(＞95%),Then,collect supernatants of culture metia,after 48 hours incubation respectively.2.The celluler morphology was observed and taken photographs by inverted phase contrast microscope.3.The cell population was identified by immunocytochemical staining with cytokeratin and prolactin.The nucleus and cytoplasm were observed by staining of streptavidin-peroxide(SP).Positive cells were more than 95%,indicating absence of contaminating elements.Cells cultured in six-well asepsis slides and treat cell to climb a basic and full, take out the slides were fixed with 4%paraformaldehyde in phosphate-buffered saline(PBS)for 30 min at room temperature,washed in PBS for 5 min. Immunohistochemistry was performed using the streptavidin-peroxide from a DAB kit.Briefly,slides were treated with 3%hydrogen peroxide at room temperature for 10 min to inhibit the activity of endogenous peroxidase,then heated for 30 min at 95℃to repair antigens and finally rinsed in PBS.Nonspecific protein staining was blocked by goat serum.The slides were incubated with 1∶100 dilution of Cytokeratin and Vimentin at 4℃overnight.The next day,they were washed with PBS and incubated at room temperature with biotinylated secondary antibody for 10 min,washed extensively,incubated with the streptavidin-peroxidase complex for 10 min4.Luminex xMAP technology was used to measure the concentrations of IL-1α,IL-1ra,IL-6,VEGF,TGF-β1,Leptin,bFGF,EGF in DSC and EVCT samples simultaneously,then compared their variety of diffrent temporo-spatial point.5.Data were evaluated using SPSS 10.0 software.Independent-samples T test was performed to analyse their difference.Partial's correlation was used to analyse their relationship.P＜0.05 was accepted as indicative of stastical significance.RESULTS1.Morphological features of two types of cells cultured in vitro were observed. Immunohistochemistry(SP)indicated DSC were positive for PRL expression,with the cytoplasm stained brown,and EVCT were positive for cytokeratin expression, with the cytoplasm stained brown.The purity of these cells prepared for this experiment was＞95%.2.Concentrations of Leptin,TGF-β1,IL-6,bFGF,IL-1ra,VEGF,EGF,IL-1αwere expressed in EVCT culture medium by turn.3.Concentrations of Leptin,TGF-β1,bFGF,IL-1ra,L-6,EGF,IL-1α,VEGF were expressed in DSC culture medium by turn.4.2 Independent Samples Tests results:IL-1α,IL-1ra,IL-6,VEGF,TGF-β1, Leptin and bFGF have significant difference,except EGF.CONCLUSIONS1.Leptin,IL-6,bFGF,IL-1ra,EGF,VEGF,IL-1α,TGF-β1 expressed in EVCT and DSC play an important role in induction and maintenance of implantation.The concentration of cytokins and growth factor have significant differenc in EVCT and DSC.The complementary balance of cytokines and growth factor between them is in favour of establish maintain the immunological success of pregnancy.2.A new technology,Luminex xMAP,facilitates the simultaneous evaluation of multiple immune mediators with advantages of higher throughput,smaller sample volume,rapider,more reliable,made it possible to measured lower concentration molecule and protein.PartⅡRegulation of IL-1βon cytokines and growth factors expressed in extravillous cytotrophoblasts and decidualized stromal cells during embryo implantationOBJECTIVETo research the effect of IL-1βon cytokines and growth factors in extravillous cytotrophoblasts and decidualized stromal cells,investigate interaction of IL- 1βand cytokines in embryo implantation and placentation.METHODS1.The EVCT and DSC were cultured in vitro as partⅠ,but serum-free medium including 10ng/ml IL-1β.2.The celluler morphology was observed and taken photographs by inverted phase contrast microscope.The cell population was identified by immunocytochemical staining with cytokeratin and prolactin.The nucleus and cytoplasm were observed by staining of streptavidin-peroxide(SP),positive cells were more than 95%,indicating absence of contaminating elements.(Methods is the same as partⅠ)3.Luminex xMAP technology was used to measure the concentrations of IL- 1α,IL-1ra,IL-6,VEGF,TGF-β1,Leptin,bFGF,EGF in DSC and EVCT samples simultaneously,then compared their variety afte using 10ng/ml IL-1βserum-free medium.4.Data were evaluated using SPSS 10.0 software.Paired -samples T test was performed to analyse their difference.P＜0.05 was accepted as indicative of stastical significance.RESULTS1.Morphological features of two types of cells cultured in vitro were observed. Immunohistochemistry(SP)indicated DSC were positive for PRL expression,with the cytoplasm stained brown,and EVCT were positive for cytokeratin expression, with the cytoplasm stained brown.The purity of these cells prepared for this experiment was＞95%.2.IL-1βdown- regulated the levels ofIL-1ra,IL-6,VEGF,TGF-β1,bFGF,and up-regulated the levels ofIL-1α,Leptin,EGF secreted by EVCT culture system.The change of IL-1ra,IL-6,VEGF,TGF-β1,bFGF,Leptin have siganificant difference. 3.IL-1βdown- regulated the levels of IL-1ra,Leptin,and up-regulated the levels ofIL-1α,IL-6,VEGF,TGF-β1,bFGF,EGF secreted by DSC culture system.The change of IL-1ra,IL-6,bFGF,VEGF,Leptin have significant difference.CONCLUSIONSIn network of cytokines and growth factors,every factor play influential fuction in embryo implantation process.The interaction between IL-1βand cytokines and growth factors expressed in EVCT and DSC is in favor of maintain of pregnancy and placentation.PartⅢRegulation of human chorionic gonadotrophinon on cytokines and growth factors expressed in extravillous cytotrophoblasts and decidualized stromal cells during embryo implantationOBJECTIVETo study the regulation of human chorionic gonadotrophinon on cytokines and growth factors expressed in EVCT and DSC during embryo implantation, investigate the molecula mechanism of HCG using for tocolysis.METHODS1.The EVCT and DSC were cultured in vitro as partⅠ,but serum-free medium including 25U/ml human chorionic gonadotrophinon.2.The celluler morphology was observed and taken photographs by inverted phase contrast microscope.3.The cell population was identified by immunocytochemical staining with eytokeratin and prolactin.The nucleus and cytoplasm were observed by staining of streptavidin-peroxide(SP).Positive cells were more than 95%,indicating absence of contaminating elements.(Methods is the same as partⅠ)4.Luminex xMAP technology was used to measure the concentrations of IL-1α,IL-1ra,IL-6,VEGF,TGF-β1,Leptin,bFGF,EGF in DSC and EVCT samples simultaneously,then compared their variety afte using 10ng/ml IL-1βserum-free medium.5.Data were evaluated using SPSS 10.0 software.Paired -samples T test was performed to analyse their difference.P＜0.05 was accepted as indicative of stastical significance.RESULTS1.Human chorionic gonadotrophinon down- regulated the levels of IL-1ra,IL-6,TGF-β1,FGF,Leptin and up-regulated the levels of IL-1α,VEGF,EGF secreted by EVCT culture system.The change of IL-1α,IL-1ra,IL-6,VEGF,TGF-β1,Leptin,FGF have siganificant difference.2.Human chorionic gonadotrophinon down- regulated the levels of IL-1α,TGF-β1,Leptin,FGF and up-regulated the levels of IL-1ra,IL-6,VEGF,EGF secreted by pure DSC system.The change of IL-1α,IL-1ra,IL-6,Leptin,FGF have significant difference.CONCLUSIONSHuman chorionic gonadotrophinon maybe play a very important role in control vascularization,placentation and invasion of trophoblast,maintaining the balance of cytokines network.