Expression Of Human Cytokines During Implantation And Effect Of Mifepristone And Cyclosporine A On Them

Embryo implantation is one of the most complicated and mysterious process during the whole life, the regulation of which is the topic for biologists trying to explore. Embryo implantation is to be thought highly of a process with certain similarities to tumor cells invasion. But the invasion of normal trophoblast is restricted to one third of the maternal endometria.The temporal and spatial regulation of trophoblast invasion is regulated by a precisely and complicated joint action network system, Several factors have been studied, including steroid hormones, eytokines, growth factors, angiogenesis factors, protease activation/inhibition factors and apoptosis regulating factors as known. But the understanding is still at onset.Miferpristone is a kind of norethisterone ramification which can end early pregnancy and use as an urgent contraceptive, its clinical effect has been generally consented, however, the exact mechanism of action is in investigating further. The research has been witnessed that miferpristone improve the expression of CD4＋lymphocyte, CD56＋CD16＋natural kill cell and TNF-αand destroy the local immunity microenvironment. Sideu deems that miferpristone influence the genetic expression of MCP-1, GM-CSF, G-CSF and many other adhesion molecules on suppressing the activity of NF-κB. All this indicates that miferpristone maybe influence the number of inflammatory cell, the phenotype of lymphocyte and some cytokines. Severals research has been carried out ,including the effect of miferpristone on endometrium, cervix, decidual and the apoptosis of villous. But how does miferpristone regulate the cytokines of embryo-maternal interface, how does the effect contribute between cytokines and the relationship of villous invasion and embryo implantation is still onset.Cyclosporsine is a kind of ring type polypeptide compound and the most effective drug in inhibiting graft rejective reaction. It can integrate cyclosporine binding protein, inhibit T lymphocyte and prevent reject reaction. It can also inhibit antigen presentation cell, including B cell, macrophage, dendritic cell and IL-12. At the same time it can regulate the balance between Thl and Th2 and induce immune tolerance by damaging the immigration andmaturity of dendritic cell. In 2004, USA National Transplantation Pregnancy Registry made a statistics on pregnancy outcome of 1319 organ transplantation since 1991, about 70 percent possessed healthy baby, so it was considered that cyclosporine can use in pregnancy safely. There are some research indicate that cyclosporine have no toxic effect on endothelial cell and enhance the ability of survival and inhibiting apoptosis of endothelial cell. So we presume that low dose cyclosporine is of protecting the embryo development at the time of embryo implantation, however, the exact mechanism of action in immune torlerance is onset.The embryo implantation is so complicated and the research of single or several cytokines is so limited.Liquichip is a multivariate analysis which can study proteomics and posses many good quality including simple operation, high sensibility, good signal to noise ratio. Using the model of decidual cells (DSC) and cytotrophoblasts (CTB) in vitro, our study has been exploring potential interactions among cytokines and growth factors in this process by Luminex xMAP and examining the expression strength and characteristics of same several factors in the sample in the system from the proteome for the first time in the normal gestation, near to the circumstance of the human body more. it highlights some of our recent observations on the roles of cytokines and their interactions in embryo-uterine "cross-talk" during implantation, to search the decisive molecules in the process of the embryo implantation in the meantime and provide a basis for the best composition combination that the embryo culture medium and provide the theories basis for the smooth progress of the gestation.PARTâ… EXPRESSION OF CYTOKINES IN THE CYTOTROPHOBLASTS AND DECIDUAL STROMAL CELLS OF HUMAN EARLY PREGNANCYBY LUMINEX XMAPOBJECTIVE1. To study the expression of cytokines and growth factors in the decidual stromal cells (DSC) and extravillous cytotrophoblast cells (EVCT)of normal early pregnancy.2. To explore effect of cytokines and growth factors on the embryo implantationMETHODS1. Collect the Decidua and Villi of 7 the Women Early Pregnancy which from 6-9weeks legal voluntary interruption pregenncy were mechanically isolated, Then, collect supernatants of culture metia, after 48 hours incubation respectively.DSC and EVCT were prepared as previously described with minor modifications. Briefly, tissue was washed in Ca2+- and Mg2+- free Hanks balanced salt solution supplemented with 100 IU/ml penicillin and 120μg/ml streptomycin, the Decidua and Villi fragments were excised with curved scissors, carefully removing blood vessels and clots, then rinsed and submitted to mild enzymatic digestion using collagenase, and trypsin-DNase, for 35 min at 37℃without agitation. After sedimentation, supematants were collected and the remaining tissue was rinsed four times with Hanks solution. The sedimentation supernatants were pooled and filtered (100μm pore size). Trypsin activity was neutralized with 10% fetal bovine serum (FBS). Cells were centrifuged and washed once in Hanks. Cells were then separated by a discontinuous BSA gradient centrifuge. Then DSC and EVCT preparation was further purified and resuspended in F12/DMEM-10% FCS by plating in plastic dishes. After incubation overnight in humidified air-5% CO₂ at 37℃.2. The celluler morphology was observed and taken photographs by inverted phase contrast microscope.3. The cell population was identified by immunocytochemical staining with cytoke(?)atin and vimentin, 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 30min at room temperature,washed in PBS for 5min.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 rain 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 min, and detected with DAB. For the control, the antibody was replaced by PBS. After immunoperoxidase staining, the slides were counterstained with hemoxylin for lmin at room temperature, redehydrated and mounted. The slides were observed by light microscopy, cells were observed per view with high magnification. Cells showing a brown-yellow color in the cytoplasm were identified as positives.4. The cytokines and growth factors (IL-1α, IL-1β, IL-1ra, IL-6, TGF-β, FGF, EGF, IFN-γ, IL-17, VEGF, Leptin) proteins expression in DSC and EVCT was detected by the the Luminex xMAP system simultaneously.5. Data were evaluated using SPSS 10.0 software. Independent-samples T test was performed to analyse their variety in decidualization.RESULTS 1. DSC and EVCT normal growth in the 24 Well-Cell Culture Cluster. The cell population was identified more than 95%, and then use to test.2. Leptin, TGF-β, IL-6, IL-Iγα, IL-1βwere produced in the greater quantity than other cytokines in DSC and EVCT, which may play specific roles in regulating embryo implantation during early pregnancy.3. Coneentrations of IL-1α, IL-1β, IL-1ra, IL-6, Leptin in EVCT culture medium were significantly higher than in DSC, while IL-17, VEGF, FGF were significantly lower. TGF-β, EGF, IFN-γ, did not differ significantly between DSC and EVCT.CONCLUSIONS1. To establish the isolation and purification model of DSC and EVCT successfully.2. Cytokines were secreted by DSC and EVCT. Diverse cytokiness play an important role in proliferation and embryo implantation. Th1 type pro-inflammatory cytokines like IFN-γ, IL-17 decreased to inhibit immunological rejection and IL-1ra, IL-6, Leptin increased to protect fetus. All cytokines establish a balance to co-regulate the success of pregnancy.3. 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.PARTâ…¡EFFECT OF MIFEPRISTONE ON CYTOKINES EXPRESSED IN THE DECIDUAL CELLS AND EXTRAVILLOUS CYTOTROPHOBLASTS INVITROOBJECTIVE.To investigate the effect of mifepristone on cytokines in DSC and EVCT and to approach the molecule mechanism of stopping early pregnancy.METHODS1. The DSC and EVCT system were cultured in vitro as Partâ… .2. Both DSC and EVCT system from the same source were divided into two groups, one was incubated with 1×10^-6mol/L mifepristone(RU486) for 48h, another was cultured without RU486 as control. Collected the culture media and detected the concentration of 11-cytokines by Luminex xMAP. Paired-samples T test was performed to demonstrate RU486's effect on cytokines of fetal-maternal interface.RESULTS1. RU486 down-regulated the levels of IL-1α, IL-1β,IL-1ra, IL-6, Leptin, TGF-βand up-regulated the levels of FGF secreted by EVCT culture system. RU486 had no effect on IL-17, VEGF, IFN-γ, EGE.2. RU486 down-regulated the levels of IL-1β, TGF-β, EGF, Leptin and up-regulated the levels of IL-1γα, IL-6, VEGF, FGF secreted by DSC culture system. RU486 had no effect on IL-1α, IFN-γ, IL-17.CONCLUSIONSMifepristone can affect the levels of cytokines and breakdown the balance between Th1 and Th2. It can induce the predominance Th1 type cells which may enhance immune rejection to conceptus and cease growth of villus and deciduas. Such lead to the termination of pregnancy.PARTâ…¢EFFECT OF CYCLOSPORINE ON CYTOKINES EXPRESSED IN THE DECIDUAL CELLS AND EXTRAVILLOUS CYTOTROPHOBLASTS INVITROOBJECTIVETo investigate the effect of cyclosporine on cytokines in DSC and EVCT and to approach the molecular biology mechanism in early pregnancy.METHODS1. The DSC and EVCT system were cultured in vitro as Partâ… .2. Both DSC and EVCT system from the same source were divided into two groups, one was incubated with 1×10^-6mol/L cyclosporine(CSA) for 48h, another was cultured without CSA as control. Collected the culture media and detected the concentration of 11-cytokines by Luminex xMAP. Paired-samples T test was performed to demonstrate CSA's effect on cytokines of fetal-maternal interface. RESULTS2.3 CSA down-regulated the levels of IL-1α, IL-1β, IL-1ra, IL-6 and up-regulated the levels of IL-17, FGF, Leptin secreted by EVCT culture system. CSA had no effect on IFN-γ, VEGF, EGF, TGF-β.2.3 CSA down-regulated the levels of IL-1β, IL-6, VEGF, Leptin and had no effect on IL-1α, IL-lra, IL-17, TGF-β, IFN-γ, EGF, FGF secreted by DSC culture system.CONCLUSIONSCyclosporine can affect the levels of cytokines and enhance the maintenance of pregnancy.