| Cyclosporine A(CsA)is an immunosuppressive drug for organ transplantation and autoimmune diseases.Since its application in the 1970 s,CsA has played an important role as an immunosuppressant?CsA is currently recognized as the most effective drug inhibit graft rejection.Beside,CsA can promote the migration,invasion and proliferation of a variety of tumor cells and non-tumor cells.In recent years,it has been found that CsA can obviously promote the proliferation of tumor cells,and the biological effects of early pregnancy cytotrophoblast are similar to those of tumor cells.In early pregnancy,low dose of CsA can not only induce the maternal immune tolerance to embryo antigen,but also promote the proliferation of trophoblast cells,inhibit the apoptosis of trophoblast cells,and enhance their ability of movement,migration and invasion.The results showed that under oxidative stress,CsA could activate MAPK / Erk Signal Pathway,reduce the damage of trophoblast and improve the proliferation,migration and invasion of trophoblast.In the study of early in vitro mouse embryo culture,we found that different concentrations of CsA at different stages could promote the development of mouse embryos at 2-cell stage,and the concentration of CsA was within certain range In a dose-dependent manner,blastocyst formation and hatching of mouse embryos were promoted in vitro.CsA could obviously improve the pregnancy outcome of the pregnancy failure model mice after 5 mg / kg administration.Therefore,it is hypothesized that CsA can improve the function of trophoblast cells,promote the proliferation,migration and invasion of trophoblast cells,and improve the implantation ability of embryos with slight trophoblast defects.ObjectiveThe aim of this study was to investigate the effect of CsA on the implantation of mouse embryos with different quality at implantation stage,and to explore whether CsA could increase the implantation rate of embryos with slight defect of trophoblast cells.In order to improve the trophoblast cells in the slight defects of the embryo implantation rate and utilization rate to find an effective method in IVF-ET? Methods1.Detection of serum concentration of CsA IN MICE30 ICR female mice aged 6-8 weeks were randomly divided into 10 groups,3 in each group were injected with CsA 5 mg / kg intraperitoneally.The blood concentration of each group was measured at different time points,and the mean value of each group was taken as the blood concentration at that time point.2.Constructing a recipient femaleFemale rats of 6-10 weeks old ICR were mated with male rats after ligation,and the next day at 7:30,the plug was found to be 0.5dpc.See plug mice as recipient mice.3.Construct pseudo-pregnant mice and surrogate rats.The 7-to 10-week-old ICR female rats were selected,and the male mice were caged at 17:00 in the afternoon of the stimulating mice.The rats were caged at 7:30 in the morning,and the sling was 0.5dpc.3.Embryo acquisition,evaluation and transplantation3-4 weeks of ICR female mice,after superovulation,the cages were mated,and the next day,7:30 am,the plug was found,see the embolization as 0.5dpc.2 cell embryos were collected at 1.5 dpc and sequentially cultured.Embryos were evaluated when the blastocyst stage was 3-5(3.5dpc).The cell cluster in class A is grade A or grade B,the trophoblast cell is grade A;the cell cluster in class B is grade A or grade B,the trophoblast is graded as B+;the cell cluster in class C is grade A or B.Grade,trophoblast cells are rated B-grade.Different types of embryos were transplanted into the 2.5dpc recipient mouse uterus using non-surgical transplantation.4.CsA interventionAt 19:00 on the day after embryo transfer,mice transplanted with the same type of embryos were placed in the CsA experimental group and the olive oil control group,and 5 mg/kg of CsA injection and an equal-divorage olive oil injection were administered according to body weight.After 12 hours,a second injection was performed.5.Implantation observationWhen the recipient mice were 5.5 dpc,the recipient mice were sacrificed,and embryo implantation was observed and recorded.6.Preparation and treatment of maternal fetal interface specimensAfter the completion of the counting,the embryos at the implantation site and the uterus tissue were cryopreserved and stored at-80 ° C.7.LIF mRNA detectionThe preserved tissues were subjected to RNA extraction and reverse transcription,and then real-time quantitative PCR(RT-qPCR)was used to detect the expression of leukemia inhibitory factor(LIF)mRNA in maternal-fetal interface under different conditions.8.Statistical methodsStatistical analysis was performed on the experimental data using SPSS 23.0 statistical software.Qualitative data were analyzed by chi-square test;mRNA expression levels were determined by independent sample T test using “mean±standard error(X±SE)”;all images were prepared using GraphPad Prism 7.0 software.? = 0.05 is the test level,P < 0.05 is the difference is significant.Result1.Blood concentration of CsA in miceThe dose of 5 mg/kg was administered in mice,and the drug concentration peaked at 3-10 h after administration,which was(994-1158)ng/ml.The blood concentration at the peak after conversion was compared with the in vitro conditions to promote nourishment during embryo culture.The optimal concentration of CsA(1 ?mol/L)for layer cell proliferation was close.2.Effect of CsA on mouse embryo implantationAfter implantation of different grades of embryos in the pre-implantation period,intraperitoneal injection of CsA and olive oil(control group),the implantation rate of type A embryos was 56.5% and 65.2%,respectively,and the implantation rate of class B embryos was 73.9%,50%,respectively.The embryo implantation rate was 39.1% and 30.4%,respectively.Statistical analysis was performed on the implantation of the three groups of embryos.The difference between the two groups was significant(P=0.018),and the other groups were not significantly different(P>0.05).3.LIF mRNA expressionThe LIF mRNA was detected in the embryos of the implantation site together with the uterus tissue.The expression of LIF mRNA was compared with the control group.The experiment was repeated 3 times.After the transplantation of A,B and C embryos,the expression of LIF mRNA in each experimental group was followed.There were no significant differences in the three groups of 1.1±0.88(P=0.863),4.46±2.24(P=0.237),and 0.74±0.58(P=0.905).ConclusionWhen administered at a dose of 5 mg/kg in vivo,CsA can improve the implantation of mouse embryos with slight defects in trophoblast morphology. |
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