The Effect Of Cyclosporin A On The Expression Of Integrin αvβ3 And Of Its Effect On Embryo Adhesion To Fibronectin During Embryo Pre-implantation

Embryo implantation is one of the important steps for women to get pregnancy,and its successful adherenc to endometrium is one of the keys.It has been found that the combination of integrin αvβ3 and fibronectin binding could directly affect embryo adhesion,thereby affecting the embryo implantation.The treatment of cyclosporine A was first applied to immune rejection,but in recent years it can be applied to trophoblast cells,which promote the proliferation 、 migration and invasion of trophoblast cells,what is more,cyclosporine A target the embryonic cells during embryo pre-implantation,promoting the function of adhesion and invasion.Our previous studies showed that cyclosporine A can enhance the proliferation of embryonic trophoblast cells during embryo pre-implantation and improve the adhesion and invasion of trophoblast cells through upregulation the expression of integrin αvβ3,which has potential value for improving outcomes of embryo implantation.Objective:Using the embryos during pre-implantation of the ICR mouse as a experimental model in vitro,cultured in embryo culture supernatants,which contain different concentration of cyclosporine A,and then we detected the expression of integrin αvβ3and the binding ability of integrin αvβ3 and fibronectin,exploring the possibility of cyclosporin A on improving the ability of embryo implantation,laying the foundation for further clinical transformation.Methods:1.Embryos were divided into four groups,respectively,the embryos were cultured sequentially in embryo culture supernatants,which contain different concentration of cyclosporine A:0 μM、0.1 μM、1 μM、10 μM,and the development of embryo were observed and recorded.2.Making dishes coated with Laminin,which is simulated a adhesion modle of endometrium,6.5 dpc embryo were divided into four groups,and cultured in dishes coated with Laminin until to 8.5 dpc,observing and recording the adhesion area of embryo.3.Detected transcription and expression of integrin αvβ3 by q PCR technology.4.Detected the location of the expression of integrin αvβ3 by immunofluorescence technology.5.6.5 dpc embryo were co-incubated with the FN-120-fluorescent microspheres,observing the adhesion of integrin αvβ3 and fibronectin through Confocal laser scanning microscopy.Result:1.1.5dpc embryo were placed in G1 and G2 medium containing different concentrations of Cs A for sequential culture,the hatching rates of the four groups were respectively 96.22%,88.72%,92.56%,92.56%,P > 0.05,there are not significant difference.2.6.5 dpc embryo were cultured in dishes coated with Laminin,which containing different concentration of Cs A,we measured the adhesion areas of four groups of embryos were 3.4431±2.19455,4.4512±1.90197,4.7426±2.01458 and4.2295±1.97604,and 1 μM group compare with 0 μM group,there are significant differences,P < 0.05.3.1.5dpc embryo were placed in G1 and G2 medium containing different concentrations of Cs A for sequential culture,and then the transcription and expression of integrin αvβ3 were detected by q PCR technology,we found that the expression of integrin αvβ3 could be enhanced in the effective dose of Cs A,and group 1 μM compare with group 0 μM,there are significant differences,P < 0.05.4.1.5dpc embryo were placed in G1 and G2 medium containing different concentrations of Cs A for sequential culture,and then marked Itg β3 by immunofluorescence,we found that the fluorescence signal of group 1 μM is stronger than other groups.5.1.5dpc embryo were placed in G1 and G2 medium containing different concentrations of Cs A for sequential culture,and then put 6.5dpc embryos co-incubated with the FN-120-fluorescent microspheres,we measured the fluorescence intensities of four groups were 12.4936±2.02757,13.0260±3.51394,24.4045±5.71791 and 20.5230±5.11722 respectively,and group 1 μM compare with group 0.1 μM,there are significant differences,P < 0.05.Conclusions:Cs A can promote the binding of integrin αvβ3 with fibronectin and increase the adhesion ability of the embryo during the pre-implantation.Such effect of Cs A appeared in the concentration of 0.1 μmol/L,when the concentration of 1 μmol/L,it could exert strongest influence,and then begin to decline in the concentration of 10μmol/L.