Establishment Of Co - Culture Co - Culture Model Of RL95-2 - BeWo And Adhesion - Multi - Factor Regulation Of Implantation Function

Objective:This study aimed to establish an effective in vitro co-culture model of embry o implantation, and preliminarily discussed the adhesive and embed function of this mode1by detecting the expression of integrinβ3and its ligind osteopontin in endometrial epithe lium cells, as well as spheroids adhesion and expansion indexes under the regulation of estradiol (E2), progesterone (P4) and heparin-binding epidermal growth factor (HB-EGF).Methods:1. To establish an in vitro co-culture model of embryo implantation: endometrial epithelial carcinoma cells RL95-2monolayers were used to mimic Endometri al epithelium, choriocarcinoma BeWo cells were formed of spheroids to mimic embryos by the ways of serum-free culturing and low adsorption petri dish, RL95-2monolayer an d BeWo spheroids were co-cultured to imitate embryo implantation process. We observed the morphology and calculated adhesion rate of BeWo spheroids, compared the same in dexes with the trials in the training of Australia Prince Henry’s Institute (PHI)2. To investigate multi-element regulation of the adhesive and embed function of RL95-2-BeWo in vitro co-culture model:(1) Detecting integrinβ3and its ligind OPN related to embryo adhesion: The localization of integrinβ3and OPN in RL95-2monolayer, BeWo monolayer and BeWo spheroids were detected by immunofluorescence. RL95-2cells were treated with different concentrations of E2、P4、E2+P4and HB-EGF, the mRNA expressions of integrin(33and OPN were analyzed by real-time PCR.(2) BeWo spheroids attachment assay and outgrowth assay RL95-2monolayer were treated with different concentrations of E2、P4、E2+P4and HB-EGF, and co-cultured with BeWo spheroids, compared adhesion rate of every group. RL95-2monolayer were treated with high concentrations of E2、P4、E2+P4and HB-EGF, and co-cultured with BeWo spheroids, take pictures and measured the diameters of spheroids at1h and24h, compared expansion ratio of every group.Results: 1. Established RL95-2-BeWo in vitro co-culture system, there were no obvious differences in spheroids morphology and adhesion rates between our research group and PHI (P>0.05)2. Integrinβ3and OPN were expressed in cytoplasm and membrane surface of RL95-2monolayer, BeWo monolayer and BeWo spheroids.3. The mRNA expressions of integrinβ3and OPN were not significantly influenced by E2(P>0.05); The mRNA expression of OPN could be up-regulated by high concentration P4, and obviously inhibited by low concentration P4(P<0.05); there were no differences when E2and P4treating federatively or separately (P>0.05); high concentration HB-EGF could increase the mRNA expressions of integrinβ3(P>0.05) and OPN (P<0.05)4. BeWo spheroids attachment were deduced when treated with high concentration E2(P>0.05), increased when treated with high concentration P4(P>0.05), obviously raised when treated with high concentration HB-EGF (P<0.05); high concentration P4and HB-EGF could evidently promote the expansion of spheroids on the RL95-2monolayer (P<0.05), and related to the action time.Conclutions:1. Successfully established RL95-2-BeWo in vitro co-culture model of implantation.2. Integrinβ3and OPN localized apically in BeWo spheroids and the RL95-2cells, the expressions were multi-element regulated.3. High concentration E2was harm to embryo attachment, high concentration P4and HB-EGF could promote embryo attachment and implantation.