Expression Of Tyrosine Kinase Receptor EphA2 In Tubal Pregnant Tissues

Objectives1. To investigate expressions of tyrosine kinase receptor EphA2 and Phospho-EphA2 (Pho-EphA2) in both human oviductal tissues during menstrual cycle and tubal pregnant tissues.2. To compare expressions of E-cadherin (E-cad) between the implantation and the non-implantation sites for tubal pregnant tissues. The correlation of E-cad and EphA2 was analyzed in the implantation site of tubal pregnancy. To investigate expressions of integrinβ1 in both human oviductal tissues during menstrual cycle and tubal pregnant tissues. To compare expressions of Pho-focal adhesion kinase (FAK) between the implantation and the non-implantation sites for tubal pregnant tissues.3. To observe expressions of Pho-EphA2 and Pho-FAK and change on adhesion of human tubal epithelial cells in vitro culture after stimulation of EphrinAl-Fc to evaluate the mechanism of tyrosine kinase receptor EphA2 in the tubal pregnancy.Materials and MethodsExperiment 1:Expressions of EphA2 in the oviduct and tubal pregnant tissuesParaffin-embedded samples of oviductal tissues (n=29) were obtained from women undergoing abdominal hysterectomy with adnexectomy for benign disease such as uterine leiomyoma, adenomyosis in the pelvic cavity. These samples included isthmus, ampullae and umbrella, and were divided into the early-proliferative stage group (n=8), the mid-and late-proliferative stage group (n=6), the early-secretory stage group (n=7), and the mid-and late-secretory stage group (n=8) based on their endometrium histological stage. Paraffin-embedded samples of tubal pregnant tissues (n=17) were divided into the implantation site group (n=17) and the non-implantation site group (n=17). Immunohistochemical and laser cofocal scaning microscopy(LCSM) methods were used to determine the expression level of EphA2 in the tissues. Fresh samples of tubal pregnant tissues (n=14) were divided into the implantation site group (n=14) and the non-implantation site group (n=14). Fresh samples of oviductal ampullae tissues in the secretory stage as the control group (n=12) were obtained from women undergoing abdominal hysterectomy with adnexectomy for benign disease such as uterine leiomyoma, adenomyosis in the pelvic cavity. Expressions of EphA2 were determined by real-time fluoregenic quantitative polymerase chain reaction, and Western blotting methods in the implantation site group, the non-implantation site group and control group. Expressions of Pho-EphA2 were detected by Western blotting method in the three groups.Experiment 2:Expressions of E-cad in the tubal pregnant tissuesFresh samples were obtained and divided into the same groups as the experiment 1. Expressions of E-cad were determined by the immunohistochemical, real-time fluoregenic quantitative polymerase chain reaction, and Western blotting methods in the implantation site group, the non-implantation site group and control group.Experiment 3:Expressions of integrinβ1 and Pho-FAK in the tubal pregnant tissuesSamples were obtained and divided into the same groups as the experiment 1. Immunohistochemical method was used to determine the expression level of integrinβ1 in the tissues. Expressions of integrinβ1 and Pho-FAK were determined by Western blotting method.Experiment 4:Human tubal epithelial cells in vitro culture and stimulated by EphrinAl-FcHuman tubal epithelial cells were isolated by scraping method and purified by hemolysis and differential adhesion methods. Expressions of Pho-EphA2 and Pho-FAK of human tubal epithelial cells were determined by Western blotting after the cells were stimulated by EphrinAl-Fc. The change on adhesion of human tubal epithelial cells to fibronectin (Fn) was observed by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide assay(MTT) after tubal epithelial cells were stimulated by EphrinAl-Fc.Results1. Expressions of EphA2 in the oviductal and tubal pregnant tissuesExpressions of EphA2 were observed in the cytoplasm and menbrane of ciliated cells and secretory cells of oviductal tissues. In epithelial cells of the isthmus, the ampullae and the umbrella, no difference was found on expression of EphA2 in the cycle (P>0.05). In the same phase of the cycle, the expression among the isthmus, the ampullae and the umbrella showed no change (P>0.05). For tubal pregnant tissues, the expression of EphA2 in the implantation site group had higher level than that of the non-implantation site group or the secretory stage group of ampullae (P<0.05). The expression of Pho-EphA2 in the implantation site group had lower level than that of non-implantation site group or the secretory stage of ampullae (P<0.05). Expressions of EphA2 mRNA showed no difference among the three groups (P>0.05).2. Expressions of E-cad in the oviductal and tubal pregnant tissuesPositive staining of E-cad was observed in the membrane of the epithelial cells in non-implantation site and oviductal ampullae tissues. In addition, positive staining was also shown in the membrane of decidual cells, cytotrophoblast, and extravillous trophoblast in tissues of the implantation site group. Expressions of E-cad mRNA and protein in the implantation site group had lower levels than those of the non-implantation site group or the control group (P<0.05) Expressions of E-cad mRNA and protein between the non-implantation site group and control group showed no change (P>0.05). The expression of E-cad had negative correlation with the expression of EphA2 in the implantation site of tubal ectopic pregnant tissues.3. Expressions of integrinβ1 in the oviductal and tubal pregnant tissuesExpressions of integrinβ1 were observed in the cytoplasm of ciliated cells and secretory cells of oviductal tissues. The positive staining was also shown in cilia of ciliated cells. In epithelial cells of the isthmus and the ampullae, no difference was found on expression of integrinβ1 in the cycle (P>0.05). In umbrella epithelial cells, the expression of integrinβ1 in the early-secretory stage group had higher level than that of the early-proliferative stage group, the mid-and late-proliferative stage group, or the mid-and late-secretory stage group, respectively (P<0.05). In the same phase of the cycle, the expression among the isthmus, the ampullae and the umbrella showed no change (P>0.05). For tubal pregnant tissues, expressions of integrinβ1 and Pho-FAK in the implantation site group had higher level than those of the non-implantation site group or the secretory stage group of ampullae (P<0.05). Expressions of integrinβ1 and Pho-FAK had positive correlation with the expression of EphA2 and had negative correlation with the expression of Pho-EphA2 in the implantation site of tubal pregnant tissues.4. Effect of EphrinAl-Fc on tubal epithelial cells in vitro cultureHuman tubal epithelial cells were isolated and purified. The expression of Pho-EphA2 of human tubal epithelial cells increased before the cells were stimulated by EphrinAl-Fc for 10 min. After 10 min stimulation, the expression of Pho-EphA2 decreased. The expression of Pho-FAK of human tubal epithelial cells decreased before the cells were stimulated by EphrinAl-Fc for 10 min. After 10min stimulation, the expression of Pho-FAK increased. There were signifiant differences of the expressions of Pho-EphA2 and Pho-FAK between the EphrinAl-Fc group and the IgG-Fc group from 2min to 60min (P<0.05). The number of the cells adhesive to the Fn in the EphrinAl-Fc group had lower level than that in the control group or IgG-Fc group (P<0.05).Conclusions1. There was no difference on the expression of EphA2 in the isthmus, the ampullae and the umbrella during the cycle. The implantation site of tubal pregnant tissues would have higher EphA2 level and lower Pho-EphA2 level.2. The implantation site of tubal pregnant tissues would have lower E-cad level, which could be involved in aetiology of tubal pregnancy. Epithelial cells of the umbrella in the early-secretory stage would have higher integrinβ1 level. The implantation site of tubal pregnant tissues would also have higher integrinβ1 and Pho-FAK level. The role of E-cad, integrinβ1 and Pho-FAK had a correlation with the role of EphA2 in tubal pregnancy.3. After human tubal epithelial cells were stimulated by EphrinAl-Fc, EphA2 was activated and FAK was deactivated in a short time and they could return to the original level in 120min.The adhesion of human tubal epithelial cells was inhibited by EphrinAl-Fc.