Endogenous Ouabain In The Pathogenesis Of Hypertensive Disorder Complicating Pregnancy

PartⅠThe Preparation of Anti- Ouabain Polyclonal Antibody.Objective—To prepare anti-ouabain polyclonal antibody.Study Design—Six male rabbits, about 4~5-month-old and 2~2.5-kilogram-weight, were chosen for preparing antibody against ouabain. Couple the ouabain with OVA/BSA according to the methods of periodic acid sodium oxidation for the preparation of complete antigen. Immuned rabbits for several times by ouabian-BSA, and purified anti-ouabain IgG by saturated ammonium sulfate and DEAE-D52 Anion exchange chromatography. Detected the valence and purity of antibody by the methods of ELISA and SDS-PAGE after lyophillization, and cryopreserved at -20℃.Results—Ouabain and ovalbumin or bovine serum albumin was coupled successfully, and the conjunction ratios of complete antigen were 1:14 and 1:18. ouabain antiserum was obtained by ouabain-BSA immunization. The valence of purified antibody was 1:500 after purified and immunized.Conclusion—Complete antigen, Ouabain-BSA, coupled by the methods of periodic acid sodium oxidation can immunize rabbit and the ouabain antiserum can be obtained effectively; the anti-ouabain polyclonal antibody with high valence and purity can be obtained after using the purification methods of saturated ammonium sulfate and DEAE-D52 Anion exchange chromatography.PartⅡThe Dependability Study between Endogenous Ouabain and Hypertensive Disorder Complicating PregnancyObjective—To observe placenta endogenous ouabain (EO) expression and sera EO levels in different courses of hypertensive disorder complicating pregnancy (HDCP) disease, in order to confirm the dependability between placenta EO expression,sera EO levels and the development of HDCP, and approach the possibility of EO secretion by brepho-placenta.Study Design—Cases were 30 patients with a clinical diagnosis of HDCP, and controls were matched to cases based on parity. The expressions of EO in placenta were studied by S-P immunohistochemistry(IHC) and Immunofluorohistochemistry(IFH) methods. And sera EO were measured by completive ELISA.Results—Compared with the controls, the expressions and sera levels of EO were dramatically increased in the HDCP groups (P<0.01) and correlated to the severity of the disease. EO concentrations of cord sera were significantly higher in comparison with antepartum maternal sera (P<0.01). On the contrary, EO concentrations of postnatal maternal sera were significantly lower, compared with antepartum maternal sera (P<0.01).Conclusion—These findings demonstrate that as well as the advanced courses of disease, the expressions of EO are gradually reinforced, while the EO levels of maternal sera and cord sera rise significantly, and cut down in maternal sera 3 days after delivery, which suggest that EO may directly participate in the pathogenesis of HDCP. In addition, EO concentrations of cord sera are significantly higher than that of maternal sera, which suggests that brepho-placenta unit may secrete EO and raise the maternal sera EO levels.PartⅢThe Research of Endogenous Ouabain in the Pathologic Regulation of Hypertensive Disorder Complicating Pregnancy. Objective—To investigate the pathologic regulation mechanism of EO on HDCP.Study Design—Cases were 30 patients with a clinical diagnosis of HDCP, and controls were matched to cases based on parity. The expressions of NKA-α1 subunit in placenta were studied by IHC and IFH methods. Erythrocyte membrane NKA activities were assayed using the methods of fix-phosphonium. And ET-1, NO levels were detected by the methods of ELISA and nitrate reductase.Results—Compared with the controls, the expression of NKA-α1 subunit and erythrocyte membrane NKA activity were dramatically decreased (P<0.01) in the HDCP. As expected, ET-1 concentrations of maternal and cord sera in HDCP groups were significantly higher in comparison with healthy pregnant group (P<0.01). On the contrast, NO concentrations were much lower in the maternal and cord sera of HDCP groups (P<0.01). In addition, ET-1 levels of cord sera were significant higher than that of maternal sera, while NO levels of cord sera were significant lower. Moreover, there was a significant negative correlation between the expression of EO in placenta and NKA-α1 subunit expression, as well as NKA activity (P<0.01). And the same correlation was observed between sera EO level and NKA-α1 subunit expression, as well as NKA activity (P<0.01). Meanwhile, there was also a significantly positive correlation among the expression of EO, sera EO level and ET-1(P<0.01), while the correlation of EO and NO was significantly negative (P<0.01). As expected, the correlation between ET-1 and NO was negative (P<0.01).Conclusion—The activity of NKA and the expression of NKA-α1 were decreased as the disease aggravate. Furthermore, there were significant dependability between EO level and the ratio of ET-1/NO, which suggests that EO may participate in the pathogenesis of HDCP. On the one hand, EO may inhibit the expression of NKA-α1 subunit or the NKA activity; On the other hand, EO may suppress the tunica intima vasorum ET-1/NO secretion or intensify the ET-1/NO disequilibration.