The Research On The Relationship Between SEng, TGFβ1and Pathogenesis Of Hypertensive Disorder Complicating Pregnancy

Hypertensive disorder complicating pregnancy is a pregnancy-specific disease which etiology and pathogenesis has not been fully elucidated. Previous studies have been shown that angiogenic factors TGFβ1and its receptor are closely related to the central steps of hypertensive disorders in pregnancy:vascular endothelial injury and placental vascular remodeling process. This study will observe the impact of TGFβ1and its soluble receptor components (sEng) in cultured human umbilical vein endothelial cells and trophoblasts explore the roles of these two factors in the development of hypertensive disorder complicating pregnancy.Section Ⅰ. Research on the correlation between soluble endoglin. transforming growth factor β1and injury of vein endothelial cellObjectives:To discuss the correlation between soluble endoglin. transforming growth factor β1and injury of vein endothelial cell. investigating the effects of these two factors on human umbilical vein endothelial cell (HUVEC). included apoptosis. proliferation, nitric oxide (NO) production and nitric oxide synthase (eNOS) phosphoryiation.Methods:Cells (within3passages) were seeded in culture plates of96wells. There are4groups:controlled group with RPMI-1640culture medium only. sEng group. TGF(31group and (sEng-TGFβ1) group. These groups were divided into3subgroups (1、10、100μg/L) respectively on account of MTT results. Collecting the cells and cell cultures at6h.12h and24h for follow-up experiments. The concentration of soluble endoglin and TGFβ1in cell culture was evaluated by ELISA. Cell apoptosis and cell cycle were detected by flow cytometry. Cell viability was determined by methyl thiazolyl tetrazolium assay (MTT). The concentration of the metabolites of nitric oxide in each group was measured by nitrate reductase method. The expression of eNOS and eNOS-Ser(p)1177protein were detected by Western blot. eNOS mRNA in each group was detected by real-time fluorescence reverse transcription polymerase chain reaction (PCR).Results:(1) The concentration of sEng in cell culture was0at24h.48h and72h. The concentration of TGFβ1in cell culture was (3.12±0.78).(3.33±0.91). (3.08±0.87)ng/L respectively. There were no significant difference at these three time points (F=2.10, p>0.05).(2) Cell viability:The cell viability was significantly decreased in sEng group. It had negative correlation with time and medicine concentration. The cell viability were significantly increased in1μg/L TGFP1group, but decreased in10and100μg/L groups. It had negative correlation with time and medicine concentration in the latter groups. There was no significant difference between (sEng+TGFβ1) group and controlled group at any concentration and time points.(3) Cell apoptosis and cell cycle:the cell apoptosis rate and the G1phase proportion in sEng group were higher than those in other groups. It had positive correction with time and medicine concentration. The cell G1proportion were significantly decreased in1μg/L TGFβ1group, but increased in10and100μg/L groups. It had positive correlation with time and medicine concentration in the latter groups. But TGFβ1had no business of apoptosis. There was no significant difference between (sEng-TGFβ1) group and controlled group on cell apoptosis and cell cycle at any concentration and time points.(4) Nitric oxide levels in cell culture:sEng decreased the concentration of nitric oxide levels through negative correlation with stimulate time and medicine concentration.1μg/L TGFβ1increased the concentration of nitric oxide levels through positive affection with stimulate time. But10and100μg/L TGFβ1decreased the concentration of nitric oxide levels through negative affection with stimulate time and medicine concentration. There was no significant difference between (sEng+TGFβ1) group and controlled group on nitric oxide levels at any concentration and time points.(5) The expression of eNOS protein and mRNA: The expression of eNOS protein and mRNA decreased significantly in sEng groups. It also had negative correlation with stimulate time and medicine concentration.1μg/L TGFβ1increased the expression of eNOS protein and mRNA through positive affection with stimulate time. But10and100μg/L TGFβ1decreased the expression of eNOS protein and mRNA through negative affection with stimulate time and medicine concentration. There was no significant difference between (sEng+TGFβ1) group and controlled group on the expression of eNOS protein and mRNA at any concentration and time points.(6) Nitric oxide synthase phosphorylation (eNOS-Ser(p)1177/ENOS ratio):eNOS-Ser(p)1177/eNOS ratio decreased significantly in sEng groups. It also had negative correlation with stimulate time and medicine concentration.1μg/L TGFβ1increased the eNOS-Ser(p)1177/eNOS ratio through positive affection with stimulate time. But10and100μg/L TGFβ1decreased the eNOS-Ser(p)1177/eNOS ratio through negative affection with stimulate time and medicine concentration. There was no significant difference between (sEng+TGFβ1) group and controlled group on the eNOS-Ser(p)1177/eNOS ratio at any concentration and time points.Conclusions:1.sEng inhibited the proliferation of HUVEC in vitro through blocking the HUVEC in G1phase and increasing the apoptosis rate. It reduced the production of NO through inhibiting the proliferation of HUVEC and activation of eNOS. sEng participated the injury of endothelial cells.2. TGFβ1inhibited the proliferation of HUVEC in vitro through blocking the HUVEC in G1phase occasionally. The affection on HUVEC was up to medicine concentration. Low dose had promoted affection on HUVEC. But high dose had converse affections.3. sEng together with TGF(31were added in the cell culture seemed like having no affection on HUVEC. but it reflected that appropriate proportion of sEng and TGFβ1was very important to keep normal form and function of endothelial cells. All of above. sEng and TGFβ1participated the injury of endothelial cells by many ways.Section Ⅱ. Effects of soluble endoglin and TGFβ1on proliferation and invasive ability of cultured cytotrophoblasts from human early pregnancy chorionic villusObjective:To investigate the effects of soluble endoglin and TGFβ1on proliferation and invasive ability of cultured cytotrophoblasts from human early pregnancy chorionic villus.Methods:Cytotrophoblasts of normal6to8week pregnancy were cultured by trypsin digestion method. Cells (within3passages) were seeded in culture plates of96wells. There are4groups:controlled group with DMEM culture medium only. sEng group. TGFβ1group and (sEng-TGFβ1) group. The concentration of soluble endoglin and TGFβ1in cell culture was evaluated by ELISA. The medicine concentration at follow-up experiments of these two factors was determined by MTT. The proliferation ability was determined by MTT. Cell cycle was detected by flowcvtometry. The invasive ability was determined by Transwell invasion assay. The expression of MMP-2and MMP-9protein was detected by Western blot. The expression of MMP-2and MMP-9mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR).Results:The concentration of sEng in cell culture was0at24h,48h and72h. The concentration of TGFfβ1in cell culture was (5.03±0.54),(4.09±0.27). and (5.91±0.68)ng/L respectively. There were no significant difference at these three time points (F=3.09, p>0.05). Cell survival probability were degraded in sEng and TGFβ1groups at36h (71.44±6.07)%.(51.44±6.34)%and48h (62.37±8.12)%.(23.48±4.36)%versus controlled group. There was no significant difference between (sEng±TGFβ1) group and controlled group at any concentration and time points. The medicine concentration was10μg/L in every group on account of MTT. There was no significant cell apoptosis in every group. G1phase proportion in sEng group (88.24±2.11)%was higher than that of (sEng+TGFβ1)(58.11±1.25)%and control groups (55.23±1.22)%. G1phase proportion in TGFβ1group (95.63±2.98)%was higher than any other groups. There had no significant difference between (sEng+TGFβ1) group and control group. Cell numbers penetrating basement membrane was decreased significantly in sEng group (87±6.3) versus controlled group (143±41.3) but increased significantly in TGFβ1group (157±55.9). There was no significant difference between (sEng+TGF±1) group and controlled group. Compared with controlled group, the expression of MMP-2and MMP-9mRNA and protein of cytotrophoblasts was significantly lower in sEng group but higher in TGF(31group. There also was no significant difference between (sEng+TGFβ1) group and controlled group.Conclusions:sEng and TGFβ1correlated with the procedure of cytotrophoblasts proliferation through moderating G1phase proportion in vitro. They also regulated cytotrophoblasts invasive ability by affecting the expression of MMP-2and MMP-9. Keeping suitable proportion was very important for cytotrophoblasts to invasive in mvometrium with normal depth at the first trimester.