| Part One. The Role of EFNB2-induced EVT Biological Functions in Spiral Artery RemodelingObjective To investigate the expression of EFNB2in villous and decidual tissues from first trimester and trophoblast cells (primary EVTs, human extravillous trophoblast cell line HTR-8/SVneo and chorioepithelioma cell lines, JEG-3and JAR), and to study the changes in cell viability, apoptosis, migration, invasion and angiogenesis after regulating of EFNB2expression in HTR-8/SVneo cells. We also applied placental-decidual co-culture (PDC) system to observe the effect of EFNB2knock-down on spiral artery remodeling.Methods1. Localizing the expression of EFNB2in villous and decidual tissues from first trimester by IHC staining;2. Analyzing the expression of EFNB2gene at mRNA level in primary EVTs, human extravillous trophoblast cell line HTR-8/SVneo and chorioepithelioma cell lines, JEG-3and JAR by qRT-PCR.3. According to different treatment, HTR-8/SVneo cells were classified into three groups: control group (cells without transfection), inhibition group (cells transfected with sh-EFNB2plasmid) and overexpression group (cells transfected with EFNB2overexpression plasmid). In qRT-PCR analysis, two negative control groups were added.4. Applying qRT-PCR and Western blotting to testify the expression of EFNB2at transcriptional level and protein level.5. Examining cell viability by CCK-8.6. Examining cell apoptosis by Hoechst33258staining.7. Examining the expression of apoptotic proteins, Bcl-2and Bax by Western blotting.8. Examining cell migration and invasion abilities by Transwell models.9. Examining cell angiogenesis ability by Matrigel model.10. Testifying the effect of EFNB2down-regulation on spiral artery remodeling in PDC system.Results1. EFNB2showed an extensive expression in villus and decidua including trophoblasts.2. The expression of EFNB2gene at mRNA level in human primary EVT, HTR-8/SVneo cell, JEG-3cells and JAR cells was0.00067±0.00012,0.00058±0.00007,0.00042±0.00004and0.00039±0.00006, respectively. Primary EVT had a higher expression of EFNB2mRNA compared with chorioepithelioma cell lines (P<0.05) and showed no statistically difference with HTR-8/SVneo cells (P>0.05).3. More than90%of all transfected cells expressed strong green fluorescent light, indicating high transfection efficiency.4. Compared with controls, expression of EFNB2mRNA in transfected cells was0.14±0.09(inhibition group),1.06±0.06(inhibition negative controls),426.81±44.76(overexpression group) and1.01±0.26(overexpression negative controls).5. Compared with controls, expression of EFNB2protein in inhibition group and overexpression group was0.25±0.04and2.19±0.07, respectively (P<0.05).6. Compared with controls, cell viability in inhibition group and overexpression group was0.67±0.0and1.37±0.51, respectively (P<0.05).7. Compared with controls, the relative rate of apoptotic cells in inhibition group a was129.2±11.9%(P<0.05), and104.7±14.9%(P>0.05) in overexpression group.8. Compared with controls, the expression of Bcl-2decreased (0.35±0.02, P<0.05) and the expression of Bax increased (1.82±0.16, P<0.05). The expression of Bcl-2and Bax protein in overexpression group was1.15±0.03and1.01±0.04, respectively (P>0.05).9. The total number of migrating cells in controls, inhibition group and overexpression group was150.2±6.7,108.5±3.0and328.7.4±13.5, respectively (P<0.05). Migration ability decreased in inhibition group (0.69±0.03) and increased in overexpression group (1.95±0.08) compared with controls (P<0.05).10. The total number of invading cells in controls, inhibition group and overexpression group was140.8±3.4,42.5±1.3and265.7±14.7, respectively (P<0.05). Invasion ability decreased in inhibition group (0.24±0.05) and increased in overexpression group (1.80±0.21) compared with controls (P<0.05).11. Compared with controls (72.6±9.6), the branching points in overexpression group increased (100.4±20.2) and decreased in inhibition group (29.6±4.8)(P<0.05).12. IHC staining from PDC showed that sh-NC group demonstrated a more advanced remodeling process than sh-ephrin-B2group.ConclusionEFNB2was able to induce the biological functions of EVTs and take part in spiral artery remodeling. Down-regulation of EFNB2in HTR-8/SVneo cells induced cell apoptosis and impaired cell functions such as cell viability, migration, invasion and angiogenesis. Overexpression of EFNB2did not have a significant effect on cell viability and apoptosis but increased other biological functions such as migration, invasion and angiogenesis. Part Two. Effects of Notch Signaling in Regulation of EVT Biological FunctionsObjectiveTo examine changes in HTR-8/SVneo cell functions after regulating activation of Notch signaling. The expression of EFNB2was also tested after different treatment.Methods1. Analyzing cell viability of HTR-8/S Vneo cells treated with different concentration of sD114.2. Applying qRT-PCR and Western blotting to testify the expression of Notch1gene at transcriptional level and protein level after transfection.3. According to different treatment, HTR-8/SVneo cells were classified into three groups: control group (cells without transfection), inhibition group (cells transfected with sh-Notchl plasmid), drug-treated group (cells treated with4.0.5mg/mL sD114) and drug-treated inhibited group). Applying qRT-PCR and Western blotting to testify the expression of EFNB2at transcriptional level and protein level.5. Examining cell viability by CCK-8.6. Examining the expression of apoptotic proteins, Bcl-2and Bax by Western blotting.7. Examining cell migration and invasion abilities by Transwell models.8. Applying qRT-PCR and Western blotting to testify the expression of EFNB2gene at transcriptional level and protein level after regulating of Notch1signaling.9. Examining cell migration and invasion abilities by Transwell models after activating Notchl signaling in EFNB2-down-regulated cells.Results1. Compared with controls, cell viability in cells with different concentration of sD114was (0.96±0.03),(1.15±0.09),(1.16±0.10) and (1.08±0.08), respectively (P>0.05).2. Compared with controls, expression of Notchl mRNA in transfected cells was0.60±0.14(inhibition group, P<0.05) and1.06±0.06(negative controls, P>0.05); Notchl protein expression in inhibition group was0.25±0.04(P<0.05).3. Compared with controls, cell viability in inhibition group, drug-treated group and drug-treated inhibited group was0.65±0.13,1.07±0.05and0.56±0.14, respectively.4. Compared with controls, the expression of Bcl-2decreased (0.47±0.02, P<0.05) and the expression of Bax increased (2.14±0.05, P<0.05). The expression of Bcl-2and Bax protein in drug-treated group was1.01±0.09and0.93±0.04, respectively (P>0.05).5. Compared with controls, migration index in inhibition group, drug-treat group and drug-treated inhibited group was0.30±0.03,2.49±0.25and2.84±0.14, respectively(P<0.05).6. Compared with controls, migration index in inhibition group, drug-treat group and drug-treated inhibited group was0.15±0.02,2.84±0.12and2.46±0.18, respectively(P<0.05).7. Compared with controls, the relative expression of EFNB2mRNA was0.24±0.08in inhibition group and3.41±0.17in drug-treated group (P<0.05).8. Compared with controls, the relative expression of EFNB2protein was0.43±0.17in inhibition group and1.79±0.25in drug-treated group (P<0.05).1. Migration ability and invasion ability in drug-treated EFNB2-knock-down cells was7.52±3.12and7.48±3.67, respectively, compared with EFNB2-knock-down cells (P<0.05).ConclusionNotch1signaling was able to regulate the expression of EFNB2in EVT, and had a further influence on the biological functions of EVT relating to the development of placental vascular network. Part Three. Study on the Role of Notch Signaling in dNK-derived Synergistic Effect on EVTObjectiveTo examine cell viability, apoptosis, IFN-y secretion and EFNB2gene expression under sD114stimulation. To illustrate the effects of Notch signaling in dNK-derived synergistic effect on EVT biological functions.Methods1. Flow cytometry examined the purity of primary dNK cells.2. Examining cell viability by CCK-8.3. Examining cell apoptosis by caspase-3colorimetric assay.4. Applying qRT-PCR and ELSA to examine IFN-y secretion.5. Examining cell migration ability of HTR-8/SVneo cells by Transwell co-culture model.Results1. Flow cytometry assay showed that the purity of primary dNK was more than90%.2. Compared with controls, cell viability in experiment group was1.01±0.05(P>0.05).3. Compared with controls, the relative caspase-3activity in experiment group was0.79±0.14(P<0.05).4. Compared with controls, the relative expression of IFN-y mRNA was622±0.24(P<0.05). The secretion of IFN-y in controls was3175.56±141.55pg/mL, and4226.28±158.09pg/mL in experiment group (P<0.05).5. Compared with controls, the relative expression of EFNB2mRNA was2.53±0.75(P<0.05).6. Compared with controls, migration index of HTR-8/SVneo cells in drug-treated group, co-culture group and drug-treated co-culture group was0.71±0.24(P<0.05),0.85±0.22(P<0.05) and2.28±0.33(P<0.05).ConclusionD114/Notch signaling might have an influence on dNK biological functions via the regulation of EFNB2, which could possibly play a role in the synergistic effect of dNK on EVT. |
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