Objective:In this study,we aimed to explore the expression of TUSC3 in Preeclampsia and to research the potential function of TUSC3 in placental trophoblast cells.Methods:We collected 10 cases of normal placental tiss...Objective:In this study,we aimed to explore the expression of TUSC3 in Preeclampsia and to research the potential function of TUSC3 in placental trophoblast cells.Methods:We collected 10 cases of normal placental tissues and preeclampsia placental tissues,respectively.These parturient received treatment at the First Affiliated Hospital of Hainan Medical University between June 1,2020,and December 31,2022.The expression of TUSC3 in placenta was detected by immunohistochemistry.The effect of TUSC3 on the migration and invasion of HTR8/SVneo cells was analyzed by migration assay and Transwell assay.Results:The expression of TUSC3 was slightly increased in placental villis in preeclampsia.Immunohistochemistry and qRT-PCR were used to detect the expression of TUSC3 protein and mRNA in placental tissues.TUSC3 was markedly upregulated in PE placental tissues(P<0.01).The results of migration assay and Transwell assay showed that the migration rate and the number of invasive cells were significantly decreased in HTR8 overexpressing TUSC3(P<0.01).Conclusions:TUSC3 was markedly increased in PE placental tissues and inhibited trophoblast cells migration and invasion.展开更多
Objective:To investigate the effect of exosomes secreted by decidual macrophages on trophoblast cells and their molecular mechanism.Methods:The decidual tissues of patients with preeclampsia(PE)and normal-term pregnan...Objective:To investigate the effect of exosomes secreted by decidual macrophages on trophoblast cells and their molecular mechanism.Methods:The decidual tissues of patients with preeclampsia(PE)and normal-term pregnant women were collected.Macrophages were obtained by the density gradient method and then flow cell sorting,then the exosomes were extracted.The structure of the exosomes was observed by transmission electron microscope.The expression of CD63,a marker protein of the exocrine body,was detected by western blot,and the exosomes were identified.CCK-8 was used to detect the effect of exosomes on trophoblast cell viability.Transwell migration experiment was used to detect the influence on migration ability.The expression of miR-146a-5p in exosomes was detected by qPCR.The effect of exosomes on the expression of HIF1αprotein in trophoblasts was detected by western blot and detection of the binding site between miR-146a-5p and HIF1αby double luciferase reporter gene was conducted.Results:The exosomes of macrophages present a"cake"structure with a middle depression about 30-130 nm in diameter,and CD63 is highly expressed,which conforms to the characteristics of exosomes.Compared with the normal group,the exosomes of decidual macrophages in the PE group inhibited the activity and migration of trophoblast cells(P<0.001).The expression of miR-146a-5p in the exosomes of decidual macrophages in the PE decreased significantly,and after exosomes of PE decidual macrophages treating trophoblast cells,the protein expression of HIF1αin trophoblast cells was significantly increased.There are targeted binding sites between miR-146a-5p and HIF1α.Conclusion:PE decidual macrophage exosomes can inhibit the viability and migration of trophoblast cells,which may be related to the decreased expression of miR-146a-5p in exosomes,thus promoting HIF1αprotein expression of trophoblast cells.展开更多
This study investigated the expression and immune effect of TLR4 in human trophoblast cells. The expression level of TLR4 mRNA in normal and LPS-stimulated human term trophoblast cells （1 mg/L LPS, 12 h） was detecte...This study investigated the expression and immune effect of TLR4 in human trophoblast cells. The expression level of TLR4 mRNA in normal and LPS-stimulated human term trophoblast cells （1 mg/L LPS, 12 h） was detected by RT-PCR. In LPS-stimulated human term trophoblast cells of TLR4-blocked group and non-TLR4-blocked group, and normal term trophoblast cells of blank control group, apoptosis rate was measured by flow cytometry （FCM）, and the level of TNF-α determined by using enzyme linked immunosorbent assay （ELISA） respectively. RT-PCR results showed that the expression level of TLR4 mRNA in LPS-stimulated human trophoblast cells was significantly higher than that in normal cells （P〈0.01）. FCM revealed that there was significant difference in apoptosis rate of LPS-stimulated human term trophoblast cells between TLR4-blocked group and non-TLR4-blocked group （P〈0.05）, or between TLR4 antibody-blocked group and blank control group. ELISA indicated that the level of TNF-α in LPS-stimulated human trophoblast cells also had statistical differences between TLR4 antibody-blocked group and non-TLR4 antibody-blocked group （P〈0.05）. Our results suggest that TLR4 plays an important role in the immunological mechanism of apoptosis and secretion of TNF-α of human term trophoblast cells stimulated by LPS.展开更多
Human trophoblast cells were isolated and cultured in vitro in order to investigate possible pathogenesis of intrauterine infection caused by HCMV.Trophoblast cells were obtained by compound enzymes digestion and disc...Human trophoblast cells were isolated and cultured in vitro in order to investigate possible pathogenesis of intrauterine infection caused by HCMV.Trophoblast cells were obtained by compound enzymes digestion and discontinuous percoll gradient.Cells and purity were identified by using immunocytochemistry assay with anti-CK7,Vim and β-hCG antibodies.HCMV AD169 strain replication in isolated trophoblast cells and cell apoptosis were detected at different time points post infection(p.i.).The results showed tha...展开更多
Objective To investigate the effect of insuline-like growth factor-Ⅰ （IGF-Ⅰ） on progesterone genesis and regulation. Methods Cytotrophoblast cells were collected by trypsin-collagenase digestion and percoll gradie...Objective To investigate the effect of insuline-like growth factor-Ⅰ （IGF-Ⅰ） on progesterone genesis and regulation. Methods Cytotrophoblast cells were collected by trypsin-collagenase digestion and percoll gradient centrifugation for primary culture. After stimulated with different concentrations（100 μg/ml, 10 μg/ml, 1 μg/ml, 0.1 μg/ml） of IGF-Ⅰ at the same time and with different duration（12 h,24 h,48 h, 72 h） of IGF-Ⅰ with the same concentration, progesterone levels in the media were measured by radioimmunoassay. Simultaneously, semiquantitative reverse transcriptase polymerase chain reaction （RT-PCR） was applied to determine the expression of low density lipoprotein receptor （LDLR） mRNA. Results Progesterone levels correlated positively with IGF-Ⅰ along with the IGF-Ⅰ concentration increasing, progesterone level began to increase at 12 h, and reached the climax at 48 h when cultured with 100 μg/L IGF-Ⅰ. The expression of LDLR mRNA was detectable in every group and accordant with variation of progesterone level. Conclusion Progesterone secretion has time- and dose-dependent effect on IGF-Ⅰ, and IGF-1 can up-regulate the expression of LDLR mRNA. IGF-Ⅰ may play an important role in promoting secretion of progesterone in trophoblast cells.展开更多
The lifespan of mammalian trophoblast cells includes polyploidization, its degree and peculiarities are, probably, accounted for the characteristics of placenta development. The main ways of genome multiplication-endo...The lifespan of mammalian trophoblast cells includes polyploidization, its degree and peculiarities are, probably, accounted for the characteristics of placenta development. The main ways of genome multiplication-endoreduplication and reduced mitosis-that basically differ by the extent of repression of mitotic events, play, most probably, different roles in the functionally different trophoblast cells in a variety of mammalian species. In the rodent placenta, highly polyploid(512-2048c) trophoblast giant cells(TGC) undergoing endoreduplication serve a barrier with semiallogenic maternal tissues whereas series of reduced mitoses allow to accumulate a great number of low-ploid junctional zone and labyrinth trophoblast cells. Endoreduplication of TGC comes to the end with formation of numerous low-ploid subcellular compartments that show some signs of viable cells though mitotically inactive; it makes impossible their ectopic proliferation inside maternal tissues. In distinct from rodent trophoblast, deviation from(2n)c in human and silver fox trophoblast suggests a possibility of aneuploidy and other chromosome changes(aberrations, etc.). It suggests that in mammalian species with lengthy period of pregnancy, polyploidy is accompanied by more diverse genome changes that may be useful to select a more specific response to stressful factors that may appear occasionally during months of intrauterine development.展开更多
OBJECTIVE:To elucidate the regulatory effects of salvianolic acid B(Sal B)on trophoblast cells in preeclampsia(PE).METHODS:3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide(MTT)assays were used to detect th...OBJECTIVE:To elucidate the regulatory effects of salvianolic acid B(Sal B)on trophoblast cells in preeclampsia(PE).METHODS:3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide(MTT)assays were used to detect the viability of human extravillous trophoblast HTR-8/Svneo cells induced by H_(2)O_(2)following treatment with different concentrations of Sal B.The levels of oxidative stressrelated molecules,including superoxide dismutase,glutathione-Px and malondialdehyde were detected using corresponding kits.Cell apoptosis was detected using a Terminal deoxynucleotidyl transferase(Td T)d UTP NickEnd Labeling(TUNEL)assay,and the expression of apoptosis-related proteins was detected using western blot analysis.In the present study,wound healing and Transwell assays were performed to measure the levels of cell invasion and migration.Western blot analysis was also used to detect the expression levels of epithelialmesenchymal transition-related proteins.The mechanisms underlying Sal B were further investigated using reverse transcription-quantitative real-time polymerase chain reaction(RT-q PCR)and western blot analysis,to determine the expression levels of matrix metallopeptidase 9(MMP-9)and phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/protein kinase B(Akt).RESULTS:Sal B increased the activity of HTR-8/Svneo cells,inhibited oxidative damage and promoted the invasion and migration of trophoblast cells induced by H_(2)O_(2).Furthermore,the expression levels of MMP-9 and members of the PI3K/Akt signaling pathway were significantly decreased.The pathway agonist,LY294002,and MMP-9 inhibitor,GM6001,reversed the effects of Sal B on H_(2)O_(2)-induced cells.CONCLUSIONS:Sal B promoted the invasion and migration of H_(2)O_(2)-induced HTR-8/Svneo trophoblast cells by upregulating MMP-9 via the PI3K/Akt signaling pathway.展开更多
T helper 17 （Th17） cells have both regulatory and protective roles in physiological conditions. The Th17 subset and the cytokine interleukin-17A （IL-17A） have been implicated in the pathogenesis of certain autoimm...T helper 17 （Th17） cells have both regulatory and protective roles in physiological conditions. The Th17 subset and the cytokine interleukin-17A （IL-17A） have been implicated in the pathogenesis of certain autoimmune diseases, several types of cancer and allograft rejection. However, the role of Th17 cells at the maternal/fetal interface remains unknown. Here, we demonstrate that Th17 cells are present in decidua and are increased in the peripheral blood of 10 clinically normal pregnancies based on intracellular cytokine analysis. Our results suggest a potential role of Th17 cells in sustaining pregnancy in humans. Furthermore, we demonstrate that decidual stromal cells （DSCs） but not trophoblast cells recruit peripheral Th17 cells into the decidua by secreting CCL2. The recruited Th17 cells promote proliferation and invasion and inhibit the apoptosis of human trophoblast cells by secreting IL-17 during the first trimester of pregnancy. These findings indicate a novel role for Th17 cells in controlling the maternal-fetal relationship and placenta development.展开更多
Successful human pregnancy requires the maternal immune system to recognize and tolerate the semi-allogeneic fetus. Myeloid-derived suppressor cells （MDSCs）, which are capable of inhibiting T-cell responses, are hig...Successful human pregnancy requires the maternal immune system to recognize and tolerate the semi-allogeneic fetus. Myeloid-derived suppressor cells （MDSCs）, which are capable of inhibiting T-cell responses, are highly increased in the early stages of pregnancy. Although recent reports indicate a role for MDSCs in fetal-maternal tolerance, little is known about the expansion of MDSCs during pregnancy. In the present study, we demonstrated that the trophoblast cell line HTR8/SVneo could instruct peripheral CD14＋ myelomonocytic cells toward a novel subpopulation of MDSCs, denoted as CD 14 ＋ H LA-DR-/=~w cells, with suppressive activity and increased expression of I DO 1, ARG- 1, a nd COX2. After interaction with HTR8/SVneo cells, CD14＋ myelomonocytic cells secrete high levels of CCL2, promoting the expression of signal transducer and activator of transcription 3. We utilized a neutralizing monoclonal antibody to reveal the prominent role of CCL2 in the induction of CD14＋HLA-DR-/low MDSCs. In combination, the results of the present study support a novel role for the cross-talk between the trophoblast cell line HTR8/SVneo and maternal CD14＋ myelomonocytic cells in initiating MDSCs induction, prompting a tolerogenic immune response to ensure a successful pregnancy.展开更多
Division of Reproductive Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 200031, China *DEPT. Of Pharmacology, Shanghai Tiedao University, Medical College Shanghai 2000...Division of Reproductive Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 200031, China *DEPT. Of Pharmacology, Shanghai Tiedao University, Medical College Shanghai 200070, China By using the morphology and the viability of cells index, the direct effects of anordrin on serum free primary cultures of rat luteal cells, human decidual cells and trophoblast cells were observed.Meanwhile, the effect of anordrin on the secretive function of rat luteal cells was also observed. The results indicated that (1) anordrin has damaging effects on rat luteal cells, human decidual cells and trophoblast cells. The LD 50 s were 14.34±0.9 μg/ml, 17.33±4.1 μg/ml and 34.87±4.9 μg/ml respectively. (2) With nonlethal dose (5 μg/ml), the activity of progesterone secretion of rat lutein cells which was stimulated by hCG and pregnenolone was not influenced by anordrin while the stimulating activity of forskolin was inhibited remarkably.The results suggest that luteolytic action is the main mechanism of the termination of early pregnancy by anordrin and the direct damaging effects of anordrin on decidua and cytotrophoblasts also play a role in the termination of early pregnancy.展开更多
Objective:Adequate extravillous trophoblast(EVT)invasion plays a crucial role in the establishment of successful pregnancy.Insufficient trophoblast migration and invasion can result in defective placentation,which is ...Objective:Adequate extravillous trophoblast(EVT)invasion plays a crucial role in the establishment of successful pregnancy.Insufficient trophoblast migration and invasion can result in defective placentation,which is associated with a number of clinical pathological conditions of pregnancy including spontaneous abortion and preeclampsia.Galectin-9(Gal-9)has a wide variety of regulatory functions in innate and adaptive immunity during infection,tumor growth,and organ transplantation.Methods:We utilized immortalized human first-trimester EVT cells(HTR8/SVneo)for our functional study.We examined the effects of Gal-9 on viability,proliferation,and invasion of HTR8/SVneo cells,as well as on matrix metalloproteinase-2(MMP-2)production in HTR8/SVneo cells.Furthermore,we observed the effects of different MAPK-signaling pathway inhibitors on the stimulatory functions of Gal-9 on HTR8/SVneo cells’invasion.Results:We verified the secretion of Gal-9 by trophoblasts and detected a correlation between low levels of Gal-9 and spontaneous abortion.Gal-9 promoted the invasion of HTR8/SVneo cells through its interaction with Tim-3,not CD44,and subsequently increased MMP-2 production.Blockade of p38 signaling pathway inhibited Gal-9 activities in HTR8/SVneo cells.Conclusion:Gal-9 promotes human trophoblast cell invasion through MMP-2 and p38 signaling pathway in a Tim-3-dependent manner.展开更多
Objective:In this study, we investigated the in vitro binding capacity of β-HCG antibody targeted SonoVue microbubbles to trophoblasts with distinct differentiation in order to explore the possibility of utility of t...Objective:In this study, we investigated the in vitro binding capacity of β-HCG antibody targeted SonoVue microbubbles to trophoblasts with distinct differentiation in order to explore the possibility of utility of targeted SonoVue microbubbles imaging for early locating diagnosis of malignant trophoblastic cell disease. Methods:Three cell groups were included in the study: (1) choriocarcinoma cells (poorly differentiated) (JAR, n=10), (2) early gestational trophoblastic cells (ETC, n=10) and (3) late placenta trophoblastic cells (LTC, n=10). The binding efficiency of the contrast agents to the targeted cells was evaluated by counting the ring formation rate before and after rinsing with PBS. Results: The binding rate was significantly higher in JAR group 84.3±5.5% than in the ETC group 67.3±3.9% and LTC group 60.4±4.6% (P<0.05). The binding rates of different targeted cells to the related targeted microbubble contrast agent (TMCA) before and after PBS rinse did not change significantly. The JARs group exhibited the highest binding rate of (84.3±5.5)% and (82.4±3.7)% before and after PBS rinse (P>0.05). The binding rates of the targeted microbubbles labeled with fluorescence by FCM to JARs, ETC or LTC were 90.1%, 81.5% and 69.2%, respectively (P<0.05). Conclusion: This in vitro study demonstrated that β-HCG an-tibody-targeted SonoVue had different binding capacities to trophoblasts with distinct degrees of differentiation. The highest binding rate occurred with the choriocarcinoma cell line JAR. There is the possibility that the β-HCG antibody-targeted strategy could improve the discriminative ability of SonoVue, in the locating malignant trophoblastic cells.展开更多
Objective:To explore tissue factor(TF)expression and methylation regulation in differentiation of human embryonic stem cells(hESCs)into trophoblast.Methods:Differentiation of hESCs into trophoblast was induced by bone...Objective:To explore tissue factor(TF)expression and methylation regulation in differentiation of human embryonic stem cells(hESCs)into trophoblast.Methods:Differentiation of hESCs into trophoblast was induced by bone morphogenetic protein 4(BMP4).Expression of gene,protein of TF and DNA methylation at different time points during induction process was detected by RTPCT,Western blot,flow cytometry and MSP-PCR method.Results:The expression of mRNA,protein level of TF could be detected during directional differentiation of hESCs to trophoblast cells,semi methylation-semi non methylation expression appeared at TF DNA promoter region,and it showed decreased methylation level and increased non methylation level with formation of trophoblast cell and increased expression of TF.Conclusions:It shows that during differentiation of hESCs into trophoblast,the differential expression of TF is related with DNA methylation level,and it is changed with the methylation or non methylated degree.It provids new platform to furtherly explore the regulation mechanisms of specific expression of tissue factor in the process of the embryonic stem cell development.展开更多
This study examined the effect of over-expression of sFlt-1 by trophoblasts on the barrier function of glomerular endothelial cells and the role of VEGF in this process in order to explore the pathogenesis of glomerul...This study examined the effect of over-expression of sFlt-1 by trophoblasts on the barrier function of glomerular endothelial cells and the role of VEGF in this process in order to explore the pathogenesis of glomerular disease in preeclampsia. SFlt-1 expression in the human trophoblasts （TEV-1 cells） was enhanced by transfecting sFlt-1 plasmid DNA into TEV-1 cells. The monolayer barrier fimction of glomerular endothelial cells （ciGEnCs） was determined by measuring the fluorescence intensity of bovine serum albumin （BSA） that crossed the monolayer of glomerular endothelial cells. The results showed that the over-expression of sFlt-1 by TEV-1 cells led to the barrier dysfunction of ciGEnCs, and the exogenous VEGF could alleviate the ciGEnCs dysfunction resulting from the over-expression of sFlt- 1 to a certain extent. It was concluded that the dysregulation of sFlt- 1 and VEGF in preeclamptic pregnancy may contribute to the barrier dysfunction of glomerular endothelial cells, and VEGF may play an important role in maintaining the barrier function of glomerular endothelial cells, but it may not be the sole factor.展开更多
The effect of axon guidance factors ephrin-A1/EphA2 on the invasion of trophoblastic cells and the possible mechanism were investigated in this study. The expression of EphA2 in vascular endothelial cells was detected...The effect of axon guidance factors ephrin-A1/EphA2 on the invasion of trophoblastic cells and the possible mechanism were investigated in this study. The expression of EphA2 in vascular endothelial cells was detected by immunohistochemistry. The proliferation and invasion of TEV-1 cells （an extravillous trophoblastic cell line） in first trimester were determined by cell counting kit-8 （CCK-8） and Transwell invasion assay. Real-time PCR was used to detect the expression ofephrin-A1 in TEV-I cells treated with EphA2 at different concentrations （10, 50, 100, 500, 1000 and 5000 μg/L）. The results showed： （1） EphA2 was expressed in the vascular endothelial cells; （2） EphA2 could promote the proliferation of TEV-1 cells. The proliferative capacity reached a peak in TEV-1 cells treated with 100 μg/L EphA2 （P〈0.05）; （3） EphA2 could increase the invasion of TEV-1 cells. The invasive ability was the greatest in TEV-1 cells treated with 500 pg/L EphA2 （P〈0.05）; （4） in the presence of EphA2 （0-500 μg/L）, the expression of ephrin-A1 was increased concentration-dependently （P〈0.05）, but when the concentration of EphA2 was over 500 μg/L, the expression of ephrin-A 1 ceased to increase （P〉0.05）. It was concluded that EphA2 can promote the invasion and proliferation of the human extravillous trophoblastic cells probably by regulating the ephrin-A1 ligand.展开更多
Regulation of activin A in cell proliferation as well as hCG and progesterone secretion was investigated using primary cultured cytotrophoblast cells and normal placenta origin cytotrophoblast cell line-NPC cells in s...Regulation of activin A in cell proliferation as well as hCG and progesterone secretion was investigated using primary cultured cytotrophoblast cells and normal placenta origin cytotrophoblast cell line-NPC cells in serum-free system. It was shown that activin A promoted hCG and progesterone secretion in primary cultured cytotrophoblast cells as well as progesterone secretion in NPC cells, while it had no effect on cell proliferation and hCG secretion in NPC cells. lmportant evidence is provided for the autocrine regulatory mechanism of activin A on hormone secretion in placental trophoblast cells at early pregnancy.展开更多
To study the relationship between p53 protein, proliferating cell nuclear antigen (PCNA) expression and benign or malignant gestational trophoblastic disease (MGTD). Methods: The histotomic sections of 48 patients wit...To study the relationship between p53 protein, proliferating cell nuclear antigen (PCNA) expression and benign or malignant gestational trophoblastic disease (MGTD). Methods: The histotomic sections of 48 patients with gestational trophoblastic disease and 24 patients of normal chorionic villi were stained using immunohistochemistry. The monoclonal antibodies were used to determine p53 protein and PCNA. Results: The frequency of p53 and PCNA positive expression were significantly different among the chorionic villi of normal pregnancy, hydratidiform mole (HM) and MGTD. But neither p53 nor PCNA has any relation with the clinical staging or metastasis of MGTD. Conclusion: Both P53 and PCNA are valuable in diagnosis of human gestational trophoblastic disease.展开更多
Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies ...Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies regarding the nature of the differentiated cells. Our goals herein were to determine if BG02 cells form trophoblast-like cells (a) in the presence of BMP4-plus-basic fibroblast growth factor (FGF-2) and (b) upon EB formation, and (c) whether the BMP4 antagonist noggin elicits direct effects on gene expression and hormone production in the cells. Transcriptome profiling of hESC incubated with BMP4/FGF-2 showed a down-regulation of pluripotency-associated genes, an up-regulation of trophoblast-associated genes, and either a down-regulation or no change in gene expression for many markers of the three embryonic germ layers. Yet, there was up-regulation of several genes associated with mesoderm, ectoderm, and endoderm, strongly suggesting that differentiation to trophoblast-like cells under the conditions used does not yield a homogeneous cell type. Several genes, heretofore unreported, were identified that are altered in hESC in response to BMP4-mediated differentiation. The production of human chorionic gonadotropin (hCG), progesterone, and estradiol in the differentiated cells confirmed that trophoblast-like cells were obtained. Gene expression by EB was characterized by an up-regulation of a number of genes associated with trophoblast, ectoderm, endoderm, and mesoderm, and the production of hCG and progesterone confirmed that trophoblast-like cells were formed. These results suggest that, in the presence of FGF-2, BG02 cells respond to BMP4 to yield trophoblast-like cells, which are also obtained upon EB formation. Thus, BMP4-mediated differentiation of hESC represents a viable cell system for studying early developmental events post-implantation;however, up-regulation of non-trophoblast genes suggests a somewhat diverse response to BMP4/FGF-2. Noggin altered the transcription of a limited number of genes but, not surprisingly, did not lead to secretion of hormones.展开更多
基金Hainan Natural Science Foundation project(822MS175)National Nature Science Foundation of China(No.82072880,82003144,81960283,82201874)+1 种基金Supported by the Specific Research Fund of the Innovation Platform for Academicians of Hainan ProvinceProject supported by Hainan Province Clinical Medical Center。
文摘Objective:In this study,we aimed to explore the expression of TUSC3 in Preeclampsia and to research the potential function of TUSC3 in placental trophoblast cells.Methods:We collected 10 cases of normal placental tissues and preeclampsia placental tissues,respectively.These parturient received treatment at the First Affiliated Hospital of Hainan Medical University between June 1,2020,and December 31,2022.The expression of TUSC3 in placenta was detected by immunohistochemistry.The effect of TUSC3 on the migration and invasion of HTR8/SVneo cells was analyzed by migration assay and Transwell assay.Results:The expression of TUSC3 was slightly increased in placental villis in preeclampsia.Immunohistochemistry and qRT-PCR were used to detect the expression of TUSC3 protein and mRNA in placental tissues.TUSC3 was markedly upregulated in PE placental tissues(P<0.01).The results of migration assay and Transwell assay showed that the migration rate and the number of invasive cells were significantly decreased in HTR8 overexpressing TUSC3(P<0.01).Conclusions:TUSC3 was markedly increased in PE placental tissues and inhibited trophoblast cells migration and invasion.
基金Hainan Provincial Natural Science Foundation Project(821MS128,822MS164)Hainan Provincial People's Hospital National Natural Science Foundation Cultivation Project(530)(2021MSXM04)。
文摘Objective:To investigate the effect of exosomes secreted by decidual macrophages on trophoblast cells and their molecular mechanism.Methods:The decidual tissues of patients with preeclampsia(PE)and normal-term pregnant women were collected.Macrophages were obtained by the density gradient method and then flow cell sorting,then the exosomes were extracted.The structure of the exosomes was observed by transmission electron microscope.The expression of CD63,a marker protein of the exocrine body,was detected by western blot,and the exosomes were identified.CCK-8 was used to detect the effect of exosomes on trophoblast cell viability.Transwell migration experiment was used to detect the influence on migration ability.The expression of miR-146a-5p in exosomes was detected by qPCR.The effect of exosomes on the expression of HIF1αprotein in trophoblasts was detected by western blot and detection of the binding site between miR-146a-5p and HIF1αby double luciferase reporter gene was conducted.Results:The exosomes of macrophages present a"cake"structure with a middle depression about 30-130 nm in diameter,and CD63 is highly expressed,which conforms to the characteristics of exosomes.Compared with the normal group,the exosomes of decidual macrophages in the PE group inhibited the activity and migration of trophoblast cells(P<0.001).The expression of miR-146a-5p in the exosomes of decidual macrophages in the PE decreased significantly,and after exosomes of PE decidual macrophages treating trophoblast cells,the protein expression of HIF1αin trophoblast cells was significantly increased.There are targeted binding sites between miR-146a-5p and HIF1α.Conclusion:PE decidual macrophage exosomes can inhibit the viability and migration of trophoblast cells,which may be related to the decreased expression of miR-146a-5p in exosomes,thus promoting HIF1αprotein expression of trophoblast cells.
文摘This study investigated the expression and immune effect of TLR4 in human trophoblast cells. The expression level of TLR4 mRNA in normal and LPS-stimulated human term trophoblast cells （1 mg/L LPS, 12 h） was detected by RT-PCR. In LPS-stimulated human term trophoblast cells of TLR4-blocked group and non-TLR4-blocked group, and normal term trophoblast cells of blank control group, apoptosis rate was measured by flow cytometry （FCM）, and the level of TNF-α determined by using enzyme linked immunosorbent assay （ELISA） respectively. RT-PCR results showed that the expression level of TLR4 mRNA in LPS-stimulated human trophoblast cells was significantly higher than that in normal cells （P〈0.01）. FCM revealed that there was significant difference in apoptosis rate of LPS-stimulated human term trophoblast cells between TLR4-blocked group and non-TLR4-blocked group （P〈0.05）, or between TLR4 antibody-blocked group and blank control group. ELISA indicated that the level of TNF-α in LPS-stimulated human trophoblast cells also had statistical differences between TLR4 antibody-blocked group and non-TLR4 antibody-blocked group （P〈0.05）. Our results suggest that TLR4 plays an important role in the immunological mechanism of apoptosis and secretion of TNF-α of human term trophoblast cells stimulated by LPS.
基金supported by grants from National Natural Sciences Foundation of China(No.30672243,30671093)
文摘Human trophoblast cells were isolated and cultured in vitro in order to investigate possible pathogenesis of intrauterine infection caused by HCMV.Trophoblast cells were obtained by compound enzymes digestion and discontinuous percoll gradient.Cells and purity were identified by using immunocytochemistry assay with anti-CK7,Vim and β-hCG antibodies.HCMV AD169 strain replication in isolated trophoblast cells and cell apoptosis were detected at different time points post infection(p.i.).The results showed tha...
基金This study was supported by the Foundation of Scientific and Technological Development of Shanghai (02DZ19115) and Chinese Post-doctor Fund
文摘Objective To investigate the effect of insuline-like growth factor-Ⅰ （IGF-Ⅰ） on progesterone genesis and regulation. Methods Cytotrophoblast cells were collected by trypsin-collagenase digestion and percoll gradient centrifugation for primary culture. After stimulated with different concentrations（100 μg/ml, 10 μg/ml, 1 μg/ml, 0.1 μg/ml） of IGF-Ⅰ at the same time and with different duration（12 h,24 h,48 h, 72 h） of IGF-Ⅰ with the same concentration, progesterone levels in the media were measured by radioimmunoassay. Simultaneously, semiquantitative reverse transcriptase polymerase chain reaction （RT-PCR） was applied to determine the expression of low density lipoprotein receptor （LDLR） mRNA. Results Progesterone levels correlated positively with IGF-Ⅰ along with the IGF-Ⅰ concentration increasing, progesterone level began to increase at 12 h, and reached the climax at 48 h when cultured with 100 μg/L IGF-Ⅰ. The expression of LDLR mRNA was detectable in every group and accordant with variation of progesterone level. Conclusion Progesterone secretion has time- and dose-dependent effect on IGF-Ⅰ, and IGF-1 can up-regulate the expression of LDLR mRNA. IGF-Ⅰ may play an important role in promoting secretion of progesterone in trophoblast cells.
基金Supported by The Program "Molecular and Cell Biology" of the Russian Academy of Sciences
文摘The lifespan of mammalian trophoblast cells includes polyploidization, its degree and peculiarities are, probably, accounted for the characteristics of placenta development. The main ways of genome multiplication-endoreduplication and reduced mitosis-that basically differ by the extent of repression of mitotic events, play, most probably, different roles in the functionally different trophoblast cells in a variety of mammalian species. In the rodent placenta, highly polyploid(512-2048c) trophoblast giant cells(TGC) undergoing endoreduplication serve a barrier with semiallogenic maternal tissues whereas series of reduced mitoses allow to accumulate a great number of low-ploid junctional zone and labyrinth trophoblast cells. Endoreduplication of TGC comes to the end with formation of numerous low-ploid subcellular compartments that show some signs of viable cells though mitotically inactive; it makes impossible their ectopic proliferation inside maternal tissues. In distinct from rodent trophoblast, deviation from(2n)c in human and silver fox trophoblast suggests a possibility of aneuploidy and other chromosome changes(aberrations, etc.). It suggests that in mammalian species with lengthy period of pregnancy, polyploidy is accompanied by more diverse genome changes that may be useful to select a more specific response to stressful factors that may appear occasionally during months of intrauterine development.
文摘OBJECTIVE:To elucidate the regulatory effects of salvianolic acid B(Sal B)on trophoblast cells in preeclampsia(PE).METHODS:3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide(MTT)assays were used to detect the viability of human extravillous trophoblast HTR-8/Svneo cells induced by H_(2)O_(2)following treatment with different concentrations of Sal B.The levels of oxidative stressrelated molecules,including superoxide dismutase,glutathione-Px and malondialdehyde were detected using corresponding kits.Cell apoptosis was detected using a Terminal deoxynucleotidyl transferase(Td T)d UTP NickEnd Labeling(TUNEL)assay,and the expression of apoptosis-related proteins was detected using western blot analysis.In the present study,wound healing and Transwell assays were performed to measure the levels of cell invasion and migration.Western blot analysis was also used to detect the expression levels of epithelialmesenchymal transition-related proteins.The mechanisms underlying Sal B were further investigated using reverse transcription-quantitative real-time polymerase chain reaction(RT-q PCR)and western blot analysis,to determine the expression levels of matrix metallopeptidase 9(MMP-9)and phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/protein kinase B(Akt).RESULTS:Sal B increased the activity of HTR-8/Svneo cells,inhibited oxidative damage and promoted the invasion and migration of trophoblast cells induced by H_(2)O_(2).Furthermore,the expression levels of MMP-9 and members of the PI3K/Akt signaling pathway were significantly decreased.The pathway agonist,LY294002,and MMP-9 inhibitor,GM6001,reversed the effects of Sal B on H_(2)O_(2)-induced cells.CONCLUSIONS:Sal B promoted the invasion and migration of H_(2)O_(2)-induced HTR-8/Svneo trophoblast cells by upregulating MMP-9 via the PI3K/Akt signaling pathway.
文摘T helper 17 （Th17） cells have both regulatory and protective roles in physiological conditions. The Th17 subset and the cytokine interleukin-17A （IL-17A） have been implicated in the pathogenesis of certain autoimmune diseases, several types of cancer and allograft rejection. However, the role of Th17 cells at the maternal/fetal interface remains unknown. Here, we demonstrate that Th17 cells are present in decidua and are increased in the peripheral blood of 10 clinically normal pregnancies based on intracellular cytokine analysis. Our results suggest a potential role of Th17 cells in sustaining pregnancy in humans. Furthermore, we demonstrate that decidual stromal cells （DSCs） but not trophoblast cells recruit peripheral Th17 cells into the decidua by secreting CCL2. The recruited Th17 cells promote proliferation and invasion and inhibit the apoptosis of human trophoblast cells by secreting IL-17 during the first trimester of pregnancy. These findings indicate a novel role for Th17 cells in controlling the maternal-fetal relationship and placenta development.
文摘Successful human pregnancy requires the maternal immune system to recognize and tolerate the semi-allogeneic fetus. Myeloid-derived suppressor cells （MDSCs）, which are capable of inhibiting T-cell responses, are highly increased in the early stages of pregnancy. Although recent reports indicate a role for MDSCs in fetal-maternal tolerance, little is known about the expansion of MDSCs during pregnancy. In the present study, we demonstrated that the trophoblast cell line HTR8/SVneo could instruct peripheral CD14＋ myelomonocytic cells toward a novel subpopulation of MDSCs, denoted as CD 14 ＋ H LA-DR-/=~w cells, with suppressive activity and increased expression of I DO 1, ARG- 1, a nd COX2. After interaction with HTR8/SVneo cells, CD14＋ myelomonocytic cells secrete high levels of CCL2, promoting the expression of signal transducer and activator of transcription 3. We utilized a neutralizing monoclonal antibody to reveal the prominent role of CCL2 in the induction of CD14＋HLA-DR-/low MDSCs. In combination, the results of the present study support a novel role for the cross-talk between the trophoblast cell line HTR8/SVneo and maternal CD14＋ myelomonocytic cells in initiating MDSCs induction, prompting a tolerogenic immune response to ensure a successful pregnancy.
文摘Division of Reproductive Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 200031, China *DEPT. Of Pharmacology, Shanghai Tiedao University, Medical College Shanghai 200070, China By using the morphology and the viability of cells index, the direct effects of anordrin on serum free primary cultures of rat luteal cells, human decidual cells and trophoblast cells were observed.Meanwhile, the effect of anordrin on the secretive function of rat luteal cells was also observed. The results indicated that (1) anordrin has damaging effects on rat luteal cells, human decidual cells and trophoblast cells. The LD 50 s were 14.34±0.9 μg/ml, 17.33±4.1 μg/ml and 34.87±4.9 μg/ml respectively. (2) With nonlethal dose (5 μg/ml), the activity of progesterone secretion of rat lutein cells which was stimulated by hCG and pregnenolone was not influenced by anordrin while the stimulating activity of forskolin was inhibited remarkably.The results suggest that luteolytic action is the main mechanism of the termination of early pregnancy by anordrin and the direct damaging effects of anordrin on decidua and cytotrophoblasts also play a role in the termination of early pregnancy.
基金This work was supported by the National Basic Research Program of China(2015CB9433002017YFC1001400)the National Nature Science Foundation of China(81630036,91542116,31570920,31700799)+2 种基金the Program of Shanghai Academic/Technology Research Leader(17XD1400900)Innovation‑oriented Science and Technology Grant from NPFPC Key Laboratory of Reproduction Regulation(No.CX2017‑0X)the Shanghai Sailing Program(17YF1411600).
文摘Objective:Adequate extravillous trophoblast(EVT)invasion plays a crucial role in the establishment of successful pregnancy.Insufficient trophoblast migration and invasion can result in defective placentation,which is associated with a number of clinical pathological conditions of pregnancy including spontaneous abortion and preeclampsia.Galectin-9(Gal-9)has a wide variety of regulatory functions in innate and adaptive immunity during infection,tumor growth,and organ transplantation.Methods:We utilized immortalized human first-trimester EVT cells(HTR8/SVneo)for our functional study.We examined the effects of Gal-9 on viability,proliferation,and invasion of HTR8/SVneo cells,as well as on matrix metalloproteinase-2(MMP-2)production in HTR8/SVneo cells.Furthermore,we observed the effects of different MAPK-signaling pathway inhibitors on the stimulatory functions of Gal-9 on HTR8/SVneo cells’invasion.Results:We verified the secretion of Gal-9 by trophoblasts and detected a correlation between low levels of Gal-9 and spontaneous abortion.Gal-9 promoted the invasion of HTR8/SVneo cells through its interaction with Tim-3,not CD44,and subsequently increased MMP-2 production.Blockade of p38 signaling pathway inhibited Gal-9 activities in HTR8/SVneo cells.Conclusion:Gal-9 promotes human trophoblast cell invasion through MMP-2 and p38 signaling pathway in a Tim-3-dependent manner.
基金Supported by a grant from the Science-Technology Program Foundation of Guangdong Province,China (No.2006B35901009)
文摘Objective:In this study, we investigated the in vitro binding capacity of β-HCG antibody targeted SonoVue microbubbles to trophoblasts with distinct differentiation in order to explore the possibility of utility of targeted SonoVue microbubbles imaging for early locating diagnosis of malignant trophoblastic cell disease. Methods:Three cell groups were included in the study: (1) choriocarcinoma cells (poorly differentiated) (JAR, n=10), (2) early gestational trophoblastic cells (ETC, n=10) and (3) late placenta trophoblastic cells (LTC, n=10). The binding efficiency of the contrast agents to the targeted cells was evaluated by counting the ring formation rate before and after rinsing with PBS. Results: The binding rate was significantly higher in JAR group 84.3±5.5% than in the ETC group 67.3±3.9% and LTC group 60.4±4.6% (P<0.05). The binding rates of different targeted cells to the related targeted microbubble contrast agent (TMCA) before and after PBS rinse did not change significantly. The JARs group exhibited the highest binding rate of (84.3±5.5)% and (82.4±3.7)% before and after PBS rinse (P>0.05). The binding rates of the targeted microbubbles labeled with fluorescence by FCM to JARs, ETC or LTC were 90.1%, 81.5% and 69.2%, respectively (P<0.05). Conclusion: This in vitro study demonstrated that β-HCG an-tibody-targeted SonoVue had different binding capacities to trophoblasts with distinct degrees of differentiation. The highest binding rate occurred with the choriocarcinoma cell line JAR. There is the possibility that the β-HCG antibody-targeted strategy could improve the discriminative ability of SonoVue, in the locating malignant trophoblastic cells.
基金supported by National Natural Science Fund Project(No.81170477)
文摘Objective:To explore tissue factor(TF)expression and methylation regulation in differentiation of human embryonic stem cells(hESCs)into trophoblast.Methods:Differentiation of hESCs into trophoblast was induced by bone morphogenetic protein 4(BMP4).Expression of gene,protein of TF and DNA methylation at different time points during induction process was detected by RTPCT,Western blot,flow cytometry and MSP-PCR method.Results:The expression of mRNA,protein level of TF could be detected during directional differentiation of hESCs to trophoblast cells,semi methylation-semi non methylation expression appeared at TF DNA promoter region,and it showed decreased methylation level and increased non methylation level with formation of trophoblast cell and increased expression of TF.Conclusions:It shows that during differentiation of hESCs into trophoblast,the differential expression of TF is related with DNA methylation level,and it is changed with the methylation or non methylated degree.It provids new platform to furtherly explore the regulation mechanisms of specific expression of tissue factor in the process of the embryonic stem cell development.
文摘This study examined the effect of over-expression of sFlt-1 by trophoblasts on the barrier function of glomerular endothelial cells and the role of VEGF in this process in order to explore the pathogenesis of glomerular disease in preeclampsia. SFlt-1 expression in the human trophoblasts （TEV-1 cells） was enhanced by transfecting sFlt-1 plasmid DNA into TEV-1 cells. The monolayer barrier fimction of glomerular endothelial cells （ciGEnCs） was determined by measuring the fluorescence intensity of bovine serum albumin （BSA） that crossed the monolayer of glomerular endothelial cells. The results showed that the over-expression of sFlt-1 by TEV-1 cells led to the barrier dysfunction of ciGEnCs, and the exogenous VEGF could alleviate the ciGEnCs dysfunction resulting from the over-expression of sFlt- 1 to a certain extent. It was concluded that the dysregulation of sFlt- 1 and VEGF in preeclamptic pregnancy may contribute to the barrier dysfunction of glomerular endothelial cells, and VEGF may play an important role in maintaining the barrier function of glomerular endothelial cells, but it may not be the sole factor.
文摘The effect of axon guidance factors ephrin-A1/EphA2 on the invasion of trophoblastic cells and the possible mechanism were investigated in this study. The expression of EphA2 in vascular endothelial cells was detected by immunohistochemistry. The proliferation and invasion of TEV-1 cells （an extravillous trophoblastic cell line） in first trimester were determined by cell counting kit-8 （CCK-8） and Transwell invasion assay. Real-time PCR was used to detect the expression ofephrin-A1 in TEV-I cells treated with EphA2 at different concentrations （10, 50, 100, 500, 1000 and 5000 μg/L）. The results showed： （1） EphA2 was expressed in the vascular endothelial cells; （2） EphA2 could promote the proliferation of TEV-1 cells. The proliferative capacity reached a peak in TEV-1 cells treated with 100 μg/L EphA2 （P〈0.05）; （3） EphA2 could increase the invasion of TEV-1 cells. The invasive ability was the greatest in TEV-1 cells treated with 500 pg/L EphA2 （P〈0.05）; （4） in the presence of EphA2 （0-500 μg/L）, the expression of ephrin-A1 was increased concentration-dependently （P〈0.05）, but when the concentration of EphA2 was over 500 μg/L, the expression of ephrin-A 1 ceased to increase （P〉0.05）. It was concluded that EphA2 can promote the invasion and proliferation of the human extravillous trophoblastic cells probably by regulating the ephrin-A1 ligand.
文摘Regulation of activin A in cell proliferation as well as hCG and progesterone secretion was investigated using primary cultured cytotrophoblast cells and normal placenta origin cytotrophoblast cell line-NPC cells in serum-free system. It was shown that activin A promoted hCG and progesterone secretion in primary cultured cytotrophoblast cells as well as progesterone secretion in NPC cells, while it had no effect on cell proliferation and hCG secretion in NPC cells. lmportant evidence is provided for the autocrine regulatory mechanism of activin A on hormone secretion in placental trophoblast cells at early pregnancy.
文摘To study the relationship between p53 protein, proliferating cell nuclear antigen (PCNA) expression and benign or malignant gestational trophoblastic disease (MGTD). Methods: The histotomic sections of 48 patients with gestational trophoblastic disease and 24 patients of normal chorionic villi were stained using immunohistochemistry. The monoclonal antibodies were used to determine p53 protein and PCNA. Results: The frequency of p53 and PCNA positive expression were significantly different among the chorionic villi of normal pregnancy, hydratidiform mole (HM) and MGTD. But neither p53 nor PCNA has any relation with the clinical staging or metastasis of MGTD. Conclusion: Both P53 and PCNA are valuable in diagnosis of human gestational trophoblastic disease.
文摘Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies regarding the nature of the differentiated cells. Our goals herein were to determine if BG02 cells form trophoblast-like cells (a) in the presence of BMP4-plus-basic fibroblast growth factor (FGF-2) and (b) upon EB formation, and (c) whether the BMP4 antagonist noggin elicits direct effects on gene expression and hormone production in the cells. Transcriptome profiling of hESC incubated with BMP4/FGF-2 showed a down-regulation of pluripotency-associated genes, an up-regulation of trophoblast-associated genes, and either a down-regulation or no change in gene expression for many markers of the three embryonic germ layers. Yet, there was up-regulation of several genes associated with mesoderm, ectoderm, and endoderm, strongly suggesting that differentiation to trophoblast-like cells under the conditions used does not yield a homogeneous cell type. Several genes, heretofore unreported, were identified that are altered in hESC in response to BMP4-mediated differentiation. The production of human chorionic gonadotropin (hCG), progesterone, and estradiol in the differentiated cells confirmed that trophoblast-like cells were obtained. Gene expression by EB was characterized by an up-regulation of a number of genes associated with trophoblast, ectoderm, endoderm, and mesoderm, and the production of hCG and progesterone confirmed that trophoblast-like cells were formed. These results suggest that, in the presence of FGF-2, BG02 cells respond to BMP4 to yield trophoblast-like cells, which are also obtained upon EB formation. Thus, BMP4-mediated differentiation of hESC represents a viable cell system for studying early developmental events post-implantation;however, up-regulation of non-trophoblast genes suggests a somewhat diverse response to BMP4/FGF-2. Noggin altered the transcription of a limited number of genes but, not surprisingly, did not lead to secretion of hormones.
