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Background Exposure to arsenic can damage trophoblast cells and thus induce abortion, but the mechanism is not known. Objective To investigate the role of miR-145 and PTEN/AKT/mTOR pathway in arsenic-induced abortion and trophoblast cell damage in rats. Methods In the animal experiment, twenty SD pregnant rats were randomly divided into a normal control group (saline gavage) and an arsenic-induced abortion group (10.65 mg·kg−1 sodium arsenite solution was administered by gavage, and the gavage volume was 10 mL·kg−1), with 10 rats in each group. After the miscarriage occurred in the arsenic-induced abortion group (5-6 d after exposure), placental tissues were collected from the two groups. The mRNA expression levels of microRNA-145 (miR-145), phosphatase and tensin homologue (PTEN), kinase B (AKT), mammalian target of rapamycin (mTOR) were detected by real-time quantitative PCR (RT-PCR), and the protein expression levels of PTEN, AKT, mTOR, p-AKT, and p-mTOR were detected by Western blotting. For the in vitro study with immortalized human trophoblast cell line (HTR-8/SVneo cells), a control group, an arsenic exposure group, an miR-145 overexpression group, and an arsenic exposure+miR-145 overexpression group were prepared and cultured for 72 h with 37 °C and 5% CO2, at cell density of 5×105 cells per well, and the arsenic exposure concentration was 20 μmol·L−1. The MTT method was applied to detect cell viability, crystal violet staining to detect the number of monoclonal formation, flow cytometry to detect the level of apoptosis, Image J Angiogenesis Analyzer 1.8.0 plug-in to evaluate total blood vessel length and total blood vessel number; the detection indexes and methods of genes and proteins were the same as "animal experiment". Results (1) In the animal experiment, compared with the normal control group, the expression level of miR-145 mRNA in the placenta tissues of the arsenic-induced abortion group was increased (P<0.05), and the expression levels of PTEN, AKT, mTOR mRNA and proteins, and p-AKT and p-mTOR proteins were decreased (P<0.05). (2) For the in vitro study, compared with the control group, the cell viability rate, number of monoclonal formation, total vessel length, and total vessel number were decreased, and the apoptosis rate was increased in the arsenic exposure group, the miR-145 overexpression group, and the arsenic exposure+miR-145 overexpression group (P<0.05). Compared with the arsenic exposure group and the miR-145 overexpression group, the cell viability rate, number of monoclonal formation, total vessel length, and vessel number were decreased, and the apoptosis rate was increased in the arsenic exposure+miR-145 overexpression group (P<0.05). Compared with the control group, the levels of miR-145 mRNA in the arsenic exposure group, the miR-145 overexpression group, and the arsenic exposure+miR-145 overexpression group increased (P<0.05), the expression levels of PTEN, AKT, mTOR mRNA and protein and the expression levels of p-AKT and p-mTOR protein were decreased (P<0.05); compared with the arsenic exposure group and the miR-145 overexpression group, the level of miR-145 mRNA in the arsenic exposure+miR-145 overexpression group was increased (P<0.05), and the levels of PTEN, AKT, mTOR mRNA and protein as well as p-AKT and p-mTOR protein were decreased (P<0.05). Conclusion miR-145 might be related to abortion due to arsenic exposure. miR-145 could inhibit the proliferation and angiogenesis of trophoblast HTR-8/SVNEO cells, and promotes their apoptosis; the mechanism may be related to the inhibition of PTEN/AKT/mTOR pathway.
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Antibody-drug conjugates(ADCs)are commonly heterogeneous and require extensive assessment of exposure-efficacy and exposure-safety relationships in preclinical and clinical studies.In this study,we report the generation of a monoclonal antibody against monomethyl auristatin E(MMAE)and the development,validation,and application of sensitive and high-throughput enzyme-linked immunosor-bent assays(ELISA)to measure the concentrations of MMAE-conjugated ADCs and total antibodies(tAb,antibodies in ADC plus unconjugated antibodies)in cynomolgus monkey sera.These assays were suc-cessfully applied to in vitro plasma stability and pharmacokinetic(PK)studies of SMADC001,an MMAE-conjugated ADC against trophoblast cell surface antigen 2(TROP-2).The plasma stability of SMADC001 was better than that of similar ADCs coupled with PEG4-Val-Cit,Lys(m-dPEG24)-Cit,and Val-Cit linkers.The developed ELISA methods for the calibration standards of ADC and tAb revealed a correlation be-tween serum concentrations and the OD450 values,with R2 at 1.000,and the dynamic range was 0.3-35.0 ng/mL and 0.2-22.0 ng/mL,respectively;the intra-and inter-assay accuracy bias％ranged from-12.2％to-5.2％,precision ranged from-12.4％to-1.4％,and the relative standard deviation(RSD)was less than 6.6％and 8.7％,respectively.The total error was less than 20.4％.The development and validation steps of these two assays met the acceptance criteria for all addressed validation parameters,which suggested that these can be applied to quantify MMAE-conjugated ADCs,as well as in PK studies.Furthermore,these assays can be easily adopted for development of other similar immunoassays.
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Objective: To explore the expression level of protein kinase AMP-activated catalytic subunit α1 (PRKAA1) in placental tissues of gestational diabetes mellitus (GDM) women, and the influence of high glucose (HG) on PRKAA1 expression and proliferation viability of trophoblast cells in vitro. Methods: The placental samples of GDM women (n=19) and normal pregnant women (n=20) of the corresponding period were collected. Real-time qPCR and Western blotting assay were used to detect the mRNA and protein levels of PRKAA1 in these biopsies, respectively. Trophoblast cells were treated by HG in vitro and then expression level of PRKAA1 was tested. CCK8 assay was used to detect proliferation viability of the cells treated by HG medium or inhibitor of PRKAA1, dorsomorphin. Results: Comparing to normal pregnant women, both mRNA and protein levels of PRKAA1 in placental tissues of GDM women significantly decreased (both P<0.05). HG treatment drastically downregulated expression of PRKAA1 in trophoblast cells in vitro (P<0.05). Both HG medium and dorsomorphin suppressed proliferation viability of trophoblast cells (both P<0.05). Conclusion: Expression level of PRKAA1 is dampened in placental tissues of GDM women. HG suppresses proliferation viability of trophoblast cells probably via downregulating PRKAA1 level in vitro.
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Objective To explore the effect of ras-related C3 botulinum toxin substrate 3 (RAC3) on the proliferation, migration and invasion abilities of early trophoblast cells. Methods Villius samples from 20 unexplained spontaneous abortion (SA) and 20 induced abortion (IA) patients were collected between May 2015 and May 2016 in Changzheng Hospital of Navy Medical University (Second Military Medical University). qPCR was used to detect the expression of RAC3 mRNA in the villus tissues. mRNA Chip detection was performed on the placental tissues of 6.5, 14.5 and 19.5 days in mice. After interfering or overexpressing RAC3 in early human trophoblast cell line HTR-8/SVneo, the proliferation, migration and invasion abilities were detected by CCK-8 and Transwell assay, respectively. Results The expression of RAC3 mRNA was significantly lower in the villus tissue of unexplained SA patients than that in the villus tissue of IA patients (P0.05). Rac3 mRNA expressions were significantly higher in the placental tissues of 6.5 and 14.5 days in mice than that in the placental tissues of 19.5 days (both P0.05). Compared with the control group, the proliferation, migration and invasion abilities of the HTR-8/SVneo cells were significantly reduced by interfering RAC3 expression (all P0.05), and the proliferation, migration and invasion abilities were significantly enhanced by overexpressing RAC3 (all P0.01). Conclusion RAC3 plays an important role in regulating of the proliferation, migration and invasion of early trophoblast cells.
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Objective·To explore the expression level of protein kinase AMP-activated catalytic subunit α1 (PRKAA1) in placental tissues of gestational diabetes mellitus(GDM)women,and the influence of high glucose(HG)on PRKAA1 expression and proliferation viability of trophoblast cells in vitro. Methods·The placental samples of GDM women (n=19) and normal pregnant women (n=20) of the corresponding period were collected. Real-time qPCR and Western blotting assay were used to detect the mRNA and protein levels of PRKAA1 in these biopsies, respectively. Trophoblast cells were treated by HG in vitro and then expression level of PRKAA1 was tested.CCK8 assay was used to detect proliferation viability of the cells treated by HG medium or inhibitor of PRKAA1, dorsomorphin. Results·Comparing to normal pregnant women, both mRNA and protein levels of PRKAA1 in placental tissues of GDM women significantly decreased (both P<0.05). HG treatment drastically downregulated expression of PRKAA1 in trophoblast cells in vitro(P<0.05).Both HG medium and dorsomorphin suppressed proliferation viability of trophoblast cells(both P<0.05). Conclusion·Expression level of PRKAA1 is dampened in placental tissues of GDM women.HG suppresses proliferation viability of trophoblast cells probably via downregulating PRKAA1 level in vitro.
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Objective · To study the effect of miR-137 overexpressed trophoblast cells on the biological functions of vascular endothelial cells.Methods · Lentivirus vectors Up-LV-miR-137 and LV-miR-NC were constructed and transfected into the trophoblast cells HTR-8/SVneo, respectively.And transfection efficiency was verified by PCR. Vascular endothelial cells were cultured with transfected trophoblast cells or their culture medium supernatant. Proliferation, migration activity and ability to recruit monocytes U937 of endothelial cells were detected by CCK8 method, scratch test and Transwell migration test, respectively. Results · miR-137 overexpressed trophoblast cells were obtained. The results of CCK8 test showed that proliferation of vascular endothelial cells cultured with miR-137 overexpressed trophoblast cells or their culture medium supernatant was inhibited, especially in high glucose condition. The repair of endothelial cells in the transfected cells culture medium slowed down in the scratch test. Transwell migration test results showed that after the endothelial cells were cultured with miR-137 overexpressed trophoblast cells, more monocytes passed through the chambers with higher chemotactic index. Conclusion · miR-137 overexpressed trophoblast cells can regulate the biological functions of vascular endothelial cells, i.e. reduce proliferation and migration activity and enhance ability to recruit monocytes.
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Objective · To study the effect of miR-137 overexpressed trophoblast cells on the biological functions of vascular endothelial cells.Methods · Lentivirus vectors Up-LV-miR-137 and LV-miR-NC were constructed and transfected into the trophoblast cells HTR-8/SVneo, respectively.And transfection efficiency was verified by PCR. Vascular endothelial cells were cultured with transfected trophoblast cells or their culture medium supernatant. Proliferation, migration activity and ability to recruit monocytes U937 of endothelial cells were detected by CCK8 method, scratch test and Transwell migration test, respectively. Results · miR-137 overexpressed trophoblast cells were obtained. The results of CCK8 test showed that proliferation of vascular endothelial cells cultured with miR-137 overexpressed trophoblast cells or their culture medium supernatant was inhibited, especially in high glucose condition. The repair of endothelial cells in the transfected cells culture medium slowed down in the scratch test. Transwell migration test results showed that after the endothelial cells were cultured with miR-137 overexpressed trophoblast cells, more monocytes passed through the chambers with higher chemotactic index. Conclusion · miR-137 overexpressed trophoblast cells can regulate the biological functions of vascular endothelial cells, i.e. reduce proliferation and migration activity and enhance ability to recruit monocytes.
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Objective To investigate the effects of stromal cell-derived factor 1 (SDF-1) and an CXC chemokine receptor 4 (CXCR4) antagonist (AMD3100) on the invasion and migration capabilities of the huaman choriocarcinoma cell line JAR for further elucidating the role of SDF-1/CXCR4 axis in the pathogenesis of preeclampsia.Methods JAR cells were divided into four groups: SDF-1 group (treated with 50 ng/ml of SDF-1),SDF-1+AM3100 mixed group (first treated with 100 ng/ml of AMD3100 for 2 hours and then treated with 50 ng/ml of SDF-1),AMD3100 group (treated with 100 ng/ml of AMD3100) and blank control group (without any treatment).RT-PCR was performed to detect the expression of CXCR4 at mRNA level in JAR cells.Western blot assay was used to measure the expression of CXCR4 and p-AKT at protein level.MTT assay was used to analyze the effects of different concentrations of SDF-1 (10,30,50 and 100 ng/ml) on the proliferation of JAR cells at different time points (0,24,48,72 h).Transwell invasion assay and wound-healing assay were used to test the changes in invasion and migration capabilities of JAR cells after different treatments.Results (1) Results of the RT-PCR showed that the expression of CXCR4 at mRNA level in JAR cells was increased in the SDF-1 group (1.839±0.083) as compared with that in the blank control group (1.372±0.086),AMD3100 group (0.694±0.045) or SDF-1+AM3100 mixed group (0.703±0.093).Moreover,the differences between the SDF-1 group and the other three groups were statistically significant (F=30.67,P<0.05).Compared with the blank control group,the expression of CXCR4 at mRNA level in JAR cells was decreased in the AMD3100 group (P<0.01).(2) Results of the Western blot assay showed that the expression of CXCR4 and p-AKT at protein level in JAR cells were enhanced in the SDF-1 group as compared with that in the blank control group,AMD3100 group or SDF-1+AM3100 mixed group.Compared with the blank control group,the expression of CXCR4 and p-AKT at protein level in JAR cells were inhibited in the AMD3100 group.(3) Results of the MTT assay showed that SDF-1,especially at the concentration of 50 ng/ml,could enhance the proliferation of JAR cells (P<0.05) and its best effect on proliferation was seen at 48 h.(4) Results of the Transwell invasion assay showed that the number of transmembrane cells in the SDF-1 group (70.49±2.42) was more than that in the blank control group (54.36±2.26),AMD3100 group (21.68±8.31),or SDF-1+AMD3100 mixed group (28.18±4.61).The differences between the SDF-1 group and the other three groups were statistically significant (F=116.26,P<0.01).Compared with the blank control group,the number of transmembrane cells was reduced in the AMD3100 group (P<0.05).(5) Results of the wound-healing assay showed that the relative migration distance was increased in the SDF-1 group (1.162±0.034) as compared with that in the blank control group (0.823±0.101),AMD3100 group (0.160±0.047),or SDF-1+AMD3100 mixed group (0.183±0.064).The differences between the SDF-1 group and the other three groups were statistically significant (F=30.500,P<0.05).Compared with the blank control group,the relative migration distance was decreased in the AMD3100 group (P<0.01).Conclusion The invasion and migration of huaman choriocarcinoma JAR cells can be enhanced by SDF-1,but inhibited by AMD3100.This study indicates that the blocked biological axis of SDF-1/CXCR4 may play an important role in the pathogenesis of preeclampsia through inducing abnormal activation of PI3K/AKT pathway,which results in inhibited invasion and migration of trophoblast cells and placenta abnormality.
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Objective To investigate the expression of TROP2 and MMP-9 protein expression in cholangiocarcinomas and their relationship between the pathological behavior and prognosis.Methods A total of 54 patients who were diagnosed with cholangiocarcinoma in the People's Hospital of Binzhou,were retrospectively reviewed.Immunohistochemical staining and Log rank test were used to detect the expression of TROP2 and MMP-9 protein in 54 cases of cholangiocarcinomas and 18 cases of normal bile duct tissues achieved by partial hepatectomy of hepatolithiasis.Results The positive expression rate of TROP2 in cholangiocarcinoma tissues (55.6％) was higher than that of normal bile duct tissues (5.6％).The positive expression rate of MMP-9 in cholangiocarcinoma tissues (51.9％) was higher than that of normal bile duct tissues (11.1％).The differences of the expression of TROP2 and MMP-9 in cholangiocarcinoma of TNM stage,lymph node metastasis and neural invasion were significant(all P ＜ 0.05).There was significant positive correlation between TROP-2 and MMP-9 expression by using spearman correlation analysis (r =0.555,P ＜ 0.001).Survival analysis showed that TROP2 expression was an independent prognostic factor in cholangiocarcinoma.Conclusions TROP2 plays a important role in the development and metastasis of cholangiocarcinoma.Thus,TROP2 may be a prognostic indicator for cholangiocarcinoma.
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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 RT-PCT, 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.
Subject(s)
Animals , Humans , Rats , Bone Morphogenetic Protein 4 , Pharmacology , Cell Differentiation , Genetics , Cell Line , DNA Methylation , Genetics , Embryonic Stem Cells , Physiology , Thromboplastin , Genetics , Metabolism , Trophoblasts , Metabolism , PhysiologyABSTRACT
Objective To observe the effects of insulin-like growth factor-Ⅰ (IGF-Ⅰ) and transforming growth factor-β1 (TGF-β1) on the expressions of sodium iodide symporter(NIS) and pendrin mRNA in a placental villous trophoblast cell line(HPT-8) exposed to different levels of iodine.Methods HPT-8 cells were cultured in vitro in the culture flask and divided into low iodine group-Ⅰ (LI-Ⅰ),low iodine group-Ⅱ (LI-Ⅱ),control group,high iodine group-Ⅰ (HI-Ⅰ) and high iodine group-Ⅱ (HI-Ⅱ) that exposed to different concentrations of iodine (0,5,50,500,5000 μg/L).After cell cultured for 24 h,the followings were added to the culture medium:iodine plus IGF-Ⅰ(0.050 mg/L),iodine plus TGF-β1 (0.001 mg/L).After cultured for another 24 h,total RNA was extracted,the expressions of NIS and pendrin mRNA of HPT-8 cells were determined by real-time quantitative PCR.Results The expression of NIS mRNA in HPT-8 cells:at different levels of iodine,the differences of NIS mRNA expression between groups were statistically significant in group with iodine alone(F =3.612,P ＜ 0.01).The expression of NIS mRNA in LI-Ⅰ group(0.44 ± 0.21) was significantly lower than that of control group(1.25 ± 0.77,P＜ 0.01).At the same level of iodine,in LI-Ⅰ group and HI-Ⅰ group,the differences of NIS mRNA expression within groups were statistically significant (F =13.632,6.900,all P ＜ 0.01).In LI-Ⅰ group,the expressions of NIS mRNA were higher in iodine plus IGF-Ⅰ(1.13 ± 0.38) and iodine plus TGF-β1 (0.81 ± 0.34) than that of pure iodine(0.44 ± 0.21,P ＜ 0.01 or ＜ 0.05);in HI-Ⅰ group,the expression of NIS mRNA was lower in iodine plus TGF-β1 (0.62 ± 0.30) than that of pure iodine(1.23 ± 0.91,P ＜ 0.01).The expression of pendrin mRNA in HPT-8 cells:at different levels of iodine,the differences of pendrin mRNA expression between groups were statistically significant in group with iodine alone(F =12.717,P ＜ 0.01).The expression of pendrin mRNA in LI-Ⅰ group(0.59 ± 0.15) was significantly lower than that of control group(1.03 ± 0.14,P ＜ 0.01) ; HI-Ⅰ group(1.29 ± 0.31) was higher than control group(P ＜ 0.05).At the same level of iodine,the differences of pendrin mRNA expression within groups were statistically significant in LI-Ⅰ,LI-Ⅱ,control and HI-Ⅰ groups (F=12.588,4.588,8.679,8.445,all P ＜ 0.01).In LI-Ⅰ,LI-Ⅱ and control groups,the expressions of pendrin mRNA were significantly higher in iodine plus IGF-Ⅰ(1.68 ± 0.82,1.51 ± 0.79,1.50 ± 0.51) than that of pure iodine(0.59 ± 0.15,0.89 ± 0.22,1.03 ± 0.14,all P ＜ 0.01); in HI-Ⅰ group,the expression of pendrin mRNA was significantly lower in iodine plus TGF-β1 (0.78 ± 0.20) than that of pure iodine(1.29 ± 0.31,P ＜ 0.01).Conclusions In the case of iodine deficiency,the mRNA expressions of NIS and pendrin in HPT-8 cells are decreased and the iodine uptake ability is decreased; the expression of pendrin mRNA in HPT-8 cells is increased and placental iodine uptake is increased under the conditions of mild iodine excessive.IGF-Ⅰ and TGF-β1 play a role in the placental iodine uptake through increasing iodine uptake under the conditions of iodine deficiency and decreasing iodine uptake under the conditions of iodine excessive.
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The effects of leptin on cytotrophoblast proliferation and invasion activity were investigated.Immunohistochemistry was used to determine the placental expression of leptin in first-trimester preg-nancy. By using RT-PCR and quantitative real-time PCR, the expression of leptin in cytotrophoblast and the effect of leptin on cytotrophoblast secretion were detected. The potential of cell proliferation, inva-siveness and migration was assessed by MTT, Transwell invasion assay and migration assay respec-tively when the cytotrophoblast was cultured with different concentrations of leptin. The results showed that: (1) Leptin was distributed diffusely around cell membrane, in cytoplasma, and on nuclear mem-brane of cytotrophoblast; (2) Leptin mRNA was expressed in cytotrophoblast. Ten ng/mL leptin could promote the secretion of cytotrophoblast significantly (P<0.01); (3) After culture with different concen-trations of leptin for 24 h or longer, the proliferation of cytotrophoblast was inhibited, while in 24 h leptin could promote cytotrophoblast invasion and migration. Leptin at a concentration of 500 ng/mL could promote cytotrophoblast invasiveness and migration significantly as compared with controls (P<0.05). It was suggested that leptin could inhibit cytotrophoblast proliferation, and promote cytotro-phoblast invasion and migration activity.
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Objective To synthesize a multiple antigenic peptide(MAP),observe its humoral immune response and evaluate its antifertility potential.Methods Based on fusogenic epitope mimic peptide and synthetic nonnatural Pan DR T Helper Epitope(PADRE),MAP antigenic peptides were designed and synthesized.After suspending with the equivalent Freund's adjuvant,the antigens were used to immunize the female C57BL/6 mice to evalutate their humoral immune response in animals and efficiency of specific antisera on inhibiting syncytial trophoblast fusion.Results The MAP peptide was successfully synthesized.After purified with HPLC,its purity reached 95%.After immunization,the highest level of specific IgG in mice serum was 1:1 024,and the corresponding antigen of human trophoblastic cell could be identified in the antisera.In presence of mice antiserum by 1:10 dilution,forskolin-induced intercellular fusion of BeWo choriocarcinoma cells decreased remarkably(P
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By the means of immunohistology we have observed the distribution of prolactin (PRL) and human placenta lactogen (HPL) in decidua and ehorion. The results are as followe: (1) HPL was negatively stained in normal nonpregnant endometria; (2)PRL staining showed posi- tive in cytoplasm of decidual cells whereas negative in chorionic villi; (3) syncytiotrophoblast cells of chorionic villi were positive for villi; (4) some small cells scattered between decidual cells were positive for HPL; (5)extravillous trophoblastic infiltrated in decidua were positively stained with HPL; (6)it is interesting to note that HPL positive cells could be present among the cells of blood vessel wall, especially in endothelial cells of capillaries, about which there is no report so far. The significance of HPL positive cells in decidua deserves further study.