RESUMEN
BACKGROUND: Patients with recurrent implantation failure (RIF) may have more uterine contractions. Several observational studies suggested that atosiban administration around embryo transfer resulted in higher pregnancy rates in RIF patients. This study aimed to evaluate the effect of atosiban given before fresh embryo transfer on pregnancy outcomes of women with RIF. METHODS: A prospective, randomized, double-blind controlled clinical trial was performed in IVF center of Shanghai First Maternity and Infant Hospital. According to a computer-generated randomization list, 194 infertile women with RIF received fresh embryo transfer between July 2017 and December 2019 were randomly allocated into the atosiban (n = 97) and the placebo (n = 97) groups. Women in the treatment group received atosiban intravenously about 30 min before embryo transfer with a bolus dose of 6.75 mg over one minute. Those in the placebo group received only normal saline infusion for the same duration. RESULTS: There was no significant difference in the live birth rate between the atosiban and placebo groups (42.3% vs 35.1%, P = 0.302, RR = 1.206 (0.844-1.723)). No significant differences were found between the two groups in the positive pregnancy test, clinical pregnancy, ongoing pregnancy, miscarriage, multiple pregnancy, ectopic pregnancy and implantation rates. Similar results were found when stratified by the number of embryos previously transferred, number of previous failed embryo transfers, frequency of endometrial peristalsis on embryo transfer day (≥ 3 waves/min) or serum estradiol (E2) on the day of hCG above the median level. And, there was no correlation between the serum E2 level on the day of hCG and the frequency of endometrial peristalsis on embryo transfer day. The frequency of endometrial peristalsis on embryo transfer day, total FSH/HMG dosage and duration were the significant factors which independently predicted the likelihood of a live birth. CONCLUSIONS: These results suggested that atosiban treatment before fresh embryo transfer might not improve the live birth rate in RIF patients. TRIAL REGISTRATION: The study had been approved by the Institutional Review Board of the hospital (2017 ethics No.43) and was registered under Clinicaltrials.gov with an identifier NCT02893722.
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Fertilización In Vitro , Infertilidad Femenina , China , Implantación del Embrión , Femenino , Fertilización In Vitro/métodos , Humanos , Infertilidad Femenina/terapia , Nacimiento Vivo , Embarazo , Índice de Embarazo , Estudios Prospectivos , Vasotocina/análogos & derivadosRESUMEN
BACKGROUND: Trophoblast cells are required for the establishment of pregnancy and fetal development. Apoptosis is an essential feature for trophoblast invasion. Uncontrolled trophoblast apoptosis is related to some complicate pregnancies. Oxidative stress (OS) is an important inducer of trophoblast apoptosis. Cyclosporin A (CsA) has been shown to promote the activity of trophoblast cells and reduce OS-induced oxidative injury. We investigated the role and mechanism of CsA in oxidative stress-induced trophoblast cell apoptosis. METHODS: JEG-3 cells were cocultured with H2O2 and CsA. Cell viability and morphology were measured by MTT assay and DAPI staining. Cell apoptosis was tested with annexin V/PI staining. The expression of Bcl-2-associated X protein (Bax), B-cell lymphoma/leukemia-2 (Bcl-2), cleaved poly (ADP-ribose) polymerase (PARP) and pro-caspase-3 was assayed by western blotting. The protein expression and phosphorylation of p53 and mitogen-activated protein kinase (MAPK) kinases (JNK, ERK1/2 and p38) were examined by western blotting. RESULTS: CsA increased the viability, alleviated morphological injury and reduced cell apoptosis of the H2O2-treated JEG-3 cells. CsA also attenuated the activation of p53, decreased the expression of Bax and cleavage of PARP, and increased the expression of Bcl-2 and pro-caspase-3 in the JEG-3 treated with H2O2. Furthermore, CsA reduced the activation of JNK and P38 but had no significant effect on the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the H2O2-treated JEG-3 cells. Promoting the activation of JNK and p38 impaired the protective effect of CsA on OS-induced trophoblast apoptosis. CONCLUSIONS: These results suggested that CsA protected trophoblast cells from OS-induced apoptosis via the inhibition of the p53 and JNK/p38 signaling pathways.
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Apoptosis/efectos de los fármacos , Ciclosporina/farmacología , Trofoblastos/efectos de los fármacos , Línea Celular Tumoral , Citoprotección/efectos de los fármacos , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fosforilación/efectos de los fármacos , Embarazo , Sustancias Protectoras/farmacología , Transducción de Señal/efectos de los fármacos , Trofoblastos/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: To measure blood and follicular antimüllerian hormone (AMH) levels in women with polycystic ovary syndrome (PCOS) undergoing assisted reproductive technologies (ART) and to examine the direct action of insulin on AMH expression in human granulosa cells. DESIGN: Prospective clinical and experimental study. SETTING: University Hospital-based laboratory. PATIENT(S): Women with (n = 86) and without (n = 172) PCOS in ART. INTERVENTION(S): Blood, follicular fluid, and luteinized granulosa cells were collected from PCOS and non-PCOS women in ART. MAIN OUTCOME MEASURE(S): Hormone levels in blood and fluid, and gene expression in granulosa cells. RESULT(S): Serum levels of AMH were elevated and inversely correlated with embryo cleavage rate in PCOS women in ART. Significant higher levels of AMH were also found in small and large follicles collected from PCOS women compared with non-PCOS women. Luteinized granulosa cells from PCOS women showed higher expression of AMH and its receptor AMHR2. Direct effect of insulin in increasing the expression of AMH in the isolated luteinized granulosa cells was observed, with the PCOS granulosa cells responding to a high dose of insulin. Cotreatment with AMH attenuated insulin-induced aromatase expression in the luteinized granulosa cells. CONCLUSION(S): These results suggest that insulin may contribute to AMH elevation in PCOS and that AMH counteracts insulin-promoted aromatase expression in granulosa cells.
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Hormona Antimülleriana/metabolismo , Células de la Granulosa/metabolismo , Insulina/administración & dosificación , Síndrome del Ovario Poliquístico/metabolismo , Técnicas Reproductivas Asistidas , Adulto , Hormona Antimülleriana/sangre , Biomarcadores/sangre , Biomarcadores/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Células de la Granulosa/efectos de los fármacos , Humanos , Inducción de la Ovulación/métodos , Inducción de la Ovulación/tendencias , Síndrome del Ovario Poliquístico/diagnóstico , Estudios Prospectivos , Técnicas Reproductivas Asistidas/tendencias , Adulto JovenRESUMEN
Spontaneous abortion is the most common complication of pregnancy. Immune activation and the subsequent inflammation-induced tissue injury are often observed at the maternal-fetal interface as the final pathological assault in recurrent spontaneous abortion. However, the precise mechanisms responsible for spontaneous abortion involving inflammation are not fully understood. Chemokine CCL28 and its receptors CCR3 and CCR10 are important regulators in inflammatory process. Here, we examined the expression of CCL28 and its receptors in decidual stromal cells (DSCs) by immunochemistry and flow cytometry (FCM), and compared their expression level in DSCs from normal pregnancy versus spontaneous abortion, and their relationship to inflammatory cytokines production by DSCs. We further analyzed regulation of the pro-inflammatory cytokines on CCL28 expression in DSCs by real-time polymerase chain reaction, In-cell Western and FCM. The effects of CCL28-CCR3/CCR10 interaction on DSC apoptosis was investigated by Annexin V staining and FCM analysis or DAPI staining and nuclear morphology. Higher levels of the inflammatory cytokines interleukin (IL)-1ß, IL-17A and tumor necrosis factor-α, and increased CCR3/CCR10 expression were observed in DSCs from spontaneous abortion compared with normal pregnancy. Treatment with inflammatory cytokines differently affected CCL28 and CCR3/CCR10 expression in DSCs. Human recombinant CCL28 promoted DSC apoptosis, which was eliminated by pretreatment with neutralizing antibodies against CCR3/CCR10 and CCL28. However, CCL28 did not affect DSC growth. These results suggest that the inflammation-promoted up-regulation of CCL28 and its receptors interaction in DSCs is involved in human spontaneous abortion via inducing DSC apoptosis.
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Aborto Espontáneo/metabolismo , Quimiocinas CC/metabolismo , Decidua/citología , Receptores CCR10/metabolismo , Receptores CCR3/metabolismo , Células del Estroma/citología , Células del Estroma/metabolismo , Aborto Espontáneo/genética , Adulto , Apoptosis/efectos de los fármacos , Apoptosis/genética , Células Cultivadas , Quimiocinas CC/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Técnicas In Vitro , Interleucina-17/farmacología , Interleucina-1beta/farmacología , Embarazo , Receptores CCR10/genética , Receptores CCR3/genética , Células del Estroma/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Adulto JovenRESUMEN
Chemokine CCL24 is the second member of eotaxins, a group of eosinophils' selectively chemoattractants. Via binding to its only receptor CCR3, CCL24 mainly mediates atopic disorders, parasitic infections and systemic diseases. It is well-known that CCR3 is expressed at the maternal-fetal interface; nevertheless whether CCL24 is located there and which role CCL24/CCR3 axis played is unclear. In this article, we assessed the expression of CCL24 and CCR3 in decidual stromal cells (DSCs) and trophoblasts, investigated the effects of DSCs-trophoblasts contact and pregnancy-associated hormones on the expression of CCR3 by DSCs, and last examined the role of trophoblasts-derived CCL24 on the proliferation, cell numbers and apoptosis of DSCs in vitro. We found that trophoblasts secrete chemokine CCL24, whereas DSCs express receptor CCR3. DSCs and trophoblasts co-culture had an raised level of CCL24 in culture supernatants, and the expression of CCR3 on DSCs was also obviously improved. Estrogen, progesterone and hCG up-regulated the expression of CCR3 on DSCs at appropriate concentration. CCL24 increased the proliferation and apoptosis of DSCs, whereas on the whole it promoted the number of DSCs. Thus, we conclude that by secreting CCL24 trophoblasts could promote the growth of DSCs; pregnancy associated environments such as DSCs-trophoblasts contact and hormones increased local CCL24/CCR3, which means a beneficial factor for the process of decidualization in human early pregnancy.
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Apoptosis , Proliferación Celular , Quimiocina CCL24/metabolismo , Decidua/metabolismo , Comunicación Paracrina , Células del Estroma/metabolismo , Trofoblastos/metabolismo , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Gonadotropina Coriónica/farmacología , Técnicas de Cocultivo , Decidua/efectos de los fármacos , Decidua/crecimiento & desarrollo , Decidua/inmunología , Relación Dosis-Respuesta a Droga , Estrógenos/farmacología , Femenino , Edad Gestacional , Humanos , Comunicación Paracrina/efectos de los fármacos , Embarazo , Primer Trimestre del Embarazo , Progesterona/farmacología , Receptores CCR3/metabolismo , Células del Estroma/efectos de los fármacos , Células del Estroma/inmunología , Factores de Tiempo , Trofoblastos/efectos de los fármacos , Trofoblastos/inmunologíaRESUMEN
OBJECTIVE: To investigate the efficacy and mechanism of Bushen Huoxue Recipe (, BHR) in the treatment of murine autoimmune premature ovarian failure (POF). METHODS: The recombinant porcine zona pellucida 4 (pZP4) was expressed in E. coli BL21 (DE3) strain within prokaryotic plasmid pET28a (+), purified by Ni-affinity chromatography and verified by Western blot. Murine autoimmune POF model was established by immunization with pZP4 of female BALB/c mice. Fifty POF mice were randomly divided into 5 groups, which were respectively given low (3.75 mg/kg), moderate (7.5 mg/kg), and high dose (15.0 mg/kg) of BHR by gastrogavage once daily for 20 days, with 17-ß-estradiol (0.13 mg/kg) and normal saline as positive and negative control. Estrous cycles were analyzed through vaginal smears, serum estradiol (E) levels, and anti-pZP4 antibody titers were detected by ELISA. The proliferative responses in vitro of spleen lymphocytes to pZP4 antigen restimulation were measured by [(3)H]-thymidine incorporation, and the histomorphology changes of ovary were evaluated by optical microscope. RESULTS: The purified pZP4 was visible as a single lane with 14.4 kD in SDS-PAGE and Western blot. The murine POF model with lengthening estrous cycles, decreased levels of serum E2, high titers of serum anti-pZP4 antibody, and reduced ovarian follicles and corpus lutea were established by immunization with recombinant pZP4. Treatment with moderate and high dosage BHR significantly increased ovarian follicles and reduced the proliferation of spleen lymphocytes to the pZP4 antigen of POF mice (P <0.05). However, only the high dosage BHR administration significantly improved the estrous cycles, elevated the serum E levels (P <0.01), and decreased the serum anti-pZP4 antibody titers of model mice P<0.05). CONCLUSIONS: The recombinant pZP4 could evoke the antigen-specific immune response in mice and induce the autoimmune ovarian injury. It has been demonstrated that BHR was able to increase the serum E levels and protect ovarian functions from the autoimmune injury in murine POF model.
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Medicamentos Herbarios Chinos/uso terapéutico , Proteínas del Huevo/inmunología , Inmunización , Glicoproteínas de Membrana/inmunología , Insuficiencia Ovárica Primaria/tratamiento farmacológico , Insuficiencia Ovárica Primaria/inmunología , Receptores de Superficie Celular/inmunología , Proteínas Recombinantes/inmunología , Animales , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos/farmacología , Proteínas del Huevo/aislamiento & purificación , Femenino , Inmunocompetencia/efectos de los fármacos , Glicoproteínas de Membrana/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Ovario/efectos de los fármacos , Ovario/inmunología , Ovario/patología , Insuficiencia Ovárica Primaria/patología , Receptores de Superficie Celular/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Sus scrofa , Glicoproteínas de la Zona PelúcidaRESUMEN
INTRODUCTION: Our previous study has demonstrated Cyclosporin A (CsA) promotes the proliferation of human trophoblast cells. Therefore, we further investigate the intracellular signaling pathway involved in the CsA-induced proliferation of human trophoblast cells. METHODS: Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the regulation of CsA on CXCL12 secretion in human trophoblast cells. Immunofluorescence analysis and western blotting analysis were used to investigate the role of CXCL12/CXCR4 axis in the CsA-induced epidermal growth factor receptor (EGFR) phosphorylation in human trophoblast cells. 5-Bromo-2'-deoxyuridine (BrdU) cell proliferation assay was performed to analyze the involvement of EGFR and its downstream extracellular signal-regulated protein kinase (ERK) signaling pathway in the CsA-induced proliferation of human trophoblast cells. RESULTS: Low concentration of CsA promoted the secretion of CXCL12, and recombinant human CXCL12 promoted the phosphorylation of EGFR in primary human trophoblast cells and choriocarcinoma cell line JEG-3. The inhibition of CXCL12 or CXCR4 by either neutralizing antibodies or small interfering RNA (siRNA) could completely block the CsA-induced EGFR phosphorylation. The CsA-induced proliferation of human trophoblast cells was effectively abrogated by the EGFR inhibitor AG1478 as well as the ERK inhibitor U0126, but not by the PI3K/PKB inhibitor LY294002. CsA promoted the activation of ERK in JEG-3 cells, which was markedly abrogated in the presence of CXCL12 siRNA, or CXCR4 siRNA, or AG1478. CONCLUSIONS: CsA may promote EGFR activation via CXCL12/CXCR4 axis, and EGFR downstream ERK signaling pathway may be involved in the CsA-induced proliferation of human trophoblast cells.
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Proliferación Celular/efectos de los fármacos , Quimiocina CXCL12/metabolismo , Ciclosporina/farmacología , Receptores ErbB/metabolismo , Inmunosupresores/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Receptores CXCR4/metabolismo , Trofoblastos/metabolismo , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Femenino , Humanos , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Trofoblastos/citologíaRESUMEN
The regulatory mechanism of Th2 bias at the maternal/fetal interface remains unclear. In this study, we characterized cytokine production in decidual stromal cells (DSCs), decidual immune cells (DICs) and embryo-derived trophoblast cells, and investigated the regulation of CXCL12/CXCR4 interaction on Th2 bias at the maternal/fetal interface in early human pregnancy. We found differential production of Th1-type and Th2-type cytokines by trophoblasts, DSCs and DICs. The secretion of these cytokines varied in different cell cocultures, conduced to Th2 bias. Flow cytometry showed that coculture of trophoblasts with DSCs and DICs significantly increased IL-4 and IL-10 production in trophoblasts, and IL-10 production in DSCs. However, the coculture of trophoblasts with DSCs and DICs significantly increased interferon (IFN)-γ expression in DSCs, and tumor-necrosis factor (TNF)-α expression in DICs. No change was seen in Th1-type cytokine production in trophoblasts, and in Th2-type cytokine production in DICs in all cocultures. Furthermore, pre-treatment with anti-CXCR4 neutralizing antibody upregulated the production of the Th1-type cytokines IFN-γ and TNF-α, and downregulated the production of the Th2-type cytokines IL-4 and IL-10, in trophoblasts, DSCs, DICs or their cocultures. Interestingly, rhCXCL12 inhibited production of the Th1-type cytokine TNF-α and enhanced the expression of the Th2-type cytokines such as IL-4 and IL-10 in DICs; this effect was abrogated by anti-CXCR4 antibody. Our present study has elucidated the individual contributions of component cells to the shaping of Th2 bias, and uncovered a complicated cross-talk via the CXCL12/CXCR4 signal at the maternal/fetal interface in early human pregnancy.
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Quimiocina CXCL12/metabolismo , Decidua/metabolismo , Embarazo/metabolismo , Receptores CXCR4/metabolismo , Trofoblastos/metabolismo , Adulto , Células Cultivadas , Técnicas de Cocultivo , Citocinas/biosíntesis , Decidua/citología , Decidua/inmunología , Femenino , Citometría de Flujo , Humanos , Embarazo/inmunología , Primer Trimestre del Embarazo , Células del Estroma/citología , Células del Estroma/inmunología , Células del Estroma/metabolismo , Balance Th1 - Th2 , Trofoblastos/citología , Trofoblastos/inmunologíaRESUMEN
Our previous work has demonstrated that cyclosporin A (CsA) up-regulates but CD82 down-regulates the invasiveness of human trophoblasts. In the present study, we further investigated whether CsA can modulate the trophoblasts invasion through regulating the expression of CD82 in decidual stromal cells (DSCs). A co-culture model was established to investigate the effect of CsA on trophoblasts invasiveness. In-cell Western was performed to evaluate the expression of CD82, p53, ß-catenin and the phosphorylation level of NF-κB p50 in DSCs. The secretion of CXCL12 of trophoblasts and DSCs was determined by enzyme-linked immunosorbent assay (ELISA). We found that CsA could not directly change the expression of CD82 in DSCs, but the CsA-treated trophoblasts significantly enhanced CD82 expression, NF-κB p50 phosphorylation and p53 expression, and decreased ß-catenin expression in DSCs, and these effects could be abolished by anti-CXCL12 or CXCR4 neutralizing antibody. In addition, the invasiveness of trophoblast cells was markedly decreased after blocking CXCR4 of trophoblasts. Interestingly, when DSCs were pretreated with anti-CXCR4 neutralizing antibody, the invasiveness of trophoblast cells was enhanced in the coculture unit, and blocking CXCR4 on DSCs could reverse the decrease of trophoblasts invasiveness induced by CD82. Moreover, CsA further amplified these effects mediated by CXCL12 and CD82. Our results suggest that CsA not only promotes the trophoblasts invasiveness through stimulating the secretion of CXCL12, but also limits the invasiveness of trophoblasts by indirectly up-regulating the expression CD82. Therefore, CsA may contribute to the appropriate invasiveness of trophoblasts via strengthening the crosstalk between trophoblasts and DSCs.
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Comunicación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL12/metabolismo , Ciclosporina/farmacología , Decidua/efectos de los fármacos , Proteína Kangai-1/metabolismo , Células del Estroma/efectos de los fármacos , Trofoblastos/efectos de los fármacos , Anticuerpos Neutralizantes/farmacología , Western Blotting , Células Cultivadas , Quimiocina CXCL12/antagonistas & inhibidores , Técnicas de Cocultivo , Decidua/citología , Decidua/inmunología , Relación Dosis-Respuesta a Droga , Implantación del Embrión/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Proteína Kangai-1/genética , Subunidad p50 de NF-kappa B/metabolismo , Fosforilación , Placentación/efectos de los fármacos , Embarazo , Interferencia de ARN , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Células del Estroma/inmunología , Transfección , Trofoblastos/inmunología , Proteína p53 Supresora de Tumor/metabolismo , beta Catenina/metabolismoRESUMEN
AIM: To investigate the effects of high cholesterol diet on the development of osteoporosis and the underlying mechanisms in rats. METHODS: Female Sprague-Dawley rats were randomly separated into 3 groups: (1) the high cholesterol fed rats were fed a high cholesterol diet containing 77% normal diet food, 3% cholesterol and 20% lard for 3 months; (2) ovariectomised (OVX) rats were bilaterally ovariectomised and fed a standard diet; and (3) the control rats were fed the standard diet. Bone mineral density (BMD) of the rats was measured using dual-energy X-ray absorptiometry. Serum levels of oestradiol (E2), osteocalcin (BGP) and carboxy-terminal collagen crosslinks (CTX) were measured using ELISA. Gene expression profile was determined with microarray. Mouse osteoblast cells (MC3T3-E1) were used for in vitro study. Proliferation, differentiation and oxidative stress of the osteoblasts were investigated using MTT, qRT-PCR and biochemical methods. RESULTS: In high cholesterol fed rats, the femur BMD and serum BGP level were significantly reduced, while the CTX level was significantly increased. DNA microarray analysis showed that 2290 genes were down-regulated and 992 genes were up-regulated in this group of rats. Of these genes, 1626 were also down-regulated and 1466 were up-regulated in OVX rats. In total, 370 genes were up-regulated in both groups, and 976 genes were down-regulated. Some of the down-regulated genes were found to code for proteins involved in the transforming growth factor beta (TGF-ß)/bone morphogenic protein (BMP) and Wnt signaling pathways. The up-regulated genes were found to code for IL-6 and Ager with bone-resorption functions. Treatment of MC3T3-E1 cells with cholesterol (12.5-50 µg/mL) inhibited the cell proliferation and differentiation in vitro in a concentration-dependent manner. The treatment also concentration-dependently reduced the expression of BMP2 and Cbfa1, and increased the oxidative injury in MC3T3-E1 cells. CONCLUSION: The results suggest a close correlation between hypercholesterolaemia and osteoporosis. High cholesterol diet increases the risk of osteoporosis, possible via inhibiting the differentiation and proliferation of osteoblasts.
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Desarrollo Óseo , Colesterol en la Dieta/administración & dosificación , Osteoporosis/etiología , Células 3T3 , Absorciometría de Fotón , Animales , Secuencia de Bases , Densidad Ósea , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Femenino , Perfilación de la Expresión Génica , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Ovariectomía , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Tetraspanin CD82 is a wide-spectrum tumor metastasis suppressor that inhibits motility and invasiveness of cancer cells. Endometriosis is a benign gynecological disorder, but appears malignant behaviors including invasion, ectopic implantation and recurrence. This study is to elucidate the role of CD82 expression regulation in the pathogenesis of endometriosis. The short interfering RNA silence was established to analyze the roles of CD82, chemokine CCL2, and its receptor CCR2 in the invasiveness of endometrial stromal cells (ESCs). We have found that the mRNA and protein levels of CD82 in the primary normal ESCs from endometrium without endometriosis are significantly higher than that of the primary ESCs from eutopic endometrium and ectopic tissue. CD82 inhibits the invasiveness of ESCs by downregulating CCL2 secretion and CCR2 expression via mitogen-activated protein kinase (MAPK) and integrinß1 signal pathway, and in turn upregulating the expression of TIMP1 and TIMP2 in an autocrine manner. The combination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with 17ß-estradiol can promote the invasion of ESCs via suppressing CD82 expression and stimulating CCL2 secretion and CCR2 expression, and the enhanced interaction of CCL2-CCR2 recruits more macrophages into the ectopic milieu in a paracrine manner, which further downregulates CD82 expression in the ectopic ESCs. Our study has demonstrated for the first time that the abnormal lower CD82 expression in ESCs induced by TCDD and estrogen may be an important molecular basis of endometriosis pathogenesis through enhancing the CCL2 secretion and CCR2 expression and the invasion of ESCs via MAPK and integrinß1 signal pathway.
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Endometriosis/metabolismo , Endometrio/citología , Proteína Kangai-1/metabolismo , Células del Estroma/citología , Células del Estroma/metabolismo , Adulto , Anticuerpos Neutralizantes/farmacología , Western Blotting , Línea Celular , Movimiento Celular/efectos de los fármacos , Colágeno , Combinación de Medicamentos , Endometriosis/genética , Ensayo de Inmunoadsorción Enzimática , Estradiol/farmacología , Femenino , Humanos , Técnicas In Vitro , Proteína Kangai-1/genética , Laminina , Persona de Mediana Edad , Dibenzodioxinas Policloradas/farmacología , Proteoglicanos , ARN Interferente Pequeño , Receptores CCR2/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/efectos de los fármacosRESUMEN
Our previous study has demonstrated cyclosporin A (CsA) promotes the invasiveness of human first-trimester trophoblast cells. In the present study, we further investigated the intracellular signaling pathway responsible for the improvements in CsA-induced invasiveness of human trophoblast cells. We showed that CsA down-regulated E-cadherin transcription and translation in human primary cultured trophoblast cells and choriocarcinoma cell line JEG-3. U0126, an inhibitor of extracellular signal-regulated protein kinase (ERK), attenuated the CsA-induced transcriptional repressor SNAI2 (also called Slug) expression and restored E-cadherin expression inhibited by CsA in JEG-3 cells. We further demonstrated that CsA amplified epidermal growth factor (EGF)-stimulated EGF receptor (EGFR) tyrosine phosphorylation in JEG-3 cells, and inhibition of EGFR tyrosine phosphorylation by AG1478, an EGFR tyrosine kinase inhibitor, abolished the down-regulation of E-cadherin by CsA through ERK signaling pathway. Moreover, our data showed that E-cadherin expression was negatively correlated to the invasiveness of JEG-3 cells, and CsA could reverse the decreased invasiveness of JEG-3 cells that resulted from E-cadherin overexpression. In conclusion, these observations indicate that CsA may decrease E-cadherin expression via EGFR/ERK signaling pathway and, ultimately, contribute to the invasiveness improvement of human trophoblast cells.
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Cadherinas/metabolismo , Ciclosporina/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Factor de Crecimiento Epidérmico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Transducción de Señal/efectos de los fármacos , Trofoblastos/metabolismo , Análisis de Varianza , Western Blotting , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Ciclosporina/farmacología , Receptores ErbB/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Fosforilación/efectos de los fármacos , Embarazo , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos , Transfección , Trofoblastos/citología , Trofoblastos/efectos de los fármacosRESUMEN
CD82 is recognized as a wide-spectrum tumor metastasis suppressor that inhibits cancer cell motility and invasiveness. At the human maternal-fetal interface, the decidua is believed to effectively limit the inappropriate invasion of trophoblasts. Here we have found the transcription and translation of CD82 in decidual stromal cells (DSCs), whereas trophoblast cells do not express CD82. The in-cell Western analysis reveals attenuation of CD82 translation in DSCs by human chorionic gonadotropin (hCG), but not by estrogen or progesterone. It is demonstrated that silencing of CD82 by RNA interference increases integrinbeta1, decreases TIMP1 expression in DSCs, and promotes the invasion of the first-trimester human trophoblasts in the coculture. Moreover, U0126, or anti-integrinbeta1 neutralizing antibody, reverses the decreased TIMP1 expression and the increased invasiveness of trophoblast cells, and the antibody also inhibits the MAPK3/1 phosphorylation induced by CD82 silence. After transfection with CD82, the invasive index of BeWo cells decreases significantly with TIMP1 increase. The results above indicate that the DSCs-expressed CD82 up-regulates the expression of TIMP1 in an autocrine manner and inhibits the invasiveness of human first-trimester trophoblast cells partly through the integrinbeta1/MAPK/MAPK3/1 signaling pathway. Furthermore, we have found that the mRNA and protein level of CD82 in decidua of the miscarriage is significantly higher than that of the normal early pregnancy, which implies that the abnormal higher CD82 expression in decidua restricts appropriate invasion of trophoblasts that leads to early pregnancy wastage.
Asunto(s)
Decidua/metabolismo , Proteína Kangai-1/metabolismo , Sistemas de Mensajero Secundario/fisiología , Células del Estroma/metabolismo , Trofoblastos/fisiología , Aborto Espontáneo/metabolismo , Células Cultivadas , Vellosidades Coriónicas/metabolismo , Decidua/citología , Implantación del Embrión/fisiología , Femenino , Humanos , Integrina beta1/metabolismo , Intercambio Materno-Fetal/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Embarazo , Transducción de Señal/fisiología , Células del Estroma/citologíaRESUMEN
Contraceptive vaccines based on hCGbeta have not met clinical application because of poor immunogenicity. In the present study, the eukaryotic expression vectors pCI-gs-signal-6His-hCGbeta and pCI-gs-signal-6His-hCGbeta-hC3d3 were constructed, and transfected into CHO cells with aid of Lipofectaine 2000 reagent to gain the secretory recombinant protein. Isolated B cells from human peripheral blood, combined B cells with T cells, and PBMC were treated in vitro, respectively, with 1 nM, 10 nM, 100 nM hCGbeta, hCGbeta-hC3d3 or PWM for 12 days. Immunoglobulin (Ig) and anti-hCG antibody levels in the supernatant were measured by an indirect enzyme-linked immunosorbent assay (ELISA). The expressions of CD80/CD86 on B cells, and CD154/CD25 on T cells, were analyzed by flow cytometry (FCM), and IL-2 production was assayed by ELISA. It was found that the Ig levels in the B-cell supernatants, the combined B with T cells, and PBMC treated with 100 nM hCGbeta-C3d3 fusion protein were 4-fold, 10-fold and 10.9-fold more, respectively, than that of hCGbeta. The anti-hCG antibody could be produced in the combined B cells with T cells, as well as PBMC challenged with 100 nM hCGbeta-C3d3, but no anti-hCG antibody was produced in the challenge with hCGbeta. The hCGbeta-hC3d3 fusion protein enhanced the expression of CD80 and CD86 on B cells, especially CD86 (P<0.05), and significantly increased the expression of CD154 and CD25 molecules on T cells compared to that of hCGbeta (P<0.05). The hCGbeta-hC3d3 promoted human PBMC producing more IL-2 than hCGbeta. These findings indicate that the fusion of hC3d3 to hCGbeta, as a means of harnessing the adjuvant potential of the innate immune system, may contribute to a more efficient humoral immune response, and might provide a potential application of protein vaccine strategies in humans in the future.