RESUMO
Idiopathic cholangiopathies are diseases that affect cholangiocytes, and they have unknown etiologies. Currently, orthotopic liver transplantation is the only treatment available for end-stage liver disease. Limited access to the bile duct makes it difficult to model cholangiocyte diseases. In this study, by mimicking the embryonic development of cholangiocytes and using a robust, feeder- and serum-free protocol, we first demonstrate the generation of unique functional 3D organoids consisting of small and large cholangiocytes derived from human pluripotent stem cells (PSCs), as opposed to traditional 2D culture systems. At day 28 of differentiation, the human PSC-derived cholangiocytes expressed markers of mature cholangiocytes, such as CK7, CK19, and cystic fibrosis transmembrane conductance regulator (CFTR). Compared with the 2D culture system-generated cholangiocytes, the 3D cholangiocyte organoids (COs) showed higher expression of the region-specific markers of intrahepatic cholangiocytes YAP1 and JAG1 and extrahepatic cholangiocytes AQP1 and MUC1. Furthermore, the COs had small-large tube-like structures and functional assays revealed that they exhibited characteristics of mature cholangiocytes, such as multidrug resistance protein 1 transporter function and CFTR channel activity. In addition to the extracellular matrix supports, the epidermal growth factor receptor (EGFR)-mediated signaling regulation might be involved in this cholangiocyte maturation and differentiation. These results indicated the successful generation of intrahepatic and extrahepatic cholangiocytes by using our 3D organoid protocol. The results highlight the advantages of our 3D culture system over the 2D culture system in promoting the functional differentiation and maturation of cholangiocytes. In summary, in advance of the previous works, our study provides a possible concept of small-large cholangiocyte transdifferentiation of human PSCs under cost-effective 3D culture conditions. The study findings have implications for the development of effective cell-based therapy using COs for patients with cholangiopathies.
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Background/Objectives: Polycystic ovary syndrome (PCOS) and irritable bowel syndrome (IBS) share similar clinical and psychosocial features. We aimed to investigate the clinical characteristics of IBS in women with PCOS, and its relationship with obesity, metabolic and hormonal profiles, as well as sleep and psychiatric disorders. Subjects/Methods: This is a cross-sectional case-control study of 431 untreated women with PCOS and 259 healthy volunteers. All participants were assessed with a comprehensive clinical evaluation and two questionnaires: the Athens Insomnia Scale (AIS) and the Brief Symptom Rating Scale (BSRS-5). IBS was diagnosed using the Rome III criteria. Obesity was defined as a BMI ≥30 kg/m2. Anthropometric measurements, metabolic, hormonal profiles, and psychosocial morbidities were compared. Results: Women with PCOS were more likely to have IBS (10.7% vs 5.8%, p=0.029) and obesity (29% vs 4%, p<0.001) than healthy volunteers. Mixed-type IBS (IBS-M) was the most common subtype (74%) among patients with PCOS and IBS. There was a higher prevalence of psychiatric morbidities (total BSRS-5 score ≥10) in women with PCOS than in healthy women (11.4% vs 3.5%, p<0.001). Women with PCOS and IBS were more likely to have sleep difficulties (67.4% vs 30.9%, p<0.001) and psychiatric morbidities (21.7% vs 10.1%, p=0.019) than those without IBS. Anthropometrics, metabolic and hormonal profiles were similar between PCOS women with and without IBS. Among women with PCOS, those with both IBS and obesity had the highest risk of developing sleep difficulties (odds ratio: 5.91; 95% confidence interval: 1.77-19.77) and psychiatric distress (odds ratio: 4.39; 95% confidence interval: 1.26-15.29) than those without. Conclusion: Women with PCOS have increased IBS, obesity, sleep and psychiatric disturbances. The presence of IBS in PCOS women is associated with sleep and psychiatric disorders. The coexistence of obesity and IBS exacerbates sleep difficulties and psychiatric distress. Screening and management of IBS and obesity might be warranted to improve sleep and psychiatric disturbances in women with PCOS.
Assuntos
Síndrome do Intestino Irritável/complicações , Transtornos Mentais/patologia , Obesidade/complicações , Síndrome do Ovário Policístico/complicações , Transtornos do Sono-Vigília/patologia , Adulto , Estudos de Casos e Controles , Estudos Transversais , Progressão da Doença , Feminino , Humanos , Síndrome do Intestino Irritável/epidemiologia , Síndrome do Intestino Irritável/fisiopatologia , Transtornos Mentais/epidemiologia , Transtornos Mentais/etiologia , Pessoa de Meia-Idade , Obesidade/epidemiologia , Obesidade/fisiopatologia , Síndrome do Ovário Policístico/epidemiologia , Síndrome do Ovário Policístico/patologia , Sono/fisiologia , Transtornos do Sono-Vigília/epidemiologia , Transtornos do Sono-Vigília/etiologia , Taiwan/epidemiologia , Adulto JovemRESUMO
CD28 is required for T cell activation as well as the generation of CD4+Foxp3+ Treg. It is unclear, however, how CD28 costimulation affects the development of CD8+ T cell suppressive function. Here, by use of Hepa1.6.gp33 in vitro killing assay and B16.gp33 tumor mouse model we demonstrate that CD28 engagement during TCR ligation prevents CD8+ T cells from becoming suppressive. Interestingly, our results showed that ectonucleotidase CD73 expression on CD8+ T cells is upregulated in the absence of CD28 costimulation. In both murine and human tumor-bearing hosts, CD73 is upregulated on CD28-CD8+ T cells that infiltrate the solid tumor. UPLC-MS/MS analysis revealed that CD8+ T cells activation without CD28 costimulation produces elevated levels of adenosine and that CD73 mediates its production. Adenosine receptor antagonists block CD73-mediated suppression. Our data support the notion that CD28 costimulation inhibits CD73 upregulation and thereby prevents CD8+ T cells from becoming suppressive. This study uncovers a previously unidentified role for CD28 costimulation in CD8+ T cell activation and suggests that the CD28 costimulatory pathway can be a potential target for cancer immunotherapy.
Assuntos
5'-Nucleotidase/metabolismo , Antígenos CD28/metabolismo , Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária/imunologia , Melanoma Experimental/imunologia , 5'-Nucleotidase/genética , Animais , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
OBJECTIVE: Obesity has been reported to have a modulatory effect on the ovulatory functions of patients with polycystic ovary syndrome. The role of adipokines in this obesity-associated ovulatory disturbance has not been extensively explored. In this study, the relationships between obesity, adipokine production from visceral fat, and ovarian folliculogenesis were explored in a mice model of induced obesity. METHODS: Obesity was induced in female C57BL/6 mice fed ad libitum with high-fat feed and fructose water for 4 weeks. Follicular developments in the ovaries were assessed by histopathology in these diet-induced obese mice. Changes in adipokine expression in the peri-ovarian adipose tissues were screened with an adipokine array. The adipokine with the most significant increase over time was identified. The functions of the adipokine in angiogenic processes were evaluated in a cell model of endothelial proliferation. The in vivo effects of neutralizing this adipokine using specific antibodies were assessed in the same obesity model. RESULTS: A high-fat and fructose diet induced an accumulation of early ovarian follicles and a reduction in mature follicles and corpus lutea. The number of microvessels in the early follicles also decreased. The adipokine protein array of the peri-ovarian adipose tissues identified a progressive increase in IL-10 expression with the duration of the obesogenic diet. In vitro experiments in the endothelial cell model confirmed IL-10 as a disrupter of VEGF-induced angiogenesis. Administration of anti-IL-10 antibodies prevented the histopathological changes induced by the obesogenic diet and further highlighted the role of IL-10 in disrupting folliculogenesis. CONCLUSIONS: Obesity may disrupt normal folliculogenesis through increased production of IL-10 in visceral fats. This relationship may help clarify the reported association between obesity and ovulatory dysfunction, which has been found in patients with polycystic ovary syndrome. However, the duration of this study was short, which limited conclusions on the long-term reproductive outcomes.
Assuntos
Interleucina-10/metabolismo , Obesidade/metabolismo , Folículo Ovariano/metabolismo , Ovário/metabolismo , Adipocinas/metabolismo , Tecido Adiposo/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Proliferação de Células , Dieta , Feminino , Expressão Gênica , Humanos , Interleucina-10/genética , Gordura Intra-Abdominal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/genética , Ovário/efeitos dos fármacosRESUMO
Cyclophosphamide (CP) could cause severe gonadotoxicity via imbalanced activation of primordial follicles through PI3K/AKT/mTOR activation. Whether metformin, a widely prescribed anti-diabetes agent with mTOR inhibitory effect, could preserve ovarian function against CP toxicity is unknown. Female C57BL/6 mice were randomized into seven groups (n = 11), including control, CP-alone, CP + metformin, CP + sirolimus or everolimus, metformin-alone and sirolimus-alone groups. The duration of pharmaceutical treatment was 4 weeks. CP treatment significantly impaired ovarian function and fertility in mice. CP + metformin treatment significantly attenuated the gonadotoxicity comparing to CP-alone treatment (primordial follicle count: 17.6 ± 4.2 versus 10.3 ± 2.7 follicles/high-power field; P = 0.027). CP + metformin treatment also tended to increase antral follicular count (5.4 ± 1.1 versus 2.5 ± 1.6 follicles/section), serum AMH levels (4.6 ± 1.2 versus 2.0 ± 0.8 ng/ml) and the litter size (4.2 ± 1.3 versus 1.5 ± 1.0 mice per pregnancy), compared with CP-alone group. Expression of phospho-mTOR and the number of TUNEL-positive granulosa cells increased after CP treatment and decreased in the CP + metformin groups, suggesting the mTOR inhibitory and anti-apoptotic effects of metformin. In in-vitro granulosa cell experiments, the anti-apoptotic effect of metformin was blocked after inhibiting p53 or p21 function, and the expression of p53 mRNA was blocked with AMPK inhibitor, suggesting that the anti-apoptotic effect was AMPK/p53/p21-mediated. In conclusion, concurrent metformin treatment during CP therapy could significantly preserve ovarian function and fertility and could be a promising novel fertility preserving agent during chemotherapy. The relatively acceptable cost and well-established long-term safety profiles of this old drug might prompt its further clinical application at a faster pace.
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Antineoplásicos Alquilantes/efeitos adversos , Ciclofosfamida/antagonistas & inibidores , Fertilidade/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Ciclofosfamida/efeitos adversos , Everolimo/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Folículo Ovariano/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Patients with polycystic ovarian syndrome (PCOS) are associated with known alterations in mitochondria DNA copy number (mtDNA-CN). The aim of this study is to study the change in mtDNA-CN in patients with PCOS who were treated with metformin. METHODS: This is a prospective cohort of patients with PCOS, who received metformin for one year. From 2009 to 2015, 88 women diagnosed with PCOS, based on the Rotterdam criteria, were enrolled. Serial measurements of mtDNA-CN, 8-hydroxydeoxyguanosine (8-OHdG), anthropometric, metabolic, endocrine, and inflammatory markers were obtained before and after 3, 6, and 12 months of treatment. RESULTS: A significant decrease in mtDNA-CN was seen over the course of one year. Other markers, including 8-OHdG, testosterone, free androgen index, blood pressure and liver enzymes, also decreased in the same interval. On regression analysis, there was a significant association between the change in mtDNA-CN and serum total testosterone, and no association between mtDNA-CN and metabolic factors. CONCLUSIONS: Treatment with metformin is associated with a time-dependent decrease in mtDNA-CN in patients with PCOS who are treated over the course of one year. This may signify a reduction in mitochondria dysfunction. The change in mtDNA-CN corresponds to a similar change in serum total testosterone, and suggests a possible relationship between mtDNA-CN and testosterone. TRIAL REGISTRATION: ClinicalTrials.gov , NCT00172523 . Registered September 15, 2005.
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Variações do Número de Cópias de DNA , DNA Mitocondrial/genética , Metformina/uso terapêutico , Síndrome do Ovário Policístico/tratamento farmacológico , Adolescente , Adulto , Índice de Massa Corporal , Variações do Número de Cópias de DNA/efeitos dos fármacos , DNA Mitocondrial/análise , DNA Mitocondrial/efeitos dos fármacos , Feminino , Humanos , Hipoglicemiantes/uso terapêutico , Resistência à Insulina/fisiologia , Estudos Longitudinais , Síndrome do Ovário Policístico/genética , Síndrome do Ovário Policístico/patologia , Adulto JovemRESUMO
OBJECTIVE: To study whether and how the pathogenesis of polycystic ovarian syndrome (PCOS) is related to epigenetic aberrations. DESIGN: A case-control experimental study. SETTING: Tertiary university hospital. PATIENT(S): Eighteen patients with PCOS and ten non-PCOS control subjects. INTERVENTIONS(S): Patient-specific induced pluripotent stem cells (iPSCs) were obtained from skin fibroblasts through the application of nonviral episomal reprogramming and were differentiated into ovarian granulosa cells (GCs) with the use of a cocktail of growth factors. Primary ovarian GCs were collected during transvaginal oocyte retrieval surgery. MAIN OUTCOME MEASURE(S): Characterization and functional validation of iPSC-derived GCs were conducted. Whole-genomic DNA methylation profiles in women with and without PCOS in both iPSC-derived GCs and primary adult GCs were analyzed with the use of the Illumina 850K MethylationEPIC Beadchip. RESULT(S): The iPSC-derived GCs successfully expressed GC-associated genes and aromatase activity after differentiation. Whole-genomic DNA methylation analysis of the iPSC-derived GCs and adult GCs both revealed a hyperactive CREB signaling pathway in the PCOS group compared with the control group. The expression of CREB-binding protein (CBP) mRNA was significantly higher in the iPSC-derived GCs in the PCOS group, and the expression of CBP protein was also significantly higher in the primary GCs from women with PCOS. CONCLUSION(S): The combination of DNA methylomic analysis in primary adult GCs and iPSC-derived GCs showed that a preserved persistent hyperactivation of the CREB signaling pathway might be involved in the pathogenesis of PCOS. These results could have implications on the early developmental origin, inheritance nature, and environmental interaction effects of this disease.
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Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome do Ovário Policístico/genética , Síndrome do Ovário Policístico/metabolismo , Transdução de Sinais/fisiologia , Animais , Estudos de Casos e Controles , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise em Microsséries/métodos , Recuperação de Oócitos/métodos , Síndrome do Ovário Policístico/patologiaRESUMO
BACKGROUND/PURPOSE: Genetic variant of HSD3B1 1245 is known to augment androgen production at peripheral tissue as skin. This study aimed to investigate whether women with polycystic ovary syndrome inheriting this variant exhibit specific androgenic phenotypes. METHODS: A cross-sectional study of Taiwanese women with polycystic ovary syndrome, defined by Rotterdam criteria, at the reproductive endocrinology outpatient clinic in a university affiliated hospital. RESULTS: The presence of female pattern hair loss in women with polycystic ovary syndrome was significantly associated with an increased body mass index, decreased sex hormone binding globulin and high density lipoprotein cholesterol levels, elevated triglyceride levels, and increased prevalence of hypertension. Using stepwise multivariate logistic regression analysis, body mass index, triglyceride and HSD3B1 1245 AC or CC genotype were significantly related to female pattern hair loss in women with polycystic ovary syndrome after considering other variables. Overweight women with polycystic ovary syndrome had significantly higher risk of female pattern hair loss than normal-weight women with polycystic ovary syndrome. The presence of female pattern hair loss was higher in overweight women with polycystic ovary syndrome who comprised HSD3B1 AC or CC genotype compared with wild type. CONCLUSION: Carrying the HSD3B1 1245C allele and overweight are associated with the presence of female pattern hair loss in women with polycystic ovary syndrome.
Assuntos
Alopecia/genética , Complexos Multienzimáticos/genética , Sobrepeso/complicações , Síndrome do Ovário Policístico/genética , Progesterona Redutase/genética , Esteroide Isomerases/genética , Adulto , Índice de Massa Corporal , Estudos Transversais , Feminino , Humanos , Resistência à Insulina , Polimorfismo Genético , Taiwan , Adulto JovemRESUMO
BACKGROUND/PURPOSE: Abnormal folliculogenesis is one of the cardinal presentations of polycystic ovarian syndrome (PCOS) and permeability of follicular wall has been proposed to be involved in the normal follicular growth. However, whether or not there is a change in intrafollicular permeability underlies PCOS is unknown. METHODS: This was a tertiary center-based case-control study. From 2014 to 2015, thirteen patients with PCOS who underwent in vitro fertilization-embryo transfer (IVF-ET) were enrolled. Eleven normo-ovulatory patients who underwent IVF-ET due to male factor and/or tubal factor infertility were enrolled as the control group. The influence of ovarian follicular fluid (FF) on endothelial cell permeability was evaluated using a human umbilical vein endothelial cell monolayer permeability assay. The intrafollicular expression profiles of angiogenesis-related proteins were analyzed using a Human Angiogenesis Protein Array Kit. RESULTS: The FF from PCOS patients caused significantly poorer endothelial cell permeability comparing with the effect of FF from the control group (46% ± 12% vs. 58% ± 9%, P = 0.023). Among the 55 angiogenesis-related proteins tested, there was a significantly higher level of intrafollicular platelet factor 4 (PF4) and PF4/IL-8 complex in the PCOS group (p = 0.004). The anti-permeability effect of PF4 was related to the decrease in the intercellular gaps and antagonistic binding with IL-8. CONCLUSION: Our study provides the first evidence of the pathophysiologic contribution of the well-known angiostatic protein, PF4, on human reproductive biology. The increase of the intrafollicular PF4 and its anti-permeability effect might affect the formation of FF and folliculogenesis in PCOS.
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Líquido Folicular/química , Infertilidade Feminina/patologia , Fator Plaquetário 4/química , Síndrome do Ovário Policístico/patologia , Adulto , Estudos de Casos e Controles , Feminino , Fertilização in vitro , Humanos , Permeabilidade , Taiwan , Centros de Atenção TerciáriaRESUMO
BACKGROUND/PURPOSE: 45,X/46,XY mosaicism is a rare sex chromosome abnormality. Here, we present our experience in the management of 45,X/46,XY Taiwanese children. PATIENTS AND METHODS: We enrolled 19 patients from January 1981 to September 2016. The diagnosis of 45,X/46,XY mosaicism was made by karyotyping peripheral blood lymphocytes. All medical records were thoroughly reviewed. RESULTS: Of the 19 patients, 16 were reared as females and 3 as males. The age at diagnosis ranged from 1 month to 15 years and 9 months. Atypical genitalia, short stature, and Turner stigmata were common manifestations. No patient exhibited a cardiac malformation but 29% had renal malformations and 12.5% had autoimmune thyroid disease who developed thyroid dysfunction later. Nine girls with short stature received growth hormone therapy and their height standard deviation score rose from -3.4 ± 1.1 to -1.4 ± 0.9 in adulthood (P < 0.01). The gonadal phenotypes included bilateral streak gonads in nine patients, a streak gonad with contralateral gonadal agenesis in one, mixed gonadal dysgenesis in five, bilateral dysgenetic testes in two, and bilateral gonadoblastomas in one. CONCLUSION: The 45,X/46,XY phenotype varies widely and a high index of suspicion is important to ensure early diagnosis. Cardiac and renal malformations should be screened ultrasonically at diagnosis and thyroid status should be monitored annually. Growth hormone effectively improves adult height in short girls. Prophylactic gonadectomy is indicated for those with intra-abdominal streaks or dysgenetic gonads to prevent the development of a malignancy.
Assuntos
Transtornos do Desenvolvimento Sexual/tratamento farmacológico , Transtornos do Desenvolvimento Sexual/genética , Hormônio do Crescimento/uso terapêutico , Mosaicismo , Adolescente , Estatura/genética , Criança , Pré-Escolar , Feminino , Disgenesia Gonadal Mista/genética , Gonadoblastoma/genética , Humanos , Lactente , Cariotipagem , Masculino , Taiwan , Síndrome de Turner/genéticaRESUMO
Context: The long-term effects of metformin in women with polycystic ovarian syndrome (PCOS) are inadequately studied. Objective: The effects of metformin on women with PCOS during 24 months with respect to menses, hormones, and metabolic profiles are assessed. Design: Prospective cohort. Setting: A reproductive endocrinology clinic in a university-affiliated medical center. Patients: One hundred nineteen women with PCOS, defined by the Rotterdam criteria, were enrolled. Intervention: Metformin was given daily for 24 months. Main Outcome Measures: The primary outcome was the proportion of patients with regular menstruation during treatment. Changes in anthropometric, hormonal, and metabolic parameters were also assessed. Analyses were performed using segmented regression analysis with a generalized estimating equation methodology. Outcomes are expressed as magnitude of change from the baseline. Results: Both overweight (OW) and normal-weight (NW) women with PCOS had increased menstrual frequency and decreased body mass index (BMI), testosterone, and luteinizing hormone levels in the first 6 months. Further stratification showed that NW women exhibiting elevated testosterone at baseline had the largest magnitude of improvement at 6 months [odds ratio (OR), 7.21; 95% confidence interval (CI), 2.35 to 22.17], whereas OW patients with normal testosterone were most likely to achieve normal menses at 12 months (OR, 0.63; 95% CI, 0.47 to 0.77). Conclusions: Metformin was associated with improvements in the menstrual cycle and most hormonal profiles in OW and NW women with PCOS during 24 months of treatment. Most parameters reached maximal response and steady-state after 6 months. Phenotypic differences in baseline BMI and testosterone level can be used as patient selection criteria or treatment prognostics.
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Hipoglicemiantes/uso terapêutico , Menstruação/efeitos dos fármacos , Metformina/uso terapêutico , Síndrome do Ovário Policístico/tratamento farmacológico , Adulto , Antropometria/métodos , Índice de Massa Corporal , Esquema de Medicação , Feminino , Humanos , Hipoglicemiantes/administração & dosagem , Estudos Longitudinais , Hormônio Luteinizante/sangue , Metformina/administração & dosagem , Síndrome do Ovário Policístico/sangue , Síndrome do Ovário Policístico/fisiopatologia , Estudos Prospectivos , Testosterona/sangue , Adulto JovemRESUMO
BACKGROUND/PURPOSE: To investigate the M1/M2 polarity of macrophages in the endometrium among different menstrual cycles, normal and abnormal pregnancies, and unexplained recurrent spontaneous abortions (RSAs). METHODS: Endometrial tissue was obtained from 43 patients undergoing hysterectomy, either in the follicular phase (Group 1, n = 23) or in the luteal phase (Group 2, n = 20). In addition, decidual tissue was obtained from 53 pregnant women during the first trimester, either of normal pregnancies (Group 3, n = 12) or abnormal pregnancies (Group 4: spontaneous abortions, n = 20; Group 5: unexplained RSA, n = 21). Using immunofluorescence to examine the M1 and M2 macrophages in the endometrium and deciduae from cases with different menstrual phases and various pregnancy outcomes, respectively, we endeavored to learn the possible pathophysiology of abortions. RESULTS: M1 macrophages were abundant in the deciduae of spontaneous abortions and unexplained RSA, whereas the frequency of M2 macrophages was significantly higher in the endometrium of luteal phase and normal pregnancies. CONCLUSION: M2 polarization is important for early successful pregnancies in humans.
Assuntos
Aborto Habitual/imunologia , Aborto Espontâneo/imunologia , Decídua/imunologia , Macrófagos/fisiologia , Adulto , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Antígeno CD11c/análise , Polaridade Celular , Feminino , Humanos , Pessoa de Meia-Idade , GravidezRESUMO
Iron is an essential nutrient that may exert toxic effects when it accumulates in tissues. Little is known regarding its effects on gonadal function. Both Fe2+ and Fe3+ could be released from iron deposition. We employed mouse nonluteinized granulosa cell for in vitro studies and human ovarian tissues for Prussian blue and immunohistochemical staining to identify the iron deposition and effect in vivo. After treatment with FeSO4-7H2O or FeCl3 in granulosa cell cultured with follicle-stimulating hormone (FSH) for 48 h, we found that Fe2+ significantly suppressed FSH-induced granulosa cell proliferation and arrested the cell cycle at the G2/M phase by cell proliferation assay and flow cytometry. Fe2+ significantly increased intracellular reactive oxygen species (ROS) and ferritin levels of mouse granulosa cells. The increases in p21 and p53 messenger RNA and protein expression facilitated by Fe2+ treatment in mouse granulosa cells were significantly suppressed by separate treatments with p53 small interfering RNA and p38 mitogen-activated protein kinase (MAPK) inhibitors. An ROS inhibitor downregulated Fe2+-induced increases in p38MAPK expression in mouse granulosa cells. Quantitative analysis of immunohistochemical staining revealed that human ovarian tissue sections with positive Prussian blue staining had lower levels of proliferating cell nuclear antigen expression, but higher levels of p21, p53, and CDC25C expression than those with negative Prussian blue staining. Conclusively, Fe2+ could directly arrest the cell cycle and inhibit granulosa cell proliferation by regulating the ROS-mediated p38MAPK/p53/p21 pathway. Therefore, iron can directly affect female gonadal function.
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Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Ferro/farmacologia , Ovário/citologia , Proteína Supressora de Tumor p53/genética , Quinases Ativadas por p21/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Animais , Apoptose , Proliferação de Células/efeitos dos fármacos , Feminino , Ferritinas/metabolismo , Hormônio Foliculoestimulante/sangue , Camundongos , Camundongos Endogâmicos ICR , Ovário/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Espécies Reativas de Nitrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Telomerase is highly expressed in cancer and embryonic stem cells (ESCs) and implicated in controlling genome integrity, cancer formation and stemness. Previous studies identified that Krüppel-like transcription factor 4 (KLF4) activates telomerase reverse transcriptase (TERT) expression and contributes to the maintenance of self-renewal in ESCs. However, little is known about how KLF4 regulates TERT expression. Here, we discover poly(ADP-ribose) polymerase 1 (PARP1) as a novel KLF4-interacting partner. Knockdown of PARP1 reduces TERT expression and telomerase activity not only in cancer cells, but also in human and mouse ESCs. Recruitment of KLF4 to TERT promoter is reduced in PARP1-suppressed cells. The poly(ADP-ribose) polymerase activity is dispensable, while the oligo(ADP-ribose) polymerase activity is required for the PARP1- and KLF4-mediated TERT activation. Repression of Parp1 in mouse ESCs decreases expression of pluripotent markers and induces differentiation. These results suggest that PARP1 recruits KLF4 to activate telomerase expression and stem cell pluripotency, indicating a positive regulatory role of the PARP1-KLF4 complex in telomerase expression in cancer and stem cells.
Assuntos
Células-Tronco Embrionárias/metabolismo , Fatores de Transcrição Kruppel-Like/fisiologia , Neoplasias/genética , Poli(ADP-Ribose) Polimerase-1/fisiologia , Telomerase/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Embrião de Mamíferos , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Neoplasias/patologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Telomerase/metabolismoRESUMO
Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation. With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well.
Assuntos
Rastreamento de Células/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Microscopia de Fluorescência/métodos , Nanodiamantes/química , Células A549 , Animais , Materiais Biocompatíveis , Células HeLa , Humanos , Pulmão/citologia , Nanodiamantes/administração & dosagem , Albumina Sérica Humana/administração & dosagem , Albumina Sérica Humana/química , Razão Sinal-Ruído , Suínos , Porco Miniatura , Distribuição TecidualRESUMO
Women with polycystic ovary syndrome (PCOS) have a higher prevalence of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) than the general population. The link between NAFLD/NASH and PCOS is not just a coincidence. Indeed, both of these disorders comprise common risk factors, including central obesity, insulin resistance, chronic low-grade inflammation, and hyperandrogenemia. The characteristics of hyperandrogenemia in women with PCOS include elevated total and free testosterone levels and low sex hormone-binding globulin levels and are reported to be associated with NAFLD and elevated liver enzymes; however, not all elevated androgen levels in women with PCOS have the same adverse effects on the liver. With the exception of weight loss and encouraging exercise in obese women, few evidence-based effective treatments target NAFLD/NASH in women with PCOS. Selective antiandrogens and insulin sensitizers might be beneficial in treating NAFLD/NASH in women with PCOS, but further elucidation is needed.
Assuntos
Dislipidemias/metabolismo , Hiperandrogenismo/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/metabolismo , Síndrome do Ovário Policístico/metabolismo , Alanina Transaminase/metabolismo , Antagonistas de Androgênios/uso terapêutico , Aspartato Aminotransferases/metabolismo , Anticoncepcionais Orais Hormonais/uso terapêutico , Sulfato de Desidroepiandrosterona/metabolismo , Dislipidemias/complicações , Dislipidemias/tratamento farmacológico , Terapia por Exercício , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hiperandrogenismo/complicações , Hiperandrogenismo/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Inflamação , Resistência à Insulina , Metformina/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Obesidade/complicações , Sobrepeso/complicações , Sobrepeso/metabolismo , Síndrome do Ovário Policístico/complicações , Síndrome do Ovário Policístico/tratamento farmacológico , Globulina de Ligação a Hormônio Sexual/metabolismo , Testosterona/metabolismo , Redução de PesoRESUMO
OBJECTIVE: Obesity and insulin resistance are associated with increased iron stores, but have conflicting effects on ovarian reserve in women with polycystic ovary syndrome (PCOS). Iron-catalyzed oxidative stress might be detrimental to ovarian tissue and granulosa cell function. In this study we determined the association between body iron stores, obesity, and ovarian reserve in women with PCOS. MATERIALS AND METHODS: One hundred and fifty-six women diagnosed with PCOS according to Rotterdam criteria and 30 normoweight healthy control women were enrolled in this cross-sectional study. Ovarian volume, total antral follicle count, and the anti-Müllerian hormone (AMH) level were measured as an indicator of ovarian reserve. RESULTS: Ferritin and transferrin-bound iron levels were significantly higher in women with PCOS than normoweight controls. Obese women with PCOS had higher ferritin levels (p = 0.006), but lower AMH levels (p < 0.0001) than nonobese women with PCOS. Using univariate analysis, the AMH level and mean ovarian volume were inversely related to the ferritin level, homeostasis model assessment of insulin resistance, and body mass index in women with PCOS. Body mass index and ferritin level remained significantly correlated with a lower AMH level and reduced ovarian volume, respectively, after considering other confounding variables. CONCLUSION: An elevated ferritin level and obesity were negatively associated with ovarian volume and the AMH level, respectively, in women with PCOS.
Assuntos
Hormônio Antimülleriano/sangue , Ferritinas/sangue , Obesidade/complicações , Folículo Ovariano , Síndrome do Ovário Policístico/sangue , Síndrome do Ovário Policístico/complicações , Adulto , Índice de Massa Corporal , Estudos de Casos e Controles , Estudos Transversais , Feminino , Humanos , Adulto JovemRESUMO
Intraovarian hyperandrogenism is one of the determining factors of follicular arrest in women with polycystic ovary syndrome (PCOS). Using androgenized rat models, we investigated the effects of androgens on metabolism, as well as on factors involved in follicular arrest and the reduced number of estrus cycles. The dihydrotestosterone (DHT)-treated rats had fewer estrus cycles, higher numbers of large arrested follicles and an increased in body weight gain compared with the dehydroepiandrostenedione (DHEA)- and placebo-treated rats. In cultured rat granulosa cells, DHT suppressed follicle stimulating hormone (FSH)-induced granulosa cell proliferation and increased the accumulation of cells in the G2/M phase. DHT decreased phosphorylated Akt (p-Akt) and cyclin D1 levels through increasing PTEN. DHT-promoted PTEN expression was regulated by peroxisome proliferator-activated receptor gamma (PPARγ) in granulosa cells. Meanwhile, in the large follicles of the DHT-treated rats, the expressions of PPARγ and PTEN were higher, but the expression of p-Akt and proliferating cell nuclear antigen (PCNA) were lower. Conclusively, DHT and DHEA produced differential effects on metabolism in prepubertal female rats like clinical manifestations of women with PCOS. DHT treatment may affect ovarian follicular maturation by altering granulosa cell proliferation through the regulation of enhancing PPARγ dependent PTEN/p-Akt expression in the granulosa cells.
Assuntos
Proliferação de Células/efeitos dos fármacos , Di-Hidrotestosterona/farmacologia , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/efeitos dos fármacos , PPAR gama/metabolismo , Androgênios/farmacologia , Animais , Western Blotting , Peso Corporal/efeitos dos fármacos , Células Cultivadas , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Células da Granulosa/metabolismo , PPAR gama/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/efeitos dos fármacosRESUMO
Artificial bone grafting is widely used in current orthopedic surgery for bone defect problems. Unfortunately, surgeons remain unsatisfied with the current commercially available products. One of the major complaints is that these products cannot provide sufficient mechanical strength to support the human skeletal structure. In this study, we aimed to develop a bone scaffold with better mechanical property and good cell affinity by 3D printing (3DP) techniques. A self-developed 3D printer with laser-aided gelling (LAG) process was used to fabricate bioceramic scaffolds with inter-porous structures. To improve the mechanical property of the bioceramic parts after heating, CaCO3 was added to the silica ceramic slurry. CaCO3 was blended into a homogenous SiO2-sol dispersion at weight ratios varying from 0/100 to 5/95 to 9/91 (w/w). Bi-component CaCO3/SiO2-sol was prepared as a biocomposite for the 3DP scaffold. The well-mixed biocomposite was used to fabricate the bioceramic green part using the LAG method. The varied scaffolds were sintered at different temperatures ranging from 900 to 1500°C, and the mechanical property was subsequently analyzed. The scaffolds showed good property with the composite ratio of 5:95 CaCO3:SiO2 at a sintering temperature of 1300°C. The compressive strength was 47 MPa, and the porosity was 34%. The topography of the sintered 3DP bioceramic scaffold was examined by SEM, EDS and XRD. The silica bioceramic presented no cytotoxicity and good MG-63 osteoblast-like cell affinity, demonstrating good biocompatibility. Therefore, the new silica biocomposite is viable for fabricating 3DP bone bioceramics with improved mechanical property and good cell affinity.
Assuntos
Cerâmica/química , Porosidade , Impressão Tridimensional , Alicerces Teciduais/química , Animais , Regeneração Óssea , Carbonato de Cálcio/química , Adesão Celular , Linhagem Celular Tumoral , Humanos , Camundongos , Dióxido de Silício/química , Engenharia Tecidual/métodosRESUMO
Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age. Although its aetiology and pathogenesis remain unclear, recent studies suggest that the dysfunction of granulosa cells may partly be responsible. This study aimed to use cDNA microarray technology to compare granulosa cell gene expression profiles in women with and without PCOS to identify genes that may be aetiologically implicated in the pathogenesis of PCOS. The study cohort included 12 women undergoing in vitro fertilization, six with PCOS and six without PCOS. Differential gene expression profiles were classified by post-analyses of microarray data, followed by western blot analyses to confirm the microarray data of selected genes. In total, 243 genes were differentially expressed (125 upregulated and 118 downregulated) between the PCOS and non-PCOS granulosa cells. These genes are involved in reproductive system development, amino acid metabolism and cellular development and proliferation. Comparative analysis revealed genes involved in the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) signaling pathways. Western blot analyses confirmed that mitogen-activated protein kinase kinase kinase 4 and phospho-ERK1/2 were decreased in PCOS granulosa cells. This study identified candidate genes involved in MAPK/ERK signaling pathways that may influence the function of granulosa cells in PCOS.