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1.
Placenta ; 39: 7-15, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26992668

RESUMO

INTRODUCTION: Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a reliable tool to analyse gene expression profiles. The expression of housekeeping genes generally serves as a reference for mRNA amount, assuming that it remains stable under pathophysiological and experimental conditions. To date, an empirical validation of reference genes suitable for RT-qPCR-based studies in the mouse placenta is missing. METHODS: We used NormFinder and BestKeeper statistical software to analyse the expression stability of candidate housekeeping genes quantified by RT-qPCR in mouse placentas. RESULTS: Fifteen of 32 potential candidate housekeeping genes analysed on gestation day (gd) 16.5 in mouse placentas exhibited an optimal cycle threshold (Ct). Among them B2m, Polr2a, Ubc, and Ywhaz genes showed the highest expression stability in placentas from control, but also experimentally-challenged mice. These genes as well as the currently widely used housekeeping genes Hprt1, Actb, and Gapdh were selected for further quality assessments. We quantified the Ct values of these selected genes in placental samples obtained from wild-type or genetically engineered dams at different gds, or upon selected experimental interventions known to affect placental phenotype. Among all housekeeping genes analysed, Polr2a was the most stably expressed and its expression stability excelled in combination with Ubc. DISCUSSION: Polr2a, especially in combination with Ubc, can be proposed as highly suitable endogenous reference for gene expression analysis in mouse-derived placental tissue. Moreover, the validation of both genes as a stable reference gene in human placenta-derived tissue strengthens the translational relevance of RT-qPCR findings using mouse placenta.


Assuntos
Perfilação da Expressão Gênica/normas , Genes Essenciais , Placenta/metabolismo , Animais , Antígenos de Neoplasias/genética , Feminino , Perfilação da Expressão Gênica/métodos , Marcadores Genéticos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Placenta/química , Gravidez , RNA Polimerase II/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência
2.
J Clin Invest ; 125(4): 1726-38, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25774501

RESUMO

Intrauterine growth restriction (IUGR) affects up to 10% of pregnancies in Western societies. IUGR is a strong predictor of reduced short-term neonatal survival and impairs long-term health in children. Placental insufficiency is often associated with IUGR; however, the molecular mechanisms involved in the pathogenesis of placental insufficiency and IUGR are largely unknown. Here, we developed a mouse model of fetal-growth restriction and placental insufficiency that is induced by a midgestational stress challenge. Compared with control animals, pregnant dams subjected to gestational stress exhibited reduced progesterone levels and placental heme oxygenase 1 (Hmox1) expression and increased methylation at distinct regions of the placental Hmox1 promoter. These stress-triggered changes were accompanied by an altered CD8+ T cell response, as evidenced by a reduction of tolerogenic CD8+CD122+ T cells and an increase of cytotoxic CD8+ T cells. Using progesterone receptor- or Hmox1-deficient mice, we identified progesterone as an upstream modulator of placental Hmox1 expression. Supplementation of progesterone or depletion of CD8+ T cells revealed that progesterone suppresses CD8+ T cell cytotoxicity, whereas the generation of CD8+CD122+ T cells is supported by Hmox1 and ameliorates fetal-growth restriction in Hmox1 deficiency. These observations in mice could promote the identification of pregnancies at risk for IUGR and the generation of clinical interventional strategies.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Desenvolvimento Fetal/fisiologia , Retardo do Crescimento Fetal/prevenção & controle , Heme Oxigenase-1/fisiologia , Proteínas de Membrana/fisiologia , Placenta/imunologia , Insuficiência Placentária/imunologia , Complicações na Gravidez/imunologia , Progesterona/fisiologia , Estresse Psicológico/imunologia , Animais , Metilação de DNA , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Retardo do Crescimento Fetal/imunologia , Feto/imunologia , Feto/patologia , Heme Oxigenase-1/biossíntese , Heme Oxigenase-1/genética , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Ruído/efeitos adversos , Placenta/metabolismo , Circulação Placentária , Insuficiência Placentária/etiologia , Gravidez , Complicações na Gravidez/genética , Complicações na Gravidez/psicologia , Progesterona/biossíntese , Progesterona/uso terapêutico , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Estresse Psicológico/genética
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