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Objective@#Endometrial fibrosis, the primary pathological feature of intrauterine adhesion, may lead to disruption of endometrial tissue structure, menstrual abnormalities, infertility, and recurrent pregnancy loss. At present, no ideal therapeutic strategy exists for this fibrotic disease. Eupatilin, a major pharmacologically active flavone from Artemisia, has been previously reported to act as a potent inducer of dedifferentiation of fibrotic tissue in the liver and lung. However, the effects of eupatilin on endometrial fibrosis have not yet been investigated. In this study, we present the first report on the impact of eupatilin treatment on transforming growth factor beta (TGF-β)-induced endometrial fibrosis. @*Methods@#The efficacy of eupatilin on TGF-β–induced endometrial fibrosis was assessed by examining changes in morphology and the expression levels of fibrosis markers using immunofluorescence staining and quantitative real-time reverse-transcription polymerase chain reaction. @*Results@#Eupatilin treatment significantly reduced the fibrotic activity of TGF-β–induced endometrial fibrosis in Ishikawa cells, which displayed more circular shapes and formed more colonies. Additionally, the effects of eupatilin on fibrotic markers including alpha-smooth muscle actin, hypoxia-inducible factor 1 alpha, collagen type I alpha 1 chain, and matrix metalloproteinase-2, were evaluated in TGF-β–induced endometrial fibrosis. The expression of these markers was highly upregulated by TGF-β pretreatment and recovered to the levels of control cells in response to eupatilin treatment. @*Conclusion@#Our findings suggest that suppression of TGF-β–induced signaling by eupatilin might be an effective therapeutic strategy for the treatment of endometrial fibrosis.
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Objective@#Despite extensive research on implantation failure, little is known about the molecular mechanisms underlying the crosstalk between the embryo and the maternal endometrium, which is critical for successful pregnancy. Profilin 1 (PFN1), which is expressed both in the embryo and in the endometrial epithelium, acts as a potent regulator of actin polymerization and the cytoskeletal network. In this study, we identified the specific role of endometrial PFN1 during embryo implantation. @*Methods@#Morphological alterations depending on the status of PFN1 expression were assessed in PFN1-depleted or control cells grown on Matrigel-coated cover glass. Day-5 mouse embryos were cocultured with Ishikawa cells. Comparisons of the rates of F-actin formation and embryo attachment were performed by measuring the stability of the attached embryo onto PFN1-depleted or control cells. @*Results@#Depletion of PFN1 in endometrial epithelial cells induced a significant reduction in cell-cell adhesion displaying less formation of colonies and a more circular cell shape. Mouse embryos co-cultured with PFN1-depleted cells failed to form actin cytoskeletal networks, whereas more F-actin formation in the direction of surrounding PFN1-intact endometrial epithelial cells was detected. Furthermore, significantly lower embryo attachment stability was observed in PFN1-depleted cells than in control cells. This may have been due to reduced endometrial receptivity caused by impaired actin cytoskeletal networks associated with PFN1 deficiency. @*Conclusion@#These observations definitively demonstrate an important role of PFN1 in mediating cell-cell adhesion during the initial stage of embryo implantation and suggest a potential therapeutic target or novel biomarker for patients suffering from implantation failure.
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BACKGROUND: Fucoidan is a highly sulfated glycosaminoglycan, which has a molecular structure similar to that of heparin. The antithrombotic effects of fucoidan in vitro have been widely reported, but its antithrombotic effects in vivo as well as its other biological properties in vitro have not been well investigated. METHODS: This study investigated the effects and mechanism of fucoidan from Fucus vesiculosus on thrombosis both in vitro and in vivo. A ferric chloride-induced mouse carotid artery thrombosis model was used to determine the antithrombotic effects of fucoidan in vivo. Additionally, changes in the levels of proinflammatory cytokines and chemokines were examined in vascular cells treated with fucoidan. RESULTS: In vivo studies employing a ferric chloride-induced mouse carotid artery thrombosis model indicated that fucoidan had a stronger antithrombotic activity than heparin. Further, vascular cells treated with fucoidan demonstrated a decrease in proinflammatory cytokine and chemokine production as well as inhibition of proliferation. CONCLUSION: The major findings of this study showed that fucoidan has a stronger antithrombotic effect than heparin in vivo and that fucoidan has an inhibitory effect on proinflammatory cytokine production and proliferation of vascular cells.
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Animais , Camundongos , Trombose das Artérias Carótidas , Quimiocinas , Citocinas , Fucus , Glicosaminoglicanos , Heparina , Estrutura Molecular , Polissacarídeos , TromboseRESUMO
OBJECTIVE: This study was performed to understand the influence of the methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) genotypes on the spontaneously aborted embryos. METHODS: DNA was extracted from tissue samples of 95 spontaneously aborted embryos and 100 samples of normal children randomly and 449 samples of normal adults were selected as the controls. MTHFR genotypes were determined by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. RESULTS: The aborted embryo group had higher frequency of MTHFR 677CC type (p=0.014) and lower 677CT type (p=0.063) than the controlled child group. The frequency of MTHFR 677CT type was drastically lower than that of controlled adult group (p=0.032). In the MTHFR C677T/A1298C combination, 677CC/1298AC genotype of the aborted embryo was significantly higher (p=0.034) than that of controlled child group, but it was not statistically significant in controlled adult group (p=0.063). CONCLUSION: MTHFR 677CC and MTHFR 677CC/1298AC genotypes may represent genetic markers for the risk of spontaneously aborted embryos at least in Koreans.
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Adulto , Criança , Humanos , Feto Abortado , DNA , Marcadores Genéticos , Genótipo , Homocisteína , Metilenotetra-Hidrofolato Redutase (NADPH2)RESUMO
Germ-line mutations of the BRCA1 gene confer an increased risk for breast and ovarian cancers. BRCA1 in female cells is directly related with the maintenance of the inactive X chromosome (Xi). The effect by the loss of the BRCA1 function on the X chromosome gene expression remains unclear in cancer cells. We attempted to investigate the expression pattern of the X-linked genes by performing BRCA1 knockdown via RNA interference in the MCF 7 breast cancer cell line. The transcriptional and translational levels of BRCA1 were decreased over 95% in the MCF 7 cells after BRCA1 knockdown. The expression patterns of one hundred ninety X-linked genes were profiled by the X chromosome-specific cDNA arrays. A total of seven percent of the X-linked genes (14/190) were aberrantly expressed by over 2-fold in the MCF7-BRCA1 knockdown cells, which contained two up-regulated genes (2/190, 1%) and 12 downregulated genes (12/190, 6.3%). It is interesting that 72% of the aberrantly expressed X-linked genes were located on the Xq (10/14,) region. Our data suggests that BRCA1 may not be important to maintain X chromosome inactivation in cancer because the BRCA1 knockdown did increase the expression of the only one percent of X-linked genes in the human breast cancer cells.
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Feminino , Humanos , Mama , Neoplasias da Mama , Linhagem Celular , Expressão Gênica , Genes BRCA1 , Genes Ligados ao Cromossomo X , Mutação em Linhagem Germinativa , Células MCF-7 , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas , Interferência de RNA , Cromossomo X , Inativação do Cromossomo XRESUMO
No abstract available.
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Animais , Camundongos , Epitélio , Expressão Gênica , Microdissecção e Captura a Laser , Análise em Microsséries , FenobarbitalRESUMO
OBJECTIVE: Recently, microdissection of tissue sections has been used increasingly for the isolation of morphologically identified homogeneous cell populations, thus overcoming the obstacle of tissue complexity for the analysis cell-specific expression of macromolecules. The aim of the present study was to establish the minimal conditions required for the RNA extraction and amplification from the cells captured by the laser captured microdissection. METHODS: Mouse ovaries were fixed and cut into serial sections (7 micrometer thickness). Oocytes were captured by laser captured microdissection (LCM) method by using PixCell IITM system. The frozen sections were fixed in 70% ethanol and stained with hematoxylin and eosin, while the paraffin sections were stained with Multiple stain. Sections were dehydrated in graded alcohols followed by xylene and air-dried for 20 min prior to LCM. All reactions were performed in ribonuclease free solutions to prevent RNA degradation. After LCM, total RNA extraction from the captured oocytes was performed using the guanidinium isothiocyanate (GITC) solution, and subsequently evaluated by reverse transcriptase -polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). RESULTS: With the frozen sections, detection of the GAPDH mRNA expression in the number of captured 25 oocytes were not repeatable, but the expression was always detectable from 50 oocytes. With 25 oocytes, at least 27 PCR cycles were required, whereas with 50 oocytes, 21 cycles were enough to detect GAPDH expression. Amount of the primary cDNA required for RT-PCR was reduced down to at least 0.25 microl with 50 oocytes, thus the resting 19.75 microl cDNA can be used for the testing other interested gene expression. Tissue-to-slide, tissue-to-tissue forces were very high in the paraffin sections, thus the greater number of cell procurement was required than the frozen sections. CONCLUSION: We have described a method for analyzing gene expression at the RNA level with the homogeneously microdissected cells from the small amount of tissues with complexity. We found that LCM coupled with RT-PCR could detect housekeeping gene expression in 50 oocytes captured. This technique can be easily applied for the study of gene expression with the small amount of tissues.
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Animais , Feminino , Camundongos , Álcoois , DNA Complementar , Amarelo de Eosina-(YS) , Etanol , Secções Congeladas , Expressão Gênica , Genes Essenciais , Guanidina , Hematoxilina , Microdissecção , Oócitos , Ovário , Parafina , Reação em Cadeia da Polimerase , Ribonucleases , Estabilidade de RNA , RNA , RNA Mensageiro , DNA Polimerase Dirigida por RNA , XilenosRESUMO
OBJECTIVE: This study was performed to determine whether the FSH receptor mutation is present in infertile Korean patients with 46,XX premature ovarian failure (POF) women. METHODS: The variant of FSH receptor exon 10 in thirteen 46, XX idiopathic POF and 4 healthy fertile (control) women were studied. Missense mutation in Exon 10 was detected in POF patients and healthy fertile women by polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP). RESULTS: The variant types of FSH receptor exon 10 (Thr307Ala; A919G) were found in healthy fertile (control) and POF women. CONCLUSIONS: This mutation may not be specific in POF patients and further study is needed in fertile (control) and POF women.
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Feminino , Humanos , Éxons , Mutação de Sentido Incorreto , Insuficiência Ovariana Primária , Receptores do FSHRESUMO
No abstract available.
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Humanos , Reação em Cadeia da Polimerase Multiplex , PapilomaRESUMO
This study was performed to determine whether the LHbeta -subunit gene missense mutation is present in Korean infertile patients with 46,XX POF women. The variants of LHbeta exon 2 (Trp 8Arg; TGG to CGG and Ile15Thr; ATC ti ACC) were studied in forty-four 46.XX idiopathic POF and 54 nonpregnant women. The LHbeta exon 2 variants were more frequent in POF patients (20.5%) than nonpregnant( 16.7%) women (p>0.05). POF patients with the variant was slightly higher than nonpregnant women with the variant.
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Feminino , Humanos , Éxons , Coreia (Geográfico) , Mutação de Sentido Incorreto , Insuficiência Ovariana PrimáriaRESUMO
No abstract available.
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Feminino , Humanos , Endometriose , Éxons , Síndrome do Ovário PolicísticoRESUMO
Determmation of the chomosomal constitution of human spermatozoa has been camed out though the human-hamster interspecific in vitro fertilization(IVF) system. In recent years, the introduction of fluorescence in-situ hybridization(FISH) technique has provided an alternative approach to evaluate the cbmmosomal constitution of human spermatozoa. The nuclei of mature spermatozoa are highly condensed with interpmtamine disulphide bridges, therefore the success of FISH on interphase human spermatozoa relies on partial decondensation of the sperm chromatin. In early studies, dithioothreitol(DTT) has been known as an efficient decondensation agent. Since then, several different decondensation methods using D1T have been establisdhed, and in terms of decondensation, we were tried to fix the optimal decondensation protocol using DlT. In our study, the optimal concentration and treatment time were 1-mM and 30 min, respectively. We examined chromosome complements of human sperm to investigate the effect of paternal age on the hequency of nondisjunction in human sperm. We investgated sperm karyotypes ftom two diffaent age groups)28+/-0.5, 46+/-6), A minimum of 1000 spermatozoa for one patient were analyzed. The mean frequencies of YY, XX, XY, 21-disamy spermatozoa ware 0.04%, 0.45%, 0.40%, 0.45% respectively in young age group and 1.06%, 0.62%, 1.06%, 0.76% in old ages. The mean frequency of disomy spermatozoa was higher in old age poup compare with those of young age group.
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Humanos , Cromatina , Aberrações Cromossômicas , Proteínas do Sistema Complemento , Constituição e Estatutos , Fluorescência , Interfase , Cariótipo , Idade Paterna , EspermatozoidesRESUMO
No abstract available.
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Humanos , Injeções de Esperma Intracitoplásmicas , Espermatozoides , Células VeroRESUMO
No abstract available.