ABSTRACT
The aim of this study is to examine the changes in the amniotic membrane diagnosed with gestational diabetes mellitus. In this study, as a control group human amnion membrane from normotensive pregnancies was collected from diabetic women at 2835 weeks of gestation. Gestational diabetes (n= 6) and normal amnion membrane (n= 6) for a total of 12 units were received. Amniotic membrane thickness was measured (p<0.0001) and it was significantly higher in GDM groups compared to control group. The diameter of the amniotic epithelial cell nuclei was measured (p=0.0022). Gestational diabetes results show that there was weakening between amniotic epithelial cell-cell junction. This study showed that structural changes in epithelial cells of amniotic membrane were formed due to diabetes. The membrane thickness has led to structural changes in diameter and in diabetes group cause extracellular matrix to increase, thus leading to MMP-9 expression increase eventually disrupting matrix balance. In addition, with cd44 increase angiogenesis has been induced and thought to influence material pass between fetus and mother.
El objetivo de este estudio fue examinar los cambios en la membrana amniótica diagnosticada con diabetes mellitus gestacional (DMG). En este estudio, como grupo control, se recogió la membrana amniótica de embarazos normotensos de mujeres diabéticas a las 28 y 35 semanas de gestación. La muestra consistió en 6 casos con diabetes gestacional (n = 6) y 6 casos de membrana amniótica normal (n = 6), para un total de 12 casos. El espesor de la membrana amniótica se midió (p <0,0001) y fue significativamente mayor en los grupos de DMG en comparación con el grupo control. Se midió el diámetro de los núcleos de las células epiteliales amnióticas (p = 0,0022). Los resultados demostraron que en la DMG hubo debilitamiento entre la célula epitelial amniótica-célula de unión. Este estudio mostró que los cambios estructurales en las células epiteliales de la membrana amniótica se presentaron debido a la DMG. El espesor de la membrana ha dado lugar a cambios estructurales en el diámetro y en el grupo de DMG debido a un aumento de la matriz extracelular, lo que condujo al aumento de la expresión de MMP-9, eventualmente interrumpiendo el equilibrio de la matriz. Además, el aumento de cd44 indujo la angiogénesis y se cree que también influye en el material que se comparte entre el feto y la madre.
Subject(s)
Humans , Female , Pregnancy , Amnion/metabolism , Amnion/pathology , Diabetes, Gestational/metabolism , Diabetes, Gestational/pathology , ImmunohistochemistryABSTRACT
OBJETIVO: Comparar, por microscopia eletrônica, a integridade anatômica e a presença de fatores de crescimento e citocinas da membrana amniótica preservada com glicerol/MEM (1:1) e dimetilsulfóxido puro. MÉTODOS: As membranas amnióticas preservadas em glicerol/MEM (1:1) ou dimetilsulfóxido puro foram processadas para microscopia eletrônica de transmissão e varredura. Como controle, membrana amniótica fresca foi imediatamente fixada após coleta e processada para microscopia eletrônica. As citocinas e os fatores de crescimento avaliados foram: TGF-beta- fator transformador de crescimento beta; TGF-beta ativ- fator transformador de crescimento beta ativado; EGF- fator recombinante de crescimento epitelial humano; FGF-4- fator de crescimento fibroblástico 4; FGF-beta- fator de crescimento fibroblástico básico; IL-4- interleucina 4; PGE2- prostaglandina E2; IL-10- interleucina 10; KGF- fator de crescimento de queratinócito; HGF- fator de crescimento de hepatócito. RESULTADOS: As membranas amnióticas do grupo controle apresentavam epitélio íntegro, com microvilos na superfície e complexos juncionais entre as células e a membrana basal. As membranas amnióticas preservadas em glicerol/MEM tinham aspecto semelhante às do controle, com maior altura das células epiteliais. Já as membranas amnióticas preservadas em dimetilsulfóxido mostraram redução das junções intercelulares e destacamento do epitélio da membrana basal. As citocinas e fatores de crescimento não apresentaram diferenças entre os grupos, exceto FGF-4, FGF-beta, PGE2 e KGF. CONCLUSÕES: A membrana amniótica preservada em meio glicerol/MEM apresentou melhor integridade tecidual, com menor desprendimento do epitélio da membrana basal, em comparação com a preservada no dimetilsulfóxido puro. Os fatores de crescimento e citocinas estavam, em sua maior parte, preservados com as duas técnicas de preservação.
PURPOSE: To compare the anatomical structure and the presence of growth factors and cytokines of amniotic membrane preserved in glycerol/MEM (1:1) or undiluted dimethyl sulfoxide through electron microscopy. METHODS: Amniotic membrane preserved in glycerol/MEM (1:1) or undiluted dimethyl sulfoxide were processed for transmission and scaning electron microscopy. As control, freshly collected amniotic membrane was fixed and processed for electron microscopy. The cytokines and growth factors assessed were: TGF-beta (transforming growth factor beta); TGF-b activ (activated transforming growth factor beta); EGF (epidermal growth factor); FGF-4 (fibroblast growth factor 4); bFGF (basic fibroblast growth factor); IL-4 (interleukin 4); PGE2 (prostaglandin E2); IL-10 (interleukin 10); KGF (keratinocyte growth factor); HGF (hepatocyte growth factor). RESULTS: Amniotic membrane from the control group showed intact epithelium, with surface microvilli and junctional complexes between the cells and the basal membrane. Glycerol/MEM preserved amniotic membrane had similar aspect to the control, with higher epithelial cells. Those amniotic membranes preserved in dimethyl sulfoxide disclosed less intercellular junction and detachment of the epithelium from the basal membrane. The cytokines and growth factors did not disclose significant differences, except for FGF-4, bFGF, PGE2 and KGF. CONCLUSIONS: Amniotic membrane preserved in glycerol/MEM showed a better tissue structure, with less detachment of the epithelium from the basal membrane, in comparison to undiluted dimethyl sulfoxide. The majority of the growth factors and cytokines were kept with both techniques of preservation.
Subject(s)
Humans , Amnion/metabolism , Amnion/ultrastructure , Cryopreservation/methods , Cryoprotective Agents/pharmacology , Dimethyl Sulfoxide/pharmacology , Glycerol/pharmacology , Amnion/drug effects , Cell Proliferation , Cells, Cultured , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/ultrastructure , Microscopy, Electron, Transmission , Transforming Growth Factor beta/metabolismABSTRACT
OBJECTIVE: To characterize the effect of IL-1 beta, on connective tissue metabolism in a human chorioamniotic membrane tissue culture (CAM). TYPE OF STUDY: Experimental, in an in vitro model. MATERIAL AND METHODS: CAM explants obtained from cesarean sections were cultured. The presence of local infection was excluded by microbiological methods. An XTT viability essay of the explants was carried out. Explants were stimulated with different doses of IL-1 beta within a 0-10 ng/mL range. After the stimulation, protein content was measured, MMP-9 production was determined by zymography, and each explant was divided in two parts: one was used for collagen measurement and the other analyzed by electronic microscopy. RESULTS: CAMs kept adequate viability and functionality. IL-1 beta stimulation produced an increase in the amount of MMP-9 expressed, as determined by the zymography method with a maximum effect 36 hours after stimulation. Collagen content decreased in a progressive manner after IL-1 beta stimulation and reached its minimum after 36 hours. The characteristic pattern of collagen fibers gradually lost its organization, and could not be observed any more after 36 hours. CONCLUSIONS: The information presented here allows us to conclude that IL-1 beta is capable of inducing an enzymatic expression affecting connective tissue, thus confirming its participation in membrane degradation processes under inflammatory conditions.
Subject(s)
Humans , Amnion/drug effects , Chorion , Collagen/drug effects , Connective Tissue , Interleukin-1 , Matrix Metalloproteinase 9 , Amnion/metabolism , Chorion , Collagen/metabolism , Connective Tissue , Culture TechniquesABSTRACT
No presente trabalho, foi investigada a produçäo de interferon nos tecidos da placenta humana induzidos por diferentes vírus. A membrana amniótica mostrou ser mais eficiente produtora de interferon quando infectada pelo vírus da doença de Newcastle (NDV) que a membrana ou as vilosidades coriônicas. A irradiaçäo do NDV com a luz ultravioleta (UV) diminuiu sua capacidade indutora nos três tecidos placentários. Os níveis de interferon encontrados em culturas da membrana amniótica ou coriônica, induzidos por vírus para-influenza I (Sendai), foram muito diferentes, dependendo da placenta utilizada, tanto com o vírus vivo como o tratado com a UV. Quando vilosidades coriônicas foram infectadas com este vírus, näo foi encontrada atividade de IFN. Testes comparativos mostraram que o NDV é melhor indutor de interferon do que o vírus Sendai em membrana amniótica. Os vírus Sindbis e Oriboca (vivos ou inativados pela UV) näo induziram títulos mensuráveis de interferon em qualquer dos tecidos mencionados. Concluiu-se que o sistema NDV - membrana amniótica é o mais eficiente na induçäo de IFN e merece investigaçöes mais abrangentes