RESUMO
This study aimed to develop an in vitro model for the analysis of the bovine endometrium. Immunofluorescent staining revealed that the hetero-spheroids and the cultured explants showed almost similar structure in the localization of bovine endometrial epithelial cells and endometrial stromal cells, except the glandular-like structure of the epithelial cells inside the explants. Gelatin zymography revealed that the hetero-spheroids did not express matrix metalloproteinases (MMPs) after 4 days of culture, but strong MMP expressions were observed in the cultured explants until 7 days of culture. Additionally, expression of progesterone receptor (PR), estrogen receptor alpha (ERα), type I interferon receptor 1 (IFNAR1) and 2 (IFNAR2) messenger RNA was observed both in the homo- and hetero-spheroids. The expression of oxytocin receptor (OTR) mRNA in E2 and E2+P4 (1,3,5(10)-Estratrien-3, 17ß-diol + 4-Pregnen-3, 20-dinone) treated groups were significantly (P < 0.05) higher than that of the control group of spheroids. In case of cultured explants, the expression of PR and OTR mRNA were significantly (P < 0.05) higher in E2 treated groups compared to the control groups. Hepatocyte growth factor (HGF) mRNA expression was also higher in P4 treated groups at 10 days in culture (P < 0.05). In a nutshell, the in vitro model developed in this study for the analysis of the endometrium may provide a new platform for extensive research on bovine endometrial function.
Assuntos
Endométrio/citologia , Endométrio/metabolismo , Modelos Biológicos , Animais , Bovinos , Células Cultivadas , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Imunofluorescência , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Esferoides Celulares/metabolismo , Coloração e RotulagemRESUMO
The formation of three-dimensional cell microspheres such as spheroids, embryoid bodies, and neurospheres has attracted attention as a useful culture technique. In this study, we investigated a technique for effective cell microsphere production by using specially prepared microchip. The basic chip design was a multimicrowell structure in triangular arrangement within a 100-mm(2) region in the center of a polymethylmethacrylate (PMMA) plate (24x24 mm(2)), the surface of which was modified with polyethylene glycol (PEG) to render it nonadhesive to cells. We also designed six similar chips with microwell diameters of 200, 300, 400, 600, 800, and 1000 microm to investigate the effect of the microwell diameter on the cell microsphere diameter. Rat hepatocytes, HepG2 cells, mouse embryonic stem (ES) cells, and mouse neural progenitor/stem (NPS) cells formed hepatocyte spheroids, HepG2 spheroids, embryoid bodies, and neurospheres, respectively, in the microwells within 5 days of culture. For all the cells, a single microsphere was formed in each microwell under all the chip conditions, and such microsphere configurations remained throughout the culture period. Furthermore, the microsphere diameters of each type of cell were strongly positively correlated with the microwell diameters of the chips, suggesting that microsphere diameter can be factitiously controlled by using different chip conditions. Thus, this chip technique is a promising cellular platform for tissue engineering or regenerative medicine research, pharmacological and toxicological studies, and fundamental studies in cell biology.