| ObjectiveAs one of saliveiy glands, submandibulary gland plays an important role not only in the physiological process, but also in the pathological research. Now more and more researchers are interested in its theraputiz value of treatment.As a part of SMG, the construction for the duct of tissue engineering SMG is an important aspect. Its principle is that the ductal cells are isolated and cultured in vitro, and then seeded in the three - dimensional scaffold. After the cells adhere to each other, proliferate and secret the matrix, new tissue and organ with certain structure and function is shaped. However, cells cultured in vitro lose the support from other cells and modification of all sorts of growth factors. This difference of living condition between in vitro and vivo results in slow growth rate and poor proliferous ability and has adverse influence on tissue engineering.So it is essential to improve the culture conditions and enhance the proliferous ability of the ductal cells, so that they can adhere to the scaffold and keep activity well, and then develop into an optimum tissue engineering product.In this study, the ductal cells of SMG were isolated and purified by density - gradient centrifugation and then cultured in vitro, so as to establish an ideal and effective cultivation system, and to investigate the effects of EGF on the proliferous ability of the ductal cells. Futhermore, they were seeded on the surface of scaffold and their growing character was observed so as to provide theoretical groundwork to form the model in vitro of tissue engineering duct of SMG.Methods1. The ductal cells of SMG were isolated and purified by density - gradient centrifugation and then were cultured and subcultured in DMEM with 20% fetal bovine serum.2. Observe shapes and structures of the cultured and subcultured cells with phase contrast microscope and transmission electron microscope, and then explore the growth character of the ductal cells of SMG.3. Use MTT colorimetric method to determine numeric value and to estimate proliferous ability of the ductal cells.4. The ductal cells were subjected to immunom'stochemistry research which could prove where the ductal cells were and the level of isolation and purification was evaluated by assay of amylase activity.5. To explore the effects of EGF on growth and differentiation of these cultured cells, the cells were cultured in the medium with EGF of different degree. The relations of dose - response and time - course were studied by MTT colorimetric method.6. The cells were cultured in the medium with EGF and then the change of intercellular calcium and cAMP were measured so that the mechanism of EGF exerting on the cells could be investigated.7. The ductal cells of the second generation were seeded in the medium of sponge scaffolds with collagen perfusion and cultured under physiologically conditions for 7 days. The shapes and structures of cultured cells were observed with phase contrast microscope, microscopic and transmission electron microscope.ResultsThe Isolation and Culture of The Ductal Cells of SMG in vitro 1. observation of culture cells with phase contrast microscopeThe cells cultured are polygon epithelium cells, and could go down to 3 to 4 generations and live up to 3 - 4w in the medium ( DMEM) with 20% fetal bovine serum conditions.(l)Primary culture;The shape of cells was observed with phase contrast microscope as spherical shape when it was just isolated and suspended in the medium. Cytoplasm showed uniform and the contour was clear and smooth. After 24 - 36 hours, most of cells attached to the glass wall and became flat and refraction capacity declined. After 48 hours their bodies began to stretch, all the cells became flat totally and the shape was seen as polygon at 4th -5th day. At 7th -9th day, neonatal cells were fused together and showed homogeneous.(2 ) Subculture:Cells of subcultured grew faster and could attach to the wall after 24 hours. The cellular shapes of t... |
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