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[ ABSTRACT] AIM:To investigate the biological characteristics of newborn rabbit tracheal chondrocytes in vitro. METHODS:Newborn rabbit tracheal chondrocytes were obtained by the method of enzyme digestion, and then cultured in monolayer in vitro.Morphological and growth observations were performed under inverted phase contrast microscope.The ultrastructures of the cells were observed under scanning electron microscope and transmission electron microscope.The bi-ological characteristics of secreted extracellular matrix components were detected by real-time PCR, immunocytochemistry staining and toluidine blue staining.RESULTS: Newborn rabbit tracheal chondrocytes isolated and cultured in vitro showed short triangular or irregular shapes, and adherent growth very well.The ultrastructures of the cells showed pore and abundant cytoplasm and organelles, with a lot of protein secretions in the cells.The chondrocytes expressed the mRNA of collagen I, collagen II and proteoglycans, mainly collagen II and proteoglycans.Immunocytochemistry staining showed col-lagen II and SOX9 positive, and collagen I weakly positive.Toluidine blue staining was also positive.CONCLUSION:Enzyme digestion and monolayer culture are suitable method to obtain newborn rabbit tracheal chondrocytes.These cells, secreting extracellular matrix components, are able to be selected as seed cells for tissue engineering of trachea in vitro, and used to study the therapeutic method for neonatal rabbit tracheal stenosis.
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Objective To localize the sinoatrial node (SAN) of the newborn rabbits in vivo and cut it for purifying cultivation and study the morophologic characters of primary cultured pacemaker cells of SAN under light microscope and transmissional electron microscope. Methods Hearts of the newborn rabbits were embedded in paraffin for HE-staining and observed the location, form of SAN under optical microscope; SAN cells isolated from neonatal rabbits cultured and purified with the method of differential attachment and BrdU-treatment.Results SAN localized in the anterior wall of the superior vena cava and the posterior-lateral atrial wall.There was about 0.32 mm between its lowest point and sulcus terminalis. Three distinctly different types of cells were observed among the cultured cells of SAN: spindle, araneiform and polygon. The spindle cells covered the greatest proportion of the cultured cells of SAN (59.6%?7.3%). The frequency of spontaneous contraction of spindle cells was the highest among the constrcting cells (145 ?9)time/min. The results of ultrastructure observation showed that myofibrils and other organelles in spindle cells were poorly organized and significantly decreased in number compared with araneiform cells. There was no significant difference between araneiform cells isolated from SAN and from atrial muscle.Conclusion Among the cultured cells from neonatal rabbits SAN, the spindle cells are the pacemaker cells of SAN.