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目的:采用经改良的取材和差速贴壁法培养纯化嗅黏膜嗅鞘细胞(OECs),以探求更加简单、高效的嗅黏膜OECs取材和培养纯化方法。方法:成年雄性SD大鼠6只,采用经改良的方法剥取嗅黏膜,然后用改良差速贴壁法培养纯化嗅黏膜OECs。倒置相差显微镜下观察细胞生长情况以及形态并照相,培养7d、14d细胞行NGFRp75免疫细胞化学染色鉴定,并根据免疫细胞化学染色结果计算细胞纯度。结果:体外培养的嗅黏膜OECs形态主要有扁圆形或油煎蛋形、梭形或双极、多突起形。嗅黏膜OECsNGFRp75免疫细胞化学染色阳性。培养7d嗅黏膜OECs纯度为90%,培养14d嗅黏膜OECs纯度为85%。嗅黏膜OECs最长可以存活35d。结论:经改良的取材和差速贴壁法培养纯化嗅黏膜OECs具有简单、高效的优点,培养的嗅黏膜OECs的纯度完全可以达到细胞移植的要求。
OBJECTIVE: To culture and purify olfactory mucosa olfactory ensheathing cells (OECs) by using improved and differential adherence methods to explore more simple and efficient OECs extraction and culture purification methods. Methods: Six adult male Sprague-Dawley rats were used to strip the olfactory mucosa by modified method. Then, the cultured olfactory mucosa OECs were cultured and purified by modified differential adherence method. Cell growth and morphology were observed under inverted phase contrast microscope. The cells were identified by NGFRp75 immunocytochemistry staining on day 7 and 14, and the cell purity was calculated according to immunocytochemical staining. Results: The morphology of OECs cultured in vitro mainly consisted of oblate or fried egg-shaped, fusiform or bipolar, multi-protuberances. OECsNGFRp75 positive for immunostaining of olfactory mucosa. The purity of OECs cultured on day 7 was 90% and that of OECs cultured on day 14 was 85%. The OECs of olfactory mucosa can survive for up to 35 days. Conclusion: It is simple and effective to culture and purify olfactory mucosa OECs with improved material and differential adherence method. The purity of OECs in cultured olfactory mucosa can reach the requirement of cell transplantation.