鸡胚盲肠上皮细胞的传代培养

Subculture of Chicken Embryo Cecum Epithelial Cell

本研究旨在探讨鸡胚盲肠上皮细胞传代培养条件及其特性,为E.tenella损伤机制及抗球虫药的研究提供体外模型。分别用胰酶-EDTA联合消化和分步消化2种方法分离纯化原代鸡胚盲肠上皮细胞,通过测定贴壁细胞覆盖率选择细胞传代的适宜消化方式;筛选了传代细胞培养液中L-GLN和胰岛素的最佳浓度,通过贴壁差进一步纯化盲肠上皮细胞,探索其合适的培养条件。通过形态学观察、透射电镜、免疫组织化学技术、组织化学技术、糖原染色、流式细胞术的方法对传代细胞特性进行了鉴定。结果表明:胰酶-EDTA联合消化、15min贴壁差纯化除去成纤维细胞,72.5mg.L-1 L-GLN、0.05mg.L-1胰岛素和5%FBS的传代细胞培养液更有利于盲肠上皮细胞的生长;经形态学和透射电镜观察,AKP、Vimentin及PAS染色,所培养的传代细胞具有典型的上皮细胞特征,在传代后24～72h盲肠上皮细胞纯度达95%以上,贴壁细胞覆盖率达85%以上;细胞凋亡测定表明,细胞连传5代,活性较好,第6代细胞凋亡率显著增加,贴壁细胞覆盖率降低。本研究提示用该法传代可获得稳定、数量大、活性高、纯度高的鸡胚盲肠上皮细胞。

Conditions and characteristics of chicken embryonic cecum epithelial cells（CEC） in subculture were studied in order to provide an in vitro model for the research of E.tenella＇s injury mechanism and coccidiostats.Primary chicken embryonic CEC were isolated by digestion combined or step by step with trypsin and EDTA.The digestion method was selected by evaluating the coverage rate of adherent cells.The optimal concentrations of L-GLN and insulin in subculture medium were identified.The CEC were further purified by differential attachment.Characteristics of passage cells were investigated by morphological and SEM observation,immunohistochemistry,histochemistry,PAS,and flow cytometry.Under the conditions of digestion combined with trypsin and EDTA and removal of fibroblasts using differential attachment for 15 min,the CEC grew better in subculture medium with 72.5 mg·L-1 L-GLN,0.05 mg·L-1 insulin,and 5% FBS.The cultured cells had the typical morphological features of epithelium.24-72 h after passage,the cell purity was more than 95%,and the coverage rate of adherent cells was more than 85%.Results of apoptosis detection showed that high viability was obtained on 5 passages.While the apoptosis rate was significantly increased and coverage rate of adherent cells was decreased after the 6th passage.The subculture conditions were optimized for harvest stable chicken embryonic CEC with large quantity,high viability and purity.