| 摘要: |
| 【目的】分离并培养山羊输卵管上皮细胞，研究其生长特性。【方法】采用胰酶、胶原酶及机械刮取方法分离输卵管上皮细胞并分析培养效果；选择免疫细胞化学染色对细胞进行鉴定；向培养液中添加下丘脑粗提物、胰岛素、EGF和血清，探讨其对输卵管上皮细胞体外培养的影响。【结果】胰酶对山羊输卵管上皮细胞的分离效果较差，胶原酶次之，机械刮取方法较好。机械刮取法分离的细胞以细胞团居多，接种后8～12 h开始贴壁生长，生长2～3 d细胞呈现明显的铺路石形态。免疫细胞化学染色显示，细胞角蛋白阳性率在95%以上。上皮细胞培养体系中含少量血清及人EGF、胰岛素，能有效促进原代和传代输卵管上皮细胞的生长，以体积分数5%血清+EGF的培养效果较好。【结论】采用机械刮取法分离培养的输卵管上皮细胞，在体外可连续传到第8代，传代细胞活性强、纯度高，可为输卵管上皮细胞相关功能的研究提供材料。 |
| 关键词: 山羊 输卵管上皮细胞 胶原酶 |
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| 基金项目:教育部博士点基金项目（200807120022）；陕西省科技发展项目（2008K02-01） |
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| Isolation and culture of goat oviduct epithelial cells |
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| Abstract: |
| 【Objective】The study was to establish a method for isolation and culture of goat oviduct epithelial cells in vitro,and to analyse some major growth characteristics of the cells.【Method】The effects of different cell culture methods,trypsin digesting,collagenase digesting and mechanical scraping were compared.The cultured goat oviduct epithelial cells were identified by immunocytochemistry.To select optimal cultivating conditions of oviduct epithelial cells,four factors including hypothalamus extract,insulin,EGF and serum were analysed by adding matters into culturefluid.【Result】The mechanical method showed better effect than trypsin and collagenase.Most of cells gained by mechanical method were clumps,which began to adhere to the wall at 8-12 hours after inoculation,and showed classical flagstone morphology in 2-3 d.Cells expressed keratin in immunocytochemical staining,the positive rate being about 95%.Low density serum,EGF and insulin in the basic culture medium ensured the oviduct epithelial cells’ growth and passage;adding 5% serum and EGF in medium had better culture effect than adding others.【Conlusion】The oviduct epithelial cells separated in this study could be propagated 8 times successively,which would provide basic materials for further study such as immortalization and relative functions. |
| Key words: goat oviduct epithelial cell collagenase |
