树鼩角膜原代上皮细胞的分离培养、纯化与鉴定

Primary Isolation,Culture,Purification and Identification of Corneal Epithelial Cells in Tree Shrew

目的建立树嗣角膜上皮细胞(CECs)稳定的体外培养技术,为人类角膜疾病研究提供新的实验材料。方法使用改进的双酶消化法取得树嗣CECs,周转化生长因子-β(TGF-β)抑制剂配合梯度消化对树鼩CECs进行纯化,角蛋白3/12抗体对CECs进行免疫荧光检测及鉴定。结果优化的酶消化法可以快速、有效得到高纯度的树鼩CECs。TGF-β抑制剂及梯度消化法可以达到良好的纯化目的,纯化的CECs在传代过程中仍保持良好形态。树鼩CECs免疫荧光染色显示角蛋白3/12单克隆抗体阳性。结论成功建立了一种有效、简单、经济适用的树鼩角膜上皮原代细胞的体外培养技术,所获得的原代细胞具有较好的上皮细胞形态特征并可以连续传代,为眼科疾病的研究提供一种新材料。

Objectives To establish a stable technique for primary isolation, culture and purification and identification of tree shrew （Tupaia belangeri） corneal epithelial cells, and to provide a new experimental material for reserch of human ophthalmic corneal diseases. Methods The primary corneal epithelial cells from tree shrew were obtained by improved double digests method, the cells were purifed by method of cornea peeled off with transforming growth factor（TGF）-β inhibitors, achieve the purpose of identification by the method of immunofluorescence with keratin 3/12 antibody. Results The primary corneal epithelial cells from tree shrew with high activity and purity were obtained by double digests method. The corneal epithelial cells were purified food by TGF-β inhibitors and gradient digestion, and the purified cells was maintained a good epithelial cells shape in the subculture. The result of tree shrew corneal epithelial cells by staining with keratin 3/12 monoclonal antibodies were shown positive. Conclusion An efficiency, simple and economic method was estabilshed for in vitro tree shrew corneal epithelial cells. The primary corneal epithelial cells from tree shrew with high activity and purity were obtained by double digests method, the subcultured cells was maintained a good epithelial cells shape. It will supply a new materials for eye diseases research.