原代奶牛子宫内膜上皮细胞体外培养方法的改进

doi:10.16431/j.cnki.1671-7236.2020.03.036

摘要/Abstract

摘要： 目前利用奶牛子宫内膜上皮细胞体外培养技术，建立奶牛子宫内膜炎模型从而减少不可控因素来研究奶牛子宫内膜炎疾病已成为国内外普遍使用的一类方法，因此获得高纯度、同一性的子宫内膜上皮细胞是体外研究的关键环节，国内外所使用方法主要是酶消化法、组织块贴壁法和组织块消化贴壁法。本文旨对先前的方法进行改良，建立一种简便易行、培养周期短、细胞活性及形态良好的原代奶牛子宫内膜上皮细胞体外培养技术。采用健康未孕奶牛子宫为试验材料，取下子宫内膜组织，加入5 mL含2%双抗、40%胰酶的DMEM/F12培养基，4 ℃消化12 h，再将组织移至25 cm²细胞培养瓶中贴壁培养。获得原代细胞后，应用角蛋白-18抗体对细胞进行免疫组化荧光鉴定，并对第3代细胞采用CCK-8法测定不同时间点的D_{450 nm}值，绘制细胞生长曲线。结果显示，培养至第8天，原代细胞基本铺满细胞培养瓶瓶底，有效地缩短了常规组织块贴壁法的周期。通过角蛋白-18免疫组化荧光鉴定，原代上皮细胞的阳性率可达98%以上，相比常规组织块贴壁法来说，纯度明显提高，省去了细胞纯化的操作步骤。细胞增殖状态，符合正常的分裂生长特性。试验结果表明将常规组织块贴壁法进行了优化改良，不仅缩短了培养周期，同时提高纯度，保持细胞活性，为原代奶牛子宫内膜上皮细胞体外培养技术的改良提供一定的参考。

关键词: 奶牛子宫内膜上皮细胞; 细胞培养; 细胞鉴定

Abstract: At present,it has become a common method to study bovine endometritis by using the in vitro culture technology of bovine endometrial epithelial cells to establish a model of bovine endometritis so as to reduce uncontrollable factors.Thus obtaining high purity and identical endometrial epithelial cells are the key link in in vitro research.The methods used at home and abroad are mainly enzymatic digestion,tissue block adherence and tissue block digestion and adherence.In this paper,the previous method was modified to establish a simple and easy-to-use technique for in vitro culture of primary bovine endometrial epithelial cells with short cell culture period and good cell activity and morphology.The healthy non-pregnant cow uterus was used as the test material.The endometrial tissue was removed,and 5 mL of DMEM/F12 medium containing 2% double antibody and 40% trypsin was added,and digested at 4 ℃ for 12 h.The tissue was transferred to a 25 cm² cell culture flask for adherent culture.After obtaining the primary cells,the cells were subjected to immunohistochemical fluorescence identification using keratin-18 antibody,and the CCK-8 method was used for the third generation cells.The detection wavelength was 450 nm,and the cell growth was drawn according to the D_{450 nm} values at different time points curve.The results showed that the primary cells were basically covered with the bottom of the cell culture flask on the 8th day,which effectively shortened the cycle of the conventional tissue block adherence method.By keratin-18 immunohistochemical fluorescence identification,the positive rate of primary epithelial cells was more than 98%.Compared with the conventional tissue block adherence method,the purity of primary epithelial cells was significantly improved,and the steps of cell purification were omitted.The proliferation of the cells was in line with the normal characteristics of division and growth.The results showed that the conventional tissue block attachment method was optimized and improved,which not only shortened the culture period,but also improved the purity and maintained the cell activity,which provided a certain reference for the improvement of the in vitro culture technique of the primary bovine endometrial epithelial cells.

Key words: bovine endometrial epithelial cells; cell culture; cell identification

中图分类号:

S823.9⁺1

王仕宇, 李博通, 刘佳玮, 倪耀娣, 刘明超. 原代奶牛子宫内膜上皮细胞体外培养方法的改进[J]. 中国畜牧兽医, 2020, 47(3): 958-964.

WANG Shiyu, LI Botong, LIU Jiawei, NI Yaodi, LIU Mingchao. Improvement of Culture Method of Primary Bovine Endometrial Epithelial Cells in Vitro[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(3): 958-964.

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