妊娠期奶牛黄体细胞的分离鉴定及培养特性

doi:10.11843/j.issn.0366-6964.2024.05.039

摘要/Abstract

摘要： 旨在研究妊娠期奶牛黄体细胞的分离、纯化、鉴定及体外培养生物学特性。本研究通过采集健康荷斯坦奶牛的妊娠期黄体组织，利用Ⅱ型与Ⅳ型胶原酶联合消化法分离奶牛黄体细胞(bovine luteal cells, BLCs)，通过Percoll不连续密度梯度离心法纯化获得高纯度的BLCs。运用3β-HSD活细胞染色法、油红O脂滴染色法鉴定BLCs的纯度，借助免疫组织化学染色法、免疫荧光染色检测BLCs特异性标志物催产素和突触素。ELISA法检测细胞培养上清液中孕酮；G显带核型分析BLCs染色体、CCK-8法测定BLC生长曲线和血清依赖性结合流式细胞术检测细胞周期分析BLCs的体外培养特性。结果表明，1)利用胶原酶消化法和Percoll不连续密度梯度离心法可一次性分离纯化获得大量高纯度的BLCs；2)3β-HSD染色、油红O染色结果显示，分离纯化所得BLCs细胞类型均匀，其形态呈不规则多边形的典型上皮细胞样，胞核大，胞质丰富，有小脂滴弥散分布；3)免疫荧光结果表明，BLCs可表达催产素和突触素等黄体细胞的特异性标志物；4)BLCs培养上清中孕酮的浓度呈时间依赖性；5)核型分析结果显示，黄体细胞由30对染色体组成，包括29对常染色体和一对XX性染色体，CCK-8及流式结果显示,BLCs在体外培养过程中具有稳定的增殖活性。综上所述，本研究成功分离纯化获得具有典型生物学特性的高纯度BLCs，并提供了一套完善的BLCs分离、纯化、鉴定及体外培养的方法，可为体外研究黄体的发育、退化及其功能调控提供稳定的细胞模型和技术参考。

关键词: 奶牛, 黄体细胞, 分离培养, 生物学特性

Abstract: The aim was to study the isolation, purification, characterization and in vitro culture biological properties of luteal cells from cows during pregnancy. In this study, luteal tissues of healthy Holstein cows during pregnancy were collected, and bovine luteal cells (BLCs) were isolated by type II and type IV collagenase digestion. High-purity BLCs were purified by Percoll discontinuous density gradient centrifugation. The purity of BLCs was identified by 3β-HSD live cell staining and oil red O lipid droplet staining. The specific marker proteins of BLCs, oxytocin and synaptophysin, were detected by immunohistochemical staining and immunofluorescence staining. Progesterone in cell culture supernatant was detected by ELISA. G-banding karyotype analysis of BLCs chromosomes, CCK-8 assay of BLC growth curve and serum-dependent flow cytometry to detect cell cycle analysis of BLCs in vitro culture characteristics. The results showed that: 1) A large number of high-purity BLCs could be obtained by one-time separation and purification using collagenase digestion and Percoll discontinuous density gradient centrifugation. 2) The results of 3β-HSD staining and oil red O staining showed that the isolated and purified BLCs had uniform cell types, and their morphology was typical epithelial cell-like cells with irregular polygons. The nucleus was large, the cytoplasm was rich, and small lipid droplets were dispersed. 3) Immunofluorescence results showed that BLCs could express specific markers of luteal cells such as oxytocin and synaptophysin; 4) The concentration of progesterone in the culture supernatant of BLCs was time-dependent. 5) Karyotype analysis showed that luteal cells were composed of 30 pairs of chromosomes, including 29 pairs of autosomes and a pair of XX sex chromosomes. CCK-8 and flow cytometry results showed that BLCs had stable proliferation activity during in vitro culture. In summary, this study successfully isolated and purified high-purity BLCs with typical biological characteristics, and sorted out a set of scientific and perfect methods for the separation, purification, identification and in vitro culture of BLCs, which can provide a stable cell model and technical reference for the study of luteal development, degeneration and functional regulation in vitro.

Key words: cow, luteal cells, isolation and culture, biological characterization

中图分类号:

S852.61

费国庆, 宁致远, 赵泽芳, 刘艳秋, 刘腾飞, 李贤, 丛日华, 陈鸿, 陈树林. 妊娠期奶牛黄体细胞的分离鉴定及培养特性[J]. 畜牧兽医学报, 2024, 55(5): 2214-2225.

FEI Guoqing, NING Zhiyuan, ZHAO Zefang, LIU Yanqiu, LIU Tengfei, LI Xian, CONG Rihua, CHEN Hong, CHEN Shulin. Isolation, Identification of Luteal Cells from Cows during Pregnancy and Investigation of Their Culture Characteristics[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2214-2225.

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