畜牧兽医学报2024，Vol.55Issue(5)：2214-2225,12.DOI:10.11843/j.issn.0366-6964.2024.05.039
妊娠期奶牛黄体细胞的分离鉴定及培养特性
Isolation,Identification of Luteal Cells from Cows during Pregnancy and Investigation of Their Culture Characteristics
摘要
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.关键词
奶牛/黄体细胞/分离培养/生物学特性Key words
cow/luteal cells/isolation and culture/biological characterization分类
农业科技引用本文复制引用
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):2214-2225,12.基金项目
国家自然科学基金项目(32172811 （）
32002242) （）
