生物学杂志

生物学杂志 ›› 2024, Vol. 41 ›› Issue (2): 110-.doi: 10.3969/j.issn.2095-1736.2024.02.110

• 技术方法 • 上一篇    下一篇

秦川牛成纤维细胞的分离培养体系优化

冯贤辀1， 陈　辉2， 贾永宏2， 刘松奇1,3， 温　飞1， 郭松茂1， 韩帅琪1， 胡建宏1   

  1. 1. 西北农林科技大学 动物科技学院， 杨凌 712100； 2. 陕西省畜牧产业试验示范中心， 泾阳 713702；
    3. 中国农业科学院 北京畜牧兽医研究所， 北京 100193
  • 出版日期:2024-04-18 发布日期:2024-04-17
  • 通讯作者: 胡建宏，博士，教授，主要研究方向为动物生殖生理调控、动物生物技术，E-mail:hjh19732008@126.com
  • 作者简介:冯贤辀，博士研究生，研究方向为动物遗传育种与繁殖，E-mail:fengxianzhou0930@ 163.com；陈辉，畜牧师，主要从事畜牧技术推广工作，E-mail:546793153@qq.com；冯贤辀和陈辉为共同第一作者
  • 基金资助:
    陕西省地方畜禽遗传材料釆集制作（陕牧函〔2022〕7号）

Optimization of the isolation and culture system of Qinchuan cattle fibroblasts

FENG Xianzhou1, CHEN Hui2, JIA Yonghong2, LIU Songqi1,3, WEN Fei1, GUO Songmao1, #br# HAN Shuaiqi1, HU Jianhong1   

  1. 1. College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China;
    2. Animal Husbandry Industry Experiment and Demonstration Center of Shaanxi Province, Jingyang 713702, China;
    3. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
  • Online:2024-04-18 Published:2024-04-17

PDF (PC)

38

摘要： 为建立秦川牛体细胞分离培养体系，分别选用组织块培养法、胰酶-胶原酶消化法和胶原酶-胰酶消化法提取秦川牛耳缘成纤维细胞。结果表明：组织块培养法获取成纤维细胞所需周期长，组织块贴壁17 d后可获得原代成纤维细胞，而双酶消化法在原代细胞贴壁至9 d时便可进行传代纯化培养；与双酶消化法Ⅱ相比，双酶消化法I获得的原代成纤维细胞浓度1.24×106/mL显著高于双酶消化法Ⅱ（0.9×106/mL）（P<0.05）；传代培养5 d后，细胞汇合度达到80%，取第三代成纤维细胞进行免疫荧光和流式细胞术鉴定，成纤维细胞纯度达到95%以上；纯化后的成纤维细胞培养至第3天，开始进入对数生长期，第8天时增殖速度下降进入平台期。因此，双酶消化法I（0.25%胰酶处理30 min，再用1%胶原酶处理60 min）为分离培养秦川牛耳缘成纤维细胞的最佳方法。

关键词: 秦川牛, 皮肤组织, 成纤维细胞, 组织块培养法, 酶消化法

Abstract: To establish the isolation and culture system of Qinchuan cattle somatic cells, this study used three different methods to extract fibroblasts from the ear margins of Qinchuan cattle. Results showed that fibroblasts could be obtained after 17 d of tissue block culture, while the double enzyme digestion method could be cultured for 9 d for passaging. Compared with that of the enzymatic digestion method Ⅱ（0.9×106/mL）, the concentration of fibroblasts obtained by the enzymatic digestion method I（1.24×106/mL）was high and the difference was significant. Third-generation fibroblast immunofluorescenceand flow cytometry identification results showed cell purity of over 95%. The purified fibroblasts were cultured until the logarithmic growth phase at 3 d, and the proliferation rate decreased at 8 d and entered the plateau phase. In conclusion, the dual enzyme digestion method I (0.25% trypsin digestion for 30 min and 1% collagenase for 60 min) was the best method for isolation and culture of Qinchuan cattle ear margin fibroblasts.

Key words: Qinchuan cattle, skin tissue, fibroblast, tissue culture method, enzyme digestion method

中图分类号: