生物学杂志

生物学杂志

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广西巴马小型猪皮肤成纤维细胞的分离培养体系优化

  

  1. 西南大学 动物科技学院， 重庆 400715
  • 出版日期:2017-04-18 发布日期:2017-04-18
  • 通讯作者: 肖雄，博士，副教授，主要从事动物胚胎工程、细胞生物学和兽医产科学方面的研究，E-mail: y1982@swu.edu.cn
  • 作者简介:张大鹏，硕士研究生，主要从事动物胚胎工程和临床兽医学方面的研究，E-mail：522672133@qq.com
  • 基金资助:
    国家自然科学基金项目（31572488）；重庆市基础与前沿研究计划项目（cstc2013jcyjA10110）；中央高校基本科研业务费项目（XDJK2014C022）；西南大学博士基金项目（SWU13002）

Isolation and culture of skin fibroblasts in Guangxi Bama mini-pig

  1. College of Animal Science and Technology, Southwest University, Chongqing 400715, China
  • Online:2017-04-18 Published:2017-04-18

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摘要： 通过比较6种方法和5个部位分离成纤维细胞的效率，旨在优化广西巴马小型猪皮肤成纤维细胞的分离培养体系。结果表明：1）采用酶消化法Ⅰ所得成纤维细胞的密度显著高于其他处理组（P＜0.05），其死亡率与方法Ⅳ处理组之间差异不显著（P＞0.05），但显著低于其余处理组（P＜0.05）；2）冷冻保存10 d后复苏的耳部皮肤组织，经方法Ⅰ酶消化后所得成纤维细胞的密度和存活率均分别极显著低于新鲜组织方法Ⅰ处理组（P＜0.01）；3）组织块培养4 d后即可从其边缘长出成纤维细胞，再经过8 d培养，细胞汇合率可达100%；4）耳部皮肤组织所获成纤维细胞的密度显著高于其他部位处理组（P＜0.05），而且贴壁生长效果良好；5）所得耳部皮肤成纤维细胞可传至10代以上，其中第3代和第4代生长最为迅速；6）生长曲线显示第3代耳部皮肤成纤维细胞呈典型的“S形”生长。因此，宜选用先加0.25%胰蛋白酶+0.04% EDTA消化30 min，再用0.2%胶原蛋白酶消化30 min的方法分离广西巴马小型猪的耳部皮肤成纤维细胞。

关键词: 广西巴马小型猪, 皮肤成纤维细胞, 酶消化法, 组织块培养法

Abstract: In order to screen appropriate methods for the isolation and culture of the skin fibroblasts from Guangxi Bama mini-pig, five enzyme digestion methods and a tissue culture method were compared in the isolation of fibroblasts of skins from different parts of mini-pig including ear, leg, abdomen, back, and tail . The results indicated that the density of fibroblasts isolated with enzyme digestion methodⅠwas significantly higher than that in other treatment groups (P<0.05), and there was no significant difference in the cell death rates between fibroblasts from the shin treated with enzyme digestion methodⅠand that with enzyme digestion method Ⅳ (P>0.05), but the cell death rates in those two treatment groups were significantly lower than that in other groups, respectively (P<0.05). Fibroblast could be isolated from the thawed ear shin tissues after frozen for 10 days with enzyme digestion methodⅠ, but the density of fibroblasts and their survival rate were significantly lower than that from the fresh ear shin tissues with the same method (p<0.01). The cell confluency rate of fibroblasts would not reach to about 100% until being cultured for 12 days. Compared to other parts of mini-pig, ear skin was more suitable to isolate the fibroblasts, because the density of fibroblasts isolated from ear skin tissues was significantly higher than that from other parts of mini-pig (P<0.05), and the attachment and growth of those fibroblasts were very well. Ear skin fibroblasts were able be passaged up to more than passage 10, but the viability of cells at passage 3 to 4 was higher than that of cells at other passage. The cell growth curve of ear skin fibroblasts at passage 3 was a typical "S" shape. Therefore, it is better to isolate the fibroblasts from the ear skin of Guangxi Bama mini-pig with the digestion of 0.25% trypsase and 0.04% EDTA for 30 min, follow by 0.2% collagenase for 30 min.

Key words: Guangxi Bama mini-pig, skin fibroblasts, enzyme digestion method, tissue culture method