犊牛脂肪干细胞的分离、培养与鉴定

doi:10.3969/j.issn.1004-1524.2019.02.09

浙江农业学报 ›› 2019, Vol. 31 ›› Issue (2): 235-241.DOI: 10.3969/j.issn.1004-1524.2019.02.09

犊牛脂肪干细胞的分离、培养与鉴定

王帅帅¹, 张才^{1, 2, *}, 孟素丹¹, 黄永志¹, 朱雪敏¹, 王玉琴^{1, 2}, 杨自军¹

1.河南科技大学动物与科技学院,河南洛阳 471000;
2.河南省肉羊繁育工程技术研究中心,河南洛阳 471000

收稿日期:2018-06-26 出版日期:2019-02-25 发布日期:2019-03-06
通讯作者: 张才,E-mail: hucaia@sina.com
作者简介:王帅帅(1994—),男,河南灵宝人,硕士研究生,研究方向为动物营养代谢与中毒病。E-mail: 1042822775@qq.com
基金资助:
国家自然科学基金(31402263;31872537); 河南省自然科学基金(182300410087)

Isolation, culture and identification of adipose-derived stem cells from calves

WANG Shuaishuai¹, ZHANG Cai^{1, 2, *}, MENG Sudan¹, HUANG Yongzhi¹, ZHU Xuemin¹, WANG Yuqin^{1, 2}, YANG Zijun¹

1. Laboratory of Environment and Livestock Product Safety, Henan University of Science and Technology, Luoyang 471000, China;
2. Henan Sheep Breeding Engineering Research Center, Luoyang 471000, China

Received:2018-06-26 Online:2019-02-25 Published:2019-03-06

摘要/Abstract

摘要： 为给奶牛营养代谢病等相关研究提供种子细胞,本试验从犊牛肾周脂肪组织中分离出脂肪干细胞(adipose-derived stem cells, ADSCs),采用组织剪碎法结合0.25%Ⅰ型胶原酶消化法消化、收集原代犊牛ADSCs,通过细胞形态学观察,使用免疫荧光染色法检测第5代ADSCs表面特异性蛋白,并使用CCK-8法测定第5、10代细胞生长曲线,观察第10代细胞的成脂、成骨分化能力。结果显示,分离出的犊牛ADSCs接种4 d后细胞呈成纤维细胞样生长;免疫荧光染色结果显示,分离的细胞Vimentin、CD34、CD44、CD90呈阳性,CD31、CD45呈阴性;该细胞在适当的诱导条件下可以定向分化为脂肪细胞和成骨细胞。本试验分离培养的细胞是犊牛脂肪干细胞,具有成脂、成骨分化能力。

关键词: 犊牛, 脂肪干细胞, 分离培养, 诱导分化

Abstract: In order to provide seed cells for related research on vegetative metabolic diseases of cows, adipose-derived stem cells (ADSCs) were eparated from perirenal adipose tissue of calves. ADSCs were collected by organization minced and 0.25% collagenase Ⅰ digestion by different times. The separated cells were proved by the following aspects, observed the morphology, passage 5 of ADSCs were used to detect the surface-specific proteins, passage 5 and 10 were used to detect the growth curve by CCK-8, passage 10 were used to detect the ability of adipogenic and osteogenic differentiation. The results showed that the ADSCs from calves were grown as fibroblast-like after 4 days of inoculation. Immunofluorescence staining showed that the isolated cells expressed Vimentin, CD34, CD44, and CD90, but did not express CD31 and CD45. The cells can be differentiated into adipocytes and osteoblasts under appropriate induction conditions. The separated cells were ADSCs with the ability of adipogenic and osteogenic differentiation.

Key words: calves, adipose stem cells, solation and cultivation, induced differentiation

中图分类号:

S828

王帅帅, 张才, 孟素丹, 黄永志, 朱雪敏, 王玉琴, 杨自军. 犊牛脂肪干细胞的分离、培养与鉴定[J]. 浙江农业学报, 2019, 31(2): 235-241.

WANG Shuaishuai, ZHANG Cai, MENG Sudan, HUANG Yongzhi, ZHU Xuemin, WANG Yuqin, YANG Zijun. Isolation, culture and identification of adipose-derived stem cells from calves[J]. , 2019, 31(2): 235-241.

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犊牛脂肪干细胞的分离、培养与鉴定

Isolation, culture and identification of adipose-derived stem cells from calves

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