绒山羊羔羊和成年羊前体脂肪细胞的原代培养及传代方法

张清月, 王雪, 刘树林, 于洋, 郭晓宇, 闫素梅

doi:10.3969/j.issn.1006-267x.2018.09.025

动物营养学报 >

2018 , Vol. 30 >Issue 9: 3551 - 3558

DOI: https://doi.org/10.3969/j.issn.1006-267x.2018.09.025

反刍动物营养 Ruminant nutrition

绒山羊羔羊和成年羊前体脂肪细胞的原代培养及传代方法

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内蒙古农业大学动物科学学院, 呼和浩特 010018

张清月(1993-),女,河南新乡人,硕士研究生,从事反刍动物营养的研究。E-mail:alicezqy@126.com

收稿日期: 2018-02-27

网络出版日期: 2018-09-20

基金资助

国家自然科学基金（31760685）

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Primary Culture and Passage Method of Preadipocytes of Cashmere Lambs and Adult Goats

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Collage of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China

Received date: 2018-02-27

Online published: 2018-09-20

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摘要

本试验主要探索绒山羊羔羊和成年羊前体脂肪细胞的培养及传代方法，为研究绒山羊的脂肪代谢机理提供细胞模型。以3月龄阿尔巴斯白绒山羊羔羊肾周脂肪组织为试验材料，采用胶原酶法直接得到羔羊前体脂肪细胞，结合其细胞形态观察、生长曲线和油红O染色进行鉴定；以绒山羊成年羊肾周脂肪组织为试验材料，采用胶原酶法和"天花板"法得到成熟脂肪细胞后，通过去分化得到前体脂肪细胞，诱导分化后利用油红O染色鉴定其向成熟脂肪细胞分化的情况。绒山羊羔羊肾周脂肪组织中前体脂肪细胞的适宜分离条件为0.1%Ⅰ型胶原酶、37℃消化1 h，250×g离心10 min；传代时分离细胞采用0.25%胰蛋白酶消化60 s。细胞形态为梭形，生长曲线呈"S"型，油红O染色结果呈阳性。成年羊采用和羔羊相同的胶原酶法获得成熟脂肪细胞后，通过改进的"天花板"法获得前体脂肪细胞，经诱导分化后，油红O染色结果呈阳性。综上，采用Ⅰ型胶原酶消化法可直接分离培养3月龄绒山羊羔羊的前体脂肪细胞；采用胶原酶法和改进后的"天花板"法分离培养成年绒山羊的前体脂肪细胞是可行的。

关键词： 绒山羊成年羊; 绒山羊羔羊; 前体脂肪细胞; 细胞培养

本文引用格式

张清月, 王雪, 刘树林, 于洋, 郭晓宇, 闫素梅 . 绒山羊羔羊和成年羊前体脂肪细胞的原代培养及传代方法[J]. 动物营养学报, 2018 , 30(9) : 3551 -3558 . DOI: 10.3969/j.issn.1006-267x.2018.09.025

Abstract

The study aimed to explore the primary culture and passage methods of preadipocytes of cashmere lambs and adult goats, and to provide a cell model for further researching the metabolic mechanism of adipocytes of cashmere goats. Perirenal adipose tissues of 3-month-old Albas cashmere lambs were selected as test materials. Preadipocytes of lambs were obtained directly by collagenase digestion method, and identified by morphological observation, growth curve and oil red O staining; perirenal adipose tissues of cashmere adult goats were selected as test materials. Mature adipocytes of adult goats were obtained by collagenase digestion and "ceiling" culture methods, and then preadipocytes were obtained by the dedifferentiation of mature adipocytes. After induction of differentiation, oil red O staining was used to identify their differentiation into mature adipocytes. The appropriate separation conditions for preadipocytes of the perirenal adipose tissues of lambs were digestion by 0.1% Ⅰ collagenase at 37℃ for 1 h, and centrifugation at 250×g for 10 min; during cell passage, digestion by 0.25% trypsin for 60 s. The preadipocytes were fusiform, grew in an "S" shape, and were positive on oil red O staining. Mature adipocytes of adult goats were obtained by collagenase digestion method in the same way as the lambs' method, and then using modified "ceiling" method obtained preadipocytes. After induction of differentiation, those preadipocytes were positive on oil red O staining. In conclusion, it is feasible to obtain preadipocytes directly by type Ⅰ collagenase digestion method from 3-month-old Albas cashmere lambs and by collagenase digestion and modified "ceiling" methods from adult cashmere goats.

Key words： cashmere adult goats; cashmere lames; preadipocytes; cell culture

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