42日龄湘东黑山羊瘤胃平滑肌细胞的分离培养和鉴定

刘思乐, 康劲翮, 谭支良, 王征

doi:10.3969/j.issn.1006-267x.2015.10.033

动物营养学报 >

2015 , Vol. 27 >Issue 10: 3249 - 3255

DOI: https://doi.org/10.3969/j.issn.1006-267x.2015.10.033

研究简报 Short Communications

42日龄湘东黑山羊瘤胃平滑肌细胞的分离培养和鉴定

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1. 湖南农业大学生物科学技术学院, 长沙 410128;
2. 中国科学院亚热带农业生态研究所, 中国科学院亚热带农业生态过程重点实验室, 湖南省农业生态过程重点实验室, 长沙 410125

刘思乐(1991—),男,湖南长沙人,硕士研究生,从事反刍动物营养研究。E-mail:349162439@qq.com

收稿日期: 2015-04-01

网络出版日期: 2015-10-15

基金资助

国家自然科学基金项目(31201826);中央驻湘科研机构技术创新发展专项(2013TF3006)

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Separation, Culture and Identification of Ruminal Smooth Muscle Cells of Xiangdong Black Goats Aged 42 Days

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1. College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China;
2. Key Laboratory of Agro-Ecological Processes in Subtropical Region, Hunan Key Laboratory of Agro-Ecological Processes, Institute of Subtropical Agriculture, The Chinese Academy of Sciences, Changsha 410125, China

Received date: 2015-04-01

Online published: 2015-10-15

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摘要

本研究旨在建立山羊瘤胃平滑肌细胞的体外培养模型。采集42日龄山羊的瘤胃肌肉组织,采用胶原酶消化法进行了山羊瘤胃平滑肌细胞的体外培养。通过光学显微镜观察了原代培养和传代培养阶段的细胞形态,采用细胞计数法检测了第5代山羊瘤胃平滑肌细胞的生长曲线及第1~11代细胞生长活性,采用细胞免疫荧光法对细胞进行了鉴定,采用蛋白质印迹(Western blotting)技术检测平滑肌细胞特异表达的α肌动蛋白(α-actin)在各代次细胞中的表达。结果表明,经0.20% Ⅱ型胶原酶消化获得的原代培养山羊瘤胃平滑肌细胞于培养1 d后开始贴壁生长,2 d开始进入对数期生长,呈典型的"波峰"状生长,5 d进入平台期,第5代细胞生长活性达到最高;免疫荧光染色显示胞浆内α-actin阳性表达,各代次间细胞α-actin表达稳定,表达量无显著差异(P>0.05)。试验表明,应用0.20% Ⅱ型胶原酶消化法可成功获得山羊瘤胃平滑肌细胞,为进一步研究营养物质对山羊瘤胃功能的影响提供了理想的细胞模型。

关键词： 山羊; 瘤胃; 平滑肌细胞; 原代培养; 鉴定; α肌动蛋白

本文引用格式

刘思乐, 康劲翮, 谭支良, 王征 . 42日龄湘东黑山羊瘤胃平滑肌细胞的分离培养和鉴定[J]. 动物营养学报, 2015 , 27(10) : 3249 -3255 . DOI: 10.3969/j.issn.1006-267x.2015.10.033

Abstract

The present study was carried out to establish a culture method for ruminal smooth muscle cells of goat.Ruminal smooth muscle tissue of goats (42 days of age) was collected, and ruminal smooth muscle cells was cultured in vitro by the method of collagenase digestion.The morphology of cells at the stages of primary culture and subculture was observed under the optical microscope, the growth curve of the 5th generation and the growth activity curve of generations of ruminal smooth muscle cells were tested using the cell counting method.Meanwhile, the ruminal smooth muscle cells were identified using cell immune fluorescence method, and the expression of α-actin in generations (1st to 11th) cells was detected using the Western blotting.The results showed that the primary cultured ruminal smooth muscle cells of goats obtained by digestion of 0.20% type Ⅱ collagenase began adherent growth after cultured for 1 day, an exponential growth started on the 2nd day resembling a "hill" in appearance, the curve stated plateau phase on the 5th day, and the growth activity of cells reach the highest in the 5th generation.The α-actin in the cytoplasm exhibited a positive reaction by immune fluorescence staining and the protein expressed stable, no significant difference in generations was found (P>0.05).The results demonstrate that ruminal smooth muscle cells of goats were obtained successfully by the methods of 0.20% type Ⅱ collagenase digestion, which provides an ideal cell model for the further study on the effects of nutrients on goats' rumen function.

Key words： goat; rumen; smooth muscle cell; primary culture; identification; α-actin

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