鹅骨骼肌卫星细胞的分离培养与鉴定

doi:10.11843/j.issn.0366-6964.2023.10.017

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (10): 4186-4195.doi: 10.11843/j.issn.0366-6964.2023.10.017

鹅骨骼肌卫星细胞的分离培养与鉴定

张力^1,2, 许加龙³, 黄锦钰², 许子月², 雷昕诺^1,2, 卢会鹏^1,2, 朱睿^1,2, 孙伟翔^1,2, 曹海月², 王安平^1,2, 朱善元^1,2*

1. 江苏农牧科技职业学院江苏省兽用生物制药高技术研究重点实验室江苏现代畜牧与新兽药工程技术中心, 泰州 225300;
2. 江苏农牧科技职业学院, 泰州 225300;
3. 南京大学医学院, 南京 210093

收稿日期:2023-03-10 出版日期:2023-10-23 发布日期:2023-10-26
通讯作者: 朱善元,主要从事动物育种与分子免疫学研究,E-mail:zsy221106@163.com
作者简介:张力(1990-),女,山东青岛人,讲师,博士,主要从事动物基因功能研究,E-mail:lzhang@jsahvc.edu.cn
基金资助:
江苏省种业振兴揭榜挂帅项目（JBGS〔2021〕030）；江苏省高等学校基础科学（自然科学）研究项目资助（23KJB230002）；江苏省高职院校青年教师企业实践培训项目资助；江苏农牧科技职业学院自然科学基金储备项目（NSF2022CB06）；江苏农牧科技职业学院科技创新团队项目（NSF2023TC02）

Isolation, Culture, and Identification of Skeletal Muscle Satellite Cells in Goose

ZHANG Li^1,2, XU Jialong³, HUANG Jinyu², XU Ziyue², LEI Xinnuo^1,2, LU Huipeng^1,2, ZHU Rui^1,2, SUN Weixiang^1,2, CAO Haiyue², WANG Anping^1,2, ZHU Shanyuan^1,2*

1. Engineering Technology Research Center for Modern Animal Science and Novel Veterinary Pharmaceutic Development, Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, China;
2. Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, China;
3. Medical School, Nanjing University, Nanjing 210093, China

Received:2023-03-10 Online:2023-10-23 Published:2023-10-26

摘要/Abstract

摘要： 旨在建立鹅骨骼肌卫星细胞体外分离、培养、纯化及鉴定的方法。本研究选取健康10周龄溆浦鹅仔鹅为试验材料，采用分散酶Dispase II和胶原酶II共同作用肌肉组织分离得到卫星细胞，利用差速贴壁法纯化卫星细胞，在体外培养过程中，含有20% FBS并添加bFGF的DMEM/F12生长完全培养基能较好维持卫星细胞的分化潜能，培养过程中涉及的培养皿需经基质胶特别包被处理；诱导分化出成熟肌管后，采用免疫荧光法和Western blotting检测肌球蛋白重链MyHC的表达，采用qRT-PCR检测Pax7、MyoD1、MyoG基因在诱导分化前（growth medium，GM）和分化后（differentiation medium，DM）的相对表达量，分化前后每组均3个重复。结果表明，新分离得到的卫星细胞体积较小，折光性强，贴壁后呈中间彭起两端针状的梭形结构，经免疫荧光鉴定呈Pax7阳性；诱导分化后可形成成熟多核肌管，免疫荧光检测成肌特异性标志MyHC呈阳性，统计分化指数高达78%（P<0.01）；qRT-PCR检测标志性基因Pax7、MyoD1、MyoG呈阳性，并且分化前Pax7基因相对表达量是分化后的2.22倍（P<0.01），而分化后标志性基因MyoD1、MyoG的相对表达量分别是分化前的1.90（P<0.01）和44.22倍（P<0.01）；Western blotting结果显示分化后MyHC蛋白表达水平明显升高。综上所述，本研究建立了一种基于胶原酶Ⅱ和分散酶Dispase II的混合酶分离鹅骨骼肌卫星细胞的方法，提供的培养体系能够使细胞在体外培养过程中维持巨大的分化潜能，为鹅肌肉组织生长发育分子调控机制的研究提供了良好的细胞模型。

关键词: 鹅, 骨骼肌卫星细胞, 分离, 培养, 鉴定

Abstract: The purpose of the study was to establish methods for isolation, culture, purification, and identification of goose skeletal muscle satellite cells in vitro. The healthy 10-week-old Xupu geese was selected as experimental materials. Satellite cells were isolated from the muscle with the action of Dispase II and collagenase II. Satellite cells were purified by differential adhesion to the culture dish. During in vitro culture, the complete DMEM/F12 growth medium containing 20% FBS and supplemented with bFGF could better maintain the differentiation potential of satellite cells, and the culture dishes involved in the culture process needed to be coated with matrigel specially. After the formation of mature myotubes, the expression of myosin heavy chain (MyHC) was detected by immunofluorescence and Western blotting. The relative expression levels of Pax7, MyoD1 and MyoG before (growth medium, GM) and after differentiation (differentiation medium, DM) were detected by qRT-PCR. There were 3 replicates in each group before and after differentiation. The results showed that the newly isolated satellite cells were small in size and strong in refraction. After adhering to the wall, they had a spindle-shaped structure with needle-like ends and puffed up in the middle. The cells were identified as Pax7 positive by immunofluorescence. Mature multinucleated myotubes could be formed after induction of differentiation. MyHC was positive by immunofluorescence detection. The statistical differentiation index was as high as 78% (P<0.01). The marker genes Pax7, MyoD1, and MyoG were detected positive by qRT-PCR. The relative expression of Pax7 gene before differentiation was 2.22 times (P<0.01) that after differentiation, while the relative expression of marker genes MyoD1 and MyoG after differentiation were 1.90 (P<0.01) and 44.22 times (P<0.01) that before differentiation, respectively. Western blotting results showed that the expression level of MyHC protein was significantly increased after differentiation. In this study, a method for the isolation and culture of goose skeletal muscle satellite cells by collagenase II and dispase II was established. The culture system can enable cells to maintain huge differentiation potential during the in vitro culture. The study results provide a good cell model for the research of the growth and development molecular regulation mechanism of goose muscle tissue.

Key words: goose, skeletal muscle satellite cells, isolation, culture, identification

中图分类号:

S835

张力, 许加龙, 黄锦钰, 许子月, 雷昕诺, 卢会鹏, 朱睿, 孙伟翔, 曹海月, 王安平, 朱善元. 鹅骨骼肌卫星细胞的分离培养与鉴定[J]. 畜牧兽医学报, 2023, 54(10): 4186-4195.

ZHANG Li, XU Jialong, HUANG Jinyu, XU Ziyue, LEI Xinnuo, LU Huipeng, ZHU Rui, SUN Weixiang, CAO Haiyue, WANG Anping, ZHU Shanyuan. Isolation, Culture, and Identification of Skeletal Muscle Satellite Cells in Goose[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4186-4195.

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鹅骨骼肌卫星细胞的分离培养与鉴定

Isolation, Culture, and Identification of Skeletal Muscle Satellite Cells in Goose

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