牛骨骼肌卫星细胞的分离鉴定和诱导分化

doi:10.16431/j.cnki.1671-7236.2020.10.024

摘要/Abstract

摘要： 为了在体外研究西门塔尔牛的肌肉生长发育过程并建立牛骨骼肌卫星细胞的原代细胞模型。本研究采用0.2%的Ⅱ型胶原酶消化后再使用0.25%胰酶消化获得牛骨骼肌卫星细胞（bovine skeletal satellite cell，BSSC），并利用差速贴壁法分离获得纯化后的BSSC，使用RT-PCR、免疫荧光染色和Western blotting等方法鉴定BSSC，使用成脂诱导剂诱导其分化为脂肪细胞，油红O染色鉴定分化后细胞的成脂能力，使用2%马血清诱导其分化为肌细胞，RT-PCR鉴定静止期PAX7基因和肌细胞的标记基因MyoG在诱导前后表达量的变化。结果发现，分离纯化得到的BSSC呈梭形或纺锤形，细胞形态饱满，折光性强，随着培养时间的延长，细胞由原来的无序生长变为有序生长；RT-PCR检测发现，BSSC表面标志基因Desmin、c-Met、Myf5和特异性标志基因PAX7均呈阳性表达；免疫荧光染色及Western blotting结果显示，PAX7和MyoD基因均呈阳性表达；成脂细胞诱导分化后被油红O大量染色并观察到大量脂滴出现；成肌细胞诱导分化后静止时期PAX7基因表达量诱导前高于诱导分化后，而成肌标记基因MyoG表达量诱导前低于诱导分化后。综上，本研究成功分离获得BSSC，并证明BSSC具有分化成脂肪细胞和肌细胞的能力，为体外研究西门塔尔牛肉制品调控机制提供原代细胞模型。

关键词: 牛骨骼肌卫星细胞; PAX7; 诱导分化; 成肌细胞; 成脂细胞

Abstract: In order to study the muscle growth and development process of Simmental cattle in vitro and establish a primary cell model of bovine skeletal muscle satellite cells.Bovine skeletal satellite cells (BSSC) were obtained by digestion with 0.2% type Ⅱ collagenase and then digested with 0.25% trypsin-EDTA,and using the differential adhesion method to separate and obtain purified BSSC,reverse transcription PCR (RT-PCR),immunofluorescence staining and Western blotting were used to identify BSSC.BSSC was induced differentiation into adipocytes by adipogenic inducers.Oil red O staining was used to identify the adipogenic capacity of differentiated cells.It was also induced differentiation into myocytes by 2% horse serum,and RT-PCR was used to identify the expression of PAX7 and MyoG marker genes in resting phase before and after induction.The results showed that the isolated and purified BSSC was fusiform or spindle-shaped,with full cell morphology and strong refraction.With the extension of the culture time,the cells changed from disordered growth to ordered growth.It was found that BSSC surface marker genes Desmin,c-Met,Myf5 and specific marker Paired box 7 (PAX7) were all positively expressed by RT-PCR detection.Immunofluorescence staining and Western blotting showed that PAX7 and MyoD gene were both positively expressed.Adipocyte induction after differentiation,it was heavily stained with oil red O and a large number of lipid droplets were observed.The expression of PAX7 gene in the resting period after induction of myoblasts was higher than that before induction,but the expression of myogenin marker gene MyoG was opposite.The above results indicated that this study successfully isolated BSSC and proved that BSSC had the ability to differentiate into adipocytes and myocytes,which provided a primary cell model for the in vitro study of the regulation mechanism of Simmental beef products.

Key words: bovine skeletal muscle satellite cells; PAX7; induced differentiation; myoblasts; adipocytes

中图分类号:

Q291

赵新艳, 郭妍婷, 陈俊贞, 李泽宇, 史慧君, 付强. 牛骨骼肌卫星细胞的分离鉴定和诱导分化[J]. 中国畜牧兽医, 2020, 47(10): 3249-3258.

ZHAO Xinyan, GUO Yanting, CHEN Junzhen, LI Zeyu, SHI Huijun, FU Qiang. Isolation,Identification and Differentiation of Bovine Skeletal Muscle Satellite Cells[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(10): 3249-3258.

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