鸡骨骼肌卫星细胞系的建立及分析

doi:10.11843/j.issn.0366-6964.2023.12.009

摘要/Abstract

摘要： 旨在通过慢病毒包装SV40-LT基因并转导PMSCs以构建鸡骨骼肌卫星细胞系。本研究采集20~30枚15胚龄健康AA鸡的胸部组织,采用混合酶消化法分离培养PMSCs。将细胞随机分为两组,每组3个重复,处理组选择SV40-LT慢病毒转染48 h后,更换含1 μg·mL^-1的嘌呤霉素筛选培养基于处理组和对照组(病毒不转染)。待对照组细胞全部死亡,对处理组细胞进行不断传代培养,最终获得鸡骨骼肌卫星细胞系。免疫荧光试验分析鸡骨骼肌卫星细胞标志基因PAX7的表达;采用CCK-8和细胞周期分析检测细胞增殖特性;血清依赖性和软琼脂分析检测其恶性转化情况;构建诱导分化模型检测其诱导分化能力。结果表明,PMSCs中有92%细胞PAX7基因呈阳性,可用于后续研究。SV40-LT基因在鸡骨骼肌卫星细胞系(IMSCs P12)中的表达是原代鸡骨骼肌卫星细胞的15倍,且连续传代至12代后,IMSCs P12与PMSCs均呈纤维样状。IMSCs P12具有更高的增殖活性且没有发生恶性转化,随后,对IMSCs P12进行诱导分化,在其增殖至70%到诱导分化1 d这一阶段,IMSCs P12与PMSCs的PAX7基因表达下调,而MyoD和MyHC基因表达上调。结果提示,本研究通过转染SV40-LT基因成功培养了鸡骨骼肌卫星细胞系,其具有与原代鸡骨骼肌卫星细胞相似的特性。该细胞系的建立为研究家禽骨骼肌相关的功能基因提供了新的平台,也为SV40-LT基因在家禽细胞永生化的研究奠定了基础。

关键词: 鸡, 骨骼肌卫星细胞, SV40-LT, 细胞系, PAX7

Abstract: The purpose of the study was to establishment of chicken skeletal satellite cell line by constructing SV40-LT gene with lentivirus and transforming primary chicken skeletal satellite cell line (PMSCs). In this study, chest tissues from 20-30 healthy AA chickens of 15 embryonic ages were collected, and a mixed enzyme digestion method was used to isolate and cultivate PMSCs. Randomly divided the cells into two groups, each with three replicates. The treatment group was transfected with SV40-LT lentivirus for 48 hours, and then replaced with medium containing 1 μg·mL^-1 puromycin for screening and cultivation based on the treatment group and the control group (virus not transfected). After all cells in the control group died, the cells in the treatment group were continuously subcultured to obtain a chicken skeletal muscle satellite cell line. Immunofluorescence assay was used to analyze the expression of the marker gene PAX7 in chicken skeletal muscle satellite cells; Cell proliferation characteristics were detected using CCK-8 and cell cycle analysis; Serum dependence and soft agar analysis were used to detect its malignant transformation status; Constructing an induced differentiation model to test its ability to induce differentiation. The 92% of cells in PMSCs were positive for the PAX7 gene, which could be used for subsequent research. Compared with primary chicken skeletal muscle satellite cells, SV40-LT was expressed 15-fold in chicken skeletal muscle satellite cell lines (IMSCs P12), and after continuous passage to 12th generation, both IMSCs P12 and PMSCs exhibited fibrous morphology. IMSCs P12 showed higher proliferative activity and did not undergo malignant transformation. Subsequently, IMSCs P12 was induced to differentiate. During the stage from 70% proliferation to 1 day of induced differentiation, the expression of PAX7 genes in IMSCs P12 and PMSCs was downregulated, while the expression of MyoD and MyHC genes was upregulated. This study successfully cultivated chicken skeletal muscle satellite cell line by transfecting the SV40-LT gene, which has similar characteristics to the primary chicken skeletal muscle satellite cells. The establishment of this cell line provided a new platform for studying functional genes related to poultry skeletal muscles, and also laid the foundation for the study of SV40-LT gene immortalization in poultry cells.

Key words: chicken, skeletal muscle satellite cells, SV40-LT, cell line, PAX7

中图分类号:

S831.2

王燕星, 张雨时, 姬海港, 刘阳, 牛玉芳, 韩瑞丽, 刘小军, 田亚东, 康相涛, 李转见. 鸡骨骼肌卫星细胞系的建立及分析[J]. 畜牧兽医学报, 2023, 54(12): 4972-4981.

WANG Yanxing, ZHANG Yushi, JI Haigang, LIU Yang, NIU Yufang, HAN Ruili, LIU Xiaojun, TIAN Yadong, KANG Xiangtao, LI Zhuanjian. Establishment and Analysis of the Chicken Skeletal Satellite Cell Line[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 4972-4981.

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