褪黑素对脂多糖刺激的滩羊骨骼肌卫星细胞炎性因子表达的影响

doi:10.16431/j.cnki.1671-7236.2021.09.029

摘要/Abstract

摘要： 试验旨在探究褪黑素（MLT）对脂多糖（LPS）诱导的滩羊骨骼肌卫星细胞炎性反应的影响。选取滩羊妊娠30日龄胎儿为研究材料，使用Ⅳ型胶原酶分离获得滩羊骨骼肌卫星细胞并进行体外培养，添加相应的诱导试剂对滩羊骨骼肌卫星细胞进行诱导分化，利用免疫荧光检测骨骼肌卫星细胞表面标记物CD29、CD44、CD73及Vimentin的表达；在无胎牛血清的培养基中添加不同浓度（0、5、8和10 μg/mL）的LPS培养骨骼肌卫星细胞，利用实时荧光定量PCR （qRT-PCR）检测白细胞介素-6（IL-6）、IL-8和肿瘤坏死因子-α（TNF-α）等炎性相关因子mRNA水平变化，筛选最佳的LPS处理浓度；在无胎牛血清的培养基中添加不同浓度（0.1和0.5 μmol/L） MLT与最佳浓度LPS共培养骨骼肌卫星细胞，利用实时荧光定量PCR检测IL-6、IL-8和干扰素-γ（IFN-γ）等炎性相关因子mRNA水平变化。免疫荧光结果显示，滩羊骨骼肌卫星细胞表面标志物CD29、CD44、CD73及Vimentin表达呈阳性，且具有诱导成肌、成脂和成骨分化的特性。实时荧光定量PCR结果显示，与对照组相比，不同浓度的LPS处理均可显著增加细胞炎性相关因子基因的表达（P<0.05），并且8 μg/mL LPS处理时炎性相关因子mRNA表达最强；当MLT与LPS共培养骨骼肌卫星细胞时，与LPS单独处理相比，0.5 μmol/L MLT与LPS共同处理组中IL-6 mRNA表达水平显著降低（P<0.05）。综上表明，MLT具有缓解LPS刺激的滩羊骨骼肌卫星细胞炎性反应的作用。本试验结果为进一步开展MLT缓解LPS诱导的骨骼肌卫星细胞炎性反应的调控机制研究奠定了基础。

关键词: 滩羊; 骨骼肌卫星细胞; 脂多糖(LPS); 褪黑素(MLT); 炎性

Abstract: The aim of this study was to investigate the effect of melatonin (MLT) on lipopolysaccharide (LPS)-induced inflammatory response in skeletal muscle satellite cells in Tan sheep. In this study, the 30-day-old fetus of Tan sheep was selected. The skeletal muscle satellite cells were isolated by collagenase Ⅳ, and cultured in vitro, the corresponding inductive reagent was added to induce differentiation of skeletal muscle satellite cells, and the expression of surface markers CD29, CD44, CD73 and Vimentin were analyzed by immunofluorescence. The skeletal muscle satellite cells were cultured at different doses (0, 5, 8 and 10 μg/mL) of LPS in the medium without fetal bovine serum, and the mRNA levels of inflammatory related factors such as interleukin-6 (IL-6), IL-8 and tumor necrosis factor-α (TNF-α) were detected by Real-time quantitative PCR (qRT-PCR), and the optimal concentration of LPS was selected. Skeletal muscle satellite cells were co-cultured with different concentrations 0.1 and 0.5 μmol/L of MLT and LPS of optimal concentration, and the mRNA levels of IL-6, IL-8 and interferon-γ (IFN-γ) were detected by qRT-PCR. The immunofluorescence results showed that the expression of the surface markers CD29, CD44, CD73 and Vimentin of skeletal muscle satellite cells were positive, and the cells had the properties of myogenic, lipogeneic and osteogenic differentiation. qRT-PCR results showed that different concentrations of LPS treatments could significantly increase the genes expression of inflammatory related factor when compared with control group (P<0.05), and the mRNA expression of inflammatory factors was the strongest when treated with 8 μg/mL LPS. When the cells were co-cultured by MLT and LPS, 0.5 μmol/L MLT could significantly decrease the expression of IL-6 induced by LPS when compared with LPS group (P<0.05). In conclusion, MLT could alleviate LPS-induced inflammatory response in skeletal muscle satellite cells in Tan sheep. The results laid a foundation for further research on the regulatory mechanism of MLT alleviating the inflammatory response of skeletal muscle satellite cells induced by LPS.

Key words: Tan sheep; skeletal muscle satellite cells; lipopolysaccharide (LPS); melatonin (MLT); inflammation

中图分类号:

S826

马思佳, 刘媛, 汪序忠, 李亭亭, 段星, 杨松柏, 宋丹, 李向臣. 褪黑素对脂多糖刺激的滩羊骨骼肌卫星细胞炎性因子表达的影响[J]. 中国畜牧兽医, 2021, 48(9): 3378-3386.

MA Sijia, LIU Yuan, WANG Xuzhong, LI Tingting, DUAN Xing, YANG Songbai, SONG Dan, LI Xiangchen. Effects of Melatonin on the Expression of Inflammatory Factors in Skeletal Muscle Satellite Cells Stimulated by LPS in Tan Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3378-3386.

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