鞘磷脂对小鼠肌卫星细胞C2C12成肌分化的影响

doi:10.16431/j.cnki.1671-7236.2021.11.017

摘要/Abstract

摘要： 研究通过检测鞘磷脂对小鼠肌卫星细胞C2C12成肌分化的影响，旨在为阐明鞘磷脂对解偶联蛋白1（UCP1）基因敲入猪骨骼肌生长的影响提供理论依据。用ELISA法检测野生型猪和UCP1敲入猪背部肌肉及血清中总鞘磷脂含量；利用CCK8法检测不同浓度（0、5、20、50和100 μg/mL）鞘磷脂对C2C12细胞增殖和毒性的影响，并通过形态学观察和分化前后细胞成肌分化标记基因生肌因子5（Myf5）、生肌决定因子（MyoD）、肌细胞生成素（Myogenin）、生肌调节因子4（MRF4）的表达检测，建立C2C12成肌分化体系；在成肌分化培养基中添加上述不同浓度的鞘磷脂，诱导分化6 d后，通过形态学和Myogenin免疫荧光染色观察肌管的形成及成肌分化标记基因的mRNA表达水平检测，确定鞘磷脂的最佳添加浓度。用筛选出的最佳鞘磷脂添加浓度诱导细胞成肌分化，在2、4和6 d收集细胞，利用实时荧光定量PCR检测周期蛋白相关基因CyclinD1、CyclinE、CDK2和CDK4的表达水平，CCK8法检测诱导2 d细胞的活力。结果发现，与野生型猪相比，UCP1-KI猪背部肌肉组织中总鞘磷脂含量显著增加（P<0.05）；血清鞘磷脂含量差异不显著（P>0.05）；不同浓度鞘磷脂对未分化C2C12细胞的增殖无显著影响（P>0.05）；成肌分化6 d后，C2C12细胞形成明显的肌管，成肌分化标记基因Myf5、MyoD、Myogenin、MRF4的mRNA和蛋白水平均极显著上调（P<0.01）；与未添加鞘磷脂的对照组相比，20 μg/mL鞘磷脂组有更多肌管形成，Myogenin阳性信号和肌管融合指数均显著增加（P<0.05），Myogenin、MRF4基因的表达量显著提高（P<0.05）。利用20 μg/mL鞘磷脂诱导细胞分化，在分化2 d时，处理组CyclinE、CDK4基因表达量显著高于对照组（P<0.05），细胞活力也显著高于对照组（P<0.05）；分化6 d后，处理组CyclinD1、CyclinE、CDK2、CDK4基因表达量均显著低于对照组（P<0.05）。本研究结果表明，20 μg/mL鞘磷脂能够提高小鼠肌卫星细胞C2C12分化早期细胞活力和成肌分化效率，可为研究鞘磷脂对骨骼肌生长的影响提供一定的参考。

关键词: 鞘磷脂; C2C12细胞; 成肌分化

Abstract: The study was aimed to provide a theoretical basis for investigating the effect of sphingomyelin on development of skeletal muscle of UCP1 knock-in (UCP1-KI) pigs, through detection the function of sphingomyelin in myogenic differentiation of C2C12 mouse satellite cells. ELISA method was used to measure the content of total sphingomyelin in the back muscle and serum from both wild type and UCP1-KI pigs. CCK8 was used to detect the effects of different concentrations of sphingomyelin (0, 5, 20, 50 and 100 μg/mL) on the proliferation of C2C12 cells. The successful differentiation was established and confirmed by morphological observation, the immunofluorescence of Myogenin and the mRNA levels of the myogenic differentiation markers, including Myf5, MyoD, Myogenin and MRF4. Then, the C2C12 mouse satellite cells were treated with different concentrations of sphingomyelin during myogenic differentiation. Cells were harvested after 6 days of differentiation and the optimal sphingomyelin concentration was determined based on the formation of myotubes, and immunofluorescence staining signal of Myogenin and the upregulation of differentiation markers (Myf5, MyoD, Myogenin and MRF4). Furthermore, the gene expression levels of cyclins (CyclinD1, CyclinE, CDK2 and CDK4) in C2C12 cells at 2, 4 and 6 d of myogenic differentiation were detected by Real-time quantitative PCR, and cell viability was detected by CCK8 at 2 d of myogenic differentiation. The results showed that the content of total sphingomyelin in the back muscle of UCP1-KI pigs was significantly increased (P<0.05) compared with those from wild-type pigs after cold stimulation. No significant difference in serum sphingomyelin content (P>0.05) was observed. Sphingomyelin had no effect on the proliferation of C2C12 cells before myogenic differentiation (P>0.05). After 6 days of myogenic differentiation, myotubes was formed obviously and the mRNA levels of myogenic differentiation markers (Myf5, MyoD, Myogenin and MRF4) were significantly up-regulated (P<0.01). Compared with control group without sphingomyelin, the Myogenin positive cells and myotube fusion index were significantly increased in the 20 μg/mL sphingomyelin group (P<0.05). Furthermore, the expression levels of Myogenin and MRF4 genes were significantly increased (P<0.05). With 20 μg/mL sphingomyelin, the expression levels of CyclinD1 and CDK4 in the treatment group were significantly higher than those from control group at 2 d of differentiation (P<0.05), and the cell viability of the treatment group was significantly higher than that of the control group (P<0.05). The mRNA expression levels of CyclinD1, CyclinE, CDK2 and CDK4 in the treatment group were significantly lower than those of control group at 6 d of differentiation (P<0.05). In this study, the results revealed that the addition of 20 μg/mL sphingomyelin could improve the cell viability at the early stage of myogenic differentiation and promote the myogenic differentiation efficiency of C2C12 cells, providing some references for the effect of sphingomyelin on muscle formation.

Key words: sphingomyelin; C2C12; myogenic differentiation

中图分类号:

侯乃鹏, 王煜, 陶聪, 王彦芳. 鞘磷脂对小鼠肌卫星细胞C2C12成肌分化的影响[J]. 中国畜牧兽医, 2021, 48(11): 4074-4083.

HOU Naipeng, WANG Yu, TAO Cong, WANG Yanfang. Effect of Sphingomyelin on Myogenic Differentiation of Mouse Muscle Satellite Cells C2C12[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(11): 4074-4083.

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