Human primary skeletal muscle-derived myoblasts and fibroblasts reveal different senescent phenotypes
Human primary skeletal muscle-derived myoblasts and fibroblasts reveal different senescent phenotypes
复制标题
人类原代骨骼肌来源的成肌细胞和成纤维细胞揭示了不同的衰老表型
DOI：
10.1002/rco2.67
复制
发表时间：
2022
影响因子：
--
通讯作者：
Francis T
中科院分区：
文献类型：
--
作者：
Francis T
背景与年龄相关的肌肉质量和质量损失（肌肉减少症）有许多影响因素，其中之一可能是细胞衰老，但这在人类骨骼肌中没有得到很好的定义。方法从健康成年男性（n = 6, 22 ± 1 年）的股外侧肌活检样本中分离原代细胞，进行分类（磁激活细胞分选）和化学诱导（阿霉素，DOX，0.2 μM）到衰老状态。这使得可以对两种主要的骨骼肌来源的细胞类型进行并行和同时的研究：卫星细胞来源的 CD56+ve/desmin+vemyoblasts（肌肉前体细胞）和 CD56−ve/TE7+ve成纤维细胞（纯度>95%）。 DOX 处理后，对两种细胞类型进行长达 35 天的跟踪，结合定量免疫细胞化学和 qRT-PCR 检测衰老标记物和衰老相关分泌表型 (SASP) 因子。结果成肌细胞和成纤维细胞在许多研究的衰老标记物中显示出时间和定量差异。 p16 蛋白表达随着时间的推移而增加 (P< 0.0001)，细胞类型之间没有差异，而在 mRNA 水平上，成肌细胞从治疗后 4 天开始显示 p16 表达增加 (FC = 3.03 ± 0.99)，而在成纤维细胞中，这种情况出现在 DOX 治疗后 10 天和 35 天（FC=8.09±2.46，P<0.0001）。成肌细胞 (FC = 8.83 ± 1.72) 和成纤维细胞 (FC = 2.33 ± 1.10) 均显示 p21 mRNA 显着增加 (P < 0.0001)，在 35 天的时间过程中，成肌细胞群中的 p21 mRNA 仍然升高，但从 DOX 治疗后 4 天起，成纤维细胞中的 p21 mRNA 恢复到基线。 DOX 治疗后 35 天内，成肌细胞和成纤维细胞的所有细胞群均达到 100% SA-β-Gal 阳性细胞（P<0.05）。 DOX 治疗后 1 天，成肌细胞中 γH2aX 表达（DNA 损伤的标志物）增加（FC = 3.3 ± 1.1，P < 0.05），但在 DOX 治疗后 4 天内恢复到基线，而成纤维细胞显示出类似的趋势，但未达到统计学显着性。 DOX 处理后 1 天，两种细胞类型中增殖标志物 Ki67 的表达均显着降低，并且在整个时间过程中均保持不变（FC = 0.11 ± 0.07，P < 0.0001）。随着时间的推移，选定的 SASP 因子（例如 PAI-1、MMP3 和 IGFBP3，P < 0.05）的 mRNA 表达也观察到显着变化。结论细胞衰老和肌少症均未完全了解。从同一组织样本中获得的人类原代成肌细胞和成纤维细胞的现有数据表明，衰老是一个复杂的、非线性的、动态的细胞过程，显示出细胞内和细胞间的变异性。
BackgroundThe age‐related loss of muscle mass and quality, sarcopenia, has many contributing factors, one of which may be cellular senescence, but this is not well defined in human skeletal muscle.MethodPrimary cells were isolated from biopsy samples of the vastus lateralis muscle from healthy adult males (n= 6, 22 ± 1 years), sorted (magnetic activated cell sorting) and chemically induced (doxorubicin, DOX, 0.2 μM) to a senescent state. This allowed the parallel and simultaneous investigation of the two main skeletal muscle‐derived cell types: satellite cell‐derived CD56+ve/desmin+vemyoblasts (muscle precursor cells) and CD56− ve/TE7+vefibroblasts (at >95% purity). Both cell types were followed for up to 35 days post DOX treatment with a combination of quantitative immunocytochemistry and qRT‐PCR for senescent markers and senescence‐associated secretory phenotype (SASP) factors.ResultsMyoblasts and fibroblasts showed temporal and quantitative differences in many of the senescence markers studied. p16 protein expression increased across the time course (P< 0.0001) with no difference between cell types, whereas at the mRNA level, myoblasts showed increased p16 expression from 4 days post treatment (FC = 3.03 ± 0.99), and in fibroblasts, this appeared later at 10 and 35 days post DOX treatment (FC = 8.09 ± 2.46,P< 0.0001). Both myoblasts (FC = 8.83 ± 1.72) and fibroblasts (FC = 2.33 ± 1.10) showed significant increases in p21 mRNA (P< 0.0001), which remained elevated in the myoblast cell populations across the 35‐day time course but returned to baseline in the fibroblasts from 4 days post DOX treatment. Within 35 days post DOX treatment, all cell populations of both myoblasts and fibroblasts had reached 100% SA‐β‐Gal‐positive cells (P< 0.05). γH2aX expression (a marker of DNA damage) increased 1 day after DOX treatment in the myoblasts (FC = 3.3 ± 1.1,P< 0.05) but returned to baseline within 4 days post DOX treatment, whereas fibroblasts showed a similar trend that did not reach statistical significance. Significant reductions in expression of the proliferation marker Ki67 1 day post DOX treatment were seen in both cell types and were maintained throughout the time course (FC = 0.11 ± 0.07,P< 0.0001). Significant changes over the time course were also observed in mRNA expression of selected SASP factors (e.g. PAI‐1, MMP3, and IGFBP3,P< 0.05).ConclusionsNeither cellular senescence nor sarcopenia is fully understood. The present data on human primary myoblasts and fibroblasts obtained from the same tissue sample show that senescence is a complex, non‐linear, and dynamic cellular process which shows intra‐ and inter‐cell variability.