Department of Integrative Medical Biology, Section for Anatomy, Umeå University, SE-901 87, Umeå, Sweden.
Department of Surgical and Perioperative Sciences, Section for Hand and Plastic Surgery, Umeå University, Umeå, Sweden.
Stem Cell Res Ther. 2021 Mar 4;12(1):162. doi: 10.1186/s13287-021-02230-y.
Recovery of muscle function after peripheral nerve injury is often poor, and this can be attributed to muscle fiber atrophy and cell death. In the current study, we have investigated the effects of stromal vascular fraction (SVF) on muscle cell apoptosis and its potential to preserve muscle tissue following denervation.
Rat gastrocnemius muscle was denervated by sciatic nerve transection. At 2 and 4 weeks after injury, muscles were examined histologically and apoptosis was measured using TUNEL assay and PCR array for a range of apoptotic genes. Additionally, an in vitro TNF-α apoptosis model was established using SVF cells co-cultured indirectly with primary rat myoblasts. Annexin V and TUNEL were used together with Western blotting to investigate the signaling pathways.
Denervated muscles showed significantly higher TUNEL reactivity at 2 and 4 weeks following nerve injury, and an increased expression of caspase family genes, mitochondria-related apoptotic genes, and tumor necrosis factor family genes. In cultured rat primary myoblasts, Annexin V labeling was significantly increased at 12 h after TNF-α treatment, and this was followed by a significant increase in TUNEL reactivity at 48 h. Western blotting showed that caspase-7 was activated/cleaved as well as the downstream substrate, poly (ADP-ribose) polymerase (PARP). Co-culture of myoblasts with SVF significantly reduced all these measures of apoptosis. Bax and Bcl-2 levels were not changed suggesting that the TNF-α-induced apoptosis occurred via mitochondria-independent pathways. The protective effect of SVF was also shown in vivo; injections of SVF cells into denervated muscle significantly improved the mean fiber area and diameter, as well as reduced the levels of TUNEL reactivity.
This study provides new insights into how adipose tissue-derived cells might provide therapeutic benefits by preserving muscle tissue.
周围神经损伤后肌肉功能的恢复往往很差，这可归因于肌肉纤维萎缩和细胞死亡。在本研究中，我们研究了基质血管成分 (SVF) 对肌肉细胞凋亡的影响及其在去神经支配后保护肌肉组织的潜力。
通过坐骨神经切断术使大鼠腓肠肌去神经支配。在损伤后 2 和 4 周，通过组织学检查和 TUNEL 测定法以及一系列凋亡基因的 PCR 阵列来测量肌肉细胞凋亡。此外，使用 SVF 细胞与原代大鼠肌母细胞间接共培养，建立 TNF-α 诱导的体外细胞凋亡模型。使用 Annexin V 和 TUNEL 以及 Western blot 一起研究信号通路。
去神经支配的肌肉在神经损伤后 2 和 4 周时 TUNEL 反应性明显升高，并且 caspase 家族基因、线粒体相关凋亡基因和肿瘤坏死因子家族基因的表达增加。在培养的大鼠原代肌母细胞中，TNF-α 处理后 12 小时 Annexin V 标记明显增加，48 小时后 TUNEL 反应性明显增加。Western blot 显示 caspase-7 被激活/切割，以及下游底物聚（ADP-核糖）聚合酶（PARP）。肌母细胞与 SVF 的共培养显著降低了所有这些凋亡指标。Bax 和 Bcl-2 水平没有改变，表明 TNF-α 诱导的凋亡是通过线粒体非依赖性途径发生的。SVF 的保护作用也在体内得到证实；将 SVF 细胞注射到去神经支配的肌肉中显著改善了平均纤维面积和直径，并降低了 TUNEL 反应性。
本研究为脂肪组织衍生细胞如何通过保护肌肉组织提供治疗益处提供了新的见解。
