中国修复重建外科杂志

中国修复重建外科杂志

脂肪干细胞来源外泌体对周围神经损伤后再生作用的实验研究

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目的 探讨脂肪干细胞(adipose-derived stem cells,ADSCs)来源外泌体对周围神经损伤后再生的影响,为周围神经损伤寻找新的治疗方法。 方法 将 36 只成年 SD 大鼠(雌雄不限,体质量 220~240 g)随机分成 3 组,每组 12 只。A 组为正常对照组,B 组为坐骨神经挤压损伤组,C 组为 ADSCs 来源外泌体治疗坐骨神经挤压损伤组。A 组仅暴露坐骨神经后直接缝合切口,B、C 组制备坐骨神经挤压损伤模型;术后次日开始,A、B 组于大鼠尾静脉注射 PBS 液 200 μL,C 组注射含 100 μg ADSCs 来源外泌体的 PBS 液 200 μL,每周注射 1 次,连续 12 周。注射结束 1 周后处死大鼠,取损伤处坐骨神经,分别行大体观察、HE 染色观察神经束情况、TUNEL 检测坐骨神经雪旺细胞(Schwann cells,SCs)凋亡情况,透射电镜观察坐骨神经超微结构和 SCs 自噬情况。 结果 大体观察示 A 组患肢无明显异常,B、C 组患肢瘫痪、肌肉萎缩,但 C 组瘫痪及肌肉萎缩程度轻于 B 组。HE 染色示,A 组神经束膜形态规则;B 组神经束形态破坏、束膜不规则,有较多无细胞结构和组织碎片;C 组神经束膜较完整,明显优于 B 组。TUNEL 检测示,B、C 组 SCs 凋亡细胞数显著多于 A 组,B 组显著多于 C 组,差异均有统计学意义(P<0.01)。透射电镜观察示,B、C 组 SCs 自噬体较 A 组明显增加,但 C 组少于 B 组。 结论 ADSCs 来源外泌体对周围神经损伤后再生有一定促进作用,其机制可能与减少 SCs 凋亡、抑制其自噬、减轻神经瓦勒变性有关。

Objective To investigate the effects of exosomes from adipose-derived stem cells (ADSCs) on peripheral nerve regeneration, and to find a new treatment for peripheral nerve injury. Methods Thirty-six adult Sprague Dawley (SD) rats (male or female, weighing 220-240 g) were randomly divided into 3 groups (n=12). Group A was the control group; group B was sciatic nerve injury group; group C was sciatic nerve injury combined with exosomes from ADSCs treatment group. The sciatic nerve was only exposed without injury in group A, and the sciatic nerve crush injury model was prepared in groups B and C. The SD rats in groups A and B were injected with pure PBS solution of 200 μL via tail veins; the SD rats in group C were injected with pure PBS solution of 200 μL containing 100 μg exosomes from ADSCs, once a week and injected for 12 weeks. At 1 week after the end of the injection, the rats were killed and the sciatic nerves were taken at the part of injury. The sciatic nerve fiber bundles were observed by HE staining; the SCs apoptosis of the sciatic nerve tissue were detected by TUNEL staining; the ultrastructure and SCs autophagy of the sciatic nerve were observed by transmission electron microscope. Results Gross observation showed that there was no obvious abnormality in the injured limbs of group A, but there were the injured limbs paralysis and muscle atrophy in groups B and C, and the degree of paralysis and muscle atrophy in group C were lighter than those in group B. HE staining showed that the perineurium of group A was regular; the perineurium of group B was irregular, and there were a lot of cell-free structures and tissue fragments in group B; the perineurium of group C was more complete, and significantly well than that of group B. TUNEL staining showed that the SCs apoptosis was significantly increased in groups B and C than in group A, in group B than in group C (P<0.01). Transmission electron microscope observation showed that the SCs autophagosomes in groups B and C were significantly increased than those in group A, but the autophagosomes in group C were significantly lower than those in group B. Conclusion The exosomes from ADSCs can promote the peripheral nerve regeneration. The mechanism may be related to reducing SCs apoptosis, inhibiting SCs autophagy, and reducing nerve Wallerian degeneration.

关键词: 外泌体; 脂肪干细胞; 周围神经损伤; 神经再生; 大鼠

Key words: Exosome; adipose-derived stem cells; peripheral nerve injury; nerve regeneration; rat

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