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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摘要
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200μL,C组注射含100μg ADSCs来源外泌体的PBS液200μL,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 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.
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 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.
引文
1Castillo-Galván ML,Martínez-Ruiz FM,de la Garza-Castro O,etal.Study of peripheral nerve injury in trauma patients.Gac MedMex,2014,150(6):527-532.
2Sadeghian H,Wolfe GI.Therapy update in nerve,neuromuscularjunction and myopathic disorders.Curr Opin Neurol,2010,23(5):496-501.
3Cooney DS,Wimmers EG,Ibrahim Z,et al.Mesenchymal stemcells enhance nerve regeneration in a rat sciatic nerve repair andhindlimb transplant model.Sci Rep,2016,6:31306.
4Chaput N,Ta?eb J,Schartz NE,et al.Exosome-based immu-notherapy.Cancer Immunol Immunother,2004,53(3):234-239.
5Yue Y,Yang X,Zhang L,et al.Low-intensity pulsed ultrasound upregulates pro-myelination indicators of Schwann cells enhanced by co-culture with adipose-derived stem cells.Cell Prolif,2016,49(6):720-728.
6李超然,黄桂林,王帅.间充质干细胞来源外泌体促进损伤组织修复与再生的应用与进展.中国组织工程研究,2018,22(1):133-139.
7Hervera A,Virgiliis De F,Palmisano I,et al.Reactive oxygen species regulate axonal regeneration through the release of exosomal NADPH oxidase 2 complexes into injured axons.Nat Cell Biol,2018,20(3):307-319.
8Xu B,Zhang Y,Du XF,et al.Neurons secrete miR-132-containing exosomes to regulate brain vascular integrity.Cell Res,2017,27(7):882-897.
9Nielsen E?,Chen L,Hansen JO,et al.Optimizing osteogenic differentiation of ovine adipose-derived stem cells by osteogenic induction medium and FGFb,BMP2,or NELL1 in vitro.Stem Cells Int,2018,2018:9781393.
10张静,易阳艳,阳水发,等.脂肪干细胞来源外泌体对人脐静脉血管内皮细胞增殖、迁移及管样分化的影响.中国修复重建外科杂志,2018,32(10):1351-1357.
11林耀发,宗海洋,胡显腾,等.大鼠坐骨神经损伤后Spastin表达变化的实验研究.中国修复重建外科杂志,2017,31(1):80-84.
12Mor D,Kendig MD,Kang JWM,et al.Peripheral nerve injury impairs the ability to maintain behavioural flexibility following acute stress in the rat.Behav Brain Res,2017,328:123-129.
13Cobianchi S,Jaramillo J,Luvisetto S,et al.Botulinum neurotoxin Apromotes functional recovery after peripheral nerve injury by increasing regeneration of myelinated fibers.Neuroscience,2017,359:82-91.
14Wang JT,Medress ZA,Barres BA.Axon degeneration:molecular mechanisms of a self-destruction pathway.J Cell Biol,2012,196(1):7-18.
15Kikuchi S,Ninomiya T,Kohno T,et al.Cobalt inhibits motility of axonal mitochondria and induces axonal degeneration in cultured dorsal root ganglion cells of rat.Cell Biol Toxicol,2018,34(2):93-107.
16Zhang Y,Chopp M,Liu XS,et al.Exosomes derived from mesenchymal stromal cells promote axonal growth of cortical neurons.Mol Neurobiol,2017,54(4):2659-2673.
17周敏,洪莉,胡鸣,等.外泌体在周围神经损伤中的研究进展.医学综述,2017,23(13):2497-2500.
18Tassew NG,Charish J,Shabanzadeh AP,et al.Exosomes mediate mobilization of autocrine Wnt10b to promote axonal regeneration in the injured CNS.Cell Rep,2017,20(1):99-111.
19Gomez-Sanchez JA,Pilch KS,van der Lans M,et al.After nerve injury,lineage tracing shows that myelin and remak Schwann cells elongate extensively and branch to form repair Schwann cells,which shorten radically on remyelination.J Neurosci,2017,37(37):9086-9099.
20Clements MP,Byrne E,Camarillo Guerrero LF,et al.The wound microenvironment reprograms Schwann cells to invasive mesenchymal-like cells to drive peripheral nerve regeneration.Neuron,2017,96(1):98-114.
21Takamatsu H,Takegahara N,Nakagawa Y,et al.Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II.Nat Immunol,2010,11(7):594-600.
2Sadeghian H,Wolfe GI.Therapy update in nerve,neuromuscularjunction and myopathic disorders.Curr Opin Neurol,2010,23(5):496-501.
3Cooney DS,Wimmers EG,Ibrahim Z,et al.Mesenchymal stemcells enhance nerve regeneration in a rat sciatic nerve repair andhindlimb transplant model.Sci Rep,2016,6:31306.
4Chaput N,Ta?eb J,Schartz NE,et al.Exosome-based immu-notherapy.Cancer Immunol Immunother,2004,53(3):234-239.
5Yue Y,Yang X,Zhang L,et al.Low-intensity pulsed ultrasound upregulates pro-myelination indicators of Schwann cells enhanced by co-culture with adipose-derived stem cells.Cell Prolif,2016,49(6):720-728.
6李超然,黄桂林,王帅.间充质干细胞来源外泌体促进损伤组织修复与再生的应用与进展.中国组织工程研究,2018,22(1):133-139.
7Hervera A,Virgiliis De F,Palmisano I,et al.Reactive oxygen species regulate axonal regeneration through the release of exosomal NADPH oxidase 2 complexes into injured axons.Nat Cell Biol,2018,20(3):307-319.
8Xu B,Zhang Y,Du XF,et al.Neurons secrete miR-132-containing exosomes to regulate brain vascular integrity.Cell Res,2017,27(7):882-897.
9Nielsen E?,Chen L,Hansen JO,et al.Optimizing osteogenic differentiation of ovine adipose-derived stem cells by osteogenic induction medium and FGFb,BMP2,or NELL1 in vitro.Stem Cells Int,2018,2018:9781393.
10张静,易阳艳,阳水发,等.脂肪干细胞来源外泌体对人脐静脉血管内皮细胞增殖、迁移及管样分化的影响.中国修复重建外科杂志,2018,32(10):1351-1357.
11林耀发,宗海洋,胡显腾,等.大鼠坐骨神经损伤后Spastin表达变化的实验研究.中国修复重建外科杂志,2017,31(1):80-84.
12Mor D,Kendig MD,Kang JWM,et al.Peripheral nerve injury impairs the ability to maintain behavioural flexibility following acute stress in the rat.Behav Brain Res,2017,328:123-129.
13Cobianchi S,Jaramillo J,Luvisetto S,et al.Botulinum neurotoxin Apromotes functional recovery after peripheral nerve injury by increasing regeneration of myelinated fibers.Neuroscience,2017,359:82-91.
14Wang JT,Medress ZA,Barres BA.Axon degeneration:molecular mechanisms of a self-destruction pathway.J Cell Biol,2012,196(1):7-18.
15Kikuchi S,Ninomiya T,Kohno T,et al.Cobalt inhibits motility of axonal mitochondria and induces axonal degeneration in cultured dorsal root ganglion cells of rat.Cell Biol Toxicol,2018,34(2):93-107.
16Zhang Y,Chopp M,Liu XS,et al.Exosomes derived from mesenchymal stromal cells promote axonal growth of cortical neurons.Mol Neurobiol,2017,54(4):2659-2673.
17周敏,洪莉,胡鸣,等.外泌体在周围神经损伤中的研究进展.医学综述,2017,23(13):2497-2500.
18Tassew NG,Charish J,Shabanzadeh AP,et al.Exosomes mediate mobilization of autocrine Wnt10b to promote axonal regeneration in the injured CNS.Cell Rep,2017,20(1):99-111.
19Gomez-Sanchez JA,Pilch KS,van der Lans M,et al.After nerve injury,lineage tracing shows that myelin and remak Schwann cells elongate extensively and branch to form repair Schwann cells,which shorten radically on remyelination.J Neurosci,2017,37(37):9086-9099.
20Clements MP,Byrne E,Camarillo Guerrero LF,et al.The wound microenvironment reprograms Schwann cells to invasive mesenchymal-like cells to drive peripheral nerve regeneration.Neuron,2017,96(1):98-114.
21Takamatsu H,Takegahara N,Nakagawa Y,et al.Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II.Nat Immunol,2010,11(7):594-600.