骨髓间充质干细胞来源的外泌体静脉移植对脊髓损伤的修复作用

Vein transplantation of exosomes released from bone marrow stem cells in repairing of spinal cord injury

中文关键词:  外泌体  脊髓损伤  骨髓间充质干细胞

英文关键词:Exosomes  Spinal cord injury  Bone marrow stem cells

中文摘要:

  【摘要】 目的：初步探讨大鼠骨髓间充质干细胞（bone marrow stem cells，BMSCs）来源的外泌体静脉移植对脊髓损伤（spinal cord injury，SCI）的修复作用。方法：全骨髓培养法培养大鼠BMSCs，收集P2代细胞上清，ExoQuick Precipitation提取法分离并纯化外泌体，通过透射电镜观察鉴定外泌体形态，采用Western blot鉴定外泌体表面标志蛋白CD9、CD63。通过脊髓法建立大鼠SCI模型，造模1h后尾静脉给予外泌体移植500μl（外泌体蛋白浓度为200μg/ml），采用随机数字表法分将30只大鼠为三个组：假手术组、对照组（SCI+磷酸盐溶液）、外泌体组（SCI+外泌体），均采用BBB评分、斜板实验于造模后1、3、7、14、21、28d评价大鼠运动功能恢复情况，并于术后28d处死取材，采用苏木精-伊红（HE）染色、髓鞘（luxol fast blue，LFB）染色观察各组脊髓组织形态学改变，尼氏（Nissl）染色观察神经元存活数目。结果：透射电镜下可见大量直径40~100nm的立体圆形或茶托形的小囊泡结构，外周可见完整的类脂质膜性结构，内含低电子密度物质。Western Blot显示CD9、CD63蛋白表达阳性。造模后假手术组各时间点的BBB评分和斜板评分均正常，对照组和外泌体组BBB评分和斜板评分均低于假手术组（P<0.05），造模后7、14、21、28d外泌体组BBB评分分别为6.30±0.95、12.70±1.57、16.60±1.08、17.00±0.67分，均高于同时间点对照组的2.50±1.08、6.90±0.99、10.50±0.85、12.50±1.08分（P<0.05）。造模后7、14、21、28d外泌体组斜板评分分别为43.00±3.50、55.50±4.38、62.50±2.64、65.00±3.33分，均高于同时间点对照组的34.00±3.16、43.00±4.22、49.00±4.59、52.50±4.25分（P<0.05）。造模后28d，假手术组脊髓组织HE染色、LFB染色、Nissl染色正常，对照组脊髓空洞形成、髓鞘排列紊乱、神经元数目减少，外泌体组与对照组比较脊髓组织损伤程度减轻，存活神经元数目增多（P<0.05）。结论：MSCs来源的外泌体可减轻脊髓损伤后的病理变化，改善运动功能，促进脊髓损伤后神经功能修复。

英文摘要:

  【Abstract】 Objectives: To investigate whether the exosomes derived from bone marrow stem cells(BMSCs) by intravenous infusion can promote the regeneration of injured spinal cord. Methods: BMSCs from adult rats were enriched and expanded by whole bone marrow culture in vitro, collecting the cell supernatant of P2 generation, by using the ExoQuick Precipitation for separation and purification of exosomes, the exosome markers, CD9, CD63 were analyzed by using Western blot. The MSCs-Exosomes morphology was assessed by transmission electron microscopy(TEM). The rats were induced by spinal cord strike machine to establish the spinal cord models． Exosomes of 500μl(final concentrate of 200μg/ml) were infused into tail vein． The rats were randomly divided into the following three groups(n=10/group): sham group, control group(SCI+PBS) and exosomes group(SCI+exosomes). Neurological functions were evaluated by the Basso-Beattie-Bresnahan(BBB) score and the slope plate experiment score at 1, 3, 7, 14, 21, 28 days after modeling. Histomorphological changes of spinal cord were observed at 28 days after modeling by hematoxylin-eosin(HE) staining and luxol fast blue(LFB) staining, Survivor number of nerve cells was observed at 28 days after modeling by Nissl staining. Results: TEM observation of MSCs-Exosomes revealed the presence of spherical vesicles with a typical cup-shape. The size distribution profile displayed a homogeneous population from 40-100nm, which was consistent with the literature description, MSCs-Exosomes were found to express high levels of CD9, CD63 by using Western blot. After modeling, BBB score and sloping plate experiment score in each time point were normal in sham group, but in control group and exosomes group were significantly lower than those in sham group(P<0.05), the scores of BBB in exosomes group were 6.30±0.95, 12.70±1.57, 16.60±1.08, 17.00±0.67 at 7, 14, 21, 28 days respectively after modeling, which were higher than those in control group of 2.50±1.08, 6.90±0.99, 10.50±0.85, 12.50±1.08(P<0.05) respectively, the scores of slope plate experiment in exosomes group were 43.00±3.50, 55.50±4.38, 62.50±2.64, 65.00±3.33 respectively at 7, 14, 21, 28 days after modeling, which were higher than those in control group of 34.00±3.16, 43.00±4.22, 49.00±4.59, 52.50±4.25(P<0.05) respectively． HE, LFB and Nissl staining showed nerve cells with normal morphology in sham group at 28 days after modeling, in control group， spinal cord porosis， disorder of myelin sheath and necrosis of nerve cells were found. The injured spinal cord was restored better and the number of neuronal survival was better in exosomes group than that in control group． TEM observation of MSCs-Exosomes revealed the presence of spherical vesicles, with a typical three-dimensional circular or cup-shape. The size distribution profile displayed a homogeneous population from 40-100nm with a complete lipid membrane structure in the peripheral region with low density content. Conclusions: Intravenous administration of MSCs-Exosomes promotes the restoration of injured spinal cord and improves motor functions， which plays a protective role in the repair of spinal cord injury．