可降解甲壳素/壳聚糖支架联合GMSCs来源外泌体对大鼠坐骨神经损伤的修复作用研究

Experimental study of degradable Chitin/Chitosan scaffold combined with GMSCs derived exosomes on the repair of sciatic nerve injury in rats

目的探究可降解甲壳素/壳聚糖支架联合牙龈间充质干细胞(GMSCs)来源外泌体对大鼠坐骨神经损伤的修复作用。方法选择2020年1月至2021年12月金华市中心医院的4名健康志愿者,在拔除智齿的过程中收集1mg牙龈组织进行原代培养GMSCs,采用超速离心法提取GMSCs来源外泌体,透射电子显微镜(TEM)和Westernblot检测外泌体的囊泡直径和标志蛋白CD9和CD63的表达。24只SD大鼠建立坐骨神经损伤模型后,采用随机数字表法分为支架组、支架+外泌体组和自体移植组,每组8只。支架组在坐骨神经损伤部分加入单纯甲壳素/壳聚糖支架凝胶;支架+外泌体组在坐骨神经损伤部分加入可降解甲壳素/壳聚糖支架+外泌体凝胶复合物;自体移植组采用自体移植的坐骨神经组织治疗。采用坐骨神经功能指数(SFI)评估大鼠坐骨神经结扎术后1、4和8周的运动功能,采用TEM、甲苯胺蓝染色和大体标本组织分析轴突组织的恢复,评估坐骨神经支配的肌肉重量和肌纤维重塑。结果TEM检查显示外泌体呈圆形结构,囊泡直径峰值为102nm。Westernblot分析结果显示外泌体标记CD9和CD63呈阳性。支架+外泌体组术后4、8周的SFI均明显高于支架组(均P<0.05),而自体移植组SFI明显高于支架+外泌体组和支架组(均P<0.05)。支架+外泌体组有髓轴突密度、有髓轴突直径和髓鞘厚度均明显高于支架组(均P<0.05)。但是支架组和支架+外泌体组的有髓轴突密度、有髓轴突直径和髓鞘厚度均明显低于自体移植组(均P<0.05)。支架+外泌体组的腓肠肌湿重比和肌纤维横截面积均明显大于支架组(均P<0.05),自体移植组的腓肠肌湿重比和肌纤维横截面积均明显大于支架组和支架+外泌体组(均P<0.05)。结论可降解甲壳素/壳聚糖支架联合GMSCs来源外泌体可以对鼠坐骨神经的损伤有一定修复作用。

Objective To investigate the effect of degradable Chitin/Chitosan scaffold combined with exosomes derived from gingival mesenchymal stem cells(GMSCs) on the repair of sciatic nerve injury in rats. Methods The 4 healthy people from Jinhua Municipal Central Hospital from January 2020 to December 2021 were collected. During the extraction of wisdom teeth, 1 mg of gingival tissue was collected for primary culture of GMSCs. The secretion of GMSCs was extracted by ultracentrifugation, and the vesicle diameter and marker protein(CD9 and CD63)expression of the secretion were detected by transmission electron microscope(TEM) and Western blot. The 24 SD rats were randomly divided into the stent group, the stent+exocrine group and the autologous transplantation group with 8 rats in each group. The sciatic nerve function index(SFI)was used to evaluate the motor function of rats at 1, 4 and 8 weeks after operation. The TEM, the toluidine blue staining and the gross sample tissue were used to analyze the recovery of axonal tissue, and the weight of muscle innervated by the sciatic nerve and muscle fiber remodeling were evaluated. Results The result of the TEM showed that the exocrine body was round.In addition, the size distribution of purified exosomes was measured with a nanoparticle tracking system, and its peak diameter was 102 nm. The result of the Western blot analysis showed that exosomes markers CD9 and CD63 were positive. In the comparison of motor function at 4 and 8 weeks after operation, the SFI of the stent+exocrine group was significantly higher than that of the stent group(P<0.05), while the SFI of the autotransplantation group was significantly higher than that of the stent+exocrine group and the stent group(both P<0.05). The density of myelinated axons, the diameter of myelinated axons and the thickness of myelin sheath in the stent+exocrine body group were also significantly higher than those in the stent group(both P<0.05). However, the density of myelinated axons, diameter of myelinated axons and thickness of myelin sheath in the stent group and the stent+exocrine group were significantly lower than those in the autotransplantation group(both P<0.05). The wet weight ratio of gastrocnemius muscle and the cross-sectional area of muscle fibers in the stent + exocrine group were significantly higher than those in the stent group(both P<0.05), and the wet weight ratio of gastrocnemius muscle and the cross-sectional area of muscle fibers in the autotransplantation group were significantly higher than those in the stent group and the stent + exocrine group(both P<0.05). Conclusion The degradable Chitin/Chitosan scaffolds combined with GMSCs derived exosomes can repair the rat sciatic nerve injury model.