脂肪干细胞来源的外泌体对大鼠肌腱愈合及对局部外周神经肽表达的影响  

Effects of adipose stem cell‑derived exosomes on rat tendon healing and its impact on the periphery neuropeptides expression

作　　者：许宏涛 张昊 沈凯 周皓 宋黄鹤[1] 郭敦明[1] 顾晓园[1] 王广玲[1] 王青[1] 殷国勇[1] Xu Hongtao;Zhang Hao;Shen Kai;Zhou Hao;Song Huanghe;Guo Dunming;Gu Xiaoyuan;Wang Guangling;Wang Qing;Yin Guoyong(Department of Orthopedics,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China;School of Biological Science and Medical Engineering,Southeast University,Nanjing 210000,China)

机构地区：[1]南京医科大学第一附属医院骨科,南京210029 [2]东南大学生物科学与医学工程学院,南京210000

出　　处：《中华医学杂志》2025年第7期544-553,共10页National Medical Journal of China

基　　金：江苏省科技计划专项资金青年基金(SBK2023041609、BK20230737);江苏省卓越博士后计划(2023ZB231);中国博士后科学基金(2023M741466);南京留学人员科技创新项目(2023)。

摘　　要：目的探讨脂肪干细胞来源的外泌体对大鼠肌腱愈合的影响,并评估愈合部位外周神经肽的时空表达变化。方法首先预混10%(质量浓度,w/v)的明胶甲基丙烯酰氯(GelMA)、0.25%(w/v)的光引发剂和脂肪干细胞来源的外泌体,然后置于405 nm波长的紫外光下照射30 s以制备可注射外泌体水凝胶备用。建立SD大鼠的跟腱横断后缝合修复模型,共分为4组,包括健康对照组(C组,选用单纯缝合修复组对侧下肢)、单纯缝合修复组(S组)、缝合修复后注射单纯水凝胶组(H组)和缝合修复后注射含有脂肪来源干细胞外泌体的水凝胶组(E组),分别在术后7、14和28 d取材。通过苏木精‑伊红(HE)染色观察肌腱愈合过程的形态学改变,免疫组化染色分析肌腱形成标记物,如Tenomodulin(TNMD)和外周神经肽,包括生长关联蛋白43(GAP43)、S100钙结合蛋白B(S100B)、神经肽Y(NPY)、降钙素基因相关肽(CGRP)和P物质(SP),在肌腱愈合过程中的变化趋势。此外,物理形态测量,包括肌腱长度、肌腱横截面积、腓肠肌重量与总体重的百分比,评估形态重塑;力学测试,包括最大失效拉力、刚度、弹性模量,评估生物力学重塑。结果E组的TNMD基因表达水平为2.12±0.43,均高于H组的1.26±0.28和S组的1.21±0.39(均P<0.05)。E组的外周神经肽GAP43表达显著增强,在术后第28天,阳性区域面积分别为11.20%±0.53%,高于H组的7.25%±0.22%和S组的8.68%±0.45%(均P<0.001);同时,在术后第28天,E组CGRP阳性区域面积为7.62%±0.50%,显著低于H组的11.16%±1.33%和S组的10.16%±0.22%(P<0.001)。与S及H组相比,E组跟腱长度恢复至(11.67±0.58)mm,跟腱横截面积恢复至(5.97±0.72)mm2,跟腱最大失效拉力恢复至(71.06±2.48)N,弹性模量恢复至(9.24±1.56)MPa(均P<0.01)。结论可注射外泌体水凝胶可促进SD大鼠跟腱肌腱愈合进程,同时能够有效调控外周神经肽的表达并且不会引起过度的神经敏化。Objective To investigate the effect of injectable adipose tissue stem cell‑derived exosome‑encapsulated hydrogel on the tendon healing in rats and to evaluate the temporal and spatial expressions of periphery neuropeptides at healing site.Methods To generate the injectable exosome‑encapsulated hydrogel,the methacryloylchloride solution(GelMA)and the photoinitiator were mixed first,then the exosome solution was added and oscillated together.Followed with exposing in the ultraviolet light in a wave length 405 nm for 30 seconds to form an injectable hydrogel.The Sprague Dawley(SD)rats were performed with full thickness transection and surgical repair administration of Achilles tendon to establish the animal model,including 4 groups:intact control(C group,selected the contralateral side of tendon transection with surgical repair group),tendon transection with surgical repair group(S group),tendon transection with surgical repair and hydrogel implantation group(H group)and tendon transection with surgical repair and exosome‑encapsulated hydrogel group(E group).The samples were harvested on Day 7,14 and 28 after the operation,respectively.Histopathological examination was performed with hematoxylin‑eosin staining(HE staining)and immunohistochemistry staining of tenogenesis marker(Tenomodulin,TNMD),periphery neuropeptide markers(growth associated protein‑43,GAP43;S100 calcium‑binding protein B,S100B;neuropeptide Y,NPY;calcitonin gene‑related peptide,CGRP;Substance P,SP).Morphological property was characterized with tendon length,cross‑sectional area(CSA),and gastrocnemius weight ratio.Biomechanical testing was measured with maximum failure load,stiffness and tensile modulus.Results The results of the quantitative polymerase chain reaction(qPCR)showed that the expression level of the TNMD gene in the E group was 2.12±0.43,which was significantly higher than that in the H group(1.26±0.28)and the S group(1.21±0.39)(both P<0.05).Based on the immunohistochemistry staining,the expression of GAP43 can be d

关 键 词：肌腱 脂肪干细胞 外泌体 可注射水凝胶 肌腱愈合 外周神经肽 

分 类 号：R686.1[医药卫生—骨科学]