中国修复重建外科杂志

中国修复重建外科杂志

  1. 期刊首页
  2. 期刊简介
  3. 投稿指南
  4. 在线期刊
  5. 本刊动态
  6. 期刊订阅
  7. 联系我们

肌外膜导管中细胞对周围神经再生影响的实验研究

冯蔚枫 , 陆海滨 , 徐筑秋 , 陈露露 , 杨晓楠 , 祁佐良

中国医学科学院整形外科医院研究中心（北京 100144）

收稿日期: 2017-12-20;  修回日期:2018-03-22; 

基金项目: 协和青年科研基金资助项目（3332015155）；国家自然科学基金资助项目（81571921、81671908）

通讯作者: 杨晓楠, Email: yxnan@aliyun.com 祁佐良, Email: qizuoliang1h@163.com

Effect of cells in the epimysium conduit on the regeneration of peripheral nerve

FENG Weifeng , LU Haibin , XU Zhuqiu , CHEN Lulu , YANG Xiaonan , QI Zuoliang

Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100144, P.R.China

Corresponding Author: YANG Xiaonan, Email: yxnan@aliyun.com QI Zuoliang, Email: qizuoliang1h@163.com

查看全文

目的探讨小鼠腹外斜肌外膜制备的肌外膜导管（epimysium conduit，EMC）中的细胞成分对坐骨神经再生影响。方法取 8 周龄雄性 C57BL/6J 增强绿色荧光蛋白（enhanced green fluorescent protein，EGFP）小鼠的腹外斜肌外膜，修剪成 5 mm×3 mm，制备成管状（即 EMC）。取部分肌外膜采用不同照射剂量（0、15、20、25、30、35 Gy）进行细胞迁移抑制处理，并计数迁移细胞，选择迁移细胞数最少的肌外膜制备 EMC；另取部分肌外膜行脱细胞处理，常规 HE 和 Masson 染色鉴定脱细胞效果后制备 EMC。取 24 只 C57BL/6J 野生型小鼠制备右后肢 3 mm 长坐骨神经缺损模型后，随机分为 3 组（n=8），分别采用 EMC（A 组）、经细胞迁移抑制处理后的 EMC（B 组）、脱细胞处理后的 EMC（C 组）修复缺损。术后 16 周取再生神经中段行大体、甲苯胺蓝染色、免疫荧光染色以及透射电镜观察。结果肌外膜细胞迁移抑制观察示，15 d 时随照射剂量增大，细胞迁移数逐渐减少；除 30 Gy 组与 35 Gy 组间比较差异无统计学意义（P>0.05）外，其余各组间比较，差异均有统计学意义（P<0.05）。选用经 35 Gy 照射剂量处理后的肌外膜进行体内实验。肌外膜脱细胞处理后均未见细胞核，且可缝合成导管状，制备 EMC。术后 16 周各组 EMC 中神经均再通，A 组修复后的坐骨神经最粗，B 组次之，C 组最细；免疫荧光染色可见 A 组 EMC 中 EGFP 细胞包绕再生轴突；甲苯胺蓝及透射电镜观察，A 组再生神经轴突数及再生有髓神经鞘厚度显著优于 B、C 组（P<0.05），B、C 组间比较差异无统计学意义（P>0.05）。结论EMC 中的细胞成分参与并促进了小鼠坐骨神经的再生。

ObjectiveTo investigate the effect of cells in the epimysium conduit (EMC) on the regeneration of sciatic nerve of mice.MethodsThe epimysium of the 8-week-old male C57BL/6J enhanced green fluorescent protein (EGFP) mouse was trimmed to a size of 5 mm×3 mm, and prepared in a tubular shape (ie, EMC). Some epimysia were treated with different irradiation doses (0, 15, 20, 25, 30, 35 Gy) to inhibit cells migration. Then the number of migrating cells were counted, and the epimysia with the least migrating cells were selected to prepare EMC. Some epimysia were subjected to decellularization treatment and prepared EMC. HE and Masson staining were used to identify the decellularization effect. Twenty-four C57BL/6J wild-type mice were used to prepare a 3-mm-long sciatic nerve defect of right hind limb model and randomly divided into 3 groups (n=8). EMC (group A), EMC after cell migration inhibition treatment (group B), and decellularized EMC (group C) were used to repair defects. At 16 weeks after operation, the midline of the regenerating nerve was taken for gross, toluidine blue staining, immunofluorescence staining, and transmission electron microscopy.ResultsAt 15 days, the number of migrating cells gradually decreased with the increase of irradiation dose. There was no significant difference between 30 Gy group and 35 Gy group (P>0.05); there were significant differences between the other groups (P<0.05). The epimysium after treatment with 35 Gy irradiation dose was selected for thein vivo experiment. After the decellularization of the epimysium, no nucleus was found in the epimysium and the epimysium could be sutured to prepare EMC. At 16 weeks after operation, the nerves in all groups were recanalized. The sciatic nerve was the thickest in group A, followed by group B, and the finest in group C. Immunofluorescence staining showed that the EGFP cells in group A were surrounded by regenerated axons. Toluidine blue staining and transmission electron microscopy observation showed that the number of regenerated axons and the thickness of regenerated myelin sheath in group A were significantly better than those in groups B and C (P<0.05). There was no significant difference between groups B and C (P>0.05).ConclusionThe cellular components of the epimysium participate in and promote the regeneration of the sciatic nerve in mice.

关键词: 肌外膜导管; 神经再生; 细胞迁移抑制; 脱细胞

Key words: Epimysium conduit; nerve regeneration; cell migration inhibition; decellularization

引用本文： 冯蔚枫, 陆海滨, 徐筑秋, 陈露露, 杨晓楠, 祁佐良. 肌外膜导管中细胞对周围神经再生影响的实验研究. 中国修复重建外科杂志, 2018, 32(5): 617-624. doi: 10.7507/1002-1892.201712092 复制

登录后 ，请手动点击刷新查看图表内容。 没有账号，
1. Hu Y, Wu Y, Gou Z, et al. 3D-engineering of cellularized conduits for peripheral nerve regeneration. Sci Rep, 2016, 6: 32184.
2. Niu Y, Li L, Chen KC, et al. Scaffolds from alternating block polyurethanes of poly (ε-caprolactone) and poly(ethylene glycol) with stimulation and guidance of nerve growth and better nerve repair than autograft. J Biomed Mater Res A, 2015, 103(7): 2355-2364.
3. Prabhakaran MP, Ghasemi-Mobarakeh L, Jin G, et al. Electrospun conducting polymer nanofibers and electrical stimulation of nerve stem cells. J Biosci Bioeng, 2011, 112(5): 501-507.
4. Binan L, Tendey C, De Crescenzo G, et al. Differentiation of neuronal stem cells into motor neurons using electrospun poly-L-lactic acid/gelatin scaffold. Biomaterials, 2014, 35(2): 664-674.
5. Junka R, Valmikinathan CM, Kalyon DM, et al. Laminin functionalized biomimetic nanofibers for nerve tissue engineering. J Biomater Tissue Eng, 2013, 3(4): 494-502.
6. 林强, 蔡杨庭, 李皓莘. 负载浓度梯度 NGF 的周围神经导管修复大鼠周围神经缺损的实验研究. 中国修复重建外科杂志, 2014, 28(2): 167-172.
7. Pereira T, Gärtner A, Amorim I, et al. Promoting nerve regeneration in a neurotmesis rat model using poly (DL-lactide-ε-caprolactone) membranes and mesenchymal stem cells from the Wharton’s jelly: in vitro and in vivo analysis. Biomed Res Int, 2014, 2014: 302659.
8. Saito K, Tamaki T, Hirata M, et al. Reconstruction of multiple facial nerve branches using skeletal muscle-derived multipotent stem cell sheet-pellet transplantation. PLoS One, 2015, 10(9): e0138371.
9. Li G, Xiao Q, Zhang L, et al. Nerve growth factor loaded heparin/chitosan scaffolds for accelerating peripheral nerve regeneration. Carbohydr Polym, 2017, 171: 39-49.
10. Cao J, Xiao Z, Jin W, et al. Induction of rat facial nerve regeneration by functional collagen scaffolds. Biomaterials, 2013, 34(4): 1302-1310.
11. Hu N, Wu H, Xue C, et al. Long-term outcome of the repair of 50 mm long median nerve defects in rhesus monkeys with marrow mesenchymal stem cells-containing, chitosan-based tissue engineered nerve grafts. Biomaterials, 2013, 34(1): 100-111.
12. Gu X, Ding F, Williams DF. Neural tissue engineering options for peripheral nerve regeneration. Biomaterials, 2014, 35(24): 6143-6156.
13. Yang XN, Jin YQ, Wei W, et al. Peripheral nerve repair with epimysium conduit. Biomaterials, 2013, 34(22): 5606-5616.
14. Iliakis G, Wang Y, Guan J, et al. DNA damage checkpoint control in cells exposed to ionizing radiation. Oncogene, 2003, 22(37): 5834-5847.
15. Rofstad EK, Mathiesen B, Galappathi K. Increased metastatic dissemination in human melanoma xenografts after subcurative radiation treatment: radiation-induced increase in fraction of hypoxic cells and hypoxia-induced up-regulation of urokinase-type plasminogen activator receptor. Cancer Res, 2004, 64(1): 13-18.
16. Nehls P, van Beuningen D, Karwowski M. After X-irradiation a transient arrest of L929 cells in G2-phase coincides with a rapid elevation of the level of O6-alkylguanine-DNA alkyltransferase. Radiat Environ Biophys, 1991, 30(1): 21-31.
17. Storme G, Mareel M, De Bruyne G. Influence of cells number on directional migration of MO4 cells in vitro. Arch Geschwulstforsch, 1981, 51(1): 45-50.
18. Weber B, Emmert MY, Schoenauer R, et al. Tissue engineering on matrix: future of autologous tissue replacement. Semin Immunopathol, 2011, 33(3): 307-315.
19. Piccoli M, Urbani L, Alvarez-Fallas ME, et al. Improvement of diaphragmatic performance through orthotopic application of decellularized extracellular matrix patch. Biomaterials, 2016, 74: 245-255.
20. Gilbert TW. Strategies for tissue and organ decellularization. J Cell Biochem, 2012, 113(7): 2217-2222.
21. Jessen KR, Mirsky R. The origin and development of glial cells in peripheral nerves. Nat Rev Neurosci, 2005, 6(9): 671-682.
22. Tamaki T, Okada Y, Uchiyama Y, et al. Clonal multipotency of skeletal muscle-derived stem cells between mesodermal and ectodermal lineage. Stem Cells, 2007, 25(9): 2283-2290.
23. Bollyky PL, Falk BA, Wu RP, et al. Intact extracellular matrix and the maintenance of immune tolerance: high molecular weight hyaluronan promotes persistence of induced CD4+CD25+ regulatory T cells. J Leukoc Biol, 2009, 86(3): 567-572.
24. Bollyky PL, Wu RP, Faik BA, et al. ECM components guide IL-10 producing regulatory T-cell (TR1) induction from effector memory T-cell precursors. Proc Natl Acad Sci U S A, 2011, 108(19): 7938-7943.
25. Morwood SR, Nicholson LB. Modulation of the immune response by extracellular matrix proteins. Arch Immunol Ther Exp (Warsz), 2006, 54(6): 367-374.
26. Brown BN, Valentin JE, Stewart-Akers AM, et al. Macrophage phenotype and remodeling outcomes in response to biologic scaffolds with and without a cellular component. Biomaterials, 2009, 30(8): 1482-1491.
27. Beattie AJ, Gilbert TW, Guyot JP, et al. Chemoattraction of progenitor cells by remodeling extracellular matrix scaffolds. Tissue Eng Part A, 2009, 15(5): 1119-1125.
28. Reing JE, Zhang L, Myers-Irvin J, et al. Degradation products of extracellular matrix affect cell migration and proliferation. Tissue Eng Part A, 2009, 15(3): 605-614.