牙髓干细胞对兔面神经损伤的修复作用及其机制

doi:10.13481/j.1671-587x.20180309

摘要/Abstract

摘要： 目的：通过手术建立兔面神经损伤模型，探讨牙髓干细胞对兔面神经损伤的修复作用，阐明其可能机制。方法：分离、培养并诱导牙髓干细胞。45只兔随机分为正常对照组、模型组和实验组，每组15只。除正常对照组外，其余各组兔沿嘴角至耳前处做长约3 cm的切口，离断面神经上颊支，建立面神经上颊支损伤的动物模型。1周后向实验组兔手术术腔部位注入0.1 mL牙髓干细胞悬液（5×10⁶个），正常对照组兔不作任何处理，模型组兔注射等量磷酸盐PBS缓冲液。术后2周观察各组兔行为表现及面部胡须运动功能评分，HE染色观察各组兔面神经组织病理形态表现，免疫组织化学染色观察兔面神经组织中脑源神经生长因子（BDNF）和睫状神经生长因子（CNTF）阳性细胞数，透射电镜观察兔面神经组织横切片中再生神经纤维数目、纤维直径和髓鞘厚度。结果：成功分离鉴定原代幼兔牙髓干细胞，大多数呈成纤维状、长梭形。HE染色后，第3代干细胞的细胞核呈深蓝色、卵圆形，呈梭形着色深细胞为成纤维细胞样细胞。与正常对照组比较，模型组兔面部胡须运动功能评分降低（P<0.05）；与模型组比较，实验组兔面部胡须运动功能评分升高（P<0.05）。与模型组比较，实验组兔治疗10周后，再生神经纤维较多，呈束状且紧密。与正常对照组比较，模型组兔面神经组织中BDNF和CNTF阳性细胞数明显降低（P<0.05）；与模型组比较，实验组兔面神经组织中BDNF和CNTF阳性细胞数明显升高（P<0.05）。与正常对照组比较，模型组兔面神经组织横切片中再生神经纤维数目、纤维直径和髓鞘厚度明显降低（P<0.05）；与模型组比较，实验组兔面神经组织横切片中再生神经纤维数目、纤维直径和髓鞘厚度数均明显升高（P<0.05）。结论：牙髓干细胞对面神经损伤具有一定的修复作用，其机制可能与上调BDNF及CNTF表达有关联。

关键词: 牙髓干细胞, 面神经损伤, 离断法, 移植

Abstract: Objective: To establish the rabbit facial nerve injury model by surgery and to explore the repair effect of dental pulp stem cells on the facial nerve injury in the rabbits,and to clarify its possible mechanism. Methods: The dental pulp stem cells were isolated,cultured and induced.A total of 45 rabbits were randomly divided into normal control group,model group and experiment group,and there were 15 rats in each group.Except for normal control group,the rabbits in the other groups were cut along the corner of mouth to the front of the ear to form a incision about 3 cm,the upper buccal branches were disconnected,and the facial nerve buccal injury models were established.After 1 week,0.1 mL dental pulp stem cell suspension (5×10⁶) was injected into the operative cavity of the rats in experiment group, and the rabbits in normal control group did not receive any treatment.The rabbits in model group were injected with the same amount of phosphate buffer (PBS).Two weeks after operation,the behavior and movement function scores of facial beard of the rabbits in various groups were evaluated;the pathomorphology of facial nerve tissue of the rabbits in various groups was observed by HE staining;immunohistochemical staining was used to detect the number of brain derived nerve growth factor (BDNF) and ciliary neurotrophic factor (CNTF) positive cells; the number of regenerated nerve fibers,the diameter of fiber and the thickness of myelin sheath were observed under transmission electron microscope. Results: The primary dental pulp stem cells of young rabbits were successfully separated and identified.Most of them were fibroblast like and long fusiform.The HE staining results showed that the nucleus of the third generation of the dental pulp stem cells showed a dark blue and oval shape,and the spindle shaped hyperchromatic cells were fibroblast like cells.Compared with normal control group,the movement function score of facial beard of the rabbits in model group was significantly decreased(P< 0.05);compared with model group,the movement function score of facial beard of the rabbits in experiment group was increased (P<0.05).Compared with model group,after treated for 10 weeks,there were more regenerated nerve fibers which were fascicular and tight.Compared with normal control group,the number of BDNF and CNTF positive cells in facial nerve tissue section of the rabbits in model group was decressed (P< 0.05);compared with model group,the number of BDNF and CNTF positive cells in facial nerve tissue section of the rabbits in experiment group was significantly increased (P<0.05). Compared with normal control group,the number of regenerated nerve fibers,the diameter of fiber and the thickness of myelin sheath of the rabbits in model group were significantly decreased(P<0.05);compared with model group,the number of regenerated nerve fibers,the diameter of fiber and the thickness of myelin sheath of the rabbits in experiment group were significantly increased (P<0.05). Conclusion: The dental pulp stem cells have a certain repair effect on the facial nerve injury,and the mechanism may be related to the up-regulation of the experessions of BDNF and CNTF.

Key words: dental pulp stem cells, transplantation, disconnection, facial nerve injury

中图分类号:

R745.12

陈彪, 张睿, 张文娟, 岳磊, 范戌辉, 刘吉伦, 刘耀强, 崔怡, 屈鹏飞, 杨威. 牙髓干细胞对兔面神经损伤的修复作用及其机制[J]. 吉林大学学报(医学版), 2018, 44(03): 504-509.

CHEN Biao, ZHANG Rui, ZHANG Wenjuan, YUE Lei, FAN Xuhui, LIU Jilun, LIU Yaoqiang, CUI Yi, QU Pengfei, YANG Wei. Repair effect of dental pulp stem cells on facial nerve injury in rabbits and its mechanism[J]. Journal of Jilin University Medicine Edition, 2018, 44(03): 504-509.

参考文献

[1] Strojny C,Boyle M,Bartholomew A,et al.Interferon gamma-treated dental pulp stem cells promote human mesenchymal stem cell migration in vitro[J].J Endod,2015,41(8):1259-1264.
[2] Kanafi M,Majumdar D,Bhonde R,et al.Midbrain cues dictate differentiation of human dental pulp stem cells towards functional dopaminergic neurons[J].J Cell Physiol,2014,229(10):1369-1377.
[3] Ronis MJ,Gomez-Acevedo H,Blackburn ML,et al.Uterine responses to feeding soy protein isolate and treatment with 17β-estradiol differ in ovariectomized female rats[J].Toxicol Appl Pharmacol,2016,297:68-80.
[4] Rataj F,Möller FJ,Jähne M,et al.Progesterone,as well as 17β-estradiol,is important for regulating AHR battery homoeostasis in the rat uterus[J].Arch Toxicol,2015,89(3):393-404.
[5] Sasaki R,Aoki S,Yamato M,et al.Neurosphere generation from dental pulp of adult rat incisor[J].Eur J Neurosci,2008,27(3):538-548.
[6] Gronthos S,Arthur A,Bartold PM,et al.A method to isolate and culture expand human dental pulp stem cells[J].Methods Mol Biol,2011,698:107-121.
[7] Lieberman DM,Jan TA,Ahmad SO,et al.Effects of corticosteroids on functional recovery and neuron survival after facial nerve injury in mice[J].Arch Facial Plast Surg,2011,13(2):117-124.
[8] Barbour JR,Yee A,Moore AM,et al.Cadaveric nerve allotransplantation in the treatment of persistent thoracic neuralgia[J].Ann Thorac Surg,2015,99(4):1414-1417.
[9] 刘浏.犬下牙槽神经切断对牙种植体周围成骨结构影响的影像学分析[D].大连:大连医科大学,2015.
[10] Goodus MT,Guzman AM,Calderon F,et al.Neural stem cells in the immature,but not the mature,subventricular zone respond robustly to traumatic brain injury[J].Dev Neurosci,2015,37(1):29-42.
[11] Shah SR,Young S,Goldman JL,et al.A composite critical-size rabbit mandibular defect for evaluation of craniofacial tissue regeneration[J].Nat Protoc,2016,11(10):1989-2009.
[12] McGregor C,Sau A,Ruddy SC,et al.Novel ligands balance estrogen receptor β and α agonism for safe and effective suppression of the vasomotor response in the ovariectomized female rat model of menopause[J].Endocrinology,2014,155(7):2480-2491.
[13] Zhu Y,Liu S,Zhou S,et al.Vascularized versus nonvascularized facial nerve grafts using a new rabbit model[J].Plast Reconstr Surg,2015,135(2):331-339.
[14] Hook V,Brennand KJ,Kim Y,et al.Human iPSC neurons display activity-dependent neurotransmitter secretion:aberrant catecholamine levels in schizophrenia neurons[J].Stem Cell Reports,2014,3(4):531-538.
[15] 庄友梅,木合塔尔·霍加,许辉,等.转化生长因子-β3和牙髓干细胞在兔面神经损伤修复中作用[J].中华实用诊断与治疗杂志,2015,29(7):637-639.
[16] 王腾,木合塔尔·霍加,白惠子.外源性转化生长因子-β_3联合兔牙髓干细胞对兔骨缺损处成骨细胞转化生长因子-β_3表达的影响[J].中华实用诊断与治疗杂志,2017,31(6):525-529.
[17] Huojia M,Wu Z,Zhang X,et al.Effect of dental pulp stem cells (DPSCs) in repairing rabbit alveolar bone defect[J].Clin Lab,2015,61(11):1703-1708.
[18] Takasugi T,Minegishi S,Asada A,et al.Two degradation pathways of the p35 Cdk5 (cyclin-dependent kinase)activation subunit,dependent and independent of ubiquitination[J].J Biol Chem,2016,291(9):4649-4657.
[19] Fu S,Shi ZY,Fan WJ,et al.Microenvironments induce iPSCs and BMSCs into neuron-like cells-Reelin's regulative role in cell differentiation and polarization[J].Sheng Li Xue Bao,2015,67(4):357-369.
[20] Barbon S,Stocco E,Negro A,et al.In vitro assessment of TAT-ciliary neurotrophic factor therapeutic potential for peripheral nerve regeneration[J].Toxicol Appl Pharmacol,2016,309:121-128.
[21] Lee N,Serbinski CR,Braunlin MR,et al.Muscle and motor neuron ciliary neurotrophic factor receptor α together maintain adult motor neuron axons in vivo[J].Eur J Neurosci,2016,44(12):3023-3034.