Roles and application values of P75 neurotrophin receptor in bone tissue engineering

doi:10.3969/j.issn.2095-4344.0590

Abstract

Abstract:

BACKGROUND: P75 neurotrophin receptor holds low affinity, which not only plays an important role in nerve growth, development and maintenance of functional integrity, but also has a close relationship with bone tissue repair and stem cell enhancement.
OBJECTIVE: To review the research progress and value of P75 neurotrophin receptor in bone tissue repair and regeneration.
METHODS: A computer-based search of CBM, CNKI, PubMed and Elsevier was performed for retrieving articles concerning P75 neurotrophin receptor in bone tissue regeneration published from September 1987 to April 2018. The keywords were “P75, P75NTR, bone repair, bone regenerate, tooth development, facial nerve, stem cell” in Chinese and English, respectively. Totally 47 eligible articles were included in final analysis.
RESULTS AND CONCLUSION: P75 neurotrophin receptor has been reported to not only promote tooth development and facial nerve repair, but also promote mineralization of bone tissue and shorten bone healing time. It also indirectly promotes bone healing by regulating the characteristics of stem cells. However, some researchers believe that apoptosis induced by P75 neurotrophin receptor may hinder fracture healing and even lead to fracture failure. Whether P75 neurotrophin receptor can promote bone repair or inhibit bone tissue repair needs further investigation. At present, its research is still limited to basic research, and there are few reports on its clinical application.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Bone and Bones, Regeneration, Receptors, Nerve Growth Factor, Tissue Engineering

CLC Number:

R318

Shen Mengjie, Yang Kun, Liu Qi. Roles and application values of P75 neurotrophin receptor in bone tissue engineering [J]. Chinese Journal of Tissue Engineering Research, 2018, 22(36): 5852-5857.

Figures/Tables 1

2.1 P75NTR生物学特性神经生长因子不仅对神经损伤的再生修复具有重要作用，而且对细胞的增殖、分化具有促进作用[8]。其具有两种受体：即具有高亲和力的酪氨酸激酶受体A(TrkA)和低亲和力的P75神经营养因子受体(P75NTR)。P75NTR是相对分子质量为 75 000的Ⅰ型膜蛋白，它的结构主要包括胞外结构域、单跨膜结构域和胞内结构域[9]。胞外结构域主要与肿瘤坏死因子受体超家族结合，负责受体构象和配体结合；跨膜结构域在受体二聚化、配体结合引起的构象变化和信号转导中起着重要作用[10]；而胞内结构域就相对比较保守，且较短，但是它有3种不同功能的结构域：①类似与肿瘤坏死因子受体相关因子结合的近膜区；②有死亡结构域；③位于C末端的蛋白结合结构域[11-12]。另外P75NTR属于肿瘤坏死因子受体，同时又是所有神经营养因子的共同受体，包括神经生长因子、神经营养素3、新生营养素等[13]。P75NTR在生物活性上具有多样性，它可以通过激活多种下游信号来调节细胞增殖、凋亡、迁移等多种细胞反应[6]。P75NTR可以单独起作用，也可以与TrkA一起协同作用。这种协同作用不仅可以促进细胞存活，而且对组织的再生修复具有非常重要的作用[14]。 2.2 P75NTR促进骨组织修复近年来，人们发现许多信号分子在骨组织再生修复过程中有所表达，而且起到了积极的作用。随着对细胞和分子生物学技术的研究不断深入，关于骨组织再生修复过程中的信号因子调节系统引起了学者们的广泛关注，其中神经生长因子开始进入了人们的视线，P75NTR在促进骨组织再生修复方面的应用也引起了很多研究者的兴趣。有报道显示P75NTR不仅可以直接在骨组织再生修复过程中起作用，而且还可以调控干细胞，使其干细胞特性更强、更稳定，从而可以通过体外筛选出优质的干细胞而间接的促进骨组织的再生修复。下面就P75NTR在骨组织工程中的应用进行阐述。 2.2.1 体外研究目前，关于P75NTR在骨组织修复方面上的应用，在基础研究中关于其作为干细胞表面标记物筛选出的P75NTR阳性的干细胞具有更优良的干细胞特性。Wen等[15]报道了使用P75NTR筛选外胚间充质干细胞，发现筛选出的P75NTR阳性的胚间充质干细胞，在细胞传代过程中呈均匀一致的成纤维细胞形态，细胞表型、增殖能力稳定。而且体外也证实P75NTR阳性的胚间充质干细胞具有分化为多种细胞类型的能力且稳定存在。同样Zhao等[16]也证明了P75NTR阳性的胚间充质干细胞具有更稳定的增殖分化能力和干细胞生物学特性。另外Rada等[17]报道了用抗P75NTR抗体分离的脂肪干细胞具有很高的成骨分化潜能。很多研究已经证明了P75NTR不仅可以作为干细胞表面的标记物，而且筛选出来的P75NTR阳性的干细胞具有更强的稳定性和干细胞特性。目前虽然还没有研究报道应用筛选出的P75NTR阳性的干细胞修复骨损伤，但体外研究证实筛选出的P75NTR阳性的干细胞具有更强的成骨分化能力，这为筛选出理想的种子细胞应用到骨组织工程奠定了坚实的理论基础。很多的文献也报道了关于P75NTR直接作用于间充质干细胞和成骨细胞，并对其关于成骨分化做了研究，但是对于P75NTR是通过什么机制起作用的目前还在探索中。Ju等[18]报道了不同胚龄的SD大鼠外胚间充质干细胞在矿化过程中P75NTR的表达量，通过流式分析发现P75NTR在12.5，15.5和18.5 d的胚间充质干细胞中均有表达，且随着时间延长P75NTR在胚间充质干细胞中阳性表达率越高，通过ARS染色和碱性磷酸酶活性检测，结果也显示在第18.5天胚间充质干细胞的成骨能力更强。同样Yang等[19]也报道了关于P75NTR对SD大鼠胚间充质干细胞成骨能力的影响，通过免疫组织化学法检测P75NTR在牙发育过程中的表达，发现大鼠神经嵴源性胚间充质干细胞的体外矿化过程中P75NTR参与其矿化，并且认为P75NTR可能是通过与Mage-D1结合来增强其的成骨分化。同时Xing等[20]将外胚层间充质干细胞在牙上皮细胞培养基进行诱导，分别在诱导4，8， 12 d后进行成骨基因和成骨蛋白以及茜素红染色检测，结果也发现P75阳性外胚间充质干细胞较P75阴性外胚间充质干细胞具有更强的成骨分化能力，而且高表达牙本质基质蛋白和牙本质涎磷蛋白，并且发现了P75NTR的矿化作用可能与TGF-β/Smad4的信号通路有关。另外Liu等[21]认为脑源性神经营养因子对人骨髓间充质干细胞的神经发生和成骨的形成具有积极的作用，将脑源性神经营养因子与人骨髓间充质干细胞共培养7 d后，发现人骨髓间充质干细胞高表达神经源性标志物P75和S100，通过茜素红染色和碱性磷酸酶活性检测证实了人骨髓间充质干细胞成骨分化的能力得到了增强；同时体内实验也发现脑源性神经营养因子不仅可以直接促进人骨髓间充质干细胞成骨，而且还可以通过增加神经的发生间接促进成骨。而Mikami等[22]却报道了相反的结果：P75NTR过表达于小鼠胚胎间充质干细胞C3 H10T1/2中时，反而抑制了干细胞的成骨矿化能力，出现这种相反结论的原因可能是P75NTR在不同种类的细胞系体外矿化过程中有不同的调控机制以及体外实验所处微环境的不同而导致的。对于P75NTR调控细胞增殖作用，认为可能与神经生长因子的存在有关系，当神经生长因子大量存在的时候能够启动TrkA存活通路，关闭P75NTR诱导的死亡通路[23]；另外P75NTR的存在能够增强神经生长因子与TrkA结合，从而可以促进细胞的生长。Akiyama等[24]将外源性P75NTR转染到MG63细胞和MC3T3-E1细胞，培养6 d后，流式细胞分析P75NTR对细胞增殖的影响，发现转染了P75NTR的MC3T3-E1细胞的增殖速度比未转染的细胞快，另外关于碱性磷酸酶活性检测，在诱导3 d后，发现转染P75NTR后的MG63细胞和MC3T3-E1细胞与对照组相比碱性磷酸酶染色均表现出强阳性，茜素红染色也得到了相似的效果。RT-PCR检测发现转染P75NTR 12 d后均高表达成骨相关基因(Runx2、OSX、BSP和OC)。另外实验还证明了P75NTR增强细胞的成骨分化可能与P75NTR结合肌钙蛋白受体激酶(Trk)基因TrkA、TrkB、TrkC和Nogo受体(NGR)有关。Mikami等[25]近期的研究也得出相类似的结论。通过对正常骨中P75NTR表达的变化以及P75NTR促进骨组织再生修复通路机制的研究，可以为骨骼疾病的治疗提供新的方案和奠定理论基础。 2.2.2 体内研究目前，由于P75NTR修复骨组织的机制原理还不是很清楚，关于应用P75NTR来修复骨缺损的实验还比较少，但是也有相关文献报道了P75NTR在骨组织的定位以及修复过程中表达的变化。Chartier 等[26]报道了P75在骨组织中有显著的表达，通过组织学检测，证实C57BL/6J小鼠股骨中P75几乎完全是由位于神经生长因子+血管附近的神经纤维表达，而P75在股骨中表达的部位是关节软骨深层区域。在骨髓和骨膜中，P75存在于具有感觉(CGRP)和交感神经(TH)神经纤维形态的神经纤维和类似TrkA神经纤维中，实验中作者对股骨进行脱钙处理，发现未脱钙的股骨相对于脱钙后的股骨高表达P75，并且对于不同鼠龄所表达P75的量也是不同的，这种老化及损伤后P75表达量的变化可以为以后治疗骨组织疾病提供新的思路。另外王莹莹等[27]利用Micro-CT对野生型小鼠和敲除了P75NTR的小鼠进行左侧股骨扫描，并对股骨的松质骨的骨量、骨小梁的数量、骨小梁的厚度、骨表面积等进行评估，结果发现：敲除了P75NTR的小鼠股骨的相关骨量相较于野生型小鼠显著下降；通过注射钙黄绿素来检测矿化沉积速率，结果也同样表明了敲除P75NTR的小鼠股骨矿化沉积速率(MRA)明显低于野生型；通过RT-PCR、Western Blot对小鼠股骨的总RNA、蛋白质以及相关基因进行检测，得到了与上述相同的结果。Yu等[28]报道了骨折后P75的表达情况，实验中制作了纯种大白兔的右下颌骨折模型，分别在术后1，3，5，7，14，28 d后取材，通过苏木精-伊红染色和免疫组织化学检测，结果表明在实验组骨折处神经生长因子受体P75的阳性率随着时间的延长逐渐增加，而对照组仅发现少量P75表达，推测P75可能通过与神经生长因子的结合来增强成骨细胞的活性，从而在骨折愈合中起到积极的修复作用。张伟等[29]也做了相类似的报道。另外有学者发现在骨折处注射神经生长因子，可以加速骨折愈合，且神经生长因子表达于骨折愈合后成骨样细胞中，这表明神经生长因子及其配体可能与促进骨组织再生修复有关[30]。 2.3 P75NTR抑制骨组织修复 P75NTR在生物活性上表现为多样性，它不仅可以调控细胞的生长、组织的再生，还可以通过激活多种下游信号参与细胞凋亡、迁移和细胞周期阻滞等多种细胞反应[5]。关于P75NTR抑制骨组织修复的机制原理还不是很清楚，但是有学者已经发现了3条关于P75NTR诱导细胞凋亡的信号通路，见表1。"

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