第二军医大学学报  2018, Vol. 39 Issue (4): 428-432   PDF    
骨形态发生蛋白2在黄韧带骨化发生和发展中的作用
袁佳滨1, 孙凯强2, 史建刚2     
1. 海军军医大学(第二军医大学)长海医院脊柱外科, 上海 200433;
2. 海军军医大学(第二军医大学)长征医院脊柱外科, 上海 200003
摘要: 黄韧带骨化是胸椎管狭窄的主要原因,其起病隐匿,出现症状时多造成脊髓严重压迫,临床治疗效果不理想,因此亟需研发针对黄韧带骨化早期诊治和预后监测的有效手段。骨形态发生蛋白2(BMP-2)属于转录生长因子β家族成员,在胚胎发育早期阶段诱导间充质干细胞成骨分化和软骨内成骨过程中发挥关键调节作用。研究表明黄韧带骨化,尤其是连续多水平的黄韧带骨化与BMP-2关系密切。本文对BMP-2在黄韧带骨化发生和发展中的作用机制作一综述,希望能为黄韧带骨化的早期诊治和预后监测提供新的思路。
关键词: 黄韧带骨化     骨形态发生蛋白质     发病机制     诊断     靶向治疗     预后    
Role of bone morphogenetic protein 2 in occurrence and development of ligamentum flavum ossification
YUAN Jia-bin1, SUN Kai-qiang2, SHI Jian-gang2     
1. Department of Spine Surgery, Changhai Hospital, Navy Medical University(Second Military Medical University), Shanghai 200433, China;
2. Department of Spine Surgery, Changzheng Hospital, Navy Medical University(Second Military Medical University), Shanghai 200003, China
Abstract: The ossification of ligamentum flavum (OLF) is a major cause of thoracic spinal stenosis. Due to insidious onset, OLF usually results in severe compression of spinal cord when discovered, and the clinical outcomes are poor. Therefore, novel and effective diagnostic and therapeutic approaches for OLF are greatly needed. Bone morphogenetic protein 2 (BMP-2), a member of the transforming growth factor β family, possesses a critical role in the differentiation of mesenchymal stem cells towards osteoblastic lineage and endochondral ossification in the early stage of embryonic development. Increasing researches revealed that OLF, especially the continuous multilevel ossification, correlates closely with BMP-2. In this review, we summarized the advances on the role of BMP-2 in the occurrence and development of OLF, and prospected the application of BMP-2 as a potential target in the diagnosis, therapy and prognosis monitoring of OLF.
Key words: ossification of ligamenta flava     bone morphogenetic protein 2     pathogenesis     diagnosis     targeted therapy     prognosis    

黄韧带骨化(ossification of ligamentum flavum,OLF)首次由Polgar教授于1920年提出[1],之后逐渐被人们关注。据统计,在胸椎管狭窄的病因中OLF占72.3%[2]。临床流行病学研究表明,胸椎OLF主要发生在东亚人群,日本、中国、韩国是高发国家[3]。这些区域性差异表明遗传因素可能在OLF发生、发展中起一定作用。OLF主要发生在下胸椎,由于其进展缓慢,早期患者一般无任何明显症状,一旦出现症状,脊髓压迫已较严重,错过保守治疗的时间窗。外科手术是OLF的有效治疗手段,然而多种并发症如脑脊液漏、神经功能恶化等仍然给临床治疗和患者预后造成巨大困难。因此目前临床亟需研发针对OLF早期诊治和预后监测的有效手段。

骨形态发生蛋白(bone morphogenetic protein,BMP)在哺乳动物早期发育成骨过程中具有关键的调控作用[4]。研究发现肿瘤坏死因子(tumor neucrosis factor β,TGF-β)/BMP基因突变与后纵韧带骨化(ossification of posterior longitudinal ligament,OPLL)有关[5]。有研究表明,BMP基因突变可能在OLF发病机制中也起着关键作用,并且已经证明重组BMP-2蛋白能够在动物体内和体外诱导OLF形成[6-8]。因此本文主要就BMP-2在OLF发生和发展中的作用机制进行综述,希望能为OLF的早期诊治和预后监测提供更多新的思路。

1 OLF发生的病理过程

从解剖学角度讲,黄韧带主要由两侧的囊状部分和中间的椎板间部分组成。正常情况下黄韧带的主要成分是弹性纤维,对脊柱提供稳定和保护作用[9]。骨化通常最先发生在韧带囊状部分,然后逐渐增厚并对脊髓造成压迫。双侧骨化块最后融合形成中央结节型骨化块[10]。OLF是一种软骨内成骨,骨化首先从成纤维细胞增殖开始,然后向软骨细胞分化,最后软骨细胞进一步转化为成骨细胞,完成整个骨化过程[11]。Okada等[12]在对患者手术切除的黄韧带组织观察中发现,骨化常常发生在囊状部分血管分布丰富的区域;骨化的黄韧带中正常弹力纤维减少且排列极不规则,而胶原纤维数量明显增加;肿胀的胶原纤维周围可见大量增殖的软骨细胞,靠近钙化区的软骨细胞增殖明显。骨化组织周围的胶原纤维主要是Ⅱ型胶原纤维,这是软骨细胞的标志,且常在软骨能被检测到之前已能够被检测到[13]

2 BMP的生物学功能

BMP属于TGF-β家族成员,主要由骨祖细胞、成骨细胞、软骨细胞和血小板等合成,在细胞增殖和分化、细胞凋亡以及成骨反应等过程中都起着关键作用。研究表明,多种BMP参与软骨内成骨,在诱导间充质干细胞向成骨细胞分化过程中起调控作用[14]。BMP-2是第1个被美国食品药品监督管理局认可用作骨移植物替代物的BMP[15]

3 BMP-2介导骨形成机制

BMP-2通过诱导间充质干细胞的骨分化实现软骨内成骨,是维持骨、软骨形成和成人骨稳态的关键调节因子,目前多用于骨折固定、关节融合和椎体骨质缺损等的治疗[16]。BMP-2蛋白直接与受体结合后启动成骨相关信号通路,骨形态发生蛋白受体(bone morphogenetic protein receptor,BMPR)Ⅱ首先被磷酸化激活,然后进一步激活BMPRⅠ。受体相关的酶使得受体调控的Smad蛋白(receptor-regulated Smad protein,R-Smad;通常为Smad1/5/8)磷酸化激活,并同时向细胞核移动。磷酸化的R-Smad与辅助型Smad(通常为Smad4)结合形成异源二聚体复合物使转导信号持久,继而转移到细胞核中结合相应的转录因子,如远端缺失同源盒5(distal-less homeobox 5,Dlx5)以启动转录[17]。BMP-2参与骨形成的另一途径是直接诱导成骨细胞中的成骨转录因子Runt相关转录因子2(Runt-related transcription factor 2,Runx2)表达激活成骨细胞,使得骨分化效应加强,同时介导Ⅱ型胶原蛋白和纤维粘连蛋白的产生[18]。研究表明,BMP-2的启动子序列中也存在Runx2结合位点,因此推测在成骨过程中两者可能存在正反馈效应[19]

4 BMP-2参与OLF

OLF、OPLL和弥漫性特发性骨质增生症(diffuse idiopathic skeletal hyperostosis,DISH)都属于脊柱韧带骨化疾病,且均属于软骨内成骨。研究发现,很多基因位点参与韧带骨化的发生和发展[20]。BMP-2是成骨的关键因子,在胚胎发育早期骨形成中发挥关键调节作用。BMP-2参与OPLL研究已经较为充分,BMP-2与OLF的关系也越来越受到研究者的重视[21]

4.1 临床病理研究

Hayashi等[22]在人体骨化黄韧带中发现,BMPR在钙化区周围成熟和未成熟的软骨细胞以及钙化组织远处的梭形细胞和圆细胞中均有表达,且与BMP-2共存于骨化的黄韧带组织中,而在对照组中仅在韧带与骨相连区域发现BMP-2及其受体少量表达。研究还发现,病变范围较大的OLF患者多合并OPLL和DISH,提示OLF也可能像其他脊柱韧带骨化一样具有基因易感性。Qu等[23]在对OLF患者基因研究中发现,在TGF-β/BMP信号通路中存在39个可能的致病基因突变,大多数突变都出现在连续多节段OLF患者组中,且有6例(6/71)患者均出现2个BMP-2(NM_001200.3)外显子区域中的错义突变,c.460C>G:p.(R154G)和c.584G>T:p.(R195M)。蛋白质印迹分析发现,突变BMP-2基因转染的细胞中成骨标志物碱性磷酸酶(alkaline phosphase,ALP)表达在第7天时上调,成骨活性明显高于对照组。

Ning等[24]对患者骨化黄韧带细胞施加循环牵力,发现多水平OLF患者黄韧带细胞BMP-2的mRNA及其蛋白表达水平明显高于单水平OLF患者;单水平OLF患者黄韧带细胞成骨相关基因表达在牵力作用时间延长后也会增加,但与对照组比较差异无统计学意义。这些提示单水平和多水平OLF可能具有不同的发病机制,这与两者不同的临床进展和预后[25]相一致。因此,我们推测BMP-2基因在多水平OLF的发生、发展中起着关键作用,可使其对机械牵力等因素的敏感性增加。对于单水平OLF,由于其多发生在下胸椎(T9~T12),此处属于胸腰关节区,脊柱后部张力大,椎体旋转范围大,相关结构更容易退化[2]。我们推测其发病可能与长期的轴向负荷过载和反复旋转牵拉使得成骨相关基因如BMP-2过度激活有关。

4.2 动物研究

Tsukamoto等[26]通过对大鼠尾椎施加反复的牵力发现,牵力负荷轻的组别以小圆细胞增殖为主,在负荷增加和时间延长后则以由编织骨形成的软骨组织为主;免疫组化染色发现,BMP-2在异位钙化组织周围的圆细胞和成纤维细胞中表达,而在成熟的软骨样细胞中不表达,表明BMP-2可能参与韧带细胞向软骨细胞或骨细胞转化过程。Hoshi等[27]向大鼠模型硬膜外间隙中注射重组人BMP-2,1周后韧带细胞开始向软骨细胞分化;第2周在Ⅱ型胶原富集区发现大量的软骨细胞,且这些软骨细胞大量表达BMPR。到第6周时韧带组织完全被骨组织取代,并且其病理变化和临床患者骨化黄韧带变化相似,说明BMP-2在OLF过程中起关键作用,且能够诱导BMPR的表达。

4.3 其他相关成骨因子

一些转录因子如性别决定相关高迁移率簇蛋白盒基因9(SRY-related high mobility group-box gene 9,Sox9)、Runx2、肌节同源异型框基因2(muscle segment homeobox homolog 2,Msx2)等也在成纤维细胞向软骨细胞和骨细胞的病理性转变时起作用。Uchida等[28]发现,在骨化黄韧带组织中Sox9主要在增殖的软骨细胞中表达,在肥大的软骨细胞中表达减少,其主要通过调节Ⅱ型胶原蛋白基因的表达参与软骨分化和软骨形成过程,同时Sox9的表达受BMP-2的调节。作为BMP-2的下游转录因子,Runx2是ALP基因启动子的关键调节因子,能增强BMP-2特异性Smad的效应[29]。研究发现Runx2也广泛存在于骨化的韧带组织中,在增殖和肥大的软骨细胞中均有表达,且Runx2在中国汉族OLF人群中具有基因多态性[30]。Msx2存在于增殖的软骨细胞中,但是在骨化韧带周围的间充质细胞中呈强阳性[28]。这些转录因子在OLF过程中均与BMP-2相互作用[31]。OLF过程伴随着血管增生,长期损伤积累导致黄韧带产生大量的毛细血管,构成韧带骨化的基础[32-33]。研究发现,钙化区肥大的软骨细胞能够通过分泌血管内皮生长因子(vascular endothelial growth factor,VEGF)调节血管生成,特别是在骨化前区更明显[34-35]。并且也通过与BMP-2之间信号转导交联方式参与成骨细胞分化[36]

根据以上研究,我们推测在OLF发生和发展过程中多种因素尤其力学因素等激活了BMP-2基因。在BMP-2的作用下,参与启动纤维细胞转变为成纤维细胞和圆细胞,随后进一步调节这2种细胞向纤维软骨细胞和软骨细胞分化。在成骨过程中BMP-2同时调控Sox9、促进软骨分化和Ⅱ型胶原蛋白形成。最后阶段BMP-2和Runx2正反馈作用协同促进软骨成熟和成骨细胞形成,而在整个骨化过程中Msx2介导成熟的间充质细胞分化、VEGF促进新生血管形成,同时与BMP-2相互作用为成骨提供相应支持作用。

5 小结

综上所述,BMP-2在OLF发生和发展中起着关键作用。在连续多水平OLF患者中BMP-2基因效应明显,分析原因可能是错义突变使BMP-2过度激活导致相关蛋白表达增加。而单水平的OLF可能与长期的轴向负荷过载和反复旋转牵拉等使BMP-2过度激活有关。临床上OLF具有发现晚和容易复发的特点,且致瘫发生率高,因此我们提出临床上可以通过检测患者血液中BMP-2基因及其产物变化,以及研制针对BMP-2的靶向药物实现OLF的早期诊治并监测预后。

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