Bone marrow mesenchymal stem cells for repair of spinal cord injury: how to promote axonal regeneration?

doi:10.3969/j.issn.2095-4344.2014.41.025

Abstract

Abstract:

BACKGROUND: Stem cells have been shown to not only replace damaged cells, but also secrete trophic factors, bringing a bright future for the treatment of clinical spinal cord injury.

OBJECTIVE: To review the latest advances of bone marrow mesenchymal stem cells in animal and clinical research.

METHODS: A computer-based search of Kjmed and Wanfang databases was done for relevant articles published from April 2004 to April 2014 using the keywords of “stem cells, spinal cord injuries, embryonic stem cells, neural stem cells, mesenchymal stem cells” in English and Chinese, respectively.

RESULTS AND CONCLUSION: Totally 2 745 articles were initially retrieved, and only 50 articles were included in result analysis. Bone marrow mesenchymal stem cells have become one of the most promising sources of stem cells in the treatment of spinal cord injury. Although the bone marrow mesenchymal stem cell in the treatment of spinal cord injury is still in its infancy, it has certain effects on the repair of spinal cord injury. The mechanism of action of bone marrow mesenchymal stem cells in the treatment of spinal cord injury is possibly related to the substitution effect, neurotrophic effects, suppression of the immune response and promoting axonal regeneration.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: spinal cord injuries, embryonic stem cells, neural stem cells, mesenchymal stem cells

CLC Number:

R318

Li Hui-li, Du Cheng-fen, Zheng Hong-mei, Hou Ping-zhi, Wang Yun, Xiang Zi-jun, Lv Gui-li, Li Meng, Yu Hai-qin, Chen Shan-shan. Bone marrow mesenchymal stem cells for repair of spinal cord injury: how to promote axonal regeneration?[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(41): 6703-6707.

Figures/Tables 1

2.1 胚胎干细胞的机制人类胚胎干细胞可以从早期囊胚细胞团中获取，具有多分化潜能，其分化潜能介于胚卵干细胞与成体干细胞之间，但通常只向特定的细胞系分化，这种细胞可以在体外增殖并具有多种细胞分化潜能，少突胶质细胞在胚胎干细胞分化中尤为重要，少突胶质细胞在脊髓轴突周围形成髓鞘，促进神经冲动及神经再生[3]。脊髓损伤后植入少突胶质祖细胞可促进髓鞘再生，进而促进脊髓运动神经元细胞再生，胚胎干细胞可利用其多种细胞分化潜能诱导其分化为少突胶质细胞，促进运动神经元生长。少突胶质祖细胞还被认为在微环境中分泌营养物质，调节局部免疫功能，加强神经保护，促进神经再生。有文献指出少突胶质祖细胞受脊髓损伤局部微环境残存神经元细胞的调节，发挥不同的功能，如果脊髓损伤为完全性脊髓横断伤则需要少突胶质细胞伴随神经元细胞一同生长[4]。 2.2 新生干细胞的机制新生干细胞同胚胎干细胞一样来源于特定的器官，脐血干细胞是惟一临床可应用的新生干细胞，人类脐血干细胞获得容易且对供者几乎无任何危害，其可在体外培养并且在低温冻存下可以长期存活，脐血干细胞的应用不存在伦理问题，但因获得数量有限，而且由于其表现出未成熟细胞的免疫原性，在移植注射时可能会有一定风险，目前，对于脐血干细胞治疗脊髓损伤的临床应用仍在讨论之中。 2.3 成体干细胞的机制 2.3.1 神经干细胞移植的机制脊髓损伤基本病理过程是目标轴突变性、神经损失及髓鞘脱失[5]，导致持久的功能残疾和瘫痪重大并发症。目前对于其治疗方式及其有限，在恢复神经功能方面大部分是无效的。干细胞移植是一种很有前途的技术，干细胞移植的原理是以取代失去的神经元连接中断的轴突连接，并提供神经因素促进脊髓损伤后的功能恢复[6]。神经干细胞是一种祖干细胞，来源于大脑、脊髓、视神经组织。多个研究证实神经干细胞移植的疗效[7]，其神经功能的显著改善与新形成的细胞迁移有关。对神经干细胞诱导预分化，使其成为少突神经胶质前体细胞，可以更多地获得胚胎源性以及成体源性神经干细胞来源的少突神经胶质细胞，无论是成体来源还是胚胎来源的少突胶质神经前体细胞，都能增强髓鞘再生。相比而言，成体来源的少突神经胶质前体细胞再生髓鞘更为迅速，发育出更多的少突神经胶质细胞，且每个少突神经胶质细胞包裹更多的宿主轴突，而胚胎阶段来源的少突胶质神经前体细胞则在移植区域迁移更为广阔[8]。 2.3.2 骨髓间充质干细胞移植的机制 20世纪70年代Friedenstein等发现一小部分骨髓中贴壁最牢固的细胞经数次传代后仍形成独特的纺锤形外观，并且具有形成软骨细胞的能力，因这些细胞呈现出成纤维细胞样的外观而被称为集落形成单位成纤维细胞，后来，卡普兰等人称这些细胞为骨髓间充质干细胞[9]。骨髓间充质干细胞具有贴壁作用，能够分化为成骨细胞、软骨细胞、脂肪细胞、肌肉细胞、神经细胞、肌腱细胞等[10]，其原因可能是由于组织的起源或培养条件不同所导致细胞表面及基因表达模式不同。人类骨髓间充质干细胞HLA-DR表面表达CD105，CD90，CD73，不表达CD79a、CD45、CD34、CD19、CD14、CD11[11]。大多数骨髓间充质干细胞或骨髓间充质成年祖细胞均形成中胚层衍生物如脂肪、骨、软骨。有研究表明，骨髓间充质干细胞可以分化为神经元及内胚层，诱导免疫调节和免疫耐受特性[12-13]。在同种异体的移植物抗宿主病中[14]，骨髓间充质干细胞直接通过可溶性细胞间相互作用或介导因素有效地抑制免疫反应调节T细胞的激活和增殖[15]，防止器官移植中的免疫排斥[16]。此外，骨髓间充质干细胞的细胞免疫调节特性用于自身免疫性疾病的治疗，如克罗恩氏病等[17]。骨髓间充质干细胞也产生大量的生长因子和细胞因子，使它们适合诱导内源性修复，例如：细胞表达成骨蛋白有效地增强软骨、骨骼、肌腱修复[18]。同样，间充质干细胞产生增强血管形成的因子，因此可用于治疗缺血性疾病如脑卒中、心肌梗死或外周动脉疾病。骨髓间充质干细胞可以直接影响调节脊髓损伤部位的微环境，迁移至损伤处的骨髓间充质细胞可以自分泌和(或)旁分泌脑源性神经营养因子、神经生长因子、血管内皮生长因子等多种神经保护性营养因子[19]，这些因子表达上调，可以促进局部微血管再生、神经再生和重构，从而使损伤细胞得以修复。有研究表明骨髓间充质细胞移植进入损伤模型后[20]，迁移至病变坏死区域并在局部微环境的作用下定向分化为神经元和胶质细胞替代受损组织，移植的骨髓间充质干细胞可以填充囊性区，减少脊髓空洞面积，阻止胶质瘢痕的进一步扩大，促进神经元轴突再生。骨髓间充质干细胞移植方式包括损伤段直接注射移植、经静脉途径注射移植、经动脉途径移植、椎管内移植、经蛛网膜下腔途径移植等方法。损伤段直接注射移植是指将体外分离培养后的骨髓间充质干细胞连同培养液多靶点直接注射到脊髓损伤区域的周围，证实对损伤部位有营养作用[21]。经蛛网膜下腔注入移植法也被证实是骨髓间充质干细胞移植治疗脊髓损伤的有效方法之一。注入蛛网膜下腔的干细胞随脑脊液可以到达损伤部位、缩小损伤范围并使神经功能得到恢复。肖以磊等[22]证实蛛网膜下隙注射和静脉注射两种途径进行自体骨髓间充质干细胞移植治疗早期脊髓损伤均安全，近期疗效确切，但蛛网膜下隙注射途径优于静脉注射途径，但远期疗效及安全性尚待进一步观察。李兵奎等[23]于脊髓损伤后1周，自大鼠尾静脉注射移植骨髓间充质干细胞悬液，对照组自大鼠尾静脉注射无血清的DMEM/F12培养液，脊髓损伤后2，4，8周移植组运动功能、神经电生理、病理学检查结果均优于对照组，说明骨髓间充质干细胞静脉移植对大鼠脊髓损伤后神经功能的恢复有明显的促进作用。王晶等[24]比较椎管内移植、静脉移植、动脉移植3种途径移植骨髓间充质干细胞联合硫酸软骨素酶修复家兔脊髓损伤的疗效差异，通过动物行为学评估、神经电生理检测、荧光示踪技术及免疫组化等方法检测结果认为3种途径移植治疗对于脊髓损伤均有明显疗效，其中肋间动脉移植途径及椎管内移植疗效更为明显。此外，细胞可能通过释放神经营养因子如神经生长因子，激活神经营养受体对脊髓内神经进行修复同时可以改变其周围环境。杜刚等[25]建立大鼠脊髓损伤模型，术后在损伤段脊髓分别注入富血小板血浆、骨髓间充质干细胞、骨髓间充质干细胞+富血小板血浆复合物，对照组不注入任何物质。术后8周各治疗组大鼠后肢功能恢复具有明显提高，骨髓间充质干细胞+富血小板血浆组大鼠后肢功能恢复最好。各治疗组脊髓的神经生长因子、脑源性神经营养因子的蛋白表达水平均有所提高。骨髓间充质干细胞还在损伤部位表达多种与细胞黏附相关的因子，如Ninjurin 1和2、Netrin 4、Robo 1和Robo 4等[26-28]。这些因子是公认的神经修复因子，能有效促进轴突的生长和细胞的迁移。骨髓间充质干细胞可以分泌膜型基质金属蛋白酶1和基质金属蛋白酶2，这两种因子可以降低硫酸软骨素的浓度，而硫酸软骨素是由胶质瘢痕分泌的一种重要轴突生长抑制因子[29]。研究发现骨髓间充质干细胞体外培养或是体内移植均具有分化为神经细胞的潜能[30]。Lee等[31]成功在体外培养的骨髓间充质干细胞中发现分化的不同亚型神经元和胶质细胞。Wu等[32]在脊髓损伤大鼠的腰椎处注射入带有绿色荧光标记的骨髓间充质干细胞，培养数日后，发现大鼠的运动和神经功能得到一定程度的改善，镜下观察到部分移植细胞转化为神经样细胞。顾卫东等[33]在脊髓损伤后7 d，于损伤中心周围移植骨髓间充质干细胞或注射PBS，结果骨髓间充质干细胞组BBB评分显著高于PBS组，而其脊髓空洞体积明显小于PBS组。电镜下可见，在脊髓损伤中心骨髓间充质干细胞组的轴突数量明显多于PBS组，细胞移植后4周，在脊髓损伤中心周围可见大量绿色荧光蛋白标记的骨髓间充质干细胞，免疫荧光染色结果表明，脊髓移植的骨髓间充质干细胞并不表达神经元、星形胶质细胞和少突胶质细胞的表面标志性蛋白。脊髓埋管实验发现，在种有骨髓间充质干细胞的导管内可见有NF阳性的神经纤维的长入，以上结果提示脊髓损伤后进行骨髓间充质干细胞移植可促进损伤脊髓功能的恢复，其机制与骨髓间充质干细胞移植促进神经元轴突再生的作用有关。大多数研究一直致力于开发新技术，可以调节炎症反应，取代丢失的神经元和少突胶质细胞，目前细胞治疗致力于受损的脊髓神经束使其功能恢复 [34]。骨髓间充质干细胞移植治疗脊髓损伤在大量动物模型实验中被证实有效[35]。学者也在对其的临床应用进行研究，国际上关于骨髓间充质干细胞移植治疗脊髓损伤的临床Ⅰ/Ⅱ期试验已有所开展，然而接受细胞移植治疗的脊髓损伤患者及对照组少，随访时间短等原因而造成其报道不多[36]。肖以磊等[37]评估自体骨髓间充质干细胞移植治疗早期脊髓损伤的近期疗效和安全性，其中干细胞移植组35例患者通过蛛网膜下隙注射方式行自体骨髓间充质干细胞移植，同期入院但未行干细胞移植患者29例作为对照组，移植后6个月，对照组患者运动、感觉功能评分均未出现明显提高；蛛网膜下隙移植组患者仍有明显恢复，与对照组比较差异有显著性意义(P < 0.05)，结果表明蛛网膜下隙注射途径进行自体骨髓间充质干细胞移植治疗早期脊髓损伤安全，近期疗效确切，但远期疗效及安全性尚待进一步观察。"

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