Stem cell therapy for degenerative disc diseases: present status and prospects

doi:10.3969/j.issn.2095-4344.2015.10.029

Abstract

Abstract:

BACKGROUND: None of the current treatment strategies has been focused on relieving or reversing the disk degeneration process after degenerative disc diseases. In recent years, more and more scientists try to treat degenerative disc diseases using stem cell therapy.

OBJECTIVE: To explore the research status and prospects of stem cell therapy for degenerative disc diseases.

METHODS: A computer-based online search of PubMed database between January 2004 and December 2014was performed to search related articles with the key words of “stem cell, intervertebral disk” in English. Literatures related to stem cell therapy for degenerative disc diseases were selected; in the same field, the articles published lately in authoritative journals were preferred.

RESULTS AND CONCLUSION: A total of 342 articles were primarily selected, and 43 articles were involved in result analysis according to inclusion criteria. Stem cell therapy is a newly treatment for degenerative disc diseases. Cells appropriate for stem cell therapy include embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, human umbilical cord mesenchymal stem cells and chondrocytes or nucleus pulposus cells. Although cell leakage, intervertebral disc infection and tumorigenesis are the main challenges, stem cell therapy for degenerative disc diseases is promising in the future.

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: Stem Cells, Intervertebral Disk, Regeneration

CLC Number:

R318

Zhou Yi-chi, Li Jing-feng, Dong Shi-shi, Jin Wei . Stem cell therapy for degenerative disc diseases: present status and prospects[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(10): 1635-1639.

Figures/Tables 1

2.1 细胞来源细胞数量的多少，获取的难易度以及分化为类软骨细胞的能力是选择候选细胞的标准。这种细胞应具备以下条件：①能适应低氧及低糖环境。②只有极低的或没有免疫反应。③低概率或不会促肿瘤生长。由于髓核细胞与软骨细胞的免疫表型及分子标记相同，能向类软骨细胞分化的细胞被认为是椎间盘再生可能的候选细胞；干细胞分化为类软骨细胞的条件需提前预设好。一篇近期的Meta分析表明，干细胞移植到动物模型的椎间盘可显着改善椎间盘退变的病程[6]。 2.1.1 胚胎干细胞胚胎干细胞是从早期冷冻胚胎中分离出来的多能干细胞；目前只有少数此类细胞适于研究[7]。胚胎干细胞能向3个胚层所有的细胞分化。它在转化生长因子β、抗坏血酸磷酸酯及类胰岛素样生长因子的作用下可向类软骨细胞分化。 2.1.2 诱导多能干细胞自从2007年Yamanaka小组和Thomson小组先后将人的体细胞重编程为诱导多能干细胞之后[8]，全世界掀起了一阵诱导多能干细胞的研究热潮。诱导多能干细胞在细胞表型、分化潜能上与胚胎干细胞相似，可向体内3个胚层来源的所有细胞分化，进而参与形成机体所有组织和器官。有文献报道它能向类软骨细胞分化[9]。另外诱导多能干细胞可避免骨髓基质干细胞每次获得数量少，传代扩增有限，定向分化受限等不可回避的问题[10]，所以诱导多能干细胞是近年来研究的热点。通过诱导异位重编程转录因子的表达，如OCT3/4，SOX1/2/3，KLF2/4，c/N/L-MYC，NANOG及LIN28而将其从体细胞中分离。MYC和KLF为癌基因。尽管有文献报道可用小分子物质替换转录因子以抑制转化生长因子β表达[11]，但肿瘤化仍然限制了胚胎干细胞及诱导多能干细胞的应用[12]。 2.1.3 间充质干细胞它是从中胚层组织和器官中分离出多潜能细胞。间充质干细胞可以向部分或全部特定类型细胞的原始组织或器官分化。在体外培育条件下，当予以合适的生长因子或指示物时，它可以向成骨细胞、脂肪细胞、软骨细胞、神经元细胞、内皮细胞及心肌细胞分化[13]。有文献报道在体内或体外条件下间充质干细胞均可向类软骨细胞分化[14]。 2.1.4 人脐带间充质干细胞人脐带间充质干细胞是多能细胞，有文献报道其能分化为软骨细胞系、脂肪细胞系及骨细胞系。目前，异体移植人脐带间充质干细胞的安全性还未得到验证。直到进一步的研究证实异体人脐带间充质干细胞移植的免疫安全性之前，其在椎间盘再生中的应用前景是有限的。 2.1.5 类软骨细胞或髓核细胞髓核的细胞成分主要是类软骨细胞；作为天然的髓核细胞，在体外条件下及移植后仍有分泌细胞外液的能力。在狗模型上的研究已证实，自体再植的髓核细胞可在体内生长并增殖，并可分泌蛋白多糖和Ⅱ型纤维蛋白[15]。因为退变的椎间盘的细胞表型改变或变为衰老状态，从而使髓核细胞产生的细胞外液量减少，故髓核细胞的应用受到了限制[16]。 2.2 分化方法体外研究表明，由间充质干细胞分化为髓核细胞的过程由“细胞-细胞”信号转导[17]、低氧[18]、生长因子如转化生长因子β1，转化生长因子β3，类胰岛素生长因子1，类胰岛素生长因子2，生长分化因子5及骨形态发生蛋白调控[14]。有学者成功地使用转化生长因子β1，转化生长因子β3及地塞米松诱导间充质干细胞分化为类软骨细胞[19]。在体外实验中，基因疗法被用于提高间充质干细胞分化为类软骨细胞的培育效率。利用此项技术，Sox-9基因编码转录因子Sox-9，其参与软骨细胞的分化被转导进入脂肪间充质干细胞，使得脂肪间充质干细胞分化为类软骨细胞的能力得以提高。原癌基因Bcl-2作用为抑制细胞凋亡，亦用来提高间充质干细胞分化为类软骨细胞的培育效率。病毒及非病毒载体(在避免导致异位感染的前提下)均可用于运载这些基因。目前尚无对照实验评估基因疗法治疗椎间盘退变性疾病的疗效。 2.3 移植技术及载体向椎间盘移植间充质干细胞有两个问题亟需解决：移植技术及载体。由于椎间隙缺乏血管，因此向椎间隙直接注射间充质干细胞可能是移植的惟一途径。在动物模型上单独移植间充质干细胞或者联合载体移植结果表明，移植后存活的类软骨细胞可产生蛋白多糖，使得椎间隙高度增加[20-22]。在人体研究中，向患者退变节段椎间盘注射间充质干细胞；随后，相比于脊柱融合或椎间盘置换，患者的髓核含水量增多，症状得以迅速改善[23]。Carragee等[24]的研究证实，进行过椎间盘造影的患者椎间盘退变速度显着加速。故而在细胞疗法应用于人体之前，向椎间盘区域直接注射干细胞是否会加速其退变需得到进一步论证。尽管能成功的移植细胞，但细胞泄露仍是值得关注的问题：泄露到脊柱峡部的间充质干细胞可能形成骨赘而造成椎管狭窄[25]。Fields等[26]的研究表明，大部分人的纤维环存在裂隙，而且这种裂隙不能被MRI发现，这种裂隙的存在增加了细胞泄露的风险。这个问题颇具挑战，因为目前最常用的移植入路是后外侧入路，其穿过了峡部的侧隐窝。从针痕或纤维环裂口泄露的间充质干细胞进入脊柱峡部后可能导致椎管狭窄。为了解决这个问题，学者们开始寻找合适的可注射细胞载体：它能快速凝胶化以避免注射泄露。细胞载体的另一个优势便是其提供了间充质干细胞增殖的三维环境。一些体外研究表明，某些支架包括胶原蛋白凝胶、水凝胶及透明质酸凝胶可满足这些条件，在这些支架中间充质干细胞仍能表达出类软骨细胞表型。目前也有关于生物载体的研究，诸如微球支架与装有持续诱导的细胞因子及增强其表型表达的支架[27]。另外，也可应用血纤蛋白黏合剂来防止泄露[28]。安全有效的载体的进一步研究对细胞疗法治疗人椎间盘退变性疾病至关重要。 2.4 细胞疗法的功效多数对照动物研究表明，干细胞移植使椎间盘退变疾病得以改善。学者们对不同的检查指标进行了评估，以对比干细胞疗法的功效。从解剖层面上来说，间充质干细胞移植组椎间盘高度及椎间盘高度指数(椎间盘高度/椎体高度)均有增高[29-30]；从组织学层面上来说，椎间盘的退变分级(分级越高，髓核细胞越少，髓核水分越少[31])有所下降，并且MRI显示T2加权像信号(椎间盘内水含量的指标)增强[32-33]；另外，从分子层面上来说，转录Ⅱ型纤维蛋白的mRNA含量增加[34]。尽管这些研究存在差异，但基于合并数据的统计显示，间充质干细胞移植能延缓、阻止甚至逆转椎间盘退变过程。椎间盘退变被认为与炎性因子有关，包括白细胞介素1β、白细胞介素6、白细胞介素8及肿瘤坏死因子α。众所周知，间充质干细胞有抗炎及免疫调节的功能，它能分泌保护因子包括细胞因子和趋化因子[35]。促炎因子可能刺激间充质干细胞分泌抗炎因子，其既能调节常驻巨噬细胞，又能减少促炎因子的下游效应[36]。纤维化在椎间盘退变疾病中亦起重要作用，通过分泌细胞因子和基质金属蛋白酶，间充质干细胞能抑制并逆转纤维化。最近有文献报道，间充质干细胞保持了胶原蛋白网和蛋白多糖间的相互作用，故其作用机制可能与给髓核提供了机械支持有关。 2.5 细胞疗法的风险主要包括：①细胞泄露引起的椎体峡部骨赘形成[25]。②椎间盘修复速度缓慢。③椎间盘感染或椎间盘炎。④因干细胞的多能性引起的肿瘤生长。为减少细胞泄露的风险，细胞载体技术得以发展：其不仅能提供间充质干细胞增殖所需的三维框架，并在注射后能改变物理性状而使干细胞不会泄露。为减少椎间盘感染的风险，细胞治疗必须强调严格的无菌性。在标准的椎间盘造影术中，每个患者椎间盘炎的发生率为0%-4.9%，每个椎间盘穿刺患者的感染率为0%-1.3%[37]。由于干细胞移植入路的扩大，应用更大的针管以适应注射剂的黏度，注射更大体积的注射物其感染率越来越高。细胞增殖过程漫长，培育期间的交叉感染可能是另一个问题。严格的细胞培育及应用合适器械的手术操作对预防感染是必须的。另外，移植过程中也应考虑预防性使用抗生素，尽管其对干细胞疗法的效果尚未得以阐明。尽管多数动物模型上的实验是成功的，但其在人体的疗效预期过于乐观。椎间盘为无血供区域，其能为移植的干细胞提供的营养相当有限。成年人髓核细胞密度为(1-5)×104 cm3[38]；即使与大型动物(如猪，羊)相比，人类髓核细胞密度仍较小[39]。故而人体椎间盘高度修复速度缓慢，不会快于实验的动物模型。所以在病变椎间盘高度及刚度恢复至正常之前，脊柱结构将持续处于不正常的生物力学负荷之中。某些动物模型研究中，胚胎干细胞和诱导多能干细胞与肿瘤的发生相关[40]。尽管间充质干细胞致肿瘤化更有争议，其在椎间盘退变性疾病治疗上的潜在致肿瘤化风险被授权评估，以阻止在人体应用后产生的灾难性后果。如果间充质干细胞增殖过程耗时过长，其染色体稳定性会受到影响，且其肿瘤化风险增加，尤其在免疫监控不可用的条件下。使用患者血清或人血小板裂解液缩短培育周期可使染色体不稳定性降低[41]。同种异体间充质干细胞移植有其独特优势，因为可减少操作步骤，并免去了等待细胞增殖的时间；但免疫反应值得关注，有报道指出，使用异体脂肪间充质干细胞移植到猪的心肌而发生了免疫反应[42]。其他人体研究表明，接受同种异体间充质干细胞治疗缺血性左心功能不全的患者发生了极小的免疫反应[43]。椎间隙发生免疫反应的风险并不清楚，大多数人认为因其缺乏血管结构，椎间盘是免疫保护性区域。到目前为止，没有任何文献描述同种异体间充质干细胞移植到椎间盘发生免疫反应的风险。在任何人体试验之前，间充质干细胞是否会在椎间隙引起强烈的免疫反应仍需得到足量动物实验的证实。"

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