Chinese Journal of Tissue Engineering Research ›› 2013, Vol. 17 ›› Issue (6): 1129-1134.doi: 10.3969/j.issn.2095-4344.2013.06.030
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Meng Xiang-peng, Sun Bao-hong, Chen Li-jie
Received:
2012-10-18
Revised:
2012-11-16
Online:
2013-02-05
Published:
2013-02-05
Contact:
Chen Li-jie, M.D., Professor, Master’s supervisor, Chief physician, Department of Neurology, Second Affiliated Hospital of Harbin Medical University, Harbin 150086, Heilongjiang Province, China chenlijie@medmail.com.cn
About author:
Meng Xiang-peng★, Master, Physician, Department of Neurology, Second Affiliated Hospital of Harbin Medical University, Harbin 150086, Heilongjiang Province, China mencius1984@163.com
CLC Number:
Meng Xiang-peng, Sun Bao-hong, Chen Li-jie. Stem cell transplantation for nerve repair[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(6): 1129-1134.
2.1 纳入文献基本情况 初检得到288篇文献,中文51篇,英文237篇。阅读标题和摘要进行初筛,排除因研究目的与此文无关199篇,内容重复性的研究57篇,共保存32篇英文文献做进一步分析。其中14篇是关于干细胞的来源、分类和特征的基础研究,18篇是关于干细胞移植在神经结构修复和功能重建方面的基础研究和临床应用。 2.2 结果描述 2.2.1 中枢神经系统 基本技术与方法:神经干细胞(neural stem cells,NSCs)是具有分化为神经元、星形胶质细胞及少突胶质细胞的能力,能自我更新并足以提供大量脑组织细胞的细胞[1-2]。在胚胎发育的早期阶段,神经干细胞以神经上皮干细胞的形式存在于胚胎神经管。有证据显示,神经干细胞在成年哺乳动物的神经形成过程当中仍发挥重要作用[3]。上述的一切表明利用神经干细胞可以修复创伤的神经,使受损的组织再生。在中枢神经系统中,神经干细胞的特异性分子标记物包括MSI-1(Musashi-1的简称,是一种相对分子质量为 39 000的RNA结合蛋白)、巢蛋白以及某些Sox蛋白家族成员。MSI-1,作为RNA结合蛋白家族的一员,在哺乳动物的胚胎期及出生后的中枢神经系统中都能够得到完美的表达。借助于MSI-1的表达,相关学者阐明神经干/祖细胞存在于成年人脑的室周区域[4]。巢蛋白亦作为一种中间产物,有选择地表达于神经干/祖细胞。 利用神经球培养技术,在包含成纤维细胞生长因子2/表皮生长因子的游离血清培养基中,神经干/祖细胞可以扩展成为一个细胞群。借助于上述培养体系,神经干/祖细胞能够以未分化的形式在体外研究中扩增。某些学者通过流动细胞计数法对上述细胞进行鉴别。例如,Uchida等[5]基于细胞免疫选择表型CD133(+)、CD34(-)及CD45(-),利用细胞表面抗原,分离出活体人胚胎神经干/祖细胞。在体外及体内实验中,上述克隆原细胞的后代能够成功分化出神经元和神经胶质细胞。 利用荧光蛋白,包括神经干细胞选择基因(包括MSI-1、巢蛋白、Sox基因及核干细胞因子)的启动子或增强子表达的绿色荧光蛋白及其诱导剂,可以构建报告基因。在转基因动物实验中,神经干细胞选择荧光蛋白报告基因的优势之一即为定位神经干/祖细胞,从而达到观察监测的目的。例如,在巢蛋白基因增强子的监控下,利用表达增强型绿色荧光蛋白的转基因鼠显示,神经干细胞以“放射状神经胶质”的形式存在于胚胎的大脑皮质[6]。 综上,通过体外实验扩增及上述分离技术获得的稳定的神经干/祖细胞治疗损伤的中枢神经系统的前景广泛,疗效可能超过预期结果。 临床应用:在过去的10年中,不断有学者证实,将神经干细胞移植入罹患神经系统疾病啮齿类动物模型的中枢神经系统后,其组织结构与神经功能会出现明显改观。Cummings等[7]证明,脊髓损伤大鼠模型在给予神经干细胞移植干预之后,不仅轴突髓鞘能够再生,神经功能也有大幅度改善。脑梗死大鼠模型在给予猴胚胎干细胞移植干预后,其偏瘫侧肢体的运动功能恢复明显[8]。另外,有报道称,将非神经干细胞移植入动物模型后,其也可转变为神经细胞。Sieber-Blum等[9]显示,脊髓损伤大鼠模型在给予表皮神经嵴干细胞移植后,其痛温觉及触觉改变显著。与神经干细胞与胚胎干细胞相比,衍自表皮神经嵴干细胞的神经嵴干细胞具有明显的优势。一方面,神经嵴干细胞能够分化为少突胶质细胞和成神经细胞且不会导致肿瘤的发生,不良反应较小。另一方面,便于获得,成本较低。另外一种既具有神经分化能力又无致瘤性的细胞是诱导多能干细胞,它的潜力在脊髓损伤大鼠模型治疗中得到完美的的体现。诱导多能干细胞在细胞植入动物模型后,参与髓鞘再生,促进轴突生长,改善运动功能;同时证明,通过鉴别和选择神经球,其能够有效预防肿瘤的形成[10]。 神经干细胞移植成功的结果是神经功能恢复,而神经功能恢复的关键在于神经保护功能的再建。神经保护伴随着生长因子(如神经生长因子及神经营养因子等)的分泌而发生。细胞移植能够提供源源不断的生长因子,较直接注射生长因子更为有利。原因在于,后者不仅易被稀释,而且半衰期也比较短。有鉴于此,有关学者致力于人工干预干细胞,使其过量表达上述生长因子,进而达到增强神经保护的目的。 Wu等[11]研究表明,衍自神经营养因子的神经胶质细胞系通过过度表达骨髓基质细胞,在帕金森病模型中发挥神经保护效应。有研究证实,在脊髓损伤模型中通过过量表达骨髓基质细胞,能够明显增强轴突再生的速度与神经功能恢复的程度[12]。Chartoff等[13]证实,将骨髓基质细胞植入到新生小鼠的侧脑室,可见骨髓基质细胞完整地分布在小脑和前脑,并且部分分化为星形胶质细胞。Mezey等[14]为了进一步证实造血干细胞在神经组织细胞增殖中的作用,将雄性小鼠造血干细胞移植到免疫功能缺陷的雌性小鼠体内,然后检测含有Y染色体的细胞,结果发现这些细胞能够表达神经元的特异性蛋白和抗体,提示骨髓基质细胞可以转化为神经元。上述发现提示来自成年骨髓的细胞具有比以往所认为的更大的可塑性,在适当的条件下,它们能够分化形成完全不同的细胞表型。 迄今为止,对于人类自身而言,临床试验获得的结论不尽相同。部分临床试验与体外实验结论一致。Meta分析显示,将胚胎中脑组织植入帕金森患者体内后,移植神经元的存活情况良好,患者的相关参数也有明显提高。其中,左旋多巴应用剂量降低37%;F-Flurodopa摄取量增加40%;帕金森病等级评定量表运动总分增高44%;运动障碍时间减少49%[15]。而某些临床试验却出现偏差,原因可能包括个体差异(年龄等)、有无相关手术史(脑深部电刺激术等)及免疫抑制药物剂量差异等因素中单一或共同造成移植物存活率的降低。 2.2.2 周围神经系统 基本技术与方法:对于周围神经系统而言,理想的干细胞来源必须符合如下条件:在客体外,便于获取与培养;在客体内,易于生存与增殖。有研究显示,将胚胎干细胞应用于客体之后,能够成功修复周围神经损伤[16]。但是,胚胎干细胞获取困难,应用范围受到极大的限制。与之相比,成人干细胞克服了上述不足,成为大多数临床周围神经损伤的最佳选择。骨髓基质细胞源自长骨,在人造神经及无细胞移植瓣领域应用广泛。对于骨髓基质细胞能否体内产生功能性髓磷脂这一问题,各国学者存在分歧。有关研究显示,骨髓基质细胞衍生的许旺细胞,完全具有髓鞘再生的能力[17]。体外实验表明,脂肪组织作为多能干细胞的“根据地”,在适当条件下,能够分化为具备髓鞘再生能力的许旺细胞[18]。皮肤及其附属相关结构是干细胞的另一来源。颈须滤泡等处发现了大量神经嵴干细胞,具有分化为神经元、平滑肌细胞、许旺细胞及黑色素细胞的潜能。有报道称,在人工干预切断啮齿类动物模型周围神经后,皮肤源性干细胞分化的许旺细胞能够将神经裂隙修复,并且促进功能的恢复[19]。 如何提高移植干细胞的生存率是目前面临的另一挑战。统计学表明,前体细胞植入客体后的生存率介于0.5%-38.0%,差异性较大,主要取决于细胞类型及评估时间点的选择[20]。免疫系统的攻击是导致外源性细胞死亡的主要原因。增高移植区域细胞数量的措施包括:基因人工操纵、预防性用药及营养支持等。Pan等[21]发现,动物模型在完成羊水干细胞移植后,如果对其进行粒系集落刺激因子干预,不仅可以增加细胞生存率,而且能够促进神经再生。另外,在种间细胞移植中,免疫抑制计划能够有效防止神经创伤位点的干细胞被过早的清除。 如何对植入周围神经系统的干细胞进行追踪探查,是各国学者面临的另一难题。以往的大多数标记物存在局限性,既缺乏稳定性又不够长效。化学标记物如双苯甲亚胺,曾用于标记植入周围神经的许旺细胞,一方面其有效期较短,另一方面还对移植细胞的活性与表型产生负面影响。Dezawa等[22]发现lacZ及绿色荧光蛋白能够克服上述缺陷,既能达到长时间追踪需要,又不危及干细胞的生长。近年来,量子学与生物学的有效结合,彻底改变了传统细胞标记技术的发展轨迹。纳米粒能够有效抵御体内代谢性降解,是长期追踪干细胞发展的理想选择[23]。 临床应用:间质干细胞因其自我更新、增殖迅速及多向分化等特点,成为细胞移植的最佳来源之一。大量研究显示,骨髓衍生性间质干细胞能够促进创伤周围神经系统的修复。骨髓间充质干细胞具有强大的增殖能力和多向分化潜能,在特定的诱导环境条件下可以向骨骼、心肌以及神经元等多胚层方向分化[24]。Chen等[25]发现,骨髓间充质干细胞分泌一系列神经营养因子;在周围神经修复过程中,植入骨髓间充质干细胞的再生潜能与上述营养因子密切相关。细胞凋亡是脊髓继发性损伤的重要形式之一,受多种凋亡蛋白的调控。有研究发现,通过静脉或局部移植的骨髓间充质干细胞,在不同时间点可不同程度的上调蛋白Bcl-2的表达,下调Caspase-3的表达,具有抗凋亡作用,可减轻脊神经的继发性损伤,Caspase家族对细胞,特别是神经元凋亡程序的启动具有核心作用,它直接水解激活与DNA断裂等凋亡特征性改变密切相关的蛋白,又称为死亡蛋白酶。在凋亡信号传导过程中,通常认为Bcl-2作用于Caspase-3的上游,通过抑制Caspase-3激活而发挥作用[26]。但是,骨髓间充质干细胞的制备过程较为复杂,骨髓间充质干细胞的数量也较为有限。 脂肪干细胞,自被报道以来,就因其独一无二的特性而成为再生医学的研究焦点。与骨髓间充质干细胞相比,脂肪干细胞在细胞表型和基因表达方面并无太多差异。脂肪干细胞的优势主要包括以下几个方面:易于获取;在脂肪组织中含量较高;免疫原性低;增殖迅速[27-28]。Kingham等[29]取大鼠腹部脂肪的第2代脂肪干细胞,加入诱导液,2周后检测神经胶质标记物神经胶质酸性蛋白、S100蛋白等的表达显著高于未诱导组,表明脂肪干细胞 向许旺细胞表型分化,从而揭示脂肪干细胞 可能用于外周神经损伤的治疗。脱细胞神经基质移植物,作为一种保留基膜导管的内源性神经段,植入客体后可以发挥“骨架”的作用,能够有效的保证细胞增殖的有效进行。Liu等[30]发现,将脂肪干细胞与脱细胞神经基质移植物共同植入客体后,能够有效的促进离断超过10 mm周围神经的修复。 另有研究发现[27],脂肪干细胞成分的多样性及成脂分化的低效率,限制了它在组织工程中的广泛应用,相比于去分化脂肪细胞的提取,脂肪干细胞在脂肪组织中的含量相对有限,同时它与骨髓干细胞一样存在特异性标记物的问题[31]。与脂肪干细胞相比,去分化脂肪细胞具有更加突出的优点,获取培养方法相对简便,含量更加丰富,易纯化,污染少,具有很高的同源性,明显优于脂肪干细胞的诱导分化效能,在体外具有与干细胞相似的扩增能力,这些都意味着去分化脂肪细胞可能比脂肪干细胞更适合作为组织工程的种子细胞[32]。 "
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