Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (45): 7358-7363.doi: 10.3969/j.issn.2095-4344.2014.45.026
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Ma Xun, Qin Jie, Xu Yu-ming
Online:
2014-11-05
Published:
2014-11-05
Contact:
Xu Yu-ming, Professor, Chief physician, Third Department of Neurology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China
About author:
Ma Xun, Third Department of Neurology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China
Supported by:
the National Natural Science Foundation of China, No. 81070920, 81471158, 81301007
CLC Number:
Ma Xun, Qin Jie, Xu Yu-ming. Research progress of induced pluripotent stem cells and its bottleneck[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(45): 7358-7363.
2.1 诱导多能干细胞研究进展 2.1.1 个体化治疗 诱导多能干细胞的主要临床应用方向是患者特异的个体化治疗。干细胞研究领域的进步极大地促进了诱导多能干细胞在临床研究中的应用,目前这方面研究涉及到多个学科领域如病理药理、心血管、神经系统相关疾病等。利用患者来源的诱导多能干细胞来研究疾病的病理机制或治疗方法,应用前景十分广阔。既往干细胞研究的重点多集中在于胚胎干细胞方面,虽然同为多能性细胞,但由于细胞为原始状态缺乏对疾病的表现能力而应用受限。患者来源的诱导多能干细胞保有疾病独特的基因型和表型,是患者自体疾病的最好表现形式,由于取材多样性、创伤小且诊断成效独特而备受支持。 诱导多能干细胞可应用于单基因遗传病,或是多因素疾病如帕金森病[9]、阿尔茨海默病[10-12],以及精神分裂症等[13]。诱导多能干细胞来源的分化成熟细胞,对于毒理试验应用也有重要意义[14]。另外,可以应用诱导多能干细胞来进行药物筛选[15]。这种患者特异性的药物筛选方式更加可靠并且灵活,使得患者免去了不断更换用药剂量方式的苦恼。虽然利用诱导多能干细胞进行个体化治疗仍处在临床前的研究实践当中,但其未来的发展必然会改变现有的医疗模式。 2.1.2 疾病模型 良好的疾病动物模型建立十分困难,而细胞模型相对来说具有一定的可重复性及易操作性。诱导多能干细胞由于其来源的特殊性,被视为是一种十分优秀的构建疾病模型的基础和途径,可用来研究各种疾病的发病机制和基因异常等。 在神经方面,诱导多能干细胞主要被用来建立神经变性病和神经发育异常疾病模型。由于诱导多能干细胞是重编程而来的具有类似胚胎干细胞多能性的原始细胞,因而被用来研究疾病的演变特别是神经变性病和神经发育异常疾病的发展过程和早期改变尤为有利。目前已有多种神经系统变性疾病和发育异常疾病的模型被构建成功。遗传性帕金森病与PINK1基因突变相关,Seibler等[16]研究工作者通过慢病毒转染的方式重编程PINK1基因突变患者的皮肤成纤维细胞为诱导多能干细胞,证实诱导多能干细胞具有该种突变,并进一步分化为多巴胺能神经元,与野生型患者相比较,证实PINK1基因缺失导致线粒体氧化应激增加、膜电位异常,影响parkin蛋白的转运。 2.1.3 基因治疗 传统的用于胚胎干细胞的基因修饰技术如同源重组、克隆扩增等费时费力、效率较低;现今研究技术的重大进步,使得干细胞研究取得了突破性进展。培养条件的改善、转染技术的进步为新的基因修饰技术的发展奠定了条件。 3种主要的技术TALEN,ZFN,CRISPR-Cas9,使得可以精确的对诱导多能干细胞做出人为修饰[17-18]。TALEN和ZFN利用可编程的、序列特异的DNA结合域与非特异的DNA片段FOK1结合形成功能二聚体[17, 19];CRISPR利用RNA引导的cas9核酸酶获得指定的DNA双链片段[20]。TALEN和CRISPR由于消耗低而可重复性好获得了更多关注。 将诱导多能干细胞技术与基因技术相结合进而发现致病基因或者对已知的基因缺陷进行基因治疗。通过联合多种基因技术,研究者可以将多种治疗作用的基因改变植入或直接修正诱导多能干细胞基因缺陷再移植来达到治疗的目的。 运动神经元病是由于某种或多种运动神经元选择性退化而引起的一类疾病,目前尚无特效治疗措施。诱导多能干细胞造模的优势在于对运动神经元病相关表型的完整体现[21-24]。SOD1基因突变是肌萎缩侧索硬化症相关的基因改变,将携带D9OA SOD1突变基因患者的成纤维细胞重编程为诱导多能干细胞,进一步分化为脊髓神经细胞,发现运动神经元中的神经丝缠结及神经突退化现象与肌萎缩侧索硬化症进展相关;通过TALEN技术对D90A突变进行校正后的诱导多能干细胞系进一步证实了基因突变与疾病的相关性[21]。 2.2 面临的问题和瓶颈 2.2.1 分化效率 虽然诱导多能干细胞的重编程技术已经得到突破,但重编程效率和培育周期并不理想,由成纤维细胞诱导为诱导多能干细胞的比例不足1%。重编程效率可通过多种方式得以提高,包括多重顺反子构造、染色体修饰、MicroRNAs或者上调/抑制一些与细胞增殖相关的信号通路[25-26]。研究者正在尝试多种非基因水平的诱导方式来提高重编程效率,包括更换载体、添加培养基质、改变生长环境等。改变培养条件或许是一种提高重编程效率的有效方式,已有尝试添加维生素C来辅助重编程并取得成功的案例[27]。但由于动物实验与人体的差异性,提高分化效率的更多重编程方案的有效性与安全性仍需要进一步研究证实。 2.2.2 致瘤性问题 最初诱导多能干细胞的重编程应用反转录或慢病毒,其应用于基因治疗或者移植治疗后可能存在一定的突变发生率,虽然在动物实验中c-Myc基因的表达受到抑制,并未见异常表现[3, 28],但病毒基因在宿主的整合引发了严重的肿瘤形成风险[29];而其应用于人体的安全性尚不可知。然而无论其风险大小,都应该避免这种可能影响宿主基因组的编程方式。Bayart等[30]的研究指出在重编程过程中,每个重编程因子都可以被替代或完全移除,而不影响重编程多能细胞形成能力,这或许在改变成瘤性方面有一定意义。载体研究方面,比较成熟的方案是应用质粒或腺病毒来作为编码载体[31],以及非病毒微环或小分子物质[32],研究期间尚未发现肿瘤形成[33],由于技术相对简单和较好的重复性使得新的方案在再生医学中广泛普及。 以microRNA导入的方式进行重编程[34],通过DNA媒介的方式避免了可能的整合和时间消耗,可以保证诱导多能干细胞无外来整合。但microRNA转染需要使用干扰素α拮抗剂、B18R等预处理目标细胞以及饲养层细胞应用,试剂昂贵,且滋养层细胞会增加病原转入风险。Yoshioka等[35]目前已研究出通过单个整合的可自我复制的RNA来诱导人体成纤维细胞为纯净的诱导多能干细胞,而避免外来物或潜在的污染源的介入,但这一方法仍需进一步验证其可靠性和可重复性。 蛋白质介导的重编程是另一种避免目标细胞基因改变的方式。最新研究显示人和鼠诱导多能干细胞均可以通过直接将蛋白导入的方式完成重编程而不经过DNA干预[36]。但这种方法的研究由于进程较缓慢、效率低下且消耗昂贵而受到限制[37]。同样的,利用小分子物质的方法也面临以上问题[38]。 由此可见诱导多能干细胞的致癌风险或许可以通过改变重编程的过程和方式来避免,但重编程的本身是否是引发成瘤性的因素仍不清楚,且一些新方法仍面临着一些技术问题。诱导多能干细胞的重编程因子是明确的致癌基因,虽然研究指出c-Myc在重编程以后的表达受到抑制,但其与诱导多能干细胞的基因异常相关性仍需进一步验证。 2.2.3 免疫原性 有相关研究表明诱导多能干细胞具有潜在的免疫原性,即使来源于同源供体,动物实验仍可检测到免疫反应的存在[39],有研究指出这种反应性并不强烈甚至微不足道[40]。Lu等[41]的研究指出了并非所有诱导多能干细胞及其衍生物都能激发免疫反应,诱导多能干细胞的免疫原性与胚胎干细胞仍有所区别,其差异性体现在细胞来源的不同及MHC分子的不同。但总的来说应用诱导多能干细胞的治疗前景仍比胚胎干细胞要好。虽然目前报道中对免疫原性的研究并不多,但免疫反应仍是不可忽视的问题,这种并不强烈的效应是否对疾病研究造成干扰仍需进一步验证,而在移植治疗等方面应用时也需要对此有更多的关注。 2.2.4 不稳定性 诱导多能干细胞面临的另一个问题是基因不稳定性。研究发现干细胞中存在着染色体的异常和更多的小突变[42-48]。基因组杂交技术在多种诱导多能干细胞系中发现染色体亚组的异常,例如染色体非整倍体改变,可引起成瘤性的增加,改变细胞的分化能力。既往研究表明这些染色体改变大多数在重编程之前的成体细胞中即可检测到,是独立于重编程过程单独出现的[49-50]。而近来研究指出,通过单核苷酸多态性高分辨率分析发现诱导多能干细胞相对于来源的成体细胞有更多的拷贝数变异[42],类似研究在成纤维细胞及胚胎干细胞方面也得到了相同的结果。更精确的分析指出诱导多能干细胞不仅需要表观遗传学也需要基因方面的修饰。 肿瘤抑制因子P53主要功能是基因监视,涉及DNA损伤应答、细胞周期终止、衰老和凋亡。目前研究表明P53是重编程的一道阻碍,抑制P53的表达可以提高重编程的效率,然而,P53的失活是否与诱导多能干细胞的基因不稳定性相关仍需进一步研究。 一些非整合的重编程技术被证明可以减少自发肿瘤形成风险并改善诱导多能干细胞的质量:其中部分技术是完全去除病毒DNA或者使用非整合的病毒[51]。未来建立在病毒基础上的重编程需要依靠DNA、蛋白质或microRNA来提高诱导多能干细胞的质量和稳定性。 因此,在利用诱导多能干细胞治疗疾病之前,必须对基因不稳定性进行理解认识及干预,以保证治疗的安全性和稳定性。 2.2.5 建立疾病模型 虽然利用诱导多能干细胞模型来模拟成人疾病已经取得了卓越进展,但依然困难重重:譬如成瘤性、基因失稳等问题都在阻碍着诱导多能干细胞向疾病应用推广;如何高效的分化纯化靶细胞、选择合适的疾病成体细胞来源、模拟病理疾病的生理特点等都需要更多的研究支持。目前来看,细胞来源是较容易解决的问题,患者的自体细胞如皮肤细胞、角质细胞或外周血单核细胞都是理想的来源;有研究机构建立诱导多能干细胞样本库的努力方向主要为外周血单个核细胞[52],也有采用皮肤或尿液。 一些报道指出,鼠模型与实际人体疾病存在一定的差异性[53]。虽然啮齿类动物模型在许多疾病研究中具有重要意义,但也存在明显的局限性:与灵长类动物模型相比,多能细胞的诱导阶段、培养环境、分化策略、行为认知评价和药物试验等都不尽相同。而灵长类动物模型则更加符合研究目的,其与人类的亲缘性更加密切,生理特点类似特别是神经系统。长时程的研究在灵长类动物上得以实现,而鼠类则难以做到。狨猴的应用在这方面较为突出,目前已有帕金森病、脑卒中等多种模型构建成功[54-55],组织学鉴定和MRI均顺利实施。 而患者自体细胞来源更适合于研究疾病的基因异常及进行药物筛选等,研究者还可在体外模拟出由于异常基因引起的环境改变来进一步模拟真实疾病。相对而言,制作动物模型和进一步细胞诱导需要大量资金支持,而患者来源的研究消耗要相对少很多,这对于疾病和药物研究都十分有利。 构建良好的疾病模型,另一个关键就需要将诱导多能干细胞分化为一个稳定的适合继续研究的细胞系,如神经细胞系。而完全分化成熟的细胞系在移植过程中的表现并不理想,因此得到一个稳定的细胞系是关键所在。以帕金森病为例,作为一种以多巴胺能神经元缺陷的疾病,被认为更适合这种细胞替代模型的构建。有研究表明不同多能细胞系的分化效率及分化条件都各有不同,神经分化的策略较多,既往成熟的方案如拟胚体形成及添加诱导小分子物质等缺乏稳定的分化效率,因此明确何种诱导多能干细胞分化为目标细胞的条件和能力对于提高分化效率和细胞质量十分重要。 体外疾病模型受到人工干预和培养条件的影响,环境中的某些因子可能会影响诱导多能干细胞的重编程及分化为靶细胞的过程,这对患者来源的疾病模型的建立造成了影响。虽然靶细胞是由稳定的诱导多能干细胞诱导来的,但环境却大有不同,其多样性问题是模拟疾病过程中的一道障碍[56]。 再者,疾病病理模型的复杂性,很难由单一细胞所表现出来,细胞间及细胞和环境间相互作用在发病过程中起到重要的作用。复杂的疾病需要更加复杂的多细胞参与和外环境支持而非单一成分[57]。因此早期诱导多能干细胞研究多着重于单基因病的研究,其更直观更容易通过模型构建来反应真实的表型。同样的,成体代谢模式的构建亦是研究诱导多能干细胞疾病模型的关键所在[58],在心血管系统相关疾病的研究中,单独应用诱导多能干细胞模型对于成人发作性疾病的体现能力有限,研究者通过构建诱导多能干细胞来源的心肌细胞,给予胚胎发育及糖代谢环境,加入相关酶学干预等代谢相关处理,获得了有效的体外成体疾病模型。神经系统领域许多疾病发展与之类似,脑和脊髓由于其特殊的代谢环境,更需要合理的构建疾病模型来了解疾病真正的演变过程,这一领域仍需要更多的研究支持。目前已有实验室成功构建三维组织器官如肠道和肝脏等,但这一技术领域发展尚不成熟[59-60]。未来的研究方向应是由分子细胞水平向组织器官整体结构的进一步发展和深化。"
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