应用基因工程技术增强间充质干细胞移植效果

张金梅 杨远荣 熊建群 丁卓玲

·综述·

应用基因工程技术增强间充质干细胞移植效果

张金梅 杨远荣 熊建群 丁卓玲

间充质干细胞（MSCs）在再生医学领域应用前景无限。它取材较容易，无论是天然的还是通过细胞工程诱导获得的均具有多向分化能力。成功的MSCs治疗依赖于有效的细胞输送和移植细胞的长期存活，以便能长久地在目标位点更多地发挥效应。在MSCs移植前应用基因工程技术对其进行修饰可取得这一效果。本文介绍了能提高移植MSCs的迁移能力、防止MSCs衰老和凋亡及提高MSCs存活率的基因工程方法策略。

间质干细胞； 基因工程； 细胞移植； 细胞运动； 细胞存活

人们对干细胞再生医药的兴趣与日俱增，基因工程技术也被充分应用于增强干细胞的治疗效果。间充质干细胞（Mesenchymal stem cells，MSCs）来源丰富，分化潜能大，在细胞治疗方面极有应用价值,但在实际应用过程中遇到许多问题，如怎样将细胞有效的传递至目标位点并维持较高的存活率。目前局部注射MSCs是最普遍的细胞移植方法，但局部注射有许多缺点，如大量的细胞沉积在精密的器官如大脑中会产生局部结构压力，导致微出血，引发炎症反应，从而增强宿主抗移植物反应。血管内注射是另一种有效的细胞移植方法，细胞随着血液输送到身体各个部位，包括大脑。静脉注射将细胞分配到全身可能会减少到达损伤部位的细胞数量。动脉注射靶向特定的身体部位。但移植细胞还是需要迁移一定的距离才能到达病变组织，细胞迁移能力弱会影响治疗效果。这可通过基因工程方法来解决。MSCs长期的体外培养会不可避免的衰老，从而导致增殖活性的丧失。基因工程技术能增加干性相关基因的表达以维持干细胞的特性，甚至可以增加干细胞体外的增殖潜能。此外，伤口部位恶劣的微环境会对移植细胞产生不利的影响，如高水平的氧化应激、局部缺氧以及促凋亡因子等都会加速移植细胞的消亡，这些都会影响治疗效果。因此，还需要采取有效措施延长移植细胞的存活时间。

一、增加MSCs迁移

SDF-1是细胞迁移过程中最强的趋化因子之一。在生理条件下，SDF-1由损伤组织合成，从受损部位释放。在外层细胞膜上表达CXCR4受体[1]，这是趋化信号。不同的MSCs外层膜上的CXCR4基底蛋白不同。在体外培养中CXCR4会发生变化[1]。在低氧环境中MSCs中CXCR4的表达会大幅增加[2-3]，充分的刺激能引起了内源性CXCR4基因的过表达[1,4]。采用基因工程增加MSCs中CXCR4基因的表达，导致高密度的表达CXCR4受体，能有效地增加MSCs向SDF-1的迁移[5-7]。

表达CXCR4的MSCs在肾脏移植中发挥有利的免疫调节作用[8]。CXCR4-遗传改造的MSCs对早期的肝再生有积极地影响，增强移植肝的归巢，促进肝细胞的增殖[9]。

MSCs中CXCR4过表达能增强急性肾损伤模型的组织修复能力[10]。与对照组MSCs相比，CXCR4-MSCs归巢到损伤部位的亲和力更高，表现出有利的旁分泌作用。修饰了CXCR4的MSCs对伤口部位有较高的亲和力，能加速伤口的愈合[11]。在大鼠缺血模型中，CXCR4-MSCs有更高的动员能力和神经保护作用[12]。除了SDF-1-CXCR4 信号轴的CXCR4修饰外，也采用过表达SDF-1的策略，Nakamura等[13]发现，SDF-1过表达DF-1/CXCR7的MSCs其体外迁移能力增强，SDF-1-MSCs被用于体内伤口愈合实验能明显缩小伤口面积。除了CXCR4与SDF-1结合外，CXC趋化因子受体7（CXCR7）也与SDF-1结合[14]。DF-1/CXCR7信号轴被用于MSCs遗传修饰。Wang等[34]在脑缺血再灌注大鼠海马模型中应用CXCR7过表达的MSCs，证实了过表达的CXCR7受体促进MSCs向SDF-1梯度迁移，与SDF-1/ CXCR4信号轴共同作用[15]。MSCs中过表达的CXCR7会导致他们向次级淋巴器官的迁移增加。CXCR7修饰的MSCs广泛存在于这些器官中，可能会抑制移植物抗宿主病的免疫系统反应，减少临床症状[16]。

CXCR1也能提高MSCs的迁移能力，CXCR1是IL-8的受体，在神经胶质瘤中表达和释放[17]，被用于提高MSCs对胶质瘤的靶向能力[18]。研究表明CXCR1-MSCs对梗死的心肌有高亲和力，移植CXCR1-MSCs的存活率也上升，为心肌损伤提供了一个新的治疗方案[19]。MSCs的迁移能力还可通过对aquaporin-1（Aqp1）基因的修饰来调节，过表达的Aqp1会增加Aqp1-MSCs向损伤部位的迁移能力[20]。Aqp1是水通道分子，转运水分子穿过细胞膜。Aqp1与β-catenin相互作用是细胞迁移的重要调节者[21]。Nur77和Nurr1两个细胞核受体也被用于提高MSCs的迁移能力[22]。高表达Nur77和Nurr1是细胞迁移能力增强的特征[23-24]。病毒转导ITGA-4足以增强MSCs的骨髓归巢能力[25]。还有为增强血管壁的迁移对MSCs进行双重靶向修饰的研究，即同时应用两个mRNAs分别修饰PSGL-1和SLeX，使MSCs产生P-选择素和E-选择素功能性的配体，改善炎症组织的MSCs归巢[26-27]。

二、基因工程修饰MSCs以抗衰老

转录因子Oct4和Sox2参与维持胚胎干细胞的多能性和自我更新，早期用来重编程体细胞为诱导多能干细胞。Fan等[28]发现骨髓来源的MSCs同时过表达Sox2和Oct4基因，能改善增殖和分化潜能。在脂肪来源的MSCs中Sox2和Oct4的过表达有同样的作用[29]。对骨髓MSCs进行Sox2基因修饰更有效，能成功地保持未分化状态。MSCs中Oct4基因的过表达导致其它干性基因如Sox2的表达增加[30]。Sox2和Oct4的过表达也可以通过同时采用leukemia inhibitory factor（LIF）和干细胞特异性miRNAs之一miR-302转染来获得[31]。MiR-302能诱导人脂肪MSCs增殖，抑制氧化剂诱导的细胞死亡[32]。

端粒酶逆转录酶（TERT）基因转染是防止培养MSCs衰老的另一种策略。TERT是RNA依赖的DNA聚合酶，它合成和延伸末端DNA，维持干细胞的永生[33]。体外扩增的MSCs缺乏TERT基因表达，为此，TERT基因工程成为逆转MSCs衰老的方法。被TERT永生化的MSCs的细胞增殖能力增强，细胞周期相关的基因表达因子上升，防止了转染MSCs的细胞周期阻滞[34]。蛋白酶体通路对维持细胞代谢有重要的作用，其功能失调会导致复制性衰老。用哺乳动物蛋白酶体复合物（PSMB5）的β亚基转染MSCs，也会抑制细胞衰老[35]。用小RNA干扰糖皮质激素受体基因[36]和脂质运载蛋白-2基因的过表达保护缺氧条件下MSCs的多能性[37]，这都可防止MSCs的衰老。生长因子基因的过表达可提高MSCs的增殖能力，而某些生长因子会严重伤害MSCs的治疗特性。

三、提高MSCs存活率

MSCs在缺氧时迁移到损伤部位，MSCs对恶劣的局部环境很敏感。治疗细胞的存活率对损伤组织缺氧的伤口部位尤其重要，如心肌梗塞与脑卒中。为此，多种促存活的方法被采纳，修饰MSCs以延长其在靶器官中的存活率，给予足够的时间以产生有益的影响。在体外培养的SDF-1β修饰的MSCs中，SDF-1β能增强细胞自噬，减少细胞凋亡，是促细胞存活因子[38]。低氧诱导因子（HIF-1α）是缺氧导致的细胞代谢变化的主要调节者[39]。HIF-1α调节一系列基因的活性包括血管生成、红细胞生成、细胞增殖、分化及凋亡，使细胞适应缺氧条件[40]。在小鼠后肢缺血模型[41]及大鼠心肌梗死模型[42]的试验中，HIF-1α基因工程MSCs取得好的效果。也可应用MiRNA技术，修饰的MSCs过表达miR-210，miR-210对HIF-1α蛋白的活性有正反馈环调节作用，能促进基因工程MSCs在缺氧条件下的存活率[43]。MSCs基因工程方法产生能抑制治疗细胞凋亡信号的蛋白质，这种蛋白质提供由Bcl-2、E1A激活基因的细胞抑制因子（CREG）、激肽释放酶（KLK1）、血管紧张素转换酶2、精氨酸脱羧酶（ADC）、整合素连接激酶（ILK）、或蛋白激酶G1α介导的抗凋亡信号。抗凋亡效应可通过小发夹RNAs（shRNA）使基因表达沉默来实现。如用pre-miRNA-155-designed caspase8 shRNA转染MSCs后，促凋亡因子caspase8基因停止表达[44]。保护MSCs不受恶血的不良影响是促移植细胞存活的一个重要方案[45]。采用MSCs细胞膜上的力生长因子（MGF-E）可保护转染细胞免受不当流体剪切力的伤害[46]。

采用自体MSCs能避免免疫排斥问题，当自体细胞供给不足时会产生严重的问题，这可通过基因工程技术增加有丝分裂次数加以解决。自体移植时细胞数量不足的问题主要发生在老年人，这些患者的MSCs治疗潜力和数量减少。采用基因工程方法能增加移植细胞的数量和治疗效果。血管内注射是创伤较小的一种方法，保护MSCs免受恶血的影响似乎是关键,但还需要良好的组织靶向性以保证在治疗细

胞在被传递到目标作用位点前细胞数量的损失降到最低，如防止细胞在肺部及淋巴器官滞留引起的意外损失等。这些细胞是用来治疗损伤部位，伤处原有的细胞已严重受损，在这个充满破坏性因素的区域，在此阶段对治疗细胞的保护似乎也很重要。

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Genetic engineering techniques to enhance therapeutic effects of mesenchymal stem cells

Zhang Jinmei, Yang Yuanrong, Xiong Jianqun, Ding Zhuoling. Department of Pharmacy in Jingzhou Centre Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Jingzhou 434020, China

Mesenchymal stem cells (MSCs) are promising in the field of regenerative medicine due to their relatively easy access and multidifferentiation capabilities, either naturally or induced through cell engineering. Successful MSCs treatment depends on efficient methods of cell delivery and the long-term survival of grafted cells at the target site. The application of genetic engineering technology to modify MSCs before transplantation can accomplish long-term survival. In this review, we describe the genetic engineering strategies to increase the migration ability, decrease senescence and apoptosis and improve the survival of transplantated MSCs.

Mesenchymal stem cells； Genetic engineering； Cell transplantation；Cell movement； Cell survival

2016-07-11）

（本文编辑：陈媛媛）

10.3877/cma.j.issn.2095-1221.2016.05.009

434020 荆州，华中科技大学同济医学院附属荆州医院药学部

杨远荣，Email:jzyyyjk@sina.com

张金梅,杨远荣,熊建群, 等.应用基因工程技术增强间充质干细胞移植效果[J/CD].中华细胞与干细胞杂志:电子版, 2016, 6(5):312-315.