Chinese Journal of Tissue Engineering Research ›› 2013, Vol. 17 ›› Issue (10): 1876-1883.doi: 10.3969/j.issn.2095-4344.2013.10.026
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Qu Yi1, 2, Sun Zheng-wei1, Yang Dong-bo1, Jiang Chuan-lu1
Received:
2012-11-06
Revised:
2013-02-02
Online:
2013-03-05
Published:
2013-03-05
Contact:
Yang Dong-bo, Doctor, Associate chief physician, Associate professor, Master’s supervisor, Department of Neurosurgery, Second Clinical Medical College of Harbin Medical University, Haerbin 150086, Heilongjiang Province, China
Ydb2112053@yahoo.com
About author:
Qu Yi★, Studying for master’s degree, Attending physician, Department of Neurosurgery, Second Clinical Medical College of Harbin Medical University, Haerbin 150086, Heilongjiang Province, China
qili99310@yahoo.com.cn
CLC Number:
Qu Yi, Sun Zheng-wei, Yang Dong-bo, Jiang Chuan-lu. Neural stem cell transplantation for treatment of focal cerebral ischemia injury in rats[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(10): 1876-1883.
2.1 神经干细胞定向分化调控机制研究 2.1.1 神经干细胞的培养分离 体外分离培养的神经干细胞有胚胎、脐血和成体来源,人神经干细胞系是最有希望用来移植治疗缺血性脑卒中的外源性干细胞,目前研究较多是来自人畸胎瘤细胞系。 分离神经干细胞的方法主要有机械分离法和胰酶消化法。机械分离法是将组织块剪碎,对神经球用吸管等反复吹打,使之分散。胰蛋白酶法是将神经球细胞分离成单个细胞,通过过滤和离心制成单细胞悬液。神经干细胞常用3种方法进行分离纯化。①反复传代法:在无血清的培养基中培养神经干细胞,同时培养基中含多种具有神经营养作用的因子,使神经干细胞在体外培养中可以稳定并有效的扩增。经过长期反复的传代培养,可以使神经干细胞得到纯化。②流式细胞分选法[12]:用一些特异性表面标记物对神经干细胞进行荧光染色,使荧光抗体与细胞表面标记结合,应用流式细胞仪对细胞悬液中的神经干细胞分选。③免疫磁珠法[13]:是将特异性的抗体包裹在磁珠的表面,与神经干细胞表面的抗原分子结成,使抗原和抗体发生免疫反应,神经干细胞表面结合的磁珠,在磁场作用下阴性细胞被洗脱掉,结合有磁珠的神经干细胞被分离出来,在脱离磁场作用后洗脱下来的细胞即是被分离纯化的神经干细胞。 神经干细胞的体外培养条件与一般细胞培养有所不同,目前,国内外较多采用无血清培养基来进行神经干细胞培养[14],并在体外培养的同时添加一些生长因子,如表皮生长因子、碱性成纤维细胞生长因子以及B27等。由于血清的成分复杂,还含有很多营养物质,如维生素、贴壁因子、矿物质等,以及一些抗体、补体等成分,这些都是普通细胞培养的常用成分,对细胞的生长具有积极的促进作用。但是,血清中的一些物质对细胞培养也起到干扰作用,不利于维持神经干细胞的未分化培养。因此,在进行神经干细胞培养时,常采用不含血清的DMEM/F12培养基来完成神经干细胞体外培养,进行诱导分化使用[15]。生长因子在神经干细胞的培养和增殖分化中起到重要作用,碱性成纤维细胞生长因子是体外培养神经干细胞最常用的促分裂增殖因子,在无血清培养基中可促进神经干细胞的分裂增殖,抑制神经干细胞的分化,在神经干细胞的培养中主要是对神经元前体细胞增殖起作用。培养基中的辅助因子如B27等,在体外培养神经干细胞过程中,还起到长期分裂增殖的作用。 体外培养神经干细胞的过程中要注意以下2个方面。首先,在细胞培养过程中如果出现死细胞、细胞碎片及细胞分解产物等要及时清理,由于这些细胞具有细胞毒性,会影响到神经干细胞的增殖以及使细胞特性降低。另外,对神经干细胞的生长密度有一定要求[16],即神经干细胞生长的密度依赖性,如细胞生长密度过低,细胞间会缺乏有效的连接,导致细胞增殖的停止和分化障碍,如果细胞生长密度过高,会导致细胞的聚集成团,这样会抑制细胞的生长繁殖。 2.1.2 神经干细胞的标记物 为了对体外培养的神经干细胞进行分离鉴定,对神经干细胞细胞表面标记物进行研究,目前研究的标记物有巢蛋白、波形蛋白1、5-溴脱氧尿嘧啶核苷、神经元特异性烯醇化酶等。 巢蛋白属于Ⅵ型中间丝纤维蛋白,是未分化状态多能干细胞的抗原标记物,是主要细胞骨架蛋白,存在于神经上皮的干细胞,巢蛋白在分裂增殖能力旺盛的神经前体细胞中高表达[17],调节其增殖分化成熟,在胚胎发育早期发挥作用[18]。巢蛋白在神经细胞形成期开始高表达,在神经细胞迁移和分化后逐渐消失,向终末细胞神经元、星形和少突胶质细胞分化为低表达,并逐渐减弱[19],因此,巢蛋白是神经干细胞特征性的生物学标记物,是原始神经细胞的标记物之一。 波形蛋白1是属于Ⅲ类中间丝蛋白,紧跟着巢蛋白在神经前体细胞表达,当细胞分化完成后低表达,并逐渐下降,是神经干细胞的一种表面标记物[20]。 5-溴脱氧尿嘧啶核苷是胸腺嘧啶的衍生物,在正常动物体内不存在,经腹腔注射,可以代替胸腺嘧啶在DNA合成期活体注射或细胞培养加入,并可与细胞核内DNA相结合,主要是用来标记分裂期的神经细胞,利用抗5-溴脱氧尿嘧啶核苷单克隆抗体来显示细胞的增殖[21]。5-溴脱氧尿嘧啶核苷的标记是用于分析培养神经细胞的分裂周期以及展示中枢神经系统细胞增殖[22]。近期的研究中发现,5-溴脱氧尿嘧啶核苷主要用于干细胞的鉴定,当干细胞增殖缓慢鉴定有利于在分裂过程中和细胞DNA进行整合[23-26]。 神经元特异性烯醇化酶是神经元特有的一种酸性蛋白酶,是神经元特异性标志蛋白,在神经细胞能量代谢过程中参与糖酵解过程的关键酶,主要存在于中枢神经系统的神经元和神经内分泌细胞内,通过返转录聚合酶链式反应半定量检测确定神经元的数目,进而判断神经干细胞是否向神经元细胞分化。胶质纤维酸性蛋白是星形胶质细胞的标志蛋白。髓磷脂碱性蛋白是单链的灵活多肽,位于致密的髓鞘与髓核中,用于少突胶质细胞的鉴定。 通过流式细胞技术可以对神经干细胞表面分子标记,神经干细胞表达不同分化抗原分子,如CD133+、CD34-、CD45-等。 2.1.3 神经干细胞的分化调节机制 神经组织在损伤后,可以通过内源性干细胞或前体细胞进行自我修复,这一过程就需要通过神经干细胞的自身调节机制来完成。神经干细胞的调节作用是由正负双重作用来实现的。负性调节是使神经干细胞不分化,对称性分裂来增加神经干细胞数量,主要通过Notch信号途径和一些生长因子来完成[27],Notch信号途径是一种抑制性的信号传导通路,可以抑制正在分化的神经干细胞以及其周围的细胞不向神经元分化,而发育成上皮细胞,使神经细胞从原来的单层细胞中分离。Notch信号其跨膜受体和配体的结合而激活。Notch受体是整合型膜蛋白,其胞外区含数量不等的表皮生长因子样重复序列,该序列与果蝇Notch配体DSL或线虫Notch配体Lag2或脊椎动物Notch配体Jagged相结合,从而使Notch受体胞内部分脱落并移入胞核,结合并激活靶基因CSL,该基因也被称作CBF-1,无毛抑制子或LAG-1,直接或间接刺激转录。 诱导神经干细胞分化的正性调节,包括参与细胞合成的gp130/JAK/Stat3和骨形态发生蛋白信号途径等。骨形态发生蛋白信号途径通过瞬时复合体Stat3/CBP/Smad的介导诱使神经干细胞向星形细胞分化[28]。神经干细胞向神经元分化是由神经元碱性螺旋-环-螺旋转录因子包括Mash1、Ngn1、Ngn2和Math家族来正性调控[29-30],骨形态发生蛋白信号可被Hes1和Hes5抑制[31]。Mash1突变鼠嗅球Notch信号途径不能被激活,不能产生感觉神经元前体细胞;Ngn1突变鼠可产生感觉神经元前体细胞,但该细胞缺乏必要调节分子的表达,因此分化受阻[32]。此外Ngn1可使CBP-Smad1转录复合体与星形细胞分化基因隔离,并抑制向胶质细胞分化所必须的STAT转录因子活性因而抑制神经干细胞向胶质细胞分化[33]。 2.2 神经干细胞移植治疗大鼠局灶性脑缺血损伤 神经干细胞移植治疗脑缺血理想的目标是将缺血区受损的神经元能被同型神经元所替代,诱导局部血管再生使移植的神经干细胞长期存活。移植神经元能与周围细胞建立突触联系。胶质细胞辅助移植神经元形成髓鞘。 2.2.1 神经干细胞移植治疗大鼠局灶性脑缺血损伤的方法[34] 神经干细胞培养:取胎龄8-10 d的Wistar胎鼠,切开脑组织,去除硬脑膜,机械剪切吹打后经80目滤网过滤成单细胞悬液,Hanks液冲洗3次,1 000 r/min离心5 min后弃上清。神经干细胞培养液重悬细胞,以体积比20︰1加入B27,青霉素100 mg/L,链霉素100 mg/L,碱性成纤维细胞生长因子20 μg/L,表皮生长因子20 μg/L,细胞计数后以1×109 L-1种植于 25 mL培养瓶中,置于含有5%二氧化碳培养箱37 ℃常规培养,三四天换液1次,7-10 d传代1次。 动物模型制备:健康成年雄性Wistar大鼠,体质量(280±20) g,10%水合氯醛0.4 g/kg腹腔注射麻醉,Longa线栓法制作右侧大脑中动脉局灶性脑缺血模型。 免疫细胞化学染色:细胞传至第5代时将细胞移植于经多聚赖氨酸处理的培养板中,培养液为含体积分数10%胎牛血清的DMEM/F12,培养六七天后丙酮固定,0.1%TritonX-100处理,相应一抗4 ℃孵育过夜,PBS冲洗后与生物化二抗在37 ℃下孵育1 h,DAB染色,苏木精复染后封固。 细胞标记和神经干细胞移植:细胞传至第5代3 d后,更换培养液,在神经干细胞培养液中加入5-溴脱氧尿嘧啶核苷至终浓度10 mg/L,待神经球再次形成时,收集并吹打使之分离,离心后弃上清待用。将模型大鼠固定于脑立体定向仪上,双侧耳棒刻度精确相同,齿棒在水平线下方2 mm,头部正中切开头皮,暴露前囟,侧脑室注射点为前囟向尾侧0.8 mm,中线偏外侧0.2 mm,注入深度3.0 mm,用微量注射器10 min注完神经干细胞,结束后留针5 min,骨蜡封闭骨窗。 2.2.2 神经干细胞移植的途径及部位 目前神经干细胞移植主要包括3种途径,即脑内移植、血液移植和脑脊液内移植。 脑内移植需将大鼠固定于脑立体定位仪,移植点可选损伤中心、损伤边缘、纹状体、海马等[35-36]。作者曾采用经枕大池及立体定向脑内移植神经干细胞对大鼠脑损伤进行修复,方法是将动物麻醉后,俯卧,消毒颈部并使之拱起,以枕外粗隆和颈椎最高点之间的中点为穿刺点,以1 mL注射器进行穿刺,方向为两耳连线的中点,当针尖穿破环枕筋膜有突破感时,回抽有少量脑脊液,穿刺成功。穿刺有突破感后进针不宜超过2 mm。立体定向移植接受细胞悬液注入[37]。脑内直接移植是将神经干细胞注入到脑损伤部位,这样可以避开血脑屏障的阻碍,使移植细胞的存活率提高,定位比较集中,是目前神经干细胞移植首选的治疗方法之一[38]。与其他移植途径相比具有减少细胞迁移的作用,同时局部的微环境不利于移植细胞诱导分化,减少移植细胞在脑外器官损失。但脑内移植的缺点是移植的同时会对脑部造成新的损伤,而且移植的干细胞数量有限。 血液内移植包括静脉移植和动脉移植[39-40]。静脉移植是通过注射细胞到股静脉和尾静脉进行移植,细胞注射到静脉内进入血液循环,迁移到脑内[41]。静脉移植主要考虑的是移植的细胞是否会通过血脑屏障,是否会顺利到达脑损伤部位,并迁移和定居。有学者研究发现在对大脑中动脉永久性闭塞大鼠进行神经干细胞移植治疗,使用股静脉和纹状体内移植的方法,研究发现通过股静脉移植大鼠的神经功能恢复比纹状体内移植大鼠的神经恢复较好[42]。可见静脉内移植神经干细胞在长时间的功能恢复方面,较纹状体内移植效果更好。Chen等[43]研究静脉移植细胞可能会被内脏器官所俘获,真正能够迁移到脑内的细胞数量很少,研究发现大鼠大脑中动脉闭塞模型通过静脉途径注入人的脐血细胞后,仅有1%数量的细胞到达脑内。动脉移植省去静脉移植后细胞长距离迁移及器官的吸收过滤作用[44],可以将移植的大量神经干细胞很快的带到脑损伤部位,治疗效果优于静脉内移植,但动脉移植操作相对复杂,危险性较高,广泛开展较难。 脑脊液内移植包括脑室移植和蛛网膜下腔内移植。蛛网膜下腔内移植具有创伤小,操作相对简单的特点,是治疗脊髓损伤中常选用的方法,在动物脑损伤模型的治疗中也可以发挥一定的功效,但由于实验操作的要求较高,还有可能引起脑水肿,因此临床推广也很难[45]。 2.2.3 神经干细胞移植时间窗 干细胞移植时间的选择具有双面性,研究表明移植神经干细胞可以向神经元分化,只是允许成功移植的时间窗非常短暂,在损伤后24 h为急性炎症期,大量炎性因子如白细胞介素1,白细胞介素6和肿瘤坏死因子α等都有神经毒性和促胶质细胞分化特性,因此该期的微环境不适于移植的神经干细胞向神经元分化,而在损伤晚期由于囊腔扩大和胶质瘢痕生成而抑制神经轴突生长,因此也不是适当的移植期。 干细胞移植研究目的主要是修复缺血梗死的神经细胞,时间窗的选择应考虑多在实验动物脑缺血两三周后。该时间段在梗死区周围皮质促进神经元生长的基因上调,进而可促进骨架蛋白的形成,轴突的锥性生长,并诱导内源性神经干细胞迁移。此时外源性干细胞的介入会起到协同作用[46-47]。当研究目的是卒中急性期的神经保护时,干细胞移植时间多在缺血后 24 h,该时期脑源性生长因子、血管内皮生长因子、胶质细胞源性生长因子等有益于移植干细胞存活,同时白细胞介素6等炎性因子对移植细胞有损害作用。 2.3 神经干细胞移植治疗大鼠神经功能检测 神经干细胞移植治疗缺血性脑损伤不仅要达到移植细胞可长期存活的目的,更重要的是移植细胞可以与宿主健存的细胞发生突触联系而达到功能恢复。有学者采用爬行计分法[48],将长2.0 m,宽2.5 cm的木杆,一端抬高45°,术前3 d训练大鼠由低向高处爬,直到熟练爬完全程。将木杆分为4段,每段6分,共24分,4段得分相加,即其最后得分。计分标准:6分,不能爬、滚落或趴于患侧;5分,患侧肢体拖行;4分,摔下或滑倒> 3次;3分,无滑落,但对侧后爪不触及木条侧面;2分,单侧跛行(肌力下降);1分,四肢支撑变宽,位于木条下;0分,正常,无明显缺陷。在脑出血造模后1,2,4,10,14 d分别进行行为学测试,各组都于14 d 在行为测试后全部处死。大鼠模型组神经缺损症状轻微,各移植组和模型组大鼠均出现活动迟缓,易激惹,左侧肢体偏瘫,立行欲倒,侧旋转爬行或拖步行走,前爪抓力减弱等。各组大鼠爬行计分在2 d时最高,4 d时开始逐渐下降;10,14 d 神经干细胞移植的4组大鼠与模型组比较差异有显著性意义(P < 0.01),而2 d移植组疗效最佳,与1 d移植组、3 d移植组、4 d移植组比较差异有显著性意义(P < 0.01) [49]。"
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