缺血性脑损伤诱导大鼠巢蛋白表达的实验研究

目的探讨缺血性脑损伤对内源性神经干细胞增殖、迁移的影响。方法选用健康雄性SD大鼠62只，随机分为正常组（6只）、脑缺血10min再灌流1、3、5、7、10、15、20d组（简称手术组，每时间点6只）、假手术对照组（14只，每时间点2只），参照Pulsinelli—Brierley方法制作短暂性全脑缺血动物模型：用SABC免疫组化法显示巢蛋白（nestin）阳性细胞；光镜下观察nestin阳性细胞的形态学变化并计数，半定量分析脑缺血损伤后内源性神经干细胞增殖、迁移的变化过程。结果手术组的nestin阳性细胞在缺血再灌流24h后表达增多，7～10d到高峰，15d时仍有显著表达；在室管膜下区的nestin阳性细胞有向皮质、海马迁移的迹象。结论缺血性脑损伤能诱导内源性神经干细胞增殖，这可能对脑损伤后的修复发挥作用。     

成年大鼠脑创伤后神经前体细胞的增殖及迁移

目的 研究液压冲击性脑损伤后成年大鼠神经前体细胞的增殖及迁移。方法 制作液压冲击性脑损伤模型，免疫组织化学方法动态检测巢蛋白（Nestin）和5溴脱氧尿苷（BrdU）的表达。BrdU标记方法确定增列殖的前体细胞；Nestin的表达用于确定神经前体细胞。结果 同正常对照组相比较，伤侧皮层、海马及室下区的Nestin阳性细胞数于伤后1d明显增多，7d达高峰,30d消失；BrdU阳性细胞数于作后3d达高峰，而7d以后逐渐减小，室下区BrdU阳性细胞及Nestin阳性细胞经胼胝体向对侧迁移。结论 液压冲击性脑损伤可激发成年大鼠神经前体细胞增殖及迁移。     

半乳凝素1对脑损伤大鼠室管膜下区内源性神经干细胞原位增殖及迁移的影响

背景：半乳凝素1表达于室管膜下区的星形胶质细胞中并诱导其分化，分化的星形胶质细胞可明显提高脑源性神经生长因子的产生。 目的：观察侧脑室注入半乳凝素1对脑缺血损伤大鼠内源性神经干细胞增殖、迁移的影响。 设计、时间及地点：细胞学体内观察实验，于2007-03/11在佳木斯大学神经科学研究所完成。 材料：纯种清洁级成年Wistar大鼠48只，随机分为模型组、药物组，24只/组。半乳凝素1为北京晶美生物工程公司产品。 方法：两组大鼠均采用线栓法制作局灶性大脑中动脉闭塞模型。造模后24 h，药物组经右侧侧脑室注入10 μL浓度为0.2 g/L的半乳凝素1，模型组注射等量生理盐水。分别于缺血再灌注后3，7，14，28 d处死大鼠，取脑组织制作石蜡切片，处死前1 d腹腔注射BrdU。 主要观察指标：免疫组织化学染色检测大脑室管膜下区BrdU和巢蛋白阳性细胞的表达。 结果：模型组缺血再灌注3 d后大脑室管膜下区BrdU、巢蛋白阳性细胞开始增加，7 d增殖达高峰，14 d后表达开始下降，28 d后下降至最低。药物组缺血再灌注3 d后大脑室管膜下区两种阳性细胞均明显增加；7 d 增殖达高峰，半定量分析BrdU、巢蛋白阳性细胞数分别是模型组的2倍和1.5倍，且BrdU阳性细胞向腹外侧迁移，巢蛋白阳性细胞胞体变大，突起增长，并有向外侧迁移进入脑实质的迹象；14 d后开始下降；28 d降至最低，但仍明显多于模型组（P < 0.05）。 结论：经侧脑室注入半乳凝素1在大鼠脑缺血后可激活内源性神经干细胞原位增殖，并存在由增殖区向外周脑实质迁移的趋势。     

2003/成年大鼠脑出血后神经再生的实验研究

目的 观察大鼠实验性脑出血后内源性神经前体细胞的增殖、迁移、分布和在出血灶周边的分化。方法 将成年SD大鼠随机分为正常组、假手术组和脑出血组;脑出血组大鼠通过立体定向术向脑内注入自体动脉血制成脑尾壳核出血模型,并按不同的再喂养时间（1、3、7、14及30 d）分为5个亚组。手术后腹腔注射5-溴脱氧核苷尿嘧啶标记新生的内源性神经前体细胞。采用免疫组化单标观察内源性神经前体细胞（BrdU阳性细胞）的增殖、迁移和分布,免疫荧光双标观察内源性神经前体细胞在出血灶周边的分化情况。结果 与正常组和假手术组相比,脑出血组大鼠的BrdU阳性细胞数显著增加,并在7～14d达高峰;BrdU阳性细胞主要分布于室管膜下层、海马齿状回、脉络丛、胼胝体腹侧、出血灶周边区、外侧隔核、斜角带、缰核和大脑皮层等处。免疫荧光双标显示在脑出血灶周边区可见BrdU/GFAP、BrdU/NSE及BrdU/Nestin三种双标阳性细胞;BrdU/Nestin双标阳性细胞随着脑出血后时间的推移逐渐减少,而BrdU/GFAP、BrdU/NF-200双标阳性细胞则增多。结论 脑出血可诱导内源性神经前体细胞增殖,并向出血灶周边区迁移,进一步分化出神经元和胶质细胞,这可能是脑出血后神经结构重塑和功能恢复的重要物质基础。     

脑缺血后内源性激活神经干细胞的机制研究

1 脑缺血后内源性神经干细胞的自身激活 近年来研究表明,脑缺血后自身的神经干细胞有大量的增殖分化[1,2],说明神经干细胞可能参与脑缺血的病理生理过程.Zhang等通过建立沙鼠大脑中动脉缺血(MCAO)模型,观察缺血再灌注后不同时期的室管膜下层(SVZ)、海马颗粒细胞层(DG)、嗅球以及梗死灶周边皮质的神经干细胞增殖、分化情况,发现7 d后在梗死同侧脑SVZ出现神经干细胞增殖高峰,14 d达最高,而DG区则无增殖,且14 d后远离梗死区的嗅球有大量的神经干细胞增殖,但28 d后,标记Brdu阳性细胞大量减少,说明脑缺血只引起短暂的神经干细胞的增殖,Zhang认为是脑缺血损伤的应急保护反应[3].     

脑缺血再灌流诱导内源性神经干细胞的增殖、分化

目的 观察脑缺血再灌注后内源性神经干细胞的分布、增殖和分化.方法 阻塞大鼠大脑中动脉2h不同时间的再灌流制成局灶性脑缺血模型,免疫组化单标、双标观察神经干细胞的增殖、分布、分化.结果 正常组和假手术组,多数脑室室管膜细胞Brdu免疫反应阳性,脑实质内有零星散在Brdu阳性细胞.再灌流1d,室管膜及室管膜下层的Brdu阳性细胞显著增多;再灌流3～10d,视前区梗死区周边区、缺血纹状体和额顶皮质的Brdu阳性细胞显著增加.再灌流3d,Brdu阳性细胞出现于海马齿状回的颗粒下层,并随再灌流时间延长,阳性细胞数量增多.免疫组化双标显示再灌流3d视前区有很少量的Brdu/GFAP双阳性细胞,随后增多.再灌流10d内,未检查到Brdu/NF双标阳性细胞.结论 成年哺乳动物的室管膜、脑实质有少量神经干细胞,脑缺血后室管膜、齿状回颗粒下层出现大量的神经干细胞增殖,这些增殖神经干细胞向缺血区迁移、分化,试图补偿丢失的细胞,重塑受损的神经组织.     

大鼠局灶性脑缺血后神经前体细胞增殖迁移的研究

目的研究成年大鼠内源性神经前体细胞在局灶性脑缺血后不同时间窗的增殖分化情况。方法用Longa线栓法制作大鼠脑缺血模型，用免疫组化的方法检测大鼠内源性神经前体细胞最佳增殖时间及最大增殖效果：j结果脑缺血半球室管膜下区、嗅球和头端迁徙渠道(rostral migratory stream，RMS)Brdu阳性细胞数在脑缺血后1d明显增多，3～7d达高峰，14d后开始下降；Brdu阳性细胞数在脑缺血半球室管膜下区和嗅球成止相关；脯缺血侧海马齿状回未见Brdu阳性细胞数明显增多；Brdu阳性细胞在脑缺血缸后第3周数量最大，从第4周开始下降。结论成年大鼠局灶性脑缺血后3～7d内源性神经前体细胞增殖达高峰，住该期进行外源性细胞移植治疗可能更有效。     

反义Noggin基因对成年大鼠海马内Nestin及GFAP表达的影响

目的探讨Noggin基因对成年大鼠海马内Nestin及GFAP表达的影响。方法反义寡核苷酸技术封闭内源性Noggin基因的表达,免疫组化法检测成年大鼠海马内Nestin与GFAP的表达。结果侧脑室连续4d注射Noggin基因的反义寡核苷酸后,可见海马齿状回(dentate gyrus,DG)内Nestin阳性细胞数与GFAP阳性细胞数较对照组显著增加;室下区GFAP阳性细胞数亦明显增加。结论Noggin对成年海马干细胞的分化有重要作用,内源性Noggin基因的表达可使神经干细胞向神经元方向分化。     

N-Methyl-D-Aspartate受体拮抗剂对脑缺血后成年大鼠神经干细胞内源性激活的实验研究

目的　研究N - Methyl- D- Aspartate(NMDA)受体拮抗剂MK- 80 1对脑缺血后神经干细胞(NSC)激活的作用。方法　将4 0只SD大鼠分成对照组和实验组,两组大鼠均采用传统线栓法作成大脑中动脉缺血再灌注模型,实验组大鼠腹腔注射MK- 80 1,对照组腹腔注射生理盐水,通过免疫组织化学技术标记鼠脑海马齿状回颗粒细胞层(SGZ)、室管膜下层(SVZ)及梗死皮质周边区注射后第3、7、11、18天的Brdu、Nestin阳性细胞数。结果　对照组大鼠Brdu、Nestin阳性细胞7d在SGZ出现一小高峰,然后迅速下降,11d阳性细胞甚少,梗死皮质周边区更少;而实验组Brdu、Nestin阳性细胞3d在SVZ明显表达,7～11d在SGZ区达高峰,并可持续至18d,同样梗死皮质区Brdu、Nestin阳性细胞7～18d表达明显,两组比较,有统计学意义(P<0 .0 1)。结论　NMDA受体拮抗剂MK- 80 1在脑缺血后,能促进NSC的增殖、分化。     

脑缺血再灌注神经干细胞原位激活的研究

目的 探讨大鼠局灶性脑缺血再灌注后不同时间、不同脑部位神经干细胞(NSC)原位激活的变化规律。方法 建立一侧大脑中动脉闭塞缺血再灌注模型,用5-溴脱氧尿苷(BrdU)标记脑内增殖细胞,免疫组化(SP法)技术检测大鼠缺血再灌注不同时间段缺血周边皮质、双侧侧脑室下区(SVZ)、海马齿状回下区(SGZ)具有增殖能力细胞BrdU表达变化。结果 对照组脑组织双侧SVZ、SGZ可见少量BrdU阳性标记细胞;单纯脑缺血2 h组脑组织BrdU阳性细胞数未见明显增加;再灌注3天组,缺血侧皮质、双侧SVZ和SGZBrdU阳性细胞明显增多(P<0.05),7天时达到高峰(P<0.001),11天时下降,且缺血侧与对侧相比,双侧SVZ呈对称分布,而SGZ以缺血侧增加为主(P<0.05)。结论 一侧大脑中动脉闭塞缺血再灌注可致成年大鼠脑内NSC原位激活,且NSC增殖于缺血后1周达到高峰,激活部位以双侧SVZ、缺血侧海马和周边皮质为主。     

Induction of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in postischemic gerbil hippocampus mainly dissociated with neural stem cell proliferation

We investigated a possible expression of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in gerbil hippocampus after 5 min of transient global ischemia in association to the proliferation of neural stem cell labeled with bromodeoxyuridine (BrdU). The number of PSA-NCAM positive cells increased in the granule cell layer (GCL) of dentate gyrus (DG) by 1.9 to 2.7-fold at 10 and 20 days after the reperfusion. The number of BrdU-labeled cells increased mainly in the subgranular zone of DG by 7.2 to 8.0-fold at 5 and 10 days after the reperfusion. Immunofluorescence for PSA-NCAM and BrdU showed that the majority of DG cells were not double labeled, while one or two cells per section were double labeled in the deepest portion of the GCL only at 10 days after the reperfusion. These results suggest different predominant spatial distribution and chronological change of PSA-NCAM positive and BrdU-labeled cells in DG after transient ischemia.     

脑缺血再灌注海马齿状回神经干细胞Mash-1的表达

背景：Mash-1在中枢神经系统和周围神经系统发育中都有表达,对神经元和神经胶质细胞的分化都有很重要的作用。 目的：检测Mash-1在大鼠脑缺血再灌注海马组织神经干细胞中的表达及其变化。 方法：建立大脑中动脉栓塞制作大鼠脑缺血再灌注模型。实验分为假手术组、缺血再灌注3,7,14,21,28 d组。 结果与结论：免疫组织化学检测结果,随着脑缺血再灌注第3天海马神经元BrdU阳性细胞明显增多,第7天达高峰,然后逐渐减少。Mash-1反应产物随脑缺血再灌注时间延长逐渐增多,第21天表达最强,以后表达逐渐减少。提示Mash-1呈时间依赖性表达,与神经干细胞增殖分化进程相吻合,说明其在神经干细胞晚期分化中起重要调控作用。     

大鼠缺血性脑损伤后室管膜下区miR-124的动态变化及意义

目的观察脑缺血后室管膜下区（SEZ区）miR-124及Sox9mRNA的表达变化,探讨miR-124对脑缺血后神经干细胞增殖分化的影响。方法12只雄性SD大鼠随机分为实验组9只和对照组3只。实验组制作大脑中动脉缺血再灌注模型．分别于造模后第1、3、7天处死3只大鼠,取SEZ区用于实验;对照组仅暴露动脉。采用免疫荧光细胞染色法检测Nestin的表达,实时PCR检测miR-124及Sox9mRNA的表达水平。结果实验组SEZ区Nestin阳性细胞数较对照组明显增加．且在造模后l、3、7d呈递增趋势（P〈0．05）;实验组miR-124表达显著上调,造模后l、3、7d分别为对照组的（1．63±0．13）、（3．67±0．28）、（2．08±0．11）倍（P〈O．05）;实验组Sox9mRNA表达显著下调,造模后1、3、7d分别为对照组的O．85±0．90、0．29±0．34、0．49±O．36（P〈0．05）。结论脑缺血可促进SEZ区神经干细胞增殖,在神经细胞新生过程中miR一124表达上调．同时其靶基因Sox9mRNA表达下调。     

人胚额叶皮层和海马干细胞的自主分化研究

目的 研究人胚胎额叶皮层和海马组织神经干细胞的自主分化特性。观察额叶皮层神经干细胞和海马神经干细胞特性的异同。方法 从人胚胎额叶皮层和海马组织分别分离提出神经干细胞，经无血清体外培养、扩增，形成神经球。神经球贴壁进行不加诱导剂的自主分化。采用细胞生长曲线检测神经干细胞的增殖能力。使用5-溴脱氧尿嘧啶核苷（BrdU）标记分裂增生的细胞，观察细胞的分裂增殖情况。免疫细胞化学法鉴定神经干细胞的自主分化能力，比较额叶皮层和海马神经干细胞的分化特点。结果 从人胚胎额叶皮层和海马分离的神经干细胞具有增殖能力，额叶皮层神经干细胞的细胞倍增时间为3．9d，海马神经干细胞的细胞倍增时间为3．2d。细胞贴壁分化后出现Nestin、GFAP、Tuj-1表达阳性的细胞。皮层和海马神经干细胞分化产生的Tuj-1阳性细胞分别是40．7％和19．3％；皮层和海马神经干细胞分化产生的GFAP阳性细胞分别是59．3％和80．7％。结论 分离培养的额叶皮层和海马神经干细胞具有自我更新和增殖能力，可以向神经元、胶质细胞分化。额叶皮层神经干细胞与海马神经干细胞的倍增时问、自主分化特点和分化为神经细胞和胶质细胞的比率各有不同。     

Increased proliferation of neural progenitor cells but reduced survival of newborn cells in the contralateral hippocampus after focal cerebral ischemia in rats.

Recent studies demonstrated that neurogenesis in the adult hippocampus increased after transient global ischemia; however, the molecular mechanism underlying increased neurogenesis after ischemia remains unclear. The finding that proliferation of progenitor cells occurred at least a week after ischemic insult suggests that the stimulus was not an ischemic insult to progenitor cells. To clarify whether focal ischemia increases the rate of neurogenesis in the remote area, the authors examined the contralateral hemisphere in rats subjected to permanent occlusion of the middle cerebral artery. In the subgranular zone of the hippocampal dentate gyrus, the numbers of bromodeoxyuridine (BrdU)-positive cells increased approximately sixfold 7 days after ischemia. In double immunofluorescence staining, more than 80% of newborn cells expressed Musashi1, a marker of neural stem/progenitor cells, but only approximately 10% of BrdU-positive cells expressed glial fibrillary acidic protein (GFAP), a marker of astrocytes. The number of BrdU-positive cells markedly decreased 28 days after BrdU administration after ischemia, but it was still elevated compared with that of sham-operated rats. In double immunofluorescence staining, 80% of newborn cells expressed NeuN, a marker of differentiated neurons, and 10% of BrdU-positive cells expressed GFAP. However, in the other areas of the contralateral hemisphere including the rostral subventricular zone, the number of BrdU-positive cells remained unchanged. These results showed that focal ischemia stimulated the proliferation of neuronal progenitor cells, but did not support survival of newborn cells in the contralateral hippocampus.     

Early expressions of hypoxia-inducible factor 1alpha and vascular endothelial growth factor increase the neuronal plasticity of activated endogenous neural stem cells after focal cerebral ischemia

Endogenous neural stem cells become "activated" after neuronal injury, but the activation sequence and fate of endogenous neural stem cells in focal cerebral ischemia model are little known. We evaluated the relationships between neural stem cells and hypoxia-inducible factor-1α and vascular endothelial growth factor expression in a photothromobotic rat stroke model using immunohistochemistry and western blot analysis. We also evaluated the chronological changes of neural stem cells by 5-bromo-2′-deoxyuridine(BrdU) incorporation. Hypoxia-inducible factor-1α expression was initially increased from 1 hour after ischemic injury, followed by vascular endothelial growth factor expression. Hypoxia-inducible factor-1α immunoreactivity was detected in the ipsilateral cortical neurons of the infarct core and peri-infarct area. Vascular endothelial growth factor immunoreactivity was detected in bilateral cortex, but ipsilateral cortex staining intensity and numbers were greater than the contralateral cortex. Vascular endothelial growth factor immunoreactive cells were easily found along the peri-infarct area 12 hours after focal cerebral ischemia. The expression of nestin increased throughout the microvasculature in the ischemic core and the peri-infarct area in all experimental rats after 24 hours of ischemic injury. Nestin immunoreactivity increased in the subventricular zone during 12 hours to 3 days, and prominently increased in the ipsilateral cortex between 3–7 days. Nestin-labeled cells showed dual differentiation with microvessels near the infarct core and reactive astrocytes in the peri-infarct area. BrdU-labeled cells were increased gradually from day 1 in the ipsilateral subventricular zone and cortex, and numerous BrdU-labeled cells were observed in the peri-infarct area and non-lesioned cortex at 3 days. BrdU-labeled cells rather than neurons, were mainly co-labeled with nestin and GFAP. Early expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor after ischemia made up the microenvironment to increase the neuronal plasticity of activated endogenous neural stem cells. Moreover, neural precursor cells after large-scale cortical injury could be recruited from the cortex nearby infarct core and subventricular zone.     

脑缺血再灌注损伤后神经细胞巢蛋白和干细胞因子基因的表达★

背景：干细胞因子是缺氧诱导的神经再生因子，可能刺激动物的神经再生。 目的：观察大鼠脑缺血再灌注损伤后神经细胞巢蛋白和干细胞因子基因表达的变化，分析两者变化的时间规律。 设计：随机对照动物实验。 单位：青岛大学医学院附属医院超声诊断科。 材料：实验选用成年健康雌性SD大鼠36只，由中国科学院上海实验动物中心提供。实验所用巢蛋白和干细胞因子 mRNA原位杂交试剂盒、DAB试剂盒均由武汉博士德生物工程有限公司提供。 方法：实验于2005-01/06在山东省脑病防治重点实验室完成。选取32只大鼠，应用线栓法经左侧颈外一颈内动脉插线建立左侧大脑中动脉阻塞再灌注模型，在缺血1.5 h再灌注后2，6 ，1 2 ，24 h，2，3，7，14 d进行观察，每个时间点4只。其余4只为假手术组：除不插线外，其余步骤同实验组。应用原位杂交技术检测脑缺血再灌注后皮质、纹状体和室旁区巢蛋白和干细胞因子mRNA的表达。 主要观察指标：大鼠皮质、纹状体和室旁区神经细胞巢蛋白和干细胞因子基因表达。 结果：进入结果分析数量保持为36只。①巢蛋白 mRNA表达：假手术组皮质、纹状体和室旁区巢蛋白 mRNA表达很弱。缺血再灌注后，在皮质中的表达除再灌注后2 h、纹状体除再灌注后2,6 h以外，室旁区除2,6 h,14 d以外各时间点均明显高于假手术组，差异有显著性意义（P < 0.05）。②干细胞因子表达:假手术组皮质、纹状体和室旁区干细胞因子表达很弱。缺血再灌注后，在皮质中的表达除2,6,12 h以外，纹状体除2,6 h以外，室旁区除2 h,14 d以外各时间点均明显高于假手术组，差异有显著性意义（P < 0.05）。 结论: 干细胞因子表达的时间规律与神经干细胞增殖的时间规律基本一致，可以提示脑缺血再灌注后干细胞因子 mRNA表达可能具有促进神经干细胞增殖作用。     

Basic fibroblast growth factor stimulates the proliferation and differentiation of neural stem cells in neonatal rats after ischemic brain injury

A little is known about the proliferation and fate of neural stem cells in the subventricular zone (SVZ) after cerebral ischemia. However, how endogenous neural stem cells are activated in the premature brain is not clear, although basic fibroblast growth factor (bFGF) is important in neurogenesis. To investigate the effect of bFGF on the proliferation and differentiation of neural stem cells after brain ischemia, we observed cellular changes in the subventricular zone (SVZ) of 3-day-old rats (approximately equivalent to premature infants) using immunofluorescence assays, Western blot analysis, and real-time quantitative PCR methods. The bilateral common carotid artery (BCCA) was occluded in 108 animals, then half received bFGF 10ng/g. Besides, 54 rats without ischemia as normal control. Proliferating cells were labeled by bromodeoxyuridine (BrdU) through intraperitoneal injection in a pulsed or a cumulative protocol. Rats were killed at 4, 7, and 14 days after ischemic injury. The number of proliferating cells in the SVZ in bFGF-treated rats was higher than that in untreated rats; bFGF also promoted neural stem cell differentiation into neurons, astrocytes, and oligodendrocytes. Western blot analysis and real-time quantitative PCR assays confirmed these results. We suggest that bFGF promotes the repair of ischemia brain injury through increasing the proliferation of neural stem cells and their differentiation into neurons, astrocytes, and oligodendrocytes.