碱性成纤维细胞生长因子缓释微球促进神经干细胞生长的体外实验研究

目的 制备一种能够负载足量碱性成纤维细胞生长因子(bFGF)并且能够以理想的速度缓慢释放的载体. 方法 构建负载bFGF的缓释微球,并检测其体外释放过程.将bFGF缓释微球与神经干细胞共培养,以常规多次添加bFGF的神经干细胞培养组作为对照,用AlamarBlue法检测培养细胞的存活情况,ELISA法检测培养上清bFGF含量,定期观察细胞形态学变化,并进行巢蛋白荧光免疫细胞化学染色,检测神经干细胞标记物. 结果 AlamarBlue法检测结果显示,培养3d、5d时bFGF缓释微球组和常规培养组之间细胞增殖率差异有统计学意义(P＜0.05).ELISA法检测结果显示,仅负载bFGF缓释微球组能测量到培养上清中bFGF含量,大致维持在200 pg/mL水平.细胞形态学观察发现bFGF缓释微球组能够在培养第3天即形成神经干细胞球,并且在培养第7天仍保持较好透光度,保持较好的细胞活性.荧光免疫细胞化学染色发现bFGF缓释微球组培养的神经干细胞球巢蛋白染色阳性. 结论 本课题组制作的bFGF缓释微球材料具有较好的生物相容性.缓慢释放的bFGF能够满足神经干细胞生长需要,并且保持神经干细胞自身的细胞特性.该bFGF缓释微球安全有效,可应用于中枢神经系统损伤的在体实验研究.     

Long-term cultivation of multipotential neural stem cells from adult rat subependyma

Cultivation of adult rat neural stem cells (RNSCs) from the ventricular subependyma has been reported to be more difficult than growth of mouse neural stem cells. This is unfortunate, because rats provide useful models of brain function and disease, and implantation of RNSCs in these models could provide critical information on allograft behavior. Growing the cells in an appropriate medium (NS-A+B27 supplement), plating at sufficient densities (>5 cells per mm²), and minimizing opportunities for detachment from the substratum made it possible to isolate and cultivate these cells for over 6 months for >50 passages with no apparent change in phenotype. Single clones could be expanded indefinitely and differentiated to form astrocytes, oligodendrocytes, and neurons, demonstrating that the cultures did indeed contain neural stem cells. The cells had a much shorter cell cycle time (13 h) than doubling time (35 h), suggesting that these cells produce post-mitotic cells in approximately two of three divisions, thus making expansion difficult. The optimization of methods to grow adult RNSCs and identification of characteristics that limit their growth should prove useful in increasing the use of RNSCs for studies of their potential role in brain health and disease.     

神经细胞粘附分子在癫痫形成过程中的表达

目的　探讨癫痫发生前即癫痫形成过程中神经细胞粘附分子 (neural cell adhesion m olecule,NCAM)的作用。方法　以戊四氮 (pentylenetetrazol,4 0 mg/ kg,腹腔注射 ,每日一次 )点燃 Wistar大鼠为对象 ,取在点燃前不同时间点的大鼠海马为标本 ,运用 RT- PCR技术测定对照组、苯巴比妥干预组和非苯巴比妥药物干预组在点燃前各时间点大鼠海马 NCAM m RNA表达。结果　非苯巴比妥药物干预组大鼠均于 17～ 2 3d点燃 ,而药物干预组在 30～ 38d点燃 ,且行为学和 EEG痫样放电均低于非药物干预组 ,苯巴比妥药物干预组 NCAM m RNA表达明显低于非干预组相应时间点 NCAM m RNA表达 ,有明显差异 (P<0 .0 1)。非干预组 NCAM m RNA表达明显高于对照组 ,有显著性差异 (P<0 .0 1)。结论　点燃前 NCAM m RNA表达量与点燃过程呈正相关 ,说明 NCAM在癫痫形成过程中起重要作用。     

Opposite effects on NCAM expression in the rat frontal cortex induced by acute vs. chronic corticosterone treatments

The temporal pattern of exposure to glucocorticoids has been reported to be a critical factor in determining the outcome of glucocorticoid actions at the brain. In this work, the effects of different regimes of subcutaneous corticosterone administration (acute-single injection-vs. chronic-daily injection for 21 days) on the expression of the neural cell adhesion molecule (NCAM) were evaluated in different rat brain regions (CA1-CA4, dentate gyrus, frontal cortex, striatum, and hypothalamus). The treatments were selected according to previous studies in which we showed biphasic effects of corticosterone on memory formation, with acute corticosterone effects being facilitating and chronic effects being deleterious. In addition, the chronic treatment was shown by others to result in structural alterations at the hippocampus. NCAM was evaluated given its cell-cell recognition and adhesion properties, and the involvement on synaptic stabilisation subserving long-term memory formation. The results showed a biphasic modulation of NCAM levels at the frontal cortex, with acute corticosterone resulting in enhanced NCAM levels at 8 h and 24 h posttraining, and the chronic treatment decreasing its expression. None of the other brain areas examined showed significant changes in NCAM expression with corticosterone treatments, except for the hypothalamus that showed reduced NCAM levels after the chronic corticosterone regime. These results support the view that NCAM regulation at the frontal cortex might be a mechanism by which corticosterone treatments influence memory formation. They also highlight the hypothalamus as a brain area particularly sensitive to NCAM regulation by prolonged exposure to elevated glucocorticoids.     

Localization of basic FGF-like immunoreactivity in the hypothalamo-hypophyseal neuroendocrine axis

We examined the localization of basic fibroblast growth factor (basic FGF) in the adult rat brain by immunohistochemical and Western blotting analysis using a specific antibody against a synthetic basic FGF fragment (N-terminal 12 residues). The antibody did not cross-react with acidic FGF. Basic FGF-like immunoreactivity was located exclusively in the neuronal elements and had very heterogenous distribution. Immunoreactive cell bodies were observed in the paraventricular, supraoptic and circular nuclei of the hypothalamus. Numerous immunoreactive neuronal processes originating from these basic FGF-positive cells extended lateroventrally and then caudally to the internal layer of the median eminence. In addition, the neurohypophysis contained a significant number of basic FGF-like immunoreactive fibers. Western-blotting analysis revealed that the hypothalamus and the hypophysis contained a main band of basic FGF immunoreactive with an apparent molecular weight of 17 kDa. These results show that the hypothalamo-hypophyseal neuroendocrine pathway contains basic FGF.     

老年大鼠局灶脑缺血半影区小胶质细胞可塑性变化和bFGF表达规律的实验研究

目的观察青年大鼠和老年大鼠局灶性脑缺血半影区小胶质细胞形态学变化及bFGF的表达规律。方法老年(20~22月龄)雄性SD大鼠36只,青年(4~5月龄)雄性SD大鼠36只;假手术组12只(老年、青年大鼠各6只),脑缺血组60只(老年大鼠和青年大鼠各30只),采用大鼠大脑中动脉闭塞(middlecerebral artery occlusion,MCAO)模型,根据缺血的时间不同分为缺血后3、7、14、21、32d5个亚组,毎亚组各6只;分别取脑,行HE染色和免疫组化染色,观察小胶质细胞的变化和BFGF的表达水平。结果在局灶脑缺血的不同时期老年大鼠半影区小胶质细胞和bFGF的表达水平均低于青年大鼠,且表达高峰延迟。结论与青年大鼠比较,老年大鼠脑缺血后局部损伤严重,修复缓慢。     

神经细胞黏附分子在GDNF保护大鼠多巴胺能神经元中的作用

目的研究神经细胞黏附分子(neural cell adhesion molecule,NCAM)在胶质细胞系源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)保护帕金森(Parkinson's disease,PD)模型大鼠受损多巴胺(dopamine,DA)能神经元中的作用。方法右侧纹状体内立体定位注射6-羟多巴胺(6-OHDA)制备早期PD模型,而后分为4组:对照组(同侧黑质内注射PBS)、NCAM组(同侧黑质内仅注射anti-NCAM抗体)、GDNF组(同侧黑质内注射GDNF)、NCAM阻断组(同侧黑质内注射anti-NCAM抗体30min后注射GDNF),采用免疫组织化学染色技术和免疫印迹技术,观察各组酪氨酸羟化酶(tyrosine hydroxylase,TH)的表达变化。结果GDNF组黑质致密部TH阳性神经元数目及表达的量明显多于PBS组,差别有统计学意义;NCAM阻断组与GDNF组相比,该处TH阳性神经元数目及表达的量明显减少,差别有统计学意义。结论NCAM参与了GDNF保护DA能神经元的作用。     

托吡酯对颞叶癫痫大鼠海马神经细胞粘附分子表达的影响

目的观察托吡酯对红藻氨酸(KA)诱导颞叶癫痫大鼠海马突触重建标记物神经细胞粘附分子(NCAM)表达的影响,进一步探讨托吡酯的抗痫作用机制。方法采用免疫组织化学染色观察KA诱导癫痫大鼠及托吡酯给药大鼠海马神经细胞粘附分子表达水平,并对两组NCAM表达进行比较。结果KA组NCAM表达水平各时间点平均NCAM阳性密度OD率均明显高于N S组和KA TPM组(P<0.01)。结论本研究提示托吡酯可能通过抑制突触重建的形成,减少痫性放电,从而控制癫痫发作。     

电针对SAMP8小鼠海马NCAM和NF-κB表达的影响

目的 研究电针对阿尔茨海默病(AD)模型小鼠海马神经细胞黏附分子(NCAM)和核转录因子κB(NF-κB)的影响,从神经细胞黏附角度探讨电针治疗AD的作用机制.方法 快速老化模型小鼠(SAMP8)24只采用随机数字表法分为模型组、模型+针刺组(简称模针组),每组12只;抗快速老化模型小鼠(SAMR1)12只为空白组,每日一次电针模针组小鼠"百会"、"涌泉"穴,连续治疗21 d.应用免疫组织化学染色、原位杂交方法 检测各组小鼠海马组织NCAM、NF-κB蛋白及mRNA的表达.结果 与模型组比较,模针组小鼠NCAM(0.231±0.007)、NF-κB蛋白(0.367±0.012)及mRNA(0.528±0.016,0.308±0.001)阳性表达明显增强,差异有统计学意义(P<0.05).结论 电针可通过提高NF-κB的表达,诱导NCAM的合成,促进神经细胞黏附.     

bFGF和BDNF对小鼠神经干细胞体外增殖分化的影响

目的观察碱性成纤维生长因子（bFGF）和脑源性神经生长因子（BDNF）对小鼠源性神经干细胞（NSCs）体外增殖及分化的影响。方法用无血清培养与单克隆技术对小鼠胚胎脑组织进行分离、培养,根据培养液中所加营养因子的不同将传代的NSCs分为bFGF组、BDNF组、bFGF＋BDNF组,观察不同组别对NSCs的定向分化作用。结果与bFGF和BDNF组相比,bFGF＋BDNF组细胞分化为神经元的比例较高（P〈0.05）。结论 bFGF可以提高BDNF诱导小鼠NSCs向神经元分化的比例。     

外源性bFGF抑制辐射诱导的C17.2神经干细胞凋亡的实验研究

目的 观察外源性碱性成纤维细胞生长因子(bFGF)对辐射诱导的C17.2神经干细胞(NSCs)凋亡的影响,探讨细胞外信号调节激酶1/2(ERK1/2)在bFGF对辐射诱导的C17.2 NSCs凋亡的抑制效应中的作用. 方法 以直线加速器照射C17.2NSCs建立离体放射性损伤模型.MTT法检测细胞活性,流式细胞仪检测不同浓度的外源性bFGF和U0126对细胞凋亡的影响. 结果 外源性bFGF浓度为0～100ng/mL时,细胞凋亡率可由(12.78±1.04)%降低至(4.83±0.31)%,组间比较差异有统计学意义(P<0.05);加入U0126后细胞凋亡率为(8.42±0.71)%.DMSO对照组为(4.71±0.42)%,差异有统计学意义(P<0.05). 结论 外源性bFGF对辐射引起的C17.2 NSCs凋亡具有抑制作用,ERK1/2参与该效应.     

Immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons induce a functional switch in the growth factor responsiveness of astroglia: involvement of basic fibroblast growth factor

Recent evidence indicates that astroglial-derived growth factors (GFs) participate in the development of luteinizing hormone-releasing hormone (LHRH) neurons, but it is still unknown whether LHRH neurons may exert a reciprocal modulation of glial cell function. Using immortalized hypothalamic LHRH (GT_1-1) neurons in co-culture with glial cells, we have recently shown that basic fibroblast growth factor (bFGF) plays a prominent role in the glial-induced acquisition of the mature LHRH phenotype by GT_1-1 cells. We have resorted to this model and combined biochemical and morphological approaches to study whether the response of glial cells to a number of GFs (including bFGF, insulin-like growth factor I, IGF-I, epidermal growth factor, EGF and insulin) expressed during LHRH neuron differentiation, is modulated by co-culture with pure LHRH neurons. Pre-treatment of hypothalamic astrocytes with an inactive (‘priming’) dose of bFGF for 12 h powerfully increased astroglia proliferative response to IGF-I (10 ng/ml), EGF (10 g/ml) and insulin (10 μg/ml), inducing a 65–100% increase in the [³H]thymidine incorporation compared to untreated cultures. When astroglial cells and developing GT_1-1 neurons were co-cultured for 5 days in vitro (DIV), the [³H]thymidine incorporation was significantly higher than in astroglial cells cultured without neurons. Application of the different GFs to the co-culture for either 12 or 24 h further stimulated DNA synthesis to various extent according to the GF applied and the time of application. Localization of the proliferating cells by dual immunohistochemical staining, followed by cell counting and bromodeoxiuridine (BrdU) labeling index calculation, revealed that the incorporation of BrdU was restricted to the nuclei of LHRH-immunopositive neurons. Such changes were accompanied by extensive morphological alterations of astroglial and LHRH fiber networks, whereas neutralization of bFGF activity in GT_1-1 neuron–glial co-cultures by a bFGF-antibody, dramatically counteracted the observed effects. The functional switch of astroglia proliferative response to GFs coupled to the potent morphological and functional modifications of developing glia and pure LHRH neurons observed in vitro, support a bidirectional interaction between immortalized LHRH neurons and astroglial cells and identify bFGF as a key player in this crosstalk.     

NILE/L1 and NCAM-polysialic acid expression on growing axons of isolated neurons

The neuron adhesion molecules NILE/L1 and NCAM may be involved in axonal guidance and cell recognition. To investigate all exposed membrane domains of single neurons, something which has not previously been done for any adhesion molecule, we used digitally processed scanning electron microscopy with a high-energy backscatter electron detector. This allowed a quantitative analysis of immunogold staining densities on all surfaces of isolated rat hippocampal neurons in culture to study NILE/L1 and NCAM expression independent of potentially inductive innervation. During early stages of neuritic extension, all growth cones showed similar NILE/L1 expression, but as soon as a single process extended farther than the others (by 20 hours), this putative axon and its growth cone generally showed a stronger level of NILE/L1 immunogold labeling than the other neurites. This is the earliest evidence of plasma membrane differentiation between axons and dendrites. With further neuritic growth, the relative NILE/L1 expression on axons and their growth cones continued to increase. In contrast to some earlier reports, NILE/L1 was expressed on axonal growth cones growing on both polylysine-coated glass and astrocyte substrates. Strong immunostaining for NCAM-related polysialic acid (PSA) was found on axonal growth cones and filopodia, suggesting that the homophilic adhesive action of NCAM may be reduced during axonal growth. PSA showed greater labeling on distal axons than on other areas of the neuron, indicating a variable NCAM-mediated adhesion on different regions of the same cell. Neither NILE/L1, NCAM, nor PSA appeared to show regional differences in axons fasciculating or defasciculating on themselves. A strong intercellular heterogeneity of NILE/L1, NCAM, and PSA expression levels on neurons in the same culture dish was found, suggesting that subsets of cells from the hippocampus may express biologically relevant differences in adhesion molecules compared to neighboring neurons. In light of the growing body of evidence pointing to the multifaceted array of homophilic and heterophilic binding interactions that NILE/L1 and NCAM may exhibit, and the functional importance of molecular densities, the quantitative data here support the hypothesis that sufficient cellular and subcellular heterogeneity exists for these molecules to be involved in some aspects of axonal guidance. © 1993 Wiley-Liss, Inc.     

局灶脑缺血恢复期半影区小胶质细胞可塑性变化和bFGF表达规律探讨

目的观察Wistar大鼠大脑中动脉永久性闭塞后不同时段,缺血半影区小胶质细胞和神经元形态学变化及bFGF在皮层和海马的表达规律。方法将雄性Wistar大鼠48只,随机分为假手术对照组和永久性局灶性脑缺血组,后者再根据缺血时间不同分5个亚组。假手术组:仅暴露大脑中动脉,2h后断头取脑。永久性局灶性脑缺血组:建立大鼠大脑中动脉闭塞模型,分别于缺血后3d、7d、14d、28d、42d断头取脑,行HE和免疫组化染色。观察梗死灶周围半影区的小胶质细胞和神经元的形态学变化和bFGF在皮层及海马部位的表达规律。结果HE染色可见脑缺血3d时半影区有少量小胶质细胞出现,14d小胶质细胞增多达高峰,42d趋于稳定。脑缺血3d梗死灶周围皮质神经元和胶质细胞开始表达bFGF,7d表达增强,14d达高峰,28d表达开始减弱,42d仍有一定表达,bFGF在海马的表达也有相同规律。结论小胶质细胞的肥大和增生性变化以及bFGF的表达,不仅发生于脑缺血早期,晚期仍显示持续性变化,表明小胶质细胞活动以及bFGF的表达贯穿于脑缺血的整个病理过程。     

全反式维甲酸对胶质瘤干细胞VEGF和bFGF表达的影响

目的 研究全反式维甲酸(ATRA)对胶质瘤干细胞(GSCs)血管内皮细胞生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)表达的影响. 方法 从人胶质母细胞瘤细胞系U87中分离培养GSCs,免疫荧光染色检测CD133、巢蛋白(nestin)的表达进行鉴定.将取GSCs分成3组分别培养:(1)ATRA组:培养基中含10 nmol/L ATRA;(2)空载体组:培养基中含与ATRA组等量的二甲基亚砜(DMSO);(3)对照组:单纯培养基,培养10 d后免疫荧光染色检测胶质纤维酸性蛋白(GFAP)、β-微管蛋白Ⅲ(β-tubulinⅢ)、半乳糖脑苷脂(GalC)的表达;CCK8法检测各组细胞的增殖;ELISA和RT-PCR分别检测各组GSCs VEGF、bFGF的分泌水平和mRNA的表达. 结果 二代细胞球表达神经干细胞(NSCs)的标记抗体CD133和nestin.免疫荧光染色检测显示分化后GSCs能够分化为多种同源子代细胞(分别表达星形胶质细胞、神经元、少突胶质细胞标志物GFAP、β-tubulinⅢ、Galc);培养10d后ATRA组细胞GFAP的阳性表达率高于对照组及空载体组,差异有统计学意义(P＜0.05).第3.～7天ATRA组细胞增殖速度较对照组和空载体组明显变缓,差异有统计学意义(P＜0.05);分化24 h后ATRA组GSCs VEGF、bFGF的分泌水平和mRNA的表达均少于对照组和空载体组,差异有统计学意义(P＜0.05). 结论 ATRA能诱导GSCs分化并抑制其增殖,其可能通过抑制VEGF和bFGF的表达发挥抗胶质母细胞瘤的作用.     

局灶脑缺血恢复期半影区小胶质细胞可塑性变化和bFGF表达规律探讨

目的 观察Wistar大鼠大脑中动脉永久性闭塞后不同时段，缺血半影区小胶质细胞和神经元形态学变化及bFGF在皮层和海马的表达规律。方法 将雄性Wistar大鼠48只，随机分为假手术对照组和永久性局灶性脑缺血组，后者再根据缺血时间不同分5个亚组。假手术组：仅暴露大脑中动脉，2h后断头取脑。永久性局灶性脑缺血组：建立大鼠大脑中动脉闭塞模型，分别于缺血后3d、7d、14d、28d、42d断头取脑，行HE和免疫组化染色。观察梗死灶周围半影区的小胶质细胞和神经元的形态学变化和bFGF在皮层及海马部位的表达规律。结果 HE染色可见脑缺血3d时半影区有少量小胶质细胞出现，14d小胶质细胞增多达高峰，42d趋于稳定。脑缺血3d梗死灶周围皮质神经元和胶质细胞开始表达bFGF，7d表达增强，14d达高峰，28d表达开始减弱，42d仍有一定表达，bFGF在海马的表达也有相同规律。结论 小胶质细胞的肥大和增生性变化以及bFGF的表达，不仅发生于脑缺血早期，晚期仍显示持续性变化，表明小胶质细胞活动以及bFGF的表达贯穿于脑缺血的整个病理过程。     

Immunoelectron-microscopic localization of the 180 kD component of the neural cell adhesion molecule N-CAM in postsynaptic membranes

In order to investigate the expression of cell adhesion molecules in synapses, we have studied the localization of the neural cell adhesion molecule N-CAM in the cerebellum and hippocampus of adult mice by immunocytological and immunochemical methods. Of the three molecular components of N-CAM with relative molecular masses (Mr) of 120, 140, and 180 kD, N-CAM 120 is not detectable in synaptosomal membranes, whereas N-CAM 140 is expressed on both pre- and postsynaptic membranes and N-CAM 180 is restricted to postsynaptic sites, with localization of the N-CAM 180-specific epitope in postsynaptic densities. Specificity of immunoreactivity is indicated by the observation that antibodies to the neural cell adhesion molecule L1 do not label synaptic membranes, whereas antibodies to two major components of postsynaptic densities, actin and erythrocyte spectrin, react with synaptic structures. Interestingly, N-CAM 180 is only detectable in subpopulations of synapses in the intact tissue. Isolated synaptosomes, opened for unimpeded accessibility of antibody by hypoosmotic treatment, also reveal a partial expression of N-CAM 180 in that 67% are labeled by antibodies to N-CAM 180, while antibodies to actin and erythrocyte spectrin react with 95% and 88% of all synaptosomes, respectively. N-CAM 180 does not appear to be differentially expressed in synapses of a particular morphological type, but is detectable in all types of synapses in the cerebellum and hippocampus, except for mossy fiber synapses and synapses between basket and Purkinje cells, which are generally N-CAM 180-negative. Since N-CAM 180 has been shown to be characteristic of stabilized or stabilizing cell contacts, possibly by its association with the cytoskeleton-membrane linker protein spectrin (Pollerberg et al.: J. Cell Biol. 101:1921-1929, '85; Nature 324:462-465, '86; Cell Tissue Res. 250:227-236, '87), we would like to suggest N-CAM 180 plays an important role in determining the stability of contacts between pre- and postsynaptic membranes and state of synaptic activity.     

Differential expression of cell adhesion molecules (CAM), neural CAM and epithelial cadherin in ependymomas and choroid plexus tumors

A series of frozen specimens of 18 ependymomas and 7 choroid plexus tumors were examined for their expression of cell adhesion molecules, such as neural cell adhesion molecule (NCAM), its polysialylated isoforms (PSA NCAM), and epithelial (E-) cadherin, and of intermediate filament proteins, such as glial fibrillary acidic protein (GFAP) and cytokeratin, using various monoclonal and polyclonal antibodies. Normal choroid plexus and ependyma were taken as controls. Anti-E-cadherin immunoreactivity was observed on the basolateral part of most adult choroid plexus and benign choroid plexus papilloma cells. However, a small number of atypical papillomas and carcinoma cells showed anti- E-cadherin immunoreactivity throughout their cell surface membrane. NCAM were not expressed by adult choroid plexus and benign papilloma cells. Only a few cells expressed NCAM and PSA NCAM in developing choroid plexus, atypical papillomas and carcinomas. Cytokeratin expression was always observed in choroid plexus and their tumors; GFAP expression was variable from case to case. In contrast, ependymal cells and their tumors never expressed E-cadherin but strongly expressed NCAM. PSA NCAM was found in ependymomas exhibiting anaplastic features. All ependymomas strongly expressed GFAP and a few demonstrated slight expression of cytokeratin. These data suggest that, besides GFAP and cytokeratin, NCAM and E-cadherin are of potential diagnostic value in distinguishing choroid plexus tumors from ependymomas. E-cadherin and NCAM may play a role in the functional organization of normal choroid plexus and ependyma, respectively. In particular, incomplete or irregular anti-E-cadherin expression in choroid plexus tumors and PSA NCAM immunoreativity in ependymomas and choroid plexus tumors correlates with the emergence of anaplastic histological features.     

碱性成纤维细胞因子在局灶性脑缺血后内源性神经干细胞增殖过程中对β-catenin的影响

BACKGROUND： The Wnt/β-catenin signaling pathway plays an important role in neural development. ,β-catenin is an important component of the Wnt/β-catenin signaling pathway. The Wnt signaling pathway has been shown to regulate the interaction of neural stem cells with the extracellular matrix.
OBJECTIVE： To investigate the effects of basic fibroblast growth factor （bFGF） on β-catenin protein and mRNA expression, and on hippocampal neural stem cell proliferation in a rat model of cerebral ischemia/reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled, neurobiology experiment was performed in Shenyang Medical College between August 2006 and August 2008. MATERIALS： A total of 72 healthy male Wistar rats, aged 3 months, were used in this study. bFGF was provided by Beijing SL Pharmaceutical Co.,Ltd., China. METHODS： Rats were randomly divided into 3 groups： sham-operated, ischemia/reperfusion, and bFGF-treated （n = 24 per group）. Focal cerebral ischemia/reperfusion was induced in rats from the ischemia/reperfusion group and the bFGF-treated group by 2 hour right middle cerebral artery occlusion and 2 hour restoration of blood flow using the suture method. The ischemia/reperfusion and bFGF-treated groups were intraperitoneally administered 500 IU/mL of bFGF, or the same volume of physiological saline, once a day at postoperative days 1 3, and once every 3 days thereafter. Simultaneously, the sham-operated group underwent experimental procedures identical to the ischemia/reperfusion and bFGF-treated groups, with the exception of ischemia/reperfusion induction and drug administration. At 2 hours, 2, 6, 13, and 20 days after ischemiaJreperfusion induction, 50 mg/kg bromodeoxyuridine （BrdU） was administered to each group, twice daily, to label proliferating neural stem cells. MAIN OUTCOME MEASURES： The effects of bFGF on BrdU labeling, and ,8 -catenin mRNA and protein expression, in neural stem cells were examined by immunohistochemistry, Western blot, RT-PCR, and in situ hybridization techniques. RESULTS： In the sham-operated group, only a few BrdU-immunoreactive neural stem cells were found. In the ischemia/reperfusion group, BrdU-immunoreactive cells began to increase from 3 days after ischemia/reperfusion induction, reached a peak level at 7 days, and gradually reduced from 21 days. At 3, 7, 14, and 21 days after ischemia/reperfusion induction, the numbers of BrdU-immunoreactive cells were significantly greater in the bFGF-treated group than in the ischemia/reperfusion group. The sham-operated group exhibited slight expression of β-catenin and β-catenin mRNA. In the ischemia/reperfusion group, the expression of β-catenin and β-catenin mRNA gradually increased with reperfusion time, peaked at 14 days after reperfusion, and gradually decreased thereafter; by 21 days, the expression was markedly lower. Following bFGF injection, the expression of hippocampal BrdU, β-catenin, and β-catenin mRNA had apparently increased in each group. CONCLUSION： bFGF promotes neural stem cell proliferation, and the expression of β-catenin and β-catenin mRNA in the ischemic brain tissue. These findings indicate that bFGF promotion of neural stem cell proliferation may be mediated by Wnt/β-catenin signaling pathway.