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1.
大鼠脑缺血再灌注诱导自体神经干细胞原位增殖的研究   总被引:3,自引:0,他引:3  
目的研究缺血性脑损伤对内源性神经干细胞增殖、迁移的影响。方法参照Pulsinelli-Brierley法制作短暂性全脑缺血动物模型,全脑缺血10min后再灌注,采用SABC免疫组化染色显示5'-溴脱氧尿嘧啶(BrdU)阳性细胞和神经巢蛋白(Nestin)阳性细胞,光镜下观察并统计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。结果脑缺血再灌流24h后,海马、齿状回和室管膜下区的BrdU阳性细胞和Nestin阳性细胞增多,7~10d达到高峰,术后20d仍有表达;在室管膜下区,BrdU阳性细胞和Nestin阳性细胞有向皮质、海马迁移的现象。结论①成年大鼠全脑缺血后7~10d,内源性神经干细胞的增殖达到高峰。②增殖的内源性神经干细胞存在由增殖区向靶区迁移的现象。  相似文献   

2.
目的探讨胎牛血清对大鼠胚胎海马神经干细胞(neural stem cells,NSCs)向表达胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)细胞分化的影响。方法体外取材、纯化、传代培养NSCs至第3代并进行细胞特异性表面标记物Nestin染色鉴定,将已鉴定好的第3代NSCs分为NSCs单独培养组(对照组)和NSCs胎牛血清诱导组(血清组)继续培养7 d,观察各组NSCs的生长变化,免疫组织化学(ICH)检测GFAP了解GFAP阳性细胞数和细胞突触长度,RT-PCR、Western blot检测突触素(synaptophysin,SYN)的表达变化。结果与对照组相比,血清组GFAP阳性细胞数增多(P0.01),细胞突触长度增长(P0.01),SYN mRNA和蛋白表达增加(P0.01)。结论胎牛血清能够诱导大鼠胚胎海马NSCs向GFAP阳性细胞分化和促进分化后的GFAP阳性细胞突触的生长,可能与胎牛血清诱导过程中促进细胞表达SYN有关。  相似文献   

3.
目的研究N-Methyl-D-Aspartate(NMDA)受体拮抗剂MK-801对大鼠神经干细胞(NSC)内源性激活的作用.方法将不同年龄阶段(10 d、3月、10月)的SD大鼠分为实验组和对照组,实验组腹腔注射MK-801,对照组腹腔注射生理盐水,用免疫组织化学方法测定两组大鼠海马齿状回颗粒层(SGZ)的Brdu阳性细胞、Nestin阳性细胞表达数.结果2组的10 d幼鼠,Brdu阳性细胞、Nestin阳性细胞增殖均不明显,2组无显著差异(P>0.05),而在3月成年鼠实验组Brdu阳性细胞7 d达高峰,Nestin阳性细胞11 d达高峰,较对照组增殖较明显(P<0.05);实验组10月老龄鼠Brdu阳性细胞、Nestin阳性细胞表达更明显,并可持续到18 d,2组比较统计学有显著意义(P<0.01).结论NMDA受体拮抗剂MK-801可明显促进3月成鼠、10月老年鼠脑中NSC的增殖、分化.  相似文献   

4.
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。  相似文献   

5.
目的探讨脑源性神经营养因子(BDNF)诱导大鼠骨髓基质细胞(BMSCs)成为神经干细胞及其分化作用。方法取成年大鼠BMSCs,分别以BDNF和BDNF+RA(维甲酸)作为诱导物诱导,于诱导3d、7d后行巢蛋白(Nestin)、神经元特异烯醇化酶(NSE)、胶质纤维酸性蛋白免疫细胞化学染色。结果诱导3天后BDNF和BDNF+RA诱导组均有大量Nestin染色阳性细胞,BDNF+RA组阳性率高于BDNF组(P<0.01)。NSE、GFAP免疫阳性细胞在诱导3d后也有少量表达。诱导7天后BDNF和BDNF+RA诱导组Nestin阳性细胞明显减少,与诱导3天后比较差异有显著性(P<0.01),而NSE、GFAP阳性细胞数增多,与诱导3天后相比差异有显著性(P<0.01),且BDNF+RA组阳性率高于BDNF组(P<0.01)。结论联合应用BDNF与RA可提高BMSCs神经转化,并促进其向神经元及星形胶质细胞细胞分化。  相似文献   

6.
目的探讨重复经颅磁刺激(rTMS)对局灶性脑缺血大鼠海马内源性神经干细胞分化的影响。方法线栓法制备大鼠大脑中动脉闭塞(MCAO)模型,随机分为脑缺血自然恢复组和rTMS治疗组,用荧光显微镜和共聚焦显微镜观察缺血14d、28d后各组大鼠海马中5-溴脱氧尿核苷(BrdU)与神经元特异核蛋白(NeuN)、神经胶质酸性蛋白(GFAP)共同标记的阳性细胞,并在高倍荧光显微镜下对双标阳性细胞计数。结果脑缺血后14d、28d,rTMS治疗组大鼠海马BrdU/NeuN双标阳性细胞数量分别为17.12±2.91、23.20±5.97,较相应自然恢复组12.96±2.79、15.92±2.52明显增加,两组同一时间点组间比较有统计学差异(P〈0.01)。而脑缺血后14d、28d,rTMS治疗组大鼠海马BrdU/GFAP双标阳性细胞数量分别为30.48±4.58、36.48±4.90,较相应自然恢复组37.44±3.58、43.60±5.96减少,两者相比有统计学差异(P〈0.05)。结论局灶性脑缺血大鼠海马增殖的内源性神经干细胞,可分化为神经元或神经胶质细胞,而rTMS可促进海马内源性神经干细胞向神经元的分化。  相似文献   

7.
目的 探讨侧脑室注射海人酸(KA)致大鼠海马损伤后Noggin的表达变化及其与颗粒细胞增殖的关系.方法 健康雄性SD大鼠32只采用随机数字表法分为实验组(16只)及对照组(16只).对照组又分为生理盐水对照组和空白对照组,各8只.实验组大鼠侧脑室注射KA,生理盐水对照组注射等剂量生理盐水.空白对照组不作处理.侧脑室注射KA 1周内,尼氏染色检测海马神经元的丢失.免疫荧光染色与原位杂交的方法检测海马齿状回BrdU标记细胞与Noggin mRNA阳性细胞的变化.结果 在侧脑室注射KA致海马损伤后1周,海马CA3、CA4区神经元丢失明显.与生理盐水对照组比较,实验组海马齿状回BrdU阳性细胞升高,差异有统计学意义(P=0.006),其中注射侧较对侧更为明显.海马Noggin mRNA阳性细胞在第3天时升高,第7天时下降.结论 侧脑室注射KA致海马损伤后.成年大鼠海马齿状回颗粒细胞异常增殖可能与Noggin表达波动有关.  相似文献   

8.
目的探讨时间因素对体外培养的神经干细胞向γ氨基丁酸(gammaaminobutyricacid,GABA)能神经元分化的影响。方法体外培养神经干细胞并加以鉴定。利用反义寡核苷酸诱导神经干细胞向GABA能神经元分化,通过免疫荧光染色计算不同时间点的GAD65阳性细胞率。结果体外培养获得神经干细胞,染色可见神经球及分化细胞中分别有nestin、NF200、GFAP阳性细胞。方差分析表明不同时间点的阳性细胞率存在的差别具有显著意义(F=702.93,P<0.05),LSDt检验表明除第7d与第9d无明显差别外,其余各时间点之间均有明显差别。其中第7天的分化率最高为87.60%±6.26%。结论本实验利用反义寡核苷酸序列特异性阻断了Hes1的表达,促进了神经干细胞向GABA神经元分化,并发现在诱导分化7d后分化率达到最高值。  相似文献   

9.
鼠脑梗死后自体神经干细胞的原位增殖、分化及其可塑性   总被引:5,自引:0,他引:5  
目的研究成年大鼠脑梗死后自体神经干细胞的原位增殖、分化及其可塑性。方法雄性Wistar大鼠共90只,分对照组(n=10)、脑梗死后1d组(n=16)、脑梗死后3d组(n=16)、脑梗死后7d组(n=16)、脑梗死后14d组(n=16)、脑梗死后28d组(n=16)。用免疫组织化学方法动态检测BrdU、GFAP、NeuN、PSA-NCAM的表达。BrdU确定神经干细胞的增殖,GFAP、NeuN确定神经干细胞的分化,PSA-NCAM确定神经干细胞的可塑性。结果与对照组相比,大鼠海马BrdU 细胞数在脑梗死后1d组开始增加,7d组达到高峰,28d组接近正常水平;BrdU /GFAP 细胞数在脑梗死前后无明显变化;BrdU /NeuN 细胞数在脑梗死后14d组开始增加,28d组最多;BrdU /PSA-NCAM 细胞数在脑梗死后7d组开始增加,14d组达到高峰,28d组开始下降,但仍高于对照组,大约占同期BrdU阳性细胞数60%。结论大鼠脑梗死激活自体神经干细胞原位增殖,大多数增殖的神经干细胞分化成神经元并且具有可塑性。  相似文献   

10.
目的观察神经干细胞在悬浮培养与贴壁培养方式下的形态学特点及分化表型。方法新生6 h内的大鼠皮质应用无血清悬浮培养与贴壁培养神经干细胞(NSCs)技术培养,观察神经干细胞的形态学特点。体外胎牛血清(FBS)诱导NSCs分化,通过巢蛋白(Nestin)和GFAP免疫荧光染色鉴定分化表型。台盼蓝活细胞计数法比较细胞增殖能力。结果新生大鼠皮质应用无血清悬浮培养可获得稳定的NSCs细胞球,应用贴壁培养形成长梭状NSCs,NSCs巢蛋白免疫荧光染色结果呈阳性。体外FBS诱导分化后,神经球内开始出现放射状的神经纤维丝,免疫荧光染色可见GFAP阳性细胞向外伸展以及Nestin阳性细胞边集。贴壁的NSCs分化成星形胶质细胞,免疫荧光染色可见Nestin和GFAP染色阳性细胞。在相同条件下,应用悬浮培养在第2~3 d时增殖速度最大,而以贴壁培养方式培养出的神经干细胞在第4~5 d时增殖速度最大。结论应用无血清培养物B27与b FGF、EGF组合的NSCs完全培养基通过悬浮培养法与贴壁培养法,都可以成功在体外培养出NSCs;悬浮培养法培养NSCs在早期增殖速率上优于贴壁培养法。  相似文献   

11.
糖皮质激素对大鼠内源性神经前体细胞增殖的影响   总被引:1,自引:0,他引:1  
目的探讨大剂量糖皮质激素(GCs)对成年大鼠内源性神经前体细胞增殖的影响.方法将25只大鼠随机分为对照组和地塞米松(DEX)作用1、3、7、14 d组,应用免疫组化方法检测神经前体细胞标记物巢蛋白(nestin)的表达,并通过5-溴脱氧尿苷(BrdU)观察神经前体细胞的增殖.结果正常大鼠海马齿状回(DG)和室下区(SVZ)存在神经前体细胞,并且其中一些细胞处于分裂增殖状态.应用大剂量GCs作用3、7、14 d组与对照组相比DG的nestin和BrdU阳性细胞数明显减少,SVZ的nestin和BrdU阳性细胞数在DEX作用7、14 d组与对照组相比明显减少,并且DG与SVZ二者阳性细胞数随着作用时间的延长而减低更为明显.结论大剂量GCs持续作用可抑制大鼠脑内的内源性神经前体细胞的增殖,DG区的神经前体细胞对GCs的反应较SVZ更为敏感.  相似文献   

12.
目的建立大鼠神经干细胞(NSCs)分离、培养方法,观察其生长、增殖和分化特点。方法利用无血清培养技术,从新生大鼠海马、室管膜下区分离NSCs,进行体外扩增培养、传代观察。采用荧光免疫细胞化学检测技术,观察鉴定NSCs及其分化结果。结果分离获取的细胞具有自我更新和增殖能力,原代及传代培养均可形成细胞克隆,克隆中的细胞巢蛋白(nestin)表达阳性,显微镜下观察见典型的干细胞特征,诱导后可分化神经元和星形胶质细胞。结论上法分离培养的细胞为具有自我更新和增殖能力的NSCs,可诱导分化为终末神经细胞。  相似文献   

13.
高压氧预处理对大鼠脊髓损伤后GFAP和巢蛋白表达的影响   总被引:5,自引:0,他引:5  
目的探讨高压氧预处理对成年大鼠脊髓损伤后不同时期巢蛋白(nestin)和胶质纤维酸性蛋白(GFAP)表达变化的影响。方法成年SD大鼠55只,雌性,体重250~300g。随机分为高压氧预处理组、正常损伤组各25只(n=5),对照组5只。采用改良Allen's法制作模型,分别于伤后1d、5d、7d、10d和14d取材,免疫组化染色及图像分析检测组织中GFAP和巢蛋白的表达。结果对照组显示除脊髓中央管周围可见到少量巢蛋白表达外,其它部位几乎不表达;GFAP在脊髓各个部位均有表达;正常脊髓损伤组:1d损伤区域巢蛋白和GFAP表达增加;5d表达显著增加;7d达高峰;10~14d逐渐下调,与对照组差异明显;高压氧预处理组:各时间段均与正常损伤组有明显差异(P〈0.05)。结论高压氧预处理可诱导巢蛋白高表达和星形胶质细胞增生,对中枢神经系统损伤起到保护作用。  相似文献   

14.
The distribution of nestin immunoreactivity was studied in the whole normal adult human forebrains using new anti-human nestin mouse monoclonal and rabbit polyclonal antiserum. The nestin immunoreactive cells could be divided into three types according to their morphological characteristics. The first type contained neuron-like nestin immunoreactive cells, distributed in CA1–3 of hippocampus, septum, the nucleus of diagonal band, amygdala and basal nucleus of Meynert. The second type contained astrocyte-like cells, distributed in the subependymal zone and subgranular layer of dentate gyrus. The third type of cells had smaller cell bodies and fewer processes, also distributed in the subependymal zone and subgranular layer of dentate gyrus. Double immunohistochemical staining showed that the nestin positive, neuron-like cells in the nucleus of diagonal band and hippocampus also expressed NSE. However, the astrocyte-like nestin immunoreactive cells of the subependymal zone and subgranular layer of dentate gyrus were not double labeled with GFAP. Although some nestin immunoreactive fibers were distributed in the infundibulum, no nestin-immunoreactive cells were detected in the cortex. These data indicate that nestin exist in the adult human brain outside of the subependymal zone and dentate gyrus and also implies that nestin-immunoreactive cells may play a role in the modulation of basal forebrain function.  相似文献   

15.
The non-angiogenic role of vascular endothelial growth factor (VEGF), and its receptors flt-1 and flk-1, together with downstream signaling pathways were examined in fetal and postnatal rat cerebral cortical organotypic explants. VEGF application in both paradigms caused a significant increase in astroglial proliferation and a dose-dependent increase in GFAP and nestin immunoreactivity. The VEGF receptor flt-1 was observed on most, though not all astrocytes, while flk-1 receptor immunoexpression was absent. Treatment with antisense oligonucleotides (AS-ODNs) to flt-1 resulted in a dramatic decrease in GFAP and nestin immunoreactivity, which further confirmed the role of flt-1 in mediating VEGF's gliotrophic effects, while AS-ODNs to flk-1 had no effect. VEGF-induced gliotrophic effects were found to be mediated by the MAPK/ERK and PI-3 kinase signaling pathways, since the both the MEK1 inhibitor, PD98059 and the PI-3 kinase inhibitor, Wortmannin abolished VEGF-induced astrocytic GFAP(+) expression. Although high dose VEGF application resulted in strong upregulation of both GFAP and nestin immunoreactivity in astrocytes, overlap of the two proteins was not observed in all cells, suggesting that some of the nestin(+) cells might be neural progenitors. Exposure to VEGF resulted in upregulation of both VEGF and bFGF mRNA at the one-day time point, and bFGF protein by 3 days; VEGF activated astrocytes expressed bFGF to a much greater degree than those in untreated explants. The increased expression of bFGF induced by VEGF, may serve in the proliferation of multipotential neural stem/progenitor cells in vitro. VEGF, an established angiogenic factor, appears to play a significant role in the growth and differentiation of astrocytes in the CNS.  相似文献   

16.
Neural stem and progenitor cells in nestin-GFP transgenic mice   总被引:17,自引:0,他引:17  
Neural stem cells generate a wide spectrum of cell types in developing and adult nervous systems. These cells are marked by expression of the intermediate filament nestin. We used the regulatory elements of the nestin gene to generate transgenic mice in which neural stem cells of the embryonic and adult brain are marked by the expression of green fluorescent protein (GFP). We used these animals as a reporter line for studying neural stem and progenitor cells in the developing and adult nervous systems. In these nestin-GFP animals, we found that GFP-positive cells reflect the distribution of nestin-positive cells and accurately mark the neurogenic areas of the adult brain. Nestin-GFP cells can be isolated with high purity by using fluorescent-activated cell sorting and can generate multipotential neurospheres. In the adult brain, nestin-GFP cells are approximately 1,400-fold more efficient in generating neurospheres than are GFP-negative cells and, despite their small number, give rise to 70 times more neurospheres than does the GFP-negative population. We characterized the expression of a panel of differentiation markers in GFP-positive cells in the nestin-GFP transgenics and found that these cells can be divided into two groups based on the strength of their GFP signal: GFP-bright cells express glial fibrillary acidic protein (GFAP) but not betaIII-tubulin, whereas GFP-dim cells express betaIII-tubulin but not GFAP. These two classes of cells represent distinct classes of neuronal precursors in the adult mammalian brain, and may reflect different stages of neuronal differentiation. We also found unusual features of nestin-GFP-positive cells in the subgranular cell layer of the dentate gyrus. Together, our results indicate that GFP-positive cells in our transgenic animals accurately represent neural stem and progenitor cells and suggest that these nestin-GFP-expressing cells encompass the majority of the neural stem cells in the adult brain.  相似文献   

17.
Transplantation of stem cells is a potential therapeutic strategy for stroke damage. The survival, migration, and differentiation of transplanted human embryonic neural stem cells in the acute post‐ischemic environment were characterized and endogenous nestin expression after transplantation was investigated. Human embryonic neural stem cells obtained from the temporal lobe cortex were cultured and labeled with fluorescent 1,1′‐dioctadecy‐6,6′‐di (4‐sulfopheyl)‐3,3,3′,3′‐tetramethylindocarbocyanin (DiI) in vitro. Labeled cells were transplanted into cortical peri‐infarction zones of adult rats 24 h after permanent middle cerebral artery occlusion. Survival, migration, and differentiation of grafted cells were quantified in immunofluorescence‐stained sections from rats sacrificed at 7, 14, and 28 days after transplantation. Endogenous nestin‐positive cells in the cortical peri‐infarction zone were counted at serial time points. The cells transplanted into the cortical peri‐infarction zone displayed the morphology of living cells and became widely located around the ischemic area. Moreover, some of the transplanted cells expressed nestin, GFAP, or NeuN in the peri‐infarction zone. Furthermore, compared with the control group, endogenous nestin‐positive cells in the peri‐infarction zone had increased significantly 7 days after cell transplantation. These results confirm the survival, migration, and differentiation of transplanted cells in the acute post‐ischemic environment and enhanced endogenous nestin expression within a brief time window. These findings indicate that transplantation of neural stem cells into the peri‐infarction zone may be performed as early as 24 h after ischemia.  相似文献   

18.
Postnatal neural stem cells (NSCs) express the "traditional" astrocyte marker, glial fibrillary acidic protein (GFAP). Here, we analyze the ontogeny of GFAP mRNA in mouse forebrain germinal zones (GZ). On embryonic day 15, mRNA distribution is highly restricted. Subsequently, expression expands to include many cells in the GZ regions adjacent to the cortex and septum but not to the striatum. Double immunostaining for GFAP and nestin did not demonstrate extensive overlap in the GZ of adult rats, suggesting that either few of the GFAP-expressing cells are stem cells, or that nestin is not a reliable marker for stem cells in the adult rat brain. The current findings indicate that while some GFAP-expressing cells in the GZ may be NSCs, most are not likely to function in a neurogenic capacity.  相似文献   

19.
The distribution of nestin immunoreactivity was studied in the whole normal adult human forebrains using new anti-human nestin mouse monoclonal and rabbit polyclonal antiserum. The nestin immunoreactive cells could be divided into three types according to their morphological characteristics. The first type contained neuron-like nestin immunoreactive cells, distributed in CA1-3 of hippocampus, septum, the nucleus of diagonal band, amygdala and basal nucleus of Meynert. The second type contained astrocyte-like cells, distributed in the subependymal zone and subgranular layer of dentate gyrus. The third type of cells had smaller cell bodies and fewer processes, also distributed in the subependymal zone and subgranular layer of dentate gyrus. Double immunohistochemical staining showed that the nestin positive, neuron-like cells in the nucleus of diagonal band and hippocampus also expressed NSE. However, the astrocyte-like nestin immunoreactive cells of the subependymal zone and subgranular layer of dentate gyrus were not double labeled with GFAP. Although some nestin immunoreactive fibers were distributed in the infundibulum, no nestin-immunoreactive cells were detected in the cortex. These data indicate that nestin exist in the adult human brain outside of the subependymal zone and dentate gyrus and also implies that nestin-immunoreactive cells may play a role in the modulation of basal forebrain function.  相似文献   

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