Chemoattractive activity of sonic hedgehog in the adult subventricular zone modulates the number of neural precursors reaching the olfactory bulb

The adult subventricular zone (SVZ) supports neural stem cell self-renewal and differentiation and continually gives rise to new neurons throughout adult life. The mechanisms orienting the migration of neuroblasts from the SVZ to the olfactory bulb (OB) via the rostral migratory stream (RMS) have been extensively studied, but factors controlling neuroblast exit from the SVZ remain poorly explored. The morphogen Sonic Hedgehog (Shh) displays proliferative and survival activities toward neural stem cells and is an axonal chemoattractant implicated in guidance of commissural axons during development. We identify here the presence of Shh protein in SVZ extracts and in the cerebrospinal fluid of adult mice, and we demonstrate that migrating neuroblasts in the SVZ and RMS express the Shh receptor Patched. We show that Shh displays a chemoattractive activity in vitro on SVZ-derived neuronal progenitors, an effect blocked by Cur61414, a Smoothened antagonist. Interestingly, Shh-expressing cells grafted above the RMS of adult mice exert a chemoattractive activity on migrating neuroblasts in vivo, thus inducing their accumulation and deviation from their normal migratory pathway. Furthermore, the adenoviral transfer of Shh into the lateral ventricle or the blocking of Shh present in the SVZ of adult mice using its physiological antagonist Hedgehog interacting protein or neutralizing Shh antibodies provides in vivo evidence that Shh can retain SVZ-derived neuroblasts. The ability to modulate the number of neuroblasts leaving the SVZ and reaching the OB through the chemoattractive activity of Shh suggests a novel degree of plasticity in cell migration of this adult stem cell niche.     

Disruption of Eph/ephrin signaling affects migration and proliferation in the adult subventricular zone

The subventricular zone (SVZ) of the lateral ventricles, the largest remaining germinal zone of the adult mammalian brain, contains an extensive network of neuroblasts migrating rostrally to the olfactory bulb. Little is known about the endogenous proliferation signals for SVZ neural stem cells or guidance cues along the migration pathway. Here we show that the receptor tyrosine kinases EphB1-3 and EphA4 and their transmembrane ligands, ephrins-B2/3, are expressed by cells of the SVZ. Electron microscopy revealed ephrin-B ligands associated with SVZ astrocytes, which function as stem cells in this germinal zone. A three-day infusion of the ectodomain of either EphB2 or ephrin-B2 into the lateral ventricle disrupted migration of neuroblasts and increased cell proliferation. These results suggest that Eph/ephrin signaling is involved in the migration of neuroblasts in the adult SVZ and in either direct or indirect regulation of cell proliferation.     

Enhanced neurogenesis in the ischemic striatum following EGF-induced expansion of transit-amplifying cells in the subventricular zone

In the subventricular zone (SVZ) of the adult mammalian brain, neural stem cells continually produce transit-amplifying precursors, which generate neuroblasts migrating into the olfactory bulb. Previous studies have suggested that SVZ cells also have the capacity to generate some striatal neurons after cerebral ischemia. The infusion of epidermal growth factor (EGF) has been demonstrated to increase the number of these regenerated neurons. However, which cell types in the SVZ are stimulated to proliferate or differentiate after EGF infusion remains unknown. In this paper, we demonstrated that cerebral ischemia results in an increase in the number of EGF receptor (EGFR)-positive transit-amplifying cells in the SVZ. EGF infusion into the ischemic brain caused the number of transit-amplifying cells to increase and the number of neuroblasts to decrease. On the other hand, after an interval of 6 days after the discontinuation of EGF infusion, a significant increase in the number of neuroblasts was found, both in the striatum and the SVZ. These results suggest that the replacement of neurons in injured striatum can be enhanced by an EGF-induced expansion of transit-amplifying cells in the SVZ.     

Tonic activation of GLUK5 kainate receptors decreases neuroblast migration in whole-mounts of the subventricular zone

In the postnatal subventricular zone (SVZ), neuroblasts migrate in chains along the lateral ventricle towards the olfactory bulb. AMPA/kainate receptors as well as metabotropic glutamate receptors subtype 5 (mGluR5) are expressed in SVZ cells. However, the cells expressing these receptors and the function of these receptors remain unexplored. We thus examined whether SVZ neuroblasts express mGluR5 and Ca(2+)-permeable kainate receptors in mouse slices. Doublecortin (DCX)-immunopositive cells (i.e. neuroblasts) immunostained positive for mGluR5 and GLU(K5-7)-containing kainate receptors. RT-PCR from approximately 10 GFP-fluorescent cell aspirates obtained in acute slices from transgenic mice expressing green fluorescent protein (GFP) under the DCX promoter showed mGluR5 and GLU(K5) receptor mRNA in SVZ neuroblasts. Patch-clamp data suggest that approximately 60% of neuroblasts express functional GLU(K5)-containing receptors. Activation of mGluR5 and GLU(K5)-containing receptors induced Ca(2+) increases in 50% and 60% of SVZ neuroblasts, respectively, while most neuroblasts displayed GABA(A)-mediated Ca(2+) responses. To examine the effects of these receptors on the speed of neuroblast migration, we developed a whole-mount preparation of the entire lateral ventricle from postnatal day (P) 20-25 DCX-GFP mice. The GABA(A) receptor (GABA(A)R) antagonist bicuculline increased the speed of neuroblast migration by 27%, as previously reported in acute slices. While the mGluR5 antagonist MPEP did not affect the speed of neuroblast migration, the homomeric and heteromeric GLU(K5) receptor antagonists, NS3763 and UB302, respectively, increased the migration speed by 38%. These data show that although both GLU(K5) receptor and mGluR5 activations increase Ca(2+) in neuroblasts, only GLU(K5) receptors tonically reduce the speed of neuroblast migration along the lateral ventricle.     

Expression of ezrin in glial tubes in the adult subventricular zone and rostral migratory stream

Ezrin is a member of the ERM (ezrin-radixin-moesin) family of membrane-cytoskeletal linking proteins. ERM proteins are involved in a wide variety of cellular functions including cell motility, signal transduction, cell-cell interaction and cell-matrix recognition. A recent in situ hybridization study showed that the mRNA encoding ezrin is expressed in neurogenic regions of the mature brain including the subventricular zone (SVZ) and rostral migratory stream (RMS); however, the specific cell types expressing ezrin and their relationship to migrating and proliferating cells in these regions have not been characterized previously. In this study, we used immunocytochemistry to perform double labeling with a variety of cell-type specific markers to characterize the expression of ezrin in the SVZ and RMS of adult mice. Ezrin was expressed at high levels in both the SVZ and RMS where ezrin-immunopositive processes formed a trabecular network surrounding the proliferating and migrating cells. Ezrin-positive cells co-labeled with the glial makers S100beta and GFAP (glial fibrillary acidic protein), but only minimally with the early neuronal markers beta III tubulin and polysialylated form of neural cell adhesion molecule 1 (PSA-NCAM), indicating that ezrin was expressed primarily in the glial tube cells. Ezrin positive cells also expressed beta-catenin, a membrane-complex protein previously implicated in the regulation of stem-cell proliferation and neuronal migration. Glial tube cells act as both precursors of, and a physical channel for, migrating neuroblasts. Bi-directional signals between glial tube cells and migrating neuroblasts have been shown to regulate the rates of both proliferation of the precursor cells and migration of the newly generated neuroblasts. Our finding that ezrin and beta-catenin are both present at the cell membrane of the glial tube cells suggests that these proteins may be involved in those signaling processes.     

Nucleotides and epidermal growth factor induce parallel cytoskeletal rearrangements and migration in cultured adult murine neural stem cells

Aim: The adult subventricular zone (SVZ) contains neural stem cells that generate neuroblasts migrating to the olfactory bulb (OB) and differentiating into interneurones. The molecular cues controlling essential functions within the neurogenesis pathway such as proliferation, short and long distance migration, functional integration and cell survival are poorly understood. We have previously shown that cultured adult neural stem cells express a considerable variety of nucleotide receptors and that nucleotides and epidermal growth factor (EGF) induce converging intracellular signalling pathways that carry potential for synergism in the control of neural stem cell proliferation and cell survival. Here we investigate the role of EGF and the nucleotides ATP, ADPβS and UTP in neural stem cell migration. Methods: Neural stem cells were prepared from adult mice and subjected to adherent culture. Labelling of F-actin was performed with tetramethylrhodamine isothiocyanate-phalloidin. Images were processed for quantitative evaluation of fluorescence labelling. Agonist-induced phosphorylation of AKT and focal adhesion kinase was analysed by quantitative Western blotting. Agonist-dependent cell migration was assayed using 48-well microchemotaxis chambers. Results: Nucleotides and EGF induce the formation of stress fibres, an increase in the cortical actin cytoskeleton and in cell spreading. This is associated with increased phosphorylation of AKT and focal adhesion kinase. Using microchemotaxis chambers we demonstrate a parallel increase in cell migration. Conclusion: Our results suggest that nucleotides and EGF acting as paracrine or autocrine signalling substances can be of relevance for structuring and maintaining the cytoarchitecture of the SVZ and the stream of neuroblasts migrating to the OB.     

Progenitor cells from the adult mouse brain acquire a neuronal phenotype in response to beta-amyloid

Neurospheres from adult mouse subventricular zone (SVZ) were grown in suspension cultures for 12-15 days. Neurospheres consisted mainly of neural precursor cells (NPCs) immunoreactive for nestin and also contained nestin-negative precursors. We used these neurospheres to determine the effects of synthetic beta-amyloid fragments (both betaAP(1-42) and betaAP(25-35)) on NPC proliferation, differentiation and survival. We show that neurospheres exposed to 25 microM betaAP(25-35) or betaAP(1-42) for 24 h (a toxic condition for mature neurons) did not undergo apoptosis. Instead, betaAP(25-35) orientated nestin-negative precursors towards nestin-positive NPCs and turned nestin-positive NPCs into neuroblasts. Intracerebroventricular infusion of full-length betaAP(1-42) increased the population of PSA-NCAM-positive cells in the SVZ, without affecting proliferation. We conclude that betaAP influences the fate of progenitor cells, driving their differentiation towards a neuronal lineage.     

Fate mapping and lineage analyses demonstrate the production of a large number of striatal neuroblasts after transforming growth factor alpha and noggin striatal infusions into the dopamine-depleted striatum

Infusion of transforming growth factor alpha (TGFalpha) into the adult dopamine (DA)-depleted striatum generates a local population of nestin(+)/proliferating cell nuclear antigen (PCNA)(+) newborn cells. The precise origin and fate of these new striatal cells are unknown, making it difficult to direct them for neural repair in Parkinson's disease. Experiments in rats using 5-bromo-2'-deoxyuridine (BrdU) to label neural progenitor cells showed that during TGFalpha infusion in the DA-depleted striatum, newborn striatal cells formed a homogeneous population of precursors, with the majority coexpressing nestin, Mash1, Olig2, and epidermal growth factor receptor, consistent with the phenotype of multipotent C cells. Upon TGFalpha pump withdrawal, the subventricular zone (SVZ) was repopulated by neuroblasts. Strikingly, during this period, numerous clusters of doublecortin(+)/polysialylated neuronal cell adhesion molecule(+) neuroblasts were also produced in the ipsilateral medial striatum. In parallel, striatal BrdU(+)/glial fibrillary acidic protein(+) astrocytes were generated, but no BrdU(+)/O4(+)/CNPase(+) oligodendrocytes were generated. Infusion of the neuralizing bone morphogenetic protein antagonist noggin after TGFalpha pump withdrawal increased the neuroblast-to-astrocyte ratio among new striatal cells by blocking glial differentiation but did not alter striatal neurogenesis. At no time or treatment condition were differentiated neurons generated, including DA neurons. Using 6-hydroxydopamine-lesioned nestin-CreER(T2)/R26R-YFP mice that allow genetic fate-mapping of SVZ nestin(+) cells, we show that TGFalpha-generated striatal cells originate from SVZ nestin(+) precursors that confirmed data from the rats on the phenotype and fate of striatal nestin(+)/PCNA(+) cells upon TGFalpha withdrawal. This work demonstrates that a large population of multipotent striatal C-like cells can be generated in the DA-depleted striatum that do not spontaneously differentiate into DA neurons.     

Beta-catenin signaling promotes proliferation of progenitor cells in the adult mouse subventricular zone

The subventricular zone (SVZ) is the largest germinal zone in the mature rodent brain, and it continuously produces young neurons that migrate to the olfactory bulb. Neural stem cells in this region generate migratory neuroblasts via highly proliferative transit-amplifying cells. The Wnt/beta-catenin signaling pathway partially regulates the proliferation and neuronal differentiation of neural progenitor cells in the embryonic brain. Here, we studied the role of beta-catenin signaling in the adult mouse SVZ. beta-Catenin-dependent expression of a destabilized form of green fluorescent protein was detected in progenitor cells in the adult SVZ of Axin2-d2EGFP reporter mice. Retrovirus-mediated expression of a stabilized beta-catenin promoted the proliferation of Mash1+ cells and inhibited their differentiation into neuroblasts. Conversely, the expression of Dkk1, an inhibitor of Wnt signaling, reduced the proliferation of Mash1+ cells. In addition, an inhibitor of GSK3 beta promoted the proliferation of Mash1+ cells and increased the number of new neurons in the olfactory bulb 14 days later. These results suggest that beta-catenin signaling plays a role in the proliferation of progenitor cells in the SVZ of the adult mouse brain.     

Antioxidant Cu/Zn SOD: expression in postnatal brain progenitor cells

Precursor cells have been shown to be affected by oxidative stress, in vivo and vitro, but little is known about the expression of antioxidant mechanisms in neuronal/glial differentiation. We have characterized the expression of Cu/Zn superoxide dismutase (Cu/Zn SOD), one of the main antioxidant proteins involved in the breakdown of superoxide, in the immature rat dorsolateral subventricular zone (SVZ), rostral migratory stream (RMS) and hippocampal subgranular zone (SGZ). Progenitor cells were identified immunohistochemically on cryostat sections by 5'Bromodeoxyuridine (BrdU) incorporation and expressing cells were further characterized using double labeling for progenitor markers. In the SVZ, only a subpopulation of BrdU+ cells, mostly found in the medial SVZ, expressed Cu/Zn SOD. These cells were mostly nestin+ and some were also vimentin+. In contrast, in the lateral SVZ few Cu/Zn SOD+/BrdU+ cells were found. These were primarily nestin+, vimentin-, showed some PSA-NCAM expression, but only a few were NG2+. In the RMS and SGZ virtually all BrdU+ progenitors were Cu/Zn SOD+ and expressed nestin and vimentin. Some RMS cells were also PSA-NCAM+. These findings show a heterogeneous expression of Cu/Zn SOD in restricted cell types in the germinative zones and suggest a role for antioxidant Cu/Zn SOD in progenitor cells of the immature rat brain.     

Expression of doublecortin (DCX) and doublecortin-like kinase (DCLK) within the developing chick brain.

Doublecortin (DCX) is a microtubule-associated protein widely expressed in the developing mammalian nervous system and important for neuronal migration. DCX is known to belong to a novel protein family defined by sequence homology and the presence of a conserved microtubule-binding domain, but the functions of other members of this family are still undefined. In this study, we describe the cloning of the chick ortholog of doublecortin-like kinase (DCLK), a member of this family, and assess the expression of DCX and DCLK in the layered regions of the developing chick brain. DCX and DCLK are widely expressed in pallial and subpallial structures, including the telencephalon, optic tectum, and cerebellum, in similar distribution patterns. In addition to their expression in migrating cells, both proteins were also detected in the ventricular zone and in postmigratory Purkinje cells. Finally, DCX and DCLK were found to be coexpressed in all areas examined. In postmigratory Purkinje cells, DCX and DCLK both colocalized to the cell membrane, although DCLK was also distributed more generally throughout the cell soma. These data are consistent with multiple roles for DCX and DCLK in the developing chicken brain and suggest that the chick cerebellum will be an intriguing system to explore the effects of DCX and DCLK on postmigratory neuronal function.     

Expression of drebrin E in migrating neuroblasts in adult rat brain: coincidence between drebrin E disappearance from cell body and cessation of migration

Migrating neuroblasts in the adult brain form the rostral migratory stream (RMS) from the lateral ventricle to the olfactory bulb (OB) and then differentiate in the OB. In this study, we immunohistochemically analyzed drebrin expression in the RMS of the adult rat brain. Although drebrin is concentrated in dendritic spines of mature neurons, drebrin-immunopositive (DIP) cell bodies were observed in the RMS. The polysialated form of a neural cell adhesion molecule (PSA-NCAM) was detected in DIP cells. K(i)-67, a marker of proliferating cells, was also detected in a subset of DIP cells; however, neither glial fibrillary acidic protein, nestin nor vimentin was detected in DIP cells. These results indicate that DIP cells in the RMS are migrating neuroblasts. An image subtraction method, based on using anti-pan-drebrin and anti-drebrin A antibodies, demonstrated that DIP migrating neuroblasts are immunopositive for drebrin E but not for drebrin A (E+A-). Furthermore, olfactory bulbectomy increased the number of cells with drebrin E+A- signals in the RMS, indicating that these cells migrate along the RMS. Drebrin E+A- cells were also found in the subgranular layer of the dentate gyrus and in the piriform cortex. Thus, detection of drebrin E+A- signals is useful for identifying migrating neuroblasts in the adult brain. In the OB, drebrin E+A- signals were observed in the cell bodies of migrating neuroblasts in the core region; however, only fibrous and punctate drebrin E+A- signals were observed in postmigratory neuroblasts at the outer layers. These data demonstrate that the disappearance of drebrin E+A- signals from the cell body coincides with the cessation of neuronal migration. The disappearance of drebrin E from the cell body may be a molecular switch for the cessation of migration in newly generated neuroblasts.     

Deprivation of sensory inputs to the olfactory bulb up-regulates cell death and proliferation in the subventricular zone of adult mice

The main olfactory bulb (MOB) is the first relay on the olfactory sensory pathway and the target of the neural progenitor cells generated in the subventricular zone (SVZ) lining the lateral ventricles and which migrate along the rostral extension of the SVZ, also called the rostral migratory stream (RMS). Within the MOB, the neuroblasts differentiate into granular and periglomerular interneurons. A reduction in the number of granule cells during sensory deprivation suggests that neurogenesis may be influenced by afferent activity. Here, we show that unilateral sensory deafferentation of the MOB by axotomy of the olfactory receptor neurons increases apoptotic cell death in the SVZ and along the rostro-caudal extent of the RMS. The vast majority of dying cells in the RMS are migrating neuroblasts as indicated by double Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling/PSA-NCAM labeling. Counting bromodeoxyuridine-labeled cells in animals killed immediately or 4 days after tracer administration showed a bilateral increase in proliferation in the SVZ and RMS which was balanced by cell death on the operated side. These data suggest that olfactory inputs are required for the survival of newborn neural progenitors. The greatest enhancement in proliferation occurred in the extension of the RMS located in the MOB, revealing a population of local precursors mitotically stimulated following axotomy. Together, these findings indicate that olfactory inputs may strongly modulate the balance between neurogenesis and apoptosis in the SVZ and RMS and provide a model for further investigation of the underlying molecular mechanisms of this activity-dependent neuronal plasticity.     

Enhanced expression of vascular endothelial growth factor receptor-3 in the subventricular zone of stroke-lesioned rats

Vascular endothelial growth factor receptor (VEGFR)-3, a receptor for VEGF-C and VEGF-D, has recently been proposed to be involved in adult hippocampal neurogenesis in response to cerebral ischemia. To identify whether VEGFR-3 is involved in poststroke neurogenesis, we investigated the temporal regulation of VEGFR-3 mRNA expression in the subventricular zone (SVZ) of rats with transient focal cerebral ischemia by in situ hybridization analysis, and identified the phenotypes of cells expressing VEGFR-3 by double- and triple-labeling techniques. In sham-operated rats, hybridization signals for VEGFR-3 mRNA were evident at a weaker intensity in the SVZ of the lateral ventricle. VEGFR-3 was transiently increased in the dorsolateral SVZ of the infarcted hemisphere on days 3–7 after reperfusion. Almost all VEGFR-3-expressing cells in the ipsilateral SVZ were colabeled with glial fibrillary acidic protein and the neural progenitor marker nestin, and were highly proliferative. In addition, a subset of VEGFR-3-labeled cells in the ipsilateral SVZ expressed the immature neuronal marker, polysialic acid-neural cell adhesion molecule. These data indicate that VEGFR-3 is upregulated in SVZ astrocytes and immature neurons after focal ischemia, suggesting that VEGFR-3 might mediate the adult neurogenesis after ischemic stroke.     

微管相关蛋白2和巢蛋白在人胚胎小肠中的表达

目的: 探讨微管相关蛋白2(MAP-2)和巢蛋白(nestin)与人胚胎小肠壁组织发育的关系。 方法: 应用免疫组织化学PV法和图像分析(NIS-DR)软件检测第2、3、4个月龄段,人胚胎小肠组织内MAP-2和nestin的表达分布规律,数据的组间比较采用单因素方差分析。 结果: 第2、3、4个月龄段,MAP-2主要表达于人胚胎小肠壁黏膜下和肌间神经丛内神经细胞和纤维,随胎龄的增大,肌间神经丛内神经细胞的阳性强度增强, 两两比较, 差异有统计学意义(P<0.05)。Nestin蛋白在小肠壁各层均有阳性表达,其黏膜下层和肌间神经丛内阳性细胞数量随胎龄增大而呈先增高再降低的趋势,两两比较, 差异有统计学意义(P<0.05)。 结论: MAP-2和nestin参与调节人胚胎小肠壁组织的生长发育过程。     

大鼠骨髓间充质干细胞神经分化蛋白的表达

目的研究大鼠骨髓间充质干细胞(MSCs)体外神经分化蛋白的表达。方法采用全骨髓贴壁筛选法分离培养大鼠骨髓MSCs。取生长状态良好的第1、3、7代细胞绘制生长曲线,免疫细胞化学法鉴定其性质,检测巢蛋白(Nestin)、神经元特异性烯醇化酶(NSE)、微管相关蛋白2(MAP2)、胶质纤维酸性蛋白(GFAP)以及酪氨酸羟化酶(TH)的表达。结果细胞传代后1-2d为滞留期,3d后达对数生长期,第6d进入平台期。免疫细胞化学法鉴定大鼠骨髓MSCsCD44呈阳性,CD34呈阴性,nestin、NSE、MAP2、GFAP、TH均呈不同程度的阳性表达,其阳性表达率分别为(25.5±5.6)%、(14.2±2.1)%、(1.4±0.5)%、(4.5±1.2)%、(3.6±1.2)%。结论大鼠骨髓MSCs在体外能自发地表达神经干细胞和神经细胞的某些标志蛋白,这种潜能使其有可能成为神经系统疾病细胞移植治疗的种子细胞。     

Ro25-6981对缺血再灌注大鼠脑室下区神经干细胞增殖的影响

目的:探讨N-甲基-D-天冬氨酸(NMDA)受体亚单位NR2B特异性拮抗剂Ro25-6981对缺血再灌注大鼠脑室下区神经干细胞增殖的影响及可能的机制。方法:线栓法制作大鼠大脑中动脉栓塞2 h再灌注模型,随机分成假手术组、缺血再灌注对照组和Ro25-6981干预组。免疫组织化学显色观察脑室下区巢蛋白和增殖细胞核抗原(PCNA)阳性细胞数,行免疫印迹对缺血侧脑组织脑源性神经营养因子(BDNF)蛋白表达水平进行定量分析。结果:缺血再灌注后脑室下区巢蛋白和PCNA阳性细胞较假手术组增加,而Ro25-6981干预组巢蛋白和PCNA阳性细胞较缺血再灌注对照组减少。缺血再灌注损伤促进了缺血侧脑组织BDNF蛋白的表达,Ro25-6981干预下调了缺血侧脑组织BDNF蛋白的表达。结论:NMDA受体亚单位NR2B能促进脑室下区神经干细胞的增殖,可能与调节BDNF表达有关。     

Hypoxia up-regulates vascular endothelial growth factor in U-87 MG cells: Involvement of TRPC1

Canonical Transient Receptor Potential (TRPC) channels play important roles in diverse physiological processes. The contribution of TRPC channels to up-regulate VEGF expression under hypoxic conditions was studied in a malignant glioma cell line, U-87 MG cells. Up-regulation of VEGF gene expression by hypoxia was markedly suppressed by a TRPC channel blocker. RT-PCR showed that U-87 MG cells expressed four TRPC isoforms in normoxia: TRPC1, 3, 4, and 5. In addition, the expression of TRPC3, 4, and 5 decreased greatly under hypoxia exposure in U-87 MG cells. In contrast, TRPC1 expression was unchanged. These results suggest TRPC channels were involved in hypoxia-induced VEGF expression, and compared with other TRPC isoforms, TRPC1 might play a different role in this process. Furthermore, we determined the function of TRPC1 by RNAi. Two different siRNAs against TRPC1 largely inhibited hypoxia-induced up-regulation of VEGF mRNA and protein levels. However, overexpression of TRPC3 or 5 neither enhanced hypoxia-induced VEGF expression, nor prevented it. Taken together, our present data suggest that TRPC1, but not TRPC3 or 5, is involved in hypoxia-induced VEGF expression in U-87 MG cells.     

成人侧脑室下区Nestin阳性细胞的免疫组化观察

目的　探讨成人侧脑室下区 (SVZ)Nestin阳性细胞的分布及其化学特点。方法　采用 3 3 1B、10C2、Rat4 0 1、GFAP、NSE和viminten等神经细胞标志物对成人SVZ区进行免疫组织化学研究。结果　SVZ区存在较多Nestin反应阳性细胞 ,GFAP免疫阳性细胞及较少的NSE免疫阳性细胞。Nestin免疫反应阳性细胞可分为两类 ,一类为卵圆形 ,胞体较大 ,少有纤维突起 ;一类为星形 ,胞体较小 ,发出多支放射状的纤维突起。Nestin免疫阳性细胞均不与GFAP及NSE发生交叉反应。成人SVZ区细胞未见有viminten的表达。结论　成人SVZ区可能存在神经前体细胞和星形胶质样Nestin免疫阳性细胞。