地高辛标记的大鼠nNOS mRNA探针的制备和应用

目的　制备大鼠神经元型一氧化氮合酶 (neuronalnitricoxidesynthase,nNOS)地高辛 (digoxigenin)标记的RNA探针 ,探讨nNOS在脊髓中的表达定位。方法　采用RT PCR方法 ,从大鼠脑组织中扩增nNOS基因mRNA部分片段 ,并经序列测定。以dig nNOSmRNA为探针 ,采用原位杂交观察成年大鼠脊髓组织中nNOSmRNA表达。结果　RT PCR法扩增出一特异产物与预期长度 2 4 0bp相符 ,T载体克隆测序与nNOS基因 10 0 %同源。原位杂交结果显示阳性信号出现在成年大鼠脊髓组织中。结论　采用RT PCR和T载体技术获得了大鼠脑组织nNOS基因克隆 ,dig nNOSmRNA探针原位杂交显示正常SD大鼠腰段脊髓组织中表达nNOSmRNA。     

大鼠脑组织中p75基因克隆及其探针制备

为了克隆大鼠p75基因并制备地高辛标记的p75 cDNA探针(dig-p75 cDNA),本研究采用RT-PCR法,从大鼠脑组织mRNA中扩增p75基因mRNA部分片段,克隆入T载体,并经序列测定。以dig-p75 cDNA为探针,采用原位杂交方法观察成年SD大鼠海马组织中p75 mRNA的表达。结果:RT-PCR法扩增出一种特异产物与预期长度386 bp相符,T载体克隆测序与p75基因100％同源。原位杂交结果显示,阳性信号出现在成年大鼠海马组织中。结论:采用RT-PCR和T载体技术获得了大鼠脑组织p75基因克隆,dig-p75 cDNA探针原位杂交显示正常SD大鼠海马组织中表达p75 mRNA。     

CNTF基因在大鼠脊髓中的表达及生后发育的变化

目的 观察CNTF基因在大鼠脊髓中的表达以及生后发育过程中的变化。方法 以地高辛标记（dig）－CNTF cDNA为探针，采用原位杂交法，观察CNTF mRNA在大鼠脊髓中的分布；采用TR－PCR法，半定量分析大鼠生后发育过程中，脊髓CNTF mRNA表达水平的变化。结果 CNTF mRNA原位杂交阳性信号存在于正常大鼠脊髓白质的腹索、外侧索周边的部分胶质细胞中；灰质中未能发现阳性杂交信号。RT－PCR结果显示，大鼠生后1d脊髓细胞中即可见CNTF mRNA表达，但表达量较低；出生15d表达量迅速增加；30d时最高；60d时的表达量有下降的趋势。结论 大鼠脊髓白质的部分胶质细胞可表达CNTF mRNA；大鼠出生后1d CNTF mRNA即有表达，之后随脊髓的发育而呈动态变化的趋势。     

大鼠大脑组织中谷氨酸脱羧酶GAD67基因的克隆和序列测定

目的　克隆大鼠脑组织中谷氨酸脱羧酶GAD6 7基因 .方法　采用RT -PCR方法 ,大鼠脑组织中的mRNA逆转录成cDNA ,再以cDNA为模板 ,扩增谷氨酸脱羧酶GAD6 7基因片段 ,克隆入T载体 ,并经序列测定 .结果　RT -PCR法扩增出一特异产物与预期长度 1795bp相符 ,T载体克隆测序与大鼠谷氨酸脱羧酶GAD6 710 0 %同源 .结论　采用RT -PCR和T载体技术获得了大鼠脑组织中的谷氨酸脱羧酶GAD6 7基因克隆 ,为该基因的体外表达打下基础 .     

大鼠脑组织中谷氨酸脱羧酶65基因的克隆和序列测定

目的探讨克隆大鼠脑组织中谷氨酸脱羧酶65基因。方法采用RT-PCR方法,将大鼠脑组织中的mRNA逆转录成cDNA,再以cDNA为模板,扩增谷氨酸脱羧酶65基因片段,克隆入T载体,并经序列测定。结果RT-PCR法扩增出一特异产物与预期长度1758bp相符,T载体克隆测序与大鼠谷氨酸脱羧酶65100%同源。结论采用RT-PCR和T载体技术获得了大鼠脑组织中的谷氨酸脱羧酶GAD65基因克隆,为该基因的体外表达打下基础。     

大鼠大脑组织中谷氨酸脱羧酶GAD67基因的克隆和序列测定

目的克隆大鼠脑组织中谷氨酸脱羧酶GAD67基因.方法采用RT-PCR方法,大鼠脑组织中的mRNA逆转录成cDNA,再以cDNA为模板,扩增谷氨酸脱羧酶GAD67基因片段,克隆入T载体,并经序列测定.结果 RT-PCR法扩增出一特异产物与预期长度1795bp相符,T载体克隆测序与大鼠谷氨酸脱羧酶GAD67 100%同源.结论采用RT-PCR和T载体技术获得了大鼠脑组织中的谷氨酸脱羧酶GAD67基因克隆,为该基因的体外表达打下基础.     

睫状神经节神经营养因子基因克隆

在构建了再生性外周神经组织cDNA文库的基础上，人工合成了睫状神经节神经营养因子（CNTF）阅读框架的引物，用PCR地高辛标记法标记了CNTF探针．筛选CDNA文库，得到了CNTF的阳性克隆，运用PCR法证实了克隆中存在着全长CNTF阅读框架，Sanger法测定了DNA序列，证实序列无误。CNTF基因克隆为从基因水平上研究CNTF的分子生物学作用机制打下基础．     

CNTF及其受体在大鼠脊髓组织中的表达

CNTF具有广泛的神经营养活性 ,可能在神经发育与再生修复中发挥重要作用。有关 CNTF在大鼠脊髓组织中的表达尚不清楚 ,我们在制备抗人 CNTF多克隆抗体的基础上 ,利用免疫组化及原位杂交的方法系统的研究了成年大鼠脊髓组织 CNTF与受体 CNTFRα的分布与细胞定位。免疫组化结果显示 :CNTF免疫反应物质存在于脊髓灰质各层神经元中 ,包括运动神经元、中间灰质神经元及部分背角小细胞 ,尤其腹角运动神经元染色明显。胞浆胞核均有染色 ,胞核染色更深 ,无核仁染色 ,灰白质中有散在胶质细胞染色。原位杂交结果表明 ,CNTF m RNA的分布与…     

大鼠Desert Hedgehog基因的克隆和表达

目的克隆大鼠Desert Hedgehog（DHH）基因,构建其真核表达载体并转染TP67细胞;同时制备DHHcRNA正义及反义探针用于检测其在细胞中的表达。方法提取SD大鼠睾丸总RNA,RT-PCR法扩增DHHcDNA片段,连接于pGEM—TEasy载体,经测序后构建真核表达载体pLXSN／DHH并转染PT67细胞;重组质粒经限制性内切酶NotⅠ和NcoⅠ酶切、回收后,进行转录标记反应,原位杂交检测DHH在嘶7细胞中的表达。结果RT-PCR扩增得到1220bp的片段;成功构建了真核表达载体pLXSN／DHH;制备DHH正义及反义探针浓度分别为150mg／L和80mg／L;DHH在PT67细胞中有表达。结论克隆的DHH基因与大鼠Sertoli细胞的DHH基因相同,成功标记了特异、敏感的DHHcRNA探针,转染的DHH基因能够在PT67细胞中表达。     

日本血吸虫卵壳蛋白基因的筛选及EST序列测定

目的 通过对日本血吸虫(Sj)成虫cDNA库的核酸杂交筛选，分离出Sj卵壳蛋白相关基因的cDNA克隆，并进一步探讨该基因的结构与功能关系。方法 用地高辛标记的特异性寡核苷酸探针对Sj成虫cDNA库进行膜原位杂交筛进，挑选出阳性克隆，用通用引物进行PCR扩增，获得cDNA插入片段，采用PCR直接序列测定法，对其部分序列进行测定，而后将EST序列输入GENEBANK进行同源性检索和分析。结果 本实验从Sj cDNA库筛选到9个不同的阳性克隆，用通用引物扩增出9十分子量大小不同的单一条带，其中两个为已知序列，其余7个为新的EST序列。结论 用寡核苷酸标记探针对Sj库中进行校酸杂交筛选是寻找特定目的基因的有效方法。     

Tenascin-C expression by neurons and glial cells in the rat spinal cord: Changes during postnatal development and after dorsal root or sciatic nerve injury

Summary We have usedin situ hybridization with a digoxigenin-labelled probe for tenascin-C mRNA and immunocytochemistry with antibodies against tenascin-C, glial fibrillary acidic protein, OX-42 and the 200 kDa neurofilament protein to study the expression, distribution and cellular relationships of tenascin-C mRNA and protein in the developing (postnatal) and adult spinal cord of rat, and the effects thereon of dorsal root, ventral root and sciatic nerve injuries. The most interesting finding was that on postnatal day 7 (P7), P14 and in the adult, but not on P0 or P3, a group of neurons in the lumbar ventral horn expressed the tenascin-C mRNA gene. They represented about 5% of ventral horn neurons in the adult and were among the smaller such neurons. Since 40–60% of such cells were lost at P13 following sciatic nerve crush on P0, some were almost certainly motor neurons. In addition, we found that at P0 and P3, mRNA-containing glial cells were widespread in grey and white matter but sparse in the developing dorsal columns; tenascin-C immunofluorescence showed a similar distribution. By P7 there were fewer mRNA-containing cells in the ventral horns and in the area of the dorsal columns containing the developing corticospinal tract where immunofluorescence was also weak. At P14 there were no glial-like mRNA-containing cells in the grey matter; such cells were confined to the periphery of the lateral and ventral white columns but were present throughout the dorsal columns where tenascin-C immunofluorescence was also strong. No glial-like mRNA-containing cells were present in the adult lumbar spinal cord and tenascin-C immunofluorescence was confined to irregular patches in the ventral horn, especially around immunonegative cell bodies of small neurons, a zone around the central canal, and a thin zone adjacent to the glia limitans. Thus the expression of tenascin-C is differentially developmentally regulated in the grey matter and in different parts of the white matter. Three days after injury of dorsal roots L4–6, many cells containing tenascin-C mRNA, some identified as glial fibrillary acidic protein-positive astrocytes, were present in the ipsilateral dorsal column, but were rare after longer survivals. Immunoreactivity, however, was elevated in the ipsilateral dorsal column at 3 days, remained high for several months and disappeared at 6.5 months. Dorsal root injury had no effect on tenascin-C mRNA or protein in the grey matter. Sciatic nerve or ventral root injury had no effect on these molecules in any part of the spinal cord.     

Survival of neural progenitor cells from the subventricular zone of the adult rat after transplantation into the host spinal cord of the same strain of adult rat

The subventricular zone (SVZ) of the lateral ventricle of the mammalian forebrain is the major site in which neural progenitor cells (NPC) persist in the adult brain. The NPC are located beneath ventricular ependymal cells and have the capacity to self-renew and continuously produce neurons and glial cells. We have shown previously that neurospheres can be obtained from the brain of deceased adult rats and that neurosphere cells survive after transplantation into the spinal cord. In the present study, we investigated whether fresh NPC from living adult rats can survive and be integrated into host tissues after transplantation into the adult rat spinal cord of the same strain. We used rats expressing transgenic green fluorescent protein (GFP) as a donor to identify the transplanted NPCs. The SVZ tissues were obtained from the striatal wall of the lateral ventricle of adult GFP-rats and were grafted into lesions of the spinal cord at the cervical level. Two to 3 weeks after grafting, NPC migrated through the host tissue 0.5-1 mm away from the implantation site, and were integrated into the white matter of the host spinal cord. Surviving NPC exhibited immunohistochemical phenotypes of astrocytes (glial fibrillary acidic protein), but not for neurons (alpha-tubulin III) or oligodendrocytes (Rip; Hybridoma Bank, Iowa City, IA, USA). Thus, NPC from the SVZ of adult rats can survive and differentiate into at least astrocytes, which can then be integrated into host tissue after transplantation into spinal cord lesions in the adult rat.     

Embryonic intermediate filament,nestin, expression following traumatic spinal cord injury in adult rats

Precursor cells in the ependyma of the lateral ventricles of adult mammalian brain have been reported in brain, and also in the spinal cord. The present study used antibody to the intermediate filament protein (nestin) as an immunohistochemical marker for neural stem cells and precursor cells in a rat model of spinal cord trauma. Male Sprague-Dawley rats (n=25) had a laminectomy at Thll-Thl2, and spinal cord contusion was created by compression with 30 g of force for 10 min. The rats were killed at 24 h, 1 week and 4 weeks after injury, and four levels of the spinal cord were examined: 5 mm and 10 mm, both rostral and caudal region to the injury center. Time- and region-dependent alterations of nestin immunoreactivity were analyzed. Revealed at 24 h post-injury, 5 mm rostral and caudal to the lesions, nestin expression was observed in ependymal cells and around the hemorrhagic and necrotic lesion located in dorsal spinal cord, peaking at 1 week after injury. Moreover, nestin expression was also observed in the white matter of ventral spinal cord, extending into arborizing processes centripetally from the pial surface toward the central canal. At 4 weeks after injury, nestin expression in ependyma decreased 10 mm from the injury site. But nestin expression in white matter increased dramatically with a 100-fold increase in nestin originating from the pial surface, and extension now to all the white matter. The latter was accompanied by glial fibrillary acidic protein positivity into very long arborizing processes, morphologically compatible with radial glia. The findings suggest two possible sources of precursor cells in adult mammalian spinal cord; ependyma of the central canal and subpial astrocytes. Subpial astrocytes may be associated with neural repair and regeneration after spinal cord injury.     

NGF、BDNF及受体trkA、trkB、trkC在正常猴脊髓的表达

采用免疫组织化学方法观察了神经生长因子 (NGF) ,脑源性神经营养因子 (BDNF)以及 NGF家族因子受体 trk A、trk B、trk C的免疫阳性反应在正常猴脊髓的分布。结果表明 :NGF免疫反应阳性的神经元在脊髓灰质各层中均有分布 ,灰、白质内也可见较多的 NGF免疫反应阳性的胶质细胞。 BDNF在脊髓各型神经无有明显的表达 ,特别是前角运动神经元。 trk A、trk B、trk C的免疫阳性反应产物主要分布在灰质的神经元及胶质细胞。本实验结果揭示了在正常猴脊髓中神经营养因子 (NGF、BDNF )及受体 trk A、trk B、trk C的表达状况 ,提示这些神经营养因子及受体在维持猴脊髓神经元的正常生理功能中具有重要作用。     

Regional specialization of the radial glial cells of the adult frog spinal cord

Summary The amphibian spinal cord is characterized by the presence of radially oriented astrocytic glial cells. These cells have their somata located in the grey matter of the spinal cord and radial processes that extend from the soma through the grey and white matters to the pial surface of the cord. Here we show that these radial glial cells are the predominant cell type labelled by horseradish peroxidase (HRP) when the marker is applied to the surface of the cord. The morphology of the HRP-labelled processes of an individual cell is different as they pass through the grey and white matter regions of the cord. By indirect immunofluorescence on frozen sections we show that the binding of an antibody raised against mammalian glial fibrillary acidic protein (GFAP) is preferentially localized in those areas of the glial process that traverse the white matter of the spinal cord. By transmission electron microscopy we confirm that there are no astrocyte cell bodies either at the pial surface or throughout the white matter region of the cord. These results demonstrate that all the astrocytes in the adult frog spinal cord can be selectively labelled through the application of HRP to the surface of the cord, and that the processes of these labelled cells display regional morphological and biochemical specializations depending on their location in the cord.We propose that these astrocytes may play an important role in setting up the grey-white matter arrangement of the amphibian spinal cord and that a single astrocyte of the frog spinal cord may combine the properties and functions of both grey and white matter mammalian astrocytes.     

用简并性引物RT—PCR技术检测大鼠舌味蕾细胞谷氨酸受体表达

为快速、有效地用ＰＣＲ检测不同组织，根据大鼠脑中谷氨酸受体家族ＤＮＡ序列，在高同源区设计出对谷氨酸家族有专一性的简并性引物。用此引物通过ＰＣＲ扩增自来大脑的多种谷氨酸受体，而味蕾细胞ＰＣＲ产物克隆后的ＤＮＡ序列，仅有脑内代谢型谷氨酸受体四型相一致；在缺少味蕾的舌上皮组织则无此种ＰＣＲ产物。以含味蕾细胞的ＰＣＲ产物为模板，合成同位素标记的ＲＮＡ探针，用ＲＮＡ酶保护法进一步检测ｍＧｌｕＲ４在不同组织中     

Spatio-temporally differential expression of genes for three members of fatty acid binding proteins in developing and mature rat brains

The chronological changes in the gene expression for three species of cytosolic fatty acid-binding proteins (FABPs) in the rat brain were examined by Northern and in situ hybridization analyses. The expression for heart(H)-FABP became evident after birth, with a gradual increase and confined to the gray matter, suggesting that the expression of H-FABP mRNA is neuron-specific in postnatal brain. The expression for brain(B)-FABP was very intense in the ventricular germinal zone, without expression in the cerebellar external granule cell layer, suggesting the dominant expression in the cells of glial lineage. B-FABP mRNA was transiently expressed in perinatal gray as well as white matter and the expression in glial cells persists only in the olfactory nerve fiber layer at the adult stage. On the other hand, the expression for skin type(S)-FABP was evident in the both ventricular germinal zone and cerebellar external granule cell layer, suggesting the expression in cells of neuronal lineage. The expression for S-FABP was evident in the prenatal gray matter and S-FABP mRNA was expressed in glial cells at early postnatal stage, whereafter the expression decreased to, but remained at weak levels in the adult brain. Discrete functions of the three FABPs were suggested in neurons and glia differentially at various developmental stages.     

Proliferation and migration of glial precursor cells in the developing rat spinal cord

The mechanisms that control the production and differentiation of glial cells during development are difficult to unravel because of displacement of precursor cells from their sites of origin to their permanent location. The two main neuroglial cells in the rat spinal cord are oligodendrocytes and astrocytes. Considerable evidence supports the view that oligodendrocytes in the spinal cord are derived from a region of the ventral ventricular zone (VZ). Some astrocytes, at least, may arise from radial glia. In this study a 5-Bromo-2'-deoxyuridine (BrdU) incorporation assay was used to identify proliferating cells and examine the location of proliferating glial precursor cells in the embryonic spinal cord at different times post BrdU incorporation. In this way the migration of proliferating cells into spinal cord white matter could be followed. At E14, most of the proliferating cells in the periventricular region were located dorsally and these cells were probably proliferating neuronal precursors. At E16 and E18, the majority of the proliferating cells in the periventricular region were located ventrally. In the white matter the number of proliferating cells increased as the animals increased in age and much of this proliferation occurred locally. BrdU labelling showed that glial precursor cells migrate from their ventral and dorsal VZ birth sites to peripheral regions of the cord. Furthermore although the majority of proliferating cells in the spinal cord at E16 and E18 were located in the ventral periventricular region, some proliferating cells remained in the dorsal VZ region of the cord.