SD大鼠偏侧帕金森病模型差异表达cDNA文库的构建

背景 帕金森病(Parkinson's disease,PD)是好发于中老年人的神经退行性疾病,随着社会的老龄化,其患病率及致残率呈上升趋势.根据流行病学调查,神经病理学研究及基因水平分析,一般认为PD的病因及其病理损伤过程是多因素的,包括遗传因素、环境毒素、氧化应激、兴奋性氨基酸毒性作用、线粒体功能障碍、细胞凋亡和神经营养因子水平降低等.对疾病分子水平变化的研究,不但为阐明疾病的发生发展提供新的资料,而且还能为其治疗开辟新的途径.然而目前对PD分子水平所知尚少,因此,揭示PD分子病理变化,具有十分重要的意义.本研究旨在构建偏侧帕金森病SD大鼠纹状体组织差异表达基因消减cDNA文库,并筛选出一些可能参与PD病理损伤过程的基因,为PD的基因治疗提供依据.方法 行SD大鼠纹状体区立体定向注射6-OHDA制备偏侧帕金森病模型;用抑制消减杂交方法分离差异表达基因的cDNA片段,连接T载体构建文库,转化扩增后随机挑取白色克隆行菌落PCR鉴定;利用反Northern杂交对重组质粒进行筛选;通过序列测定后与Genebank进行同源性比较.结果 消减文库扩增获得1500余个白色阳性克隆,随机挑取95个行PCR扩增,81%的克隆中有100～600 bp的插入片段,经反Northern杂交筛选出12个差异表达基因.结论 利用立体定向技术成功制备偏侧帕金森病SD大鼠模型;利用SSH法及T/A克隆技术成功构建偏侧帕金森病大鼠纹状体组织差异表达基因消减cDNA文库.     

改良消减杂交法克隆大鼠脊髓损伤修复相关基因

目的 克隆大鼠脊髓损伤修复相关基因，从分子水平了解中枢神经系统损伤修复的机制。方法 建立大鼠脊髓损伤模型，运用基于聚合酶链反应(PCR)的改良消减杂交技术，克隆与脊髓损伤修复相关的基因。结果 随机选取l10个克隆进行研究，排除假阳性克隆和重复序列后，筛选到47个差异表达序列，其中39个已知序列，8个未知序列。39个已知序列中，包括synuclein、clusterin等与神经系统发育及退行性病变密切相关的蛋白编码序列；8个未知序列中，69号末端含有Leu拉链结构，包含该序列的基因可能编码DNA结合蛋白。结论 运用基于PcR的改良消减杂交技术成功克隆到损伤脊髓上调表达的基因序列，为探讨中枢神经系统损伤修复的分子机制奠定了基础。     

大鼠脊髓损伤后巢蛋白表达

目的探讨成年大鼠脊髓损伤后损伤区局部巢蛋白（nestin）的表达及意义。方法应用Allen＇s法建立大鼠脊髓拟伤模型，行为学评分采用BBB评分（Basso，Beattie＆Bresnahan locomotor rating scale，BBB scale），观察局部病理组织学改变及用免疫荧光组织化学方法检测局部脊髓在不同时段nestin的阳性表达变化。结果伤后1w BBB评分最低，随后增加，到4w以后达到最高并进入平台期。常规病理学检查显示拟伤模型类似于临床常见的脊髓损伤。损伤后1W，可见损伤区附近nestin表达升高，2W达高峰，4W后nestin表达明显下调。结论脊髓损伤可诱导损伤区周围短暂的nestin阳性表达，后者可能存在脊髓损伤后的再生与修复中起重要作用。     

脊髓损伤大鼠脊髓VRI表达及意义

目的了解脊髓损伤后逼尿肌反射亢进的原因。方法建立反射亢进型神经原性膀胱SD大鼠模型,于不同时间点取L4、L5、L6、S1脊髓节段,进行VRI免疫组化染色,观察各脊髓节段VRI表达。结果 VRI阳性表达局限于脊髓后角浅层（I层、Ⅱ层）的神经纤维,在模型组的表达高于对照组。结论脊髓损伤后脊髓中VRI表达增强可能是膀胱逼尿肌反射亢进的原因。     

组织工程修复脊髓损伤

目的：就近年来组织工程修复脊髓损伤进展进行归纳总结。资料来源：检索人为第一作者,检索文献时限为1999-09/2009-09,检索数据库为PubMed数据库（http://www.ncbi.nlm.nih.gov/PubMed）及CNKI数据库（www.cnki.net/index.htm）。中文检索词为“脊髓损伤,组织工程”;英文检索词为“spinal cord injury,tissue engineering”。资料选择：纳入标准,①选取组织工程修复脊髓损伤相关的临床和实验研究论文;②组织工程修复脊髓损伤方面的相关研究进展。排除重复研究。结局评价指标：计算机初检到82篇文献,阅读标题和摘要进行初筛,排除研究目的与本文无关的文献31篇,内容重复性研究20篇,保留31篇文献进一步分析,包括综述、评述、论著及临床报道文章,对文献分析,总结研究内容。结果：组织工程修复脊髓损伤研究包括如下,组织工程种子细胞选择,组织工程脊髓对支架材料的要求,再生所需神经营养因子,再生所需特殊内环境构建。结论：选择最佳组织工程组分是修复脊髓损伤重要依据。     

应用cDNA微阵列分析脊髓损伤后基因表达的差异☆

背景：基因芯片可以大规模地平行检测分析上千个基因的表达模式,克服了传统的一次实验仅能对单个或数个基因表达水平进行分析的局限。 目的：应用含有1 176条人类全长基因的cDNA表达谱芯片,对大鼠急性脊髓损伤模型中基因表达水平的变化进行动态观察。 方法：雌性SD大鼠70只随机分成正常对照组、手术对照组、损伤4 h,24 h,3 d,7 d,10 d组7组。损伤组切除T7、T8的椎板,并用钢棒从高处自由落下致脊髓损伤。手术对照组仅进行T7、T8椎板切除。正常组和各损伤组于伤后各时间段,手术对照组于术后3 d取T6~T10段脊髓,提取总RNA,运用AtlasImageTM 2.01软件(Clontech)对放射自显影基因表达谱进行分析,各个处理组与正常组相比,灰度值差异超过3倍的基因定为有表达差异。 结果与结论：结果显示共有显著表达差异基因81条,其中表达上调的基因有46条,表达下调的基因有35条,并在国内外首次观察到神经激肽B、神经肽Y、垂体后叶加压素V2受体等数个基因在脊髓损伤中的变化。结果表明利用基因芯片技术结合实验动物模型能大规模、高通量地观察急性脊髓损伤继发性损伤的基因表达谱,筛选疾病相关基因,对进一步阐明疾病在基因水平上的发病机制,有重要的意义。     

雪旺细胞与实验性脊髓损伤修复

脊髓损伤是神经科学领域致死率、致残率最高的创伤之一,可直接导致神经元坏死、神经轴突中断,脊髓结构严重破坏.而损伤后的微环境又有诸多不利于脊髓神经轴突再生的因素,如:多种神经营养因子的缺乏以及胶质疤痕和多种抑制再生的物质存在等.雪旺细胞是外周神经系统特有的胶质细胞,包绕轴突形成髓鞘,近年发现雪旺细胞不只在轴突周围形成髓鞘,它还有许多有利于调节损伤后轴突再生的独特功能.大量的动物实验已证明雪旺细胞移植和其他方法的联合应用能够克服这些因素达到促进轴突再生、髓鞘形成及功能恢复的目的,为脊髓损伤治疗带来了新的希望.     

大鼠胚脊髓植入成鼠损伤脊髓前后光电镜观察

大鼠胚脊髓植入成鼠损伤脊髓前后光电镜观察李兵仓，廖维宏，刘枚，李应玉一、材料与方法以体重２００ｇ左右的Ｗｉｓｔａｒ大鼠脊髓为受体，以同种妊１４天（Ｅ１４）的胎鼠脊髓为供体。移植时先半切受体脊髓腰膨大左侧并吸出局部脊髓组织，然后由孕鼠作者单位：６３００...     

神经组织移植与脊髓损伤的再生修复

脊髓损伤后神经再生与功能修复研究已进行了近百年。早期的研究大多限于观察轴突断端的生长。1928年,Cajal 等研究证实,在脊髓损伤的数天内,可见轴突有新芽(Sprouting)再生,但未见轴突的延长生长,新芽的最终结局仍是变性消失。此后,又有许多学者重复了有关脊髓损伤再生的研究,结论是:脊髓损伤后轴突再生能力极其有限,即使有一定程度的再生,也难以延长穿越损伤部位的瘢痕组织。这种中枢神经系统     

Identification of four differentially expressed genes associated with acute and chronic spinal cord injury based on bioinformatics data

Complex pathological changes occur during the development of spinal cord injury(SCI), and determining the underlying molecular events that occur during SCI is necessary for the development of promising molecular targets and therapeutic strategies. This study was designed to explore differentially expressed genes(DEGs) associated with the acute and chronic stages of SCI using bioinformatics analysis. Gene expression profiles(GSE45006, GSE93249, and GSE45550) were downloaded from the Gene Expression Omnibus database. SCI-associated DEGs from rat samples were identified, and Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed. In addition, a protein-protein interaction network was constructed. Approximately 66 DEGs were identified in GSE45550 between 3–14 days after SCI, whereas 2418 DEGs were identified in GSE45006 1–56 days after SCI. Moreover, 1263, 195, and 75 overlapping DEGs were identified between these two expression profiles, 3, 7/8, and 14 days after SCI, respectively. Additionally, 16 overlapping DEGs were obtained in GSE45006 1–14 days after SCI, including Pank1, Hn1, Tmem150 c, Rgd1309676, Lpl, Mdh1, Nnt, Loc100912219, Large1, Baiap2, Slc24 a2, Fundc2, Mrps14, Slc16 a7, Obfc1, and Alpk3. Importantly, 3882 overlapping DEGs were identified in GSE93249 1–6 months after SCI, including 3316 protein-coding genes and 567 long non-coding RNA genes. A comparative analysis between GSE93249 and GSE45006 resulted in the enrichment of 1135 overlapping DEGs. The significant functions of these 1135 genes were correlated with the response to the immune effector process, the innate immune response, and cytokine production. Moreover, the biological processes and KEGG pathways of the overlapping DEGs were significantly enriched in immune system-related pathways, osteoclast differentiation, the nuclear factor-κB signaling pathway, and the chemokine signaling pathway. Finally, an analysis of the overlapping DEGs associated with both acute and chronic SCI, assessed using the expression profiles GSE93249 and GSE45006, identified four overlapping DEGs: Slc16 a7, Alpk3, Lpl and Nnt. These findings may be useful for revealing the biological processes associated with SCI and the development of targeted intervention strategies.     

EST-based identification of genes expressed in brain and spinal cord of Gekko japonicus , a species demonstrating intrinsic capacity of spinal cord regeneration

In the present study, a brain and spinal cord cDNA library of Gekko japonicus was constructed, and 2349 unique expressed sequence tags (ESTs), composed of 494 clusters and 1855 singletons, were determined from 4108 randomly selected clones. Of these sequences, 54.8% (1287/2349) ESTs are identified as known genes and 45.2% (1062/2349), as unknown genes. The identified ESTs were classified with respect to their functional categorization; both the housekeeping genes and the nervous system-related genes were well represented in the library. The complete open reading frame (ORF)-containing clones derived from the library were identified as well; 303 clones were obtained to date. Finally, two clones with sequence homology to endothelial differentiation-related factor 1 (EDF-1) and to myelin-associated glycoprotein (MAG) precursor were selected, and their expression patterns in normal and regenerating spinal cord were analyzed by RT-PCR. Both of them were expressed in the spinal cord, and the expression level decreased significantly after tail amputation. This study provides a preliminary view of the gene expression profiles of the central nervous system in gecko, and it might accelerate the process of gene discovery and function analysis in this organism, which demonstrates an, intrinsic capacity of spinal cord regeneration.     

Clustering analysis to identify key genes associated with motor neuron excitability following spinal cord injury

Abstract

Purpose: As a neurodegenerative disease, spinal cord injury can lead to the loss of autonomic function, muscle function, or sensation. This study is designed to identify the key genes implicated in the excitability of motor neurons following spinal cord injury.

Materials and methods: The GSE19701 dataset was obtained from Gene Expression Omnibus. It includes a total of 31 motor neurons after spinal cord injury (samples at day 0, 2, 7, 21 and 60 following injury). After the data were preprocessed by the Robust Multi-array Average method, soft clustering analysis was conducted by the Fuzzy C-Means method in the Mfuzz package to identify the differentially expressed genes. Afterward, the differentially expressed genes were analyzed with enrichment analysis using the DAVID online tool. Based on Cytoscape software, a protein-protein interaction network was constructed and then module analysis was carried out. Furthermore, miRNA-differentially expressed gene pairs were downloaded from the miRWalk2.0 database and the miRNA regulatory network was visualized by Cytoscape software.

Results: We found 218 upregulated genes and 526 downregulated genes. In the protein-protein interaction network, Uba3, Sumo1, and Pik3ca had higher scores, and Uba3 interacted with Sumo1. Among the eight modules identified from the protein–protein interaction network, module 1 and 8 were significantly enriched in pathways related to degenerative diseases of the nervous system. Additionally, Pdcd4 was targeted by miR-21 in the miRNA regulatory network.

Conclusion: Uba3, Sumo1, Pik3ca and miR-21 targeting Pdcd4 might be responsible for the excitability of motor neurons after spinal cord injury.     

Effect of a spinal cord photolesion injury on catalase

Ischemic injury to the spinal cord results in cell and tissue damage. Oxygen free radicals have been implicated in post-ischemic cell injury and death while free radical scavengers like superoxide dismutase and catalase are associated with an amelioration of ischemic injury. Measurement of catalase enzyme activity or protein in ischemic tissue presents mechanical problems due to extensive tissue destruction. Therefore, we looked at the effects of a photochemical lesion (which reproduces ischemic injury) on the levels of catalase mRNA in the spinal cord tissues of rodents under various experimental conditions. A significant depletion in the levels of catalase mRNA was observed in the spinal cord tissues of rats that received a severe lesion and were sacrificed 6 days post-lesion, while levels of catalase mRNA in the spinal cord tissues of similarly lesioned rats sacrificed 14 days post-lesion showed a return to control values.     

EST-based identification of genes expressed in brain and spinal cord of Gekko japonicus, a species demonstrating intrinsic capacity of spinal cord regeneration

In the present study, a brain and spinal cord cDNA library of Gekko japonicus was constructed, and 2349 unique expressed sequence tags (ESTs), composed of 494 clusters and 1855 singletons, were determined from 4108 randomly selected clones. Of these sequences, 54.8% (1287/2349) ESTs are identified as known genes and 45.2% (1062/2349), as unknown genes. The identified ESTs were classified with respect to their functional categorization; both the housekeeping genes and the nervous system-related genes were well represented in the library. The complete open reading frame (ORF)-containing clones derived from the library were identified as well; 303 clones were obtained to date. Finally, two clones with sequence homology to endothelial differentiation-related factor 1 (EDF-1) and to myelin-associated glycoprotein (MAG) precursor were selected, and their expression patterns in normal and regenerating spinal cord were analyzed by RT-PCR. Both of them were expressed in the spinal cord, and the expression level decreased significantly after tail amputation. This study provides a preliminary view of the gene expression profiles of the central nervous system in gecko, and it might accelerate the process of gene discovery and function analysis in this organism, which demonstrates an, intrinsic capacity of spinal cord regeneration.     

Key genes expressed in different stages of spinal cord ischemia/reperfusion injury

The temporal expression of microRNA atfer spinal cord ischemia/reperfusion injury is not yet fully understood. In the present study, we established a model of spinal cord ischemia in Sprague-Dawley rats by clamping the abdominal aorta for 90 minutes, before allowing reperfusion for 24 or 48 hours. A sham-operated group underwent surgery but the aorta was not clamped. The damaged spinal cord was removed for hematoxylin-eosin staining and RNA extraction. Neuronal degeneration and tissue edema were the most severe in the 24-hour reperfusion group, and milder in the 48-hour reperfusion group. RNA ampliifcation, labeling, and hybridization were used to obtain the microRNA expression proifles of each group. Bioinformatics analysis conifrmed four differentially expressed microRNAs (miR-22-3p, miR-743b-3p, miR-201-5p and miR-144-5p) and their common target genes (Tmem69 and Cxcl10). Compared with the sham group, miR-22-3p was continuously upregulated in all three ischemia groups but was highest in the group with no reperfusion, whereas miR-743b-3p, miR-201-5p and miR-144-5p were downregulated in the three ischemia groups. We have successfully identiifed the key genes expressed at different stages of spinal cord ischemia/reperfusion injury, which provide a reference for future investigations into the mechanism of spinal cord injury.     

Syntenin‐a promotes spinal cord regeneration following injury in adult zebrafish

In contrast to mammals, adult zebrafish recover locomotor function after spinal cord injury, in part due to the capacity of the central nervous system to repair severed connections. To identify molecular cues that underlie regeneration, we conducted mRNA expression profiling and found that syntenin‐a expression is upregulated in the adult zebrafish spinal cord caudal to the lesion site after injury. Syntenin is a scaffolding protein involved in mammalian cell adhesion and movement, axonal outgrowth, establishment of cell polarity, and protein trafficking. It could thus be expected to be involved in supporting regeneration in fish. Syntenin‐a mRNA and protein are expressed in neurons, glia and newly generated neural cells, and upregulated caudal to the lesion site on days 6 and 11 following spinal cord injury. Treatment of spinal cord‐injured fish with two different antisense morpholinos to knock down syntenin‐a expression resulted in significant inhibition of locomotor recovery at 5 and 6 weeks after injury, when compared to control morpholino‐treated fish. Knock‐down of syntenin‐a reduced regrowth of descending axons from brainstem neurons into the spinal cord caudal to the lesion site. These observations indicate that syntenin‐a is involved in regeneration after traumatic insult to the central nervous system of adult zebrafish, potentially leading to novel insights into the cellular and molecular mechanisms that require activation in the regeneration‐deficient mammalian central nervous system.     

Ion channel blockers and spinal cord injury

The activation of a delayed secondary cascade of unsatisfactory cellular and molecular responses after a primary mechanical insult to the spinal cord causes the progressive degeneration of this structure. Disturbance of ionic homeostasis is part of the secondary injury process and plays an integral role in the early stage of spinal cord injury (SCI). The secondary pathology of SCI is complex and involves disturbance of the homeostasis of K(+) , Na(+) , and Ca(2+) . The effect of ion channel blockers on chronic SCI has also been proved. In this Mini-Review, we provide a comprehensive summary of the effects of ion channel blockers on the natural responses after SCI. Combination therapy is based on the roles of ions and disturbance of their homeostasis in SCI. The effects of ion channel blockers suggest that they have potential in the treatment of SCI, although the complexity of their effects shows that further knowledge is needed before they can be applied clinically.     

Serum albumin improves recovery from spinal cord injury

A neuroprotective factor is shown to be present in mammalian serum. This factor is identified by Western blotting to be serum albumin. The serum factor and albumin both protected cultured spinal cord neurons against the toxicity of glutamate. The inability of K252a, a blocker of the high affinity tyrosine kinase receptor for members of the nerve growth factor family, to block the neuroprotective effect of the serum factor established that the serum factor is not a member of the nerve growth factor family. Post-injury injection of albumin intravenously or into the site of injury immediately after injury both improved significantly locomotor function according to Basso-Beattie-Bresnahan assessment and spontaneous locomotor activity recorded with a photobeam activity system. Albumin has multiple mechanisms whereby it may be neuroprotective, and it is a potentially useful agent for treating neurotraumas.