小鼠脊髓脱髓鞘病变后LINGO-1的动态表达变化

目的观察LINGO-1在溶血卵磷脂注射诱导脊髓脱髓鞘病变小鼠脊髓中的动态表达变化。方法模型组小鼠接受1%溶血卵磷脂注射至T8-9和T9-10胸椎节段的脊髓双侧腹外侧区,诱导脱髓鞘病变。甲苯胺蓝染色观察病变范围;实时定量RT-PCR和western blot检测病变区脊髓LINGO-1的表达变化。结果溶血卵磷脂注射1d后,病变区脊髓LINGO-1mRNA和蛋白表达上调均最为显著,其后逐渐下降,LINGO-1mRNA在2周后接近空白对照,而LINGO-1蛋白至4周时仍略高于空白对照。结论LINGO-1在脊髓脱髓鞘病变早期即上调并呈一定规律性改变,提示其与脊髓脱髓鞘病变有关;对于治疗脊髓脱髓鞘病变,LINGO-1拮抗剂应在损伤早期使用。     

哮喘小鼠C7-T5节段脊髓后角PKD1与PKD2的表达研究

目的探讨在哮喘发病中,蛋白激酶D1、蛋白激酶D2（proteinkinaseD1,D2,PKD1,PKD2）在哮喘小鼠c7-T5节段脊髓后角内的表达变化。方法BALB／c小鼠20只,按随机数字表法均分为正常对照组和哮喘组,利用AniRes2005肺功能仪测小鼠气道阻力、免疫荧光方法和Westernblot方法检测各组小鼠c7-T5节段脊髓后角内PKD1,PKD2的表达变化。结果哮喘组小鼠吸气阻力和呼气阻力明显高于正常组（P〈0．01）,哮喘模型建立成功。免疫荧光结果显示哮喘组c7．T5节段脊髓后角内PKD1,PKD2阳性产物的平均光密度值（MOD）显著高于正常对照组（P〈0．01）,westernblot方法检测也得到了相同的结果。结论哮喘小鼠C7-T5节段脊髓后角内PKD1,PKD2的表达升高。     

小鼠胚胎脊髓Draxin蛋白表达水平的动态变化研究

目的:探讨正常小鼠胚胎脊髓不同发育时期背侧抑制性轴突导向蛋白(Draxin)的表达部位和表达水平的动态变化过程。方法:应用免疫组织化学、Western Blot观察不同发育阶段胎鼠脊髓内Draxin的分布及表达变化特点;应用体外培养结合免疫荧光双染标记,观察不同类型神经细胞内Draxin的表达情况。结果:免疫组织化学染色结果显示,不同发育时期胎鼠脊髓内部及其周边组织内,Draxin的表达呈现明显的动态变化趋势; Western Blot检测结果显示,E11. 5开始Draxin表达量逐渐增加,至E14. 5达到高峰,此后下降并逐渐消失;体外培养结合免疫荧光双标的结果显示,部分Tuj-1阳性的未成熟神经元、Nestin阳性的神经干细胞以及GFAP阳性的星形胶质细胞均表达Draxin。结论:Draxin在胎鼠脊髓发育过程中的表达,具有明显的动态变化趋势,多种类型的神经元和星形胶质细胞表达Draxin。     

Nrf2/HO-1在肌萎缩侧索硬化症转基因小鼠脊髓中的表达变化

目的:检测不同疾病阶段肌萎缩侧索硬化症(ALS)转基因小鼠脊髓内Nrf2及HO-1表达变化,以探究抗氧化应激能力变化与ALS发病的关系。方法:应用免疫荧光双重标记和Western Blot技术检测Nrf2及其下游调节蛋白HO-1在ALS发病早期(95 d)、发病晚期(122 d)脊髓中的表达差异。结果:与同窝别野生型鼠相比较,免疫荧光双重标记结果显示:95 d、122 d ALS鼠脊髓腰段内Nrf2~+/GFAP~+星形胶质细胞增多;虽与同时间点同窝别野生型鼠比较,ALS转基因鼠脊髓中Nrf2蛋白表达略有升高,但差异无统计学意义。HO-1蛋白表达在ALS早期95 d趋势与Nrf2相同,前角神经元内表达较多;至122 d转基因鼠脊髓内HO-1~+表达多集中于星形胶质细胞内,且脊髓内HO-1蛋白总量较同窝别野生型鼠显著升高(P0.05)。结论:ALS运动神经元退变可能与星形胶质细胞激活后Nrf2及HO-1参与的氧化应激机制有关。     

PD1基因敲除加重小鼠脊髓损伤后继发性损伤的研究

目的:探索程序性死亡分子1(PD1)敲除对小鼠脊髓损伤后巨噬/小胶质细胞极化、血管生成、神经元存活、空洞形成及运动功能恢复的效应,阐明PD1在脊髓继发性损伤中的作用,为进一步研发干预措施奠定基础。方法:采用PD1基因敲除小鼠,制备脊髓挫伤模型;免疫荧光化学染色研究巨噬/小胶质细胞极化、血管生成、神经元存活及空洞形成;实时荧光定量聚合酶链式反应(real time RT-PCR)和免疫印迹实验(Western Blot)研究炎症因子、血管生长因子的表达变化; Basso小鼠运动功能评分(BMS)评估小鼠的运动功能。结果:与野生型(WT)小鼠相比,PD1敲除小鼠脊髓损伤区M1型巨噬小胶质细胞相关分子诱导型一氧化氮合酶(i NOS)、白细胞分化抗原86(CD86)、白介素-1β(IL-1β)、白介素6(IL-6)和肿瘤坏死因子-α(TFN-α)的表达水平显著上调,M2型相关分子精氨酸酶1(Arg1)、白介素-10(IL-10)、白介素-4(IL-4)和转化生长因子-β(TGF-β)显著下调;损伤中心两侧1 mm内血管密度显著下降,血管内皮生长因子(VEGF)表达水平显著下调,空洞面积显著增加,空洞周围存活神经元数量显著减少。BMS评分显示PD1敲除小鼠的自发运动功能恢复显著差于WT小鼠。结论:PD1敲除加重小鼠脊髓挫伤后的继发性损伤,增强PD1信号可能有助于促进脊髓损伤后的运动功能恢复。     

Shh/Gli1信号参与小鼠脊髓损伤后血-脊髓屏障渗透性改变及运动功能恢复

目的:探讨sonic hedgehog/Gli1(Shh/Gli1)信号在小鼠脊髓损伤后病理变化及运动功能恢复中的作用。方法:成年雄性C57野生型、Gli1lz和Gli1lz/lz小鼠行T8节段脊髓夹伤及假手术。Gli1lz小鼠脊髓损伤及假手术后3 d行X-gal染色;7 d行Shh/PDGFr-α、Shh/GFAP、LacZ/GFAP染色,观察Shh/Gli1信号在小鼠脊髓损伤后的激活。C57野生型和Gli1lz/lz小鼠脊髓夹伤后7 d行GFAP染色和实时定量RT-PCR,观察星形胶质细胞反应;术后14 d尾静脉注射伊文氏兰观察血-脊髓屏障改变;术后1、3、5 d和7 d行BMS评分评价小鼠后肢运动功能。结果:脊髓损伤7 d后,损伤区Shh表达升高;Shh/Gli1信号报告基因LacZ表达升高,主要表达于反应性星形胶质细胞;免疫组织化学及实时定量RT-PCR结果显示Gli1基因敲除不影响脊髓损伤后GFAP表达;伊文氏兰染色及其定量分析显示Gli1lz/lz小鼠脊髓损伤后脊髓组织伊文氏兰外渗增加。BMS评分显示Gli1lz/lz小鼠运动功能恢复显著差于野生型小鼠。结论:Shh/Gli1信号在小鼠脊髓损伤后激活,可能参与脊髓损伤后血-脊髓屏障渗透性的改变,并影响脊髓损伤后的运动功能恢复。     

小鼠脊髓损伤早期不同炎症因子的表达变化

目的:探讨小鼠脊髓夹伤后早期各时间点损伤区中IL-1β、IL-6、单核细胞趋化蛋白-1(MCP-1)、巨噬细胞炎性蛋白-1(MIP-1)、IL-4、IL-10、TGF-β的表达变化情况。方法:6 8周雄性BALB/c小鼠行T8节段脊髓重度夹伤模型,分别于术后6 h、12 h、24 h、3 d和7 d取以损伤区为中心共5 mm脊髓,用TRIzol试剂提取总RNA,反转录后进行实时定量PCR检测上述各基因的表达情况。结果:小鼠脊髓损伤早期,损伤区促炎因子IL-1β、IL-6及趋化因子MCP-1、MIP-1急剧升高,6 h内即达高峰,随后开始下降,3 d内归于正常;而炎症抑制因子IL-4早期不升反降,3 d降至最低;IL-10在6 h有所升高,但无统计学意义;TGF-β逐渐升高,12 h至高峰,随后下降,7 d再度升高。结论:脊髓损伤早期损伤区促炎因子表达增加,炎症抑制因子表达不变或减少,可能使损伤局部的炎症反应呈扩大趋势,从而导致继发的炎症损伤。     

Wnt抑制因子-1在肌萎缩脊髓侧索硬化症转基因小鼠中的表达变化

目的 检测Wnt抑制因子-1(Wif-1)在成年肌萎缩脊髓侧索硬化症(ALS)转基因模型小鼠脊髓内的表达变化,进一步探讨Wif-1在ALS发病中的作用.方法 选取ALS转基因鼠和同窝野生型鼠各54只,分别于鼠龄95d、108d、122d取材,部分鼠经4%多聚甲醛心脏灌注固定、分离脊髓、制备冷冻切片,应用免疫荧光染色技术...     

脑白质损伤模型中Lingo-1的动态变化

BACKGROUND:Lingo-1 has been identified as a negative regulator of oligodendrocyte differentiation and myelination, which may be closely related to the white matter damage, but there is no systematic report on the dynamic changes of Lingo-1 after white matter damage.  OBJECTIVE: Toexplore the dynamic expression of Lingo-1 at different time points after white matter injury in newborn rats. METHODS:Seventy-eight Sprague-Dawley rats aged 3 days old were equally and randomly divided into sham operation group and model group. In the model group, models of white matter injury were established by unilateral ligation of the right common carotid artery combined with hypoxia. In the sham operation group, the right common carotid artery was isolated only, without ligation or hypoxia.  RESULTS AND CONCLUSION:At 7 days after model induction, hematoxylin-eosin staining and immunohistochemical staining for myelin basic protein showed that a selective white matter injury was seen at the injury site of a rat model, suggesting successful model establishment. Fluorescent quantitative PCR and western blot assay results demonstrated that the expression levels of Lingo-1 mRNA and protein were significantly up-regulated at 1 day and reached a peak at 7 days post-surgery. After 7 days, above expression was gradually decreased and the up-regulation of Lingo-1 protein lasted to the 28 days post-surgery compared to the sham operation group. These results show that Lingo-1 protein was closely related to the brain white matter injury.     

芳香化酶在成年小鼠脊髓的表达

目的:探讨芳香化酶在正常成年小鼠脊髓组织中的表达特征。方法:取成年C57SJL小鼠,应用免疫组织化学、免疫荧光、免疫印迹检测正常雄性与雌性成年小鼠脊髓组织芳香化酶的分布特征。结果:芳香化酶在正常成年小鼠脊髓组织中的颈、胸、腰段均有表达,主要在灰质神经元中表达,尤其是第Ⅳ、Ⅴ、Ⅶ、Ⅸ层。脊髓腰段前角芳香化酶阳性运动神经元数量在雌雄间无明显的差异。结论:正常成年小鼠脊髓组织表达芳香化酶,不仅在后角感觉神经元中,前角运动神经元也确有表达,且其分布特征在雌雄间无明显差异。     

Neurogenin1在肌萎缩脊髓侧索硬化症转基因鼠脊髓中的表达变化

目的:研究neurogenin 1 (Ngn1)转录因子在成年肌萎缩脊髓侧索硬化症(ALS)转基因模型鼠脊髓内的表达变化,探讨其在ALS转基因鼠发病中的作用.方法:选取ALS转基因鼠和同窝野生型鼠各18只,分别于95、108 d和122 d取材,部分鼠经4％多聚甲醛心灌注固定,分离脊髓、制备冷冻切片;部分鼠分离脊髓、提取蛋白和RNA,分别应用免疫荧光显色、免疫印迹和RT-PCR检测Ngnl蛋白及mRNA在ALS转基因鼠发病中的变化.结果:与同窝野生型鼠比较,ALS转基因鼠Ngnl mRNA和蛋白表达在95、108 d和122 d均降低.免疫荧光实验结果显示,Ngnl阳性细胞主要分布在脊髓灰质前角,与同窝野生型鼠比较,ALS转基因鼠Ngnl阳性细胞光密度值明显减小.结论:在ALS转基因鼠发病脊髓中Ngnl阳性细胞光密度值明显减少,mRNA和蛋白表达降低,表明Ngnl与ALS的发生密切相关.     

MR-based measurement of spinal cord motion during flexion of the spine: implications for intradural spinal cord stimulator systems

This study develops a means of delivering electrical stimuli directly to the pial surface of the spinal cord for treatment of intractable pain. This intradural implant must remain in direct contact with the cord as it moves within the spinal canal. Therefore, magnetic resonance imaging was used to measure the movement of the spinal cord between neutral and flexed-back positions in a series of volunteers (n?=?16). Following flexion of the back, the mean change in the pedicle-to-spinal cord dorsal root entry zone distance at the T10-11 level was (8.5?±?6.0) mm, i.e. a 71% variation in the range of rostral-caudal movement of the spinal cord across all patients. There will be a large spectrum of spinal cord strains associated with this observed range of rostral-caudal motions, thus calling for suitable axial compliance within the electrode bearing portion of the intradural implant.     

急性不完全脊髓损伤模型大鼠相关炎症因子的表达

背景：随着现代交通和工矿事业的发展,脊髓损伤成为临床常见的多发病,严重的影响了人类身心健康和生活质量。 目的：检测在大鼠急性脊髓损伤模型中磷酸化信号转导与转录激活因子3（p-STAT3）介导的IL-1β、IL-6、IL-17等炎症因子的表达变化情况,探讨IL-1β、IL-6、IL-17等炎症因子在急性脊髓损伤后炎症反应过程中的作用。 方法：雄性成年SD大鼠75只,随机分为对照组和脊髓损伤1h、6h、24h和72h四个亚组（n=15只）。采用改良Allen氏法建立脊髓损伤模型,对照组仅行椎板全切除术。在建模之后相应的时间点获取损伤段脊髓、脾脏组织,免疫组织化学染色法检测IL-6和IL-17在损伤段脊髓组织中的分布及表达情况,蛋白免疫印迹法检测p-STAT3在损伤段脊髓组织中的表达变化,采用RT-PCR法检测IL-1βmRNA、IL-6mRNA和IL-17AmRNA在脾脏组织中的表达情况。 结果与结论：脊髓损伤后p-STAT3、IL-1β、IL-6和IL-17的表达明显高于对照组（P＜0.05）。损伤后炎症因子表达量立即升高,IL-1β和IL-6在损伤后6h小时到达高峰,随后开始下降;p-STAT3和IL-17在损伤后24h到达高峰,随后开始下降,至损伤后72h表达量仍高于对照组。损伤早期p-STAT3介导的促炎因子IL-1β和IL-6表达增加,可能导致损伤区域炎症级联放大,诱导继发性脊髓损伤;促炎因子IL-17的异常升高,可能在继发性损伤炎症反应中起着重要作用。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

Regeneration of the rat spinal cord after thoracic segmentectomy: Restoration of the anatomical integrity of the spinal cord

Segmentectomy of the spinal cord (SC) was performed in 28 rats at the level of the tenth thoracic vertebra. Scar formation at the operation site was studied, along with the completeness of the anatomical integrity of the SC, in control animals (group 1) and after filling of the defect with the neural matrix Spherogel (group 2) and with Spherogel containing embryonic nerve cells (group 3). The defect in the SC in animals of the control group was filled with fibrin masses by 1–2 weeks, while connective tissues scars were already formed by this time in rats of experimental groups 2 and 3. By 10–11 weeks, these animals showed partial recovery of movement in the three lower limb joints. The scar tissue and adjacent zones of the SC showed large quantities of regenerating fine myelinated nerve fibers. These were clearly evident in the zones of the cranial and caudal margins, to the extent of the appearance of typical SC tissue. In preparations obtained from control animals, fine myelinated fibers were noted in the immediate vicinity of SC substance, while nerve fibers were few in number in scar tissue and the cellular bands of the transitional zone. __________ Translated from Morfologiya, Vol. 127, No. 2, pp. 39–43, March–April, 2005.     

Pain reactivity of monkeys after lesions to the dorsal and lateral columns of the spinal cord

Summary Macaca speciosa monkeys were trained to escape electrical stimulation of either hindlimb, and they were given the alternative of witholding the response for 10 sec to receive food reward. The force, latency and threshold of escape responses were measured as a function of stimulation intensity before and after lesions of the spinal cord. The stimulus range was set so that pain was certainly evoked by some portion of the intensities above escape threshold, and it was inferred that pain reactivity was altered if the force or latency curves or both were shifted by a lesion. Ipsilateral dorsal column lesions decreased pain reactivity as revealed by higher latencies and lower forces of escape responses. Thresholds were generally unaffected. Ipsilateral lesions of the dorsolateral column produced hyperesthesia, or reduced latencies and increased forces. Thresholds were again unaffected. Contralateral lesions of the lateral column elevated thresholds, and this effect was magnified by inclusion of the dorsal columns in the lesion.Supported by Grant NS07261 from the National Institute of Neurological Diseases and Stroke, U.S. Department of Health, Education and Welfare, Bethesda, Maryland, and the Veterans Administration Hospital, Gainesville, Florida. The research described in this report involved animals maintained in animal care facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. The animal care was provided in part by N.I.H. grant FR00421. We thank Dorothy Robinson for technical assistance.During this investigation, Dr. Hamilton was supported by a postdoctoral fellowship from the Center for Neurobiological Sciences (grant MH10320 from the National Institutes of Mental Health).     

RhoA和ROCK2在SOD1-G93A转基因小鼠脊髓中的表达

目的:通过研究Rho A和ROCK2在SOD1-G93A转基因小鼠脊髓内的表达变化以阐明Rho/ROCK信号通路在肌萎缩侧索硬化症(ALS)病程中的作用。方法:饲养SOD1-G93A转基因小鼠和同窝野生型小鼠至发病早期、中期和晚期,部分小鼠冰上剥离新鲜脊髓组织,利用RT-PCR方法检测Rho A和ROCK2 mRNA的表达,利用Western Blot方法检测Rho A和ROCK2蛋白的表达;部分小鼠行心脏灌注并剥离其脊髓组织制成冰冻切片,利用免疫组织化学染色方法检测Rho A和ROCK2蛋白的表达。结果:在SOD1-G93A鼠发病的早期、中期和晚期,转基因小鼠脊髓中Rho A和ROCK2的mRNA及蛋白表达均上调。免疫组织化学染色实验结果显示,野生型小鼠脊髓中Rho A和ROCK2弥散分布于胞质和突起中,阳性染色浅,SOD1-G93A转基因小鼠脊髓中Rho A和ROCK2阳性染色深,大量聚集在细胞膜及细胞质。结论:Rho A和ROCK2在SOD1-G93A转基因小鼠脊髓中异常高水平表达与ALS脊髓区病变密切相关,可能参与ALS疾病进程。     

Nontumor lesions of spinal cord and spine

Nontumor lesions of the spinal cord and spine include developmental disorders, cystic tumor-like lesions, vascular disorders, infective diseases, demyelinating diseases, degenerative diseases, metabolic and toxic disorders, and spinal cord injury. In addition, diseases of the spine and extradural spaces secondarily cause spinal cord injury. Aside from tumors, these include developmental abnormalities, inflammatory diseases, nontumor space-occupying lesions, and tumor-like lesions such as lipomas, vascular malformations, and cysts. Awareness is required of hemostatic agents used during surgery and subsequently presenting as space-occupying lesions, which have to be differentiated from recurrent lesions. On the therapeutic front, stem cell transplantation into spinal cord for treatment of neurodegenerative disorders, spinal cord injury, and multiple sclerosis is a challenging prospect.     

急性脊髓损伤后差异蛋白的表达

背景：在质谱分析中,任何一种同位素标记相对和绝对定量试剂标记的不同样本中的同一蛋白质表现为相同的质荷比,而在串联质谱中,信号离子表现为不同质荷比(114-121)的峰,因此可以分析得到相关蛋白质的定量信息。 目的：建立大鼠急性脊髓损伤后脊髓组织差异蛋白质谱,应用同位素标记相对和绝对定量技术联合LC-MS/MS质谱技术从分子水平来探索脊髓差异蛋白的表达情况。 方法：取8只SD大鼠,参照Allen’s等方法建立大鼠急性脊髓损伤模型,随机分为脊髓损伤后0 h组和8 h组,每组各4只。损伤后取脊髓组织,用同位素标记相对和绝对定量技术分析大鼠急性脊髓损伤后脊髓组织的差异蛋白质。 结果与结论：共鉴定到了220个差异表达的蛋白,上调的差异蛋白数是116个,下调的差异蛋白有104个。其中相关神经再生的差异蛋白12个,其中上调的有7个,下调的有5个。提示实验中检测到的多种差异蛋白及表达明显的神经生长因子可能作为急性脊髓损伤的生物标记物或可能作为临床管理监测急性脊髓损伤的损伤进程、靶向治疗及评估疗效的有力证据。