电针对脊髓损伤后少突胶质细胞再髓鞘化的影响

目的：观察电针治疗对脊髓损伤后少突胶质细胞增生及新生轴突髓鞘再形成的影响。方法：选用成年大鼠,制作中度脊髓损伤模型,应用督脉电针治疗。电镜观察各组髓鞘和少突胶质细胞的超微结构变化;原位杂交显示髓磷脂碱性蛋白（myelin basic protein,MBP）的基因表达。结果：脊髓损伤后髓鞘明显肿胀,部分崩解;少突胶质细胞坏死溶解。1～2周后出现少量增生少突胶质细胞和厚薄不等的髓鞘。电针组髓鞘肿胀较轻,少突胶质细胞坏死少。1周后可见较多增生的少突胶质细胞和完整髓鞘。原位杂交显示电针组MBP基因表达明显高于损伤组,3d组最低,1周达高峰,此后逐渐下降。结论：脊髓损伤后电针治疗可有效防止神经纤维的溃变,促进少突胶质细胞增生和再生神经纤维髓鞘再形成。     

成年大鼠脊髓完全性横断后星形胶质细胞的时空分布及其变化

本研究以成年大鼠脊髓完全性横断模型研究反应性胶质细胞的时空分布和变化。将30只成年Wistar大鼠随机分为5组:正常对照组,T9横断伤1周、2周、4周和8周组,每组6只。利用免疫组织化学方法及图像分析系统对各组动物脊髓内星形胶质细胞的时空分布和变化进行观察和分析。结果显示:脊髓横断组胶质纤维酸性蛋白(GFAP)阳性的星形胶质细胞数目较正常对照组明显增加(P<0.05);距损伤近侧端较距损伤远侧端的GFAP阳性星形胶质细胞数目增加显著(P<0.05);脊髓横断组髓磷脂碱性蛋白(MBP)阳性的少突胶质细胞数目的时间及空间分布与正常对照组相比无统计学差异(P>0.05)。实验结果提示,星形胶质细胞是胶质瘢痕的主要成分,而少突胶质细胞在瘢痕形成过程中并非是反应活跃的成分。     

脊髓损伤模型大鼠神经胶质细胞增殖与胶质纤维酸性蛋白的表达

背景：星形胶质细胞可以通过细胞裂解释放各种神经营养因子,并可促进损伤脊髓的修复。 目的：观察脊髓损伤模型大鼠神经胶质纤维酸性蛋白的表达及对其后肢功能恢复的影响。 方法：将SD大鼠采用Allen's法撞击T9~10节段致脊髓损伤,造模成功后蛛网膜下腔移植骨形态发生蛋白7,并设置仅蛛网膜下腔移植His蛋白的正常SD大鼠做对照。用BBB评分法评估两组大鼠后肢的运动功能,用免疫组织化学染色法和Western-blot法观察各组神经胶质纤维酸性蛋白的表达。 结果与结论：BBB评分结果显示,模型组大鼠脊髓损伤后下肢功能自行恢复率达68%。模型组脊髓损伤3和7 d,损伤区域神经胶质纤维酸性蛋白表达逐渐增加(P  < 0.05),随后逐渐下降,于脊髓损伤28 d后逐渐恢复到对照组水平(P > 0.05)。脊髓损伤后1~14 d两组胶质纤维酸性蛋白表达逐渐升高(P > 0.05)。结果证实,脊髓损伤后蛛网膜下腔移植骨形态发生蛋白7可诱导星形胶质细胞增殖,神经胶质纤维酸性蛋白的表达增强,进而促进脊髓损伤大鼠后肢功能的恢复。     

脑缺血再灌流后海马区胶质纤维酸性蛋白的动态表达及意义

本研究目的在于 :观察脑缺血再灌流后海马区胶质纤维酸性蛋白的分布及动态表达 ,探讨其与缺血性神经元的联系。钳夹沙土鼠的双侧颈总动脉制造脑缺血模型 ,应用免疫荧光法染色。结果显示 :脑缺血再灌流后胶质纤维酸性蛋白的阳性反应主要分布于海马本部的始层、放射层、分子层及齿状回门区。再灌流 3 d,胶质纤维酸性蛋白反应增强 ;7～ 15 d,胶质纤维酸性蛋白反应达高峰 ;脑缺血再灌流 40 d和对照组相比胶质纤维酸性蛋白阳性反应仍维持较高水平。再灌流 3 0～ 40 d,CA1区锥体层胶质纤维酸性蛋白阳性细胞明显增强。本研究结果表明 :脑缺血再灌流后海马区星形胶质细胞活化及胶质纤维酸性蛋白表达增强长期保持在较高水平 ,星形胶质细胞的活化、增生可作为神经元受损可靠而敏感的指标     

控释神经营养因子与细胞移植减少损伤脊髓的胶质瘢痕

背景：前期研究发现控释胶质细胞源性神经营养因子与骨髓间充质干细胞源神经元样细胞联合移植可有效促进猕猴脊髓损伤后运动功能和感觉功能的恢复。 目的：观察控释胶质细胞源性神经营养因子联合骨髓间充质干细胞源神经元样细胞移植抑制猴脊髓损伤后胶质瘢痕形成的作用是否优于单纯细胞移植。 方法：取12只恒河猴,采用改良Allen氏法制作急性重度脊髓损伤模型,随机数字表法分为3组,实验组以控释胶质细胞源性神经营养因子联合自体骨髓间充质干细胞源神经元样细胞移植修复,对照组以自体骨髓间充质干细胞源神经元样细胞移植修复,空白对照组以磷酸盐缓冲液修复。修复后5个月,取出脊髓组织制成石蜡标本,应用免疫组织化学染色显示胶质瘢痕的形态特征、构成特点及瘢痕中神经纤维的再生情况,检测胶质瘢痕面积及胶质纤维酸性蛋白染色的平均吸光度值。 结果与结论：脊髓损伤部位胶质瘢痕由混合性增生的星形胶质细胞和组织细胞构成。空白对照组脊髓胶质瘢痕累及范围广,星形胶质细胞增生显著,神经丝蛋白免疫组织化学染色阴性,胶质瘢痕面积、胶质纤维酸性蛋白染色平均吸光度值高于实验组与对照组(P < 0.05);实验组、对照组脊髓胶质瘢痕累及范围较局限,神经丝蛋白免疫组织化学染色显示有少量神经纤维通过瘢痕区,并且实验组胶质瘢痕面积、胶质纤维酸性蛋白染色平均吸光度值低于对照组(P < 0.05)。结果表明,控释胶质细胞源性神经营养因子联合骨髓间充质干细胞源性神经元样细胞移植可更强抑制脊髓损伤后胶质瘢痕的形成。     

弥漫性轴索损伤后胶质细胞的反应性变化

目的 探讨大鼠弥漫性轴索损伤（DAI）后胶质细胞的反应性变化规律及与轴索继发损伤的相互关系。 方法 SD 成年大鼠随机分为对照组及DAI损伤1d、2d、3d、7d组,每组8只。参照Marmarou方法制做 DAI 模型,并对大鼠的脑干部位进行胶质纤维酸性蛋白（GFAP）、离子钙结合适配器分子1（Iba1）、少突胶质细胞系转录因子2（Olig 2）、CC-1、NG2免疫组织化学染色及TUNEL染色,并且通过透射电子显微镜进一步观察脑干超微结构变化。 结果 DAI大鼠损伤后3d脑干Iba1标记的小胶质细胞细胞数目显著增多,一直持续至伤后7d,且激活的小胶质细胞呈肥大样形态学改变;DAI大鼠伤后1周内脑干GFAP标记的星形胶质细胞数目虽有所增多,但并没有统计学意义;CC-1标记的成熟少突胶质细胞在DAI后1d即开始明显降低,且随损伤时间的延长而进一步降低。DAI大鼠脑干TUNEL标记的凋亡细胞随损伤时间延长持续增多,与成熟少突胶质细胞的丢失成正相关。Olig2标记的少突胶质细胞系表达在DAI损伤后1周内各时间点均明显增高,于损伤后3d达峰值。NG2标记的少突胶质细胞前体细胞数量于损伤后3d和7d显著增多。透射电子显微镜结果示,DAI大鼠损伤呈现由髓鞘到轴索的特点,最早表现为髓鞘松解,轴索完整;且脱髓鞘会进一步促进轴索骨架崩解,最终导致轴索变性。 结论 成熟少突胶质细胞对DAI损伤具有选择易感性,髓鞘脱失是轴索继发损伤的重要因素。少突胶质细胞前体细胞增殖,并伴随小胶质细胞的激活。探索胶质细胞的动态变化将为进一步解释轴索继发损伤的病理生理学机制奠定基础。     

脑穿刺伤灶愈合过程中大胶质细胞的变化

作者采用免疫组化、免疫荧光染色、流式细胞计数等方法研究脑穿刺损伤灶愈合过程中伤灶组织中大胶质细胞的形态和比例的变化及其规律,阐明大胶质细胞在脑损伤后胶质瘢痕增生中的作用。结果发现,脑穿刺损伤后,大量的胶质原纤维酸性蛋白（GFA）免疫组化染色阳性细胞聚集在伤灶周围,呈典型的反应性胶质化改变;流式细胞计数结果证实,GFAP阳性细胞比例显著增高,在伤后2w达高峰,为46％;半乳糖脑苷脂（GC）阳性细胞的形态与比例无明显变化。作者提出,星形胶质细胞是胶质瘢痕中增生的主要胶质细胞,少突胶质细胞在这个过程中,不是一种反应活跃的细胞成分。     

大鼠脊髓损伤后星形胶质细胞特异性表达Gc的研究

用免疫组织化学方法观察了脊髓的星形胶质细胞在损伤后出现的抗原性改变并对其改变的意义进行了探讨。实验选用Wistar大鼠 2 0只。实验组 10只 ,对脊髓 T1 0 节段进行完全横断 ;对照组 10只 ,只进行 T1 0 椎板切除术 ,不损伤脊髓。在术后第 1、3、5、7、14 d分别对 2 0只大鼠灌流固定 ,并取出 3 cm长手术段脊髓。用 anti-Galactocerebrosides( anti-Gc)和 anti-glial fibrillaryacidic protein( anti-GFAP)抗体对脊髓进行标记。结果表明 :脊髓损伤后第 7d,增生肥大的星形胶质细胞可以同时被 anti-GF AP和 anti-Gc标记 (荧光双标 )。此抗原表型改变至术后 14 d依然显现。被双标的星形胶质细胞在形态上与成熟的正常胶质细胞基本相同 ,而少突胶质细胞只为 anti-Gc单独标记。对照组脊髓星形胶质细胞和少突胶质细胞只为 anti-GFAP、anti-Gc分别标记。本实验结果提示 :大鼠脊髓受损后 ,星形胶质细胞出现 GFAP和 Gc二种抗原表型。此结果首次表明成熟哺乳动物脊髓损伤后星形胶质细胞也可出现类似少突胶质细胞特异性抗原抗体改变。这可能是星形胶质细胞对脊髓创伤的一种特异性反应。这种变化可为探索脊髓损伤区域微环境的变化对脊髓损伤修复的影响提供新的线索     

大鼠生后中枢神经系统S100B和胶质纤维酸性蛋白表达的变化

目的 探讨SD大鼠生后中枢神经系统发育过程中S100B和胶质纤维酸性蛋白(GFAP)的表达变化.方法 24只雄性SD大鼠分为生后7d、14d、21d和成年4组,用免疫组织化学方法对脑、脊髓切片进行S100B、GFAP抗体染色,观察不同时间点不同部位中两种阳性细胞平均数.结果 生后7d到成年,前额皮质、海马、纹状体、黑质和脊髓S100B阳性标记的密度和数量逐渐减少,生后2～3周时渐趋于稳定;脑内GFAP阳性星形胶质细胞(AST)随年龄增大逐渐增多,突起增粗增长,生后21d GFAP阳性细胞数量已接近成年;相反,脊髓GFAP阳性标记数则随年龄增长呈现由多到少的趋势;海马CAl区生后各年龄段GFAP和S100B免疫荧光双标显示,生后1周至成年S100B阳性细胞的数量明显减少,尤以分子层明显;随年龄增长,双标阳性细胞的比例逐渐增高,多集中分布于锥体细胞层和多形层.结论 大鼠中枢神经系统中S100B和GFAP两种星形胶质细胞蛋白存在不同的表达模式;同时S100B和GFAP蛋白的表达在发育过程中可能受不同机制的调节,并可能代表星形胶质细胞的不同亚型.     

马桑内酯所致慢性癫痫大鼠海马星形胶质细胞胶质纤维酸性蛋白的表达

用免疫组织化学方法研究了系统应用马桑内酯所致的慢性癫痫大鼠海马中星形胶质细胞的胶质纤维酸性蛋白的表达。结果证明,整个海马的胶质纤维酸性蛋白免疫反应明显增强,并可见阳性细胞增生、胞体肥大,尤以齿状回门区和海马分子腔隙层及始层为甚。此外,本实验还发现海马胶质纤维酸性蛋白的阳性反应强度随发作后不同时间间隔（２ ｈ～９ ｄ）而不同,且直至发作后９ ｄ,胶质纤维酸性蛋白阳性反应程度仍高于对照组。此结果表明,马桑内酯所致癫痫反复发作可使海马星形胶质细胞内胶质纤维酸性蛋白的表达长期保持在较高水平。     

Patterns of glial development in the human foetal spinal cord during the late first and second trimester

Summary Although the presence of radial glia, astrocytes, oligodendrocytes and microglia has been reported in the human foetal spinal cord by ten gestational weeks, neuroanatomic studies employing molecular probes that describe the interrelated development of these cells from the late first trimester through the late second trimester are few. In this study, immunocytochemical methods using antibodies to vimentin and glial fibrillary acidic protein were used to identify radial glia and/or astrocytes. An antibody to myelin basic protein was used for oligodendrocytes and myelin; and, an antibody to phosphorylated high and medium molecular weight neurofilaments identified axons. Lectin histochemistry usingRicinus communis agglutinin-I was employed to identify microglia. Vibratome sections from 35 human foetal spinal cord ranging in age from 9–20 gestation weeks were studied. By 12 gestational weeks, vimentin-positive radial glia were present at all three levels of the spinal cord. Their processes were easily identified in the dorsal two-thirds of cord sections, and reaction product for vimentin was more intense at cervical and thoracic levels than lumbosacral sections. By 15 gestational weeks, vimentin-positive processes were radially arranged in the white matter. At this time, glial fibrillary acidic protein-positive astrocytes were more obvious in both the anterior and anterolateral funiculi than in the dorsal funiculus, and the same rostral to caudal gradient was seen for glial fibrillary acidic protein as it was for vimentin. Myelin basic protein expression followed similar temporal and spatial patterns.Ricinus communis agglutinin-I labelling revealed more microglia in the white matter than in grey matter throughout the spinal cord from 10–20 gestational weeks. By 20 gestational weeks, the gradients of glial fibrillary acidic protein and vimentin expression were more difficult to discern. White matter contained more microglia than grey matter. These results suggest that astrocytes as well as oligodendrocytes follow anterior-to-posterior and rostral-to-caudal developmental patterns in the human foetus during middle trimester development.     

胚胎大鼠脊髓神经干细胞的培养及鉴定

为了探讨胚胎大鼠脊髓神经管神经干细胞体外培养、鉴定的方法,本实验在解剖显微镜下机械性分离11.5 d的胚胎大鼠脊髓神经管,并制备细胞悬液,无血清培养基培养。应用免疫荧光染色方法对原代和传代细胞进行巢蛋白(nestin)鉴定;加入胎牛血清诱导其分化后分别行神经元特异烯醇化酶(NSE)、胶质原纤维酸性蛋白(GFAP)和髓磷脂碱性蛋白(MBP)鉴定。结果显示:11.5 d胚胎大鼠脊髓神经管来源的神经干细胞经连续传代培养可形成较多克隆球,呈nestin免疫阳性;加入胎牛血清可诱导其分别向神经元、星形胶质细胞和少突胶质细胞分化。本实验结果提示,利用E11.5的胚胎大鼠脊髓神经管可成功培养出大量稳定增殖并有多向分化潜能的神经干细胞,可用于进一步的研究。     

汉黄芩苷对大鼠脊髓损伤的修复效果

目的 通过观察不同剂量汉黄芩苷对大鼠脊髓损伤后介导的炎症反应的干预情况,探讨汉黄芩苷对脊髓损伤的影响.方法 建立大鼠脊髓横断损伤模型(n=95),随机分成5组:正常组(N)、生理盐水组(NS)、汉黄芩苷低剂量组(WG12.5)、汉黄芩苷中剂量组(WG25)及汉黄芩苷高剂量组(WG50).采用ELISA检测炎性因子肿瘤坏...     

纤维蛋白支架移植对大鼠脊髓损伤后神经再生和胶质瘢痕形成的影响

观察纤维蛋白-纤粘连蛋白-层粘连蛋白复合支架移植对大鼠脊髓损伤后神经再生和胶质瘢痕形成的影响,评价该支架移植修复脊髓损伤的可行性。本研究将圆柱状复合支架移植入大鼠脊髓完全横断缺损部位,同时以该模型动物作为对照组。分别于术后4w、8w、12w对动物下肢运动功能进行BBB评分。然后取出支架/脊髓组织,用免疫荧光双标和免疫印迹技术分析损伤部位神经纤维再生和胶质细胞增生情况;并用透射电镜观察支架移植部位的组织学结构。结果显示:术后4～12w支架移植组动物BBB评分明显高于对照组(P<0.05)。于术后4w,移植组可见支架内有少量神经纤维,此后神经纤维逐渐增多,而胶质细胞增生不明显;对照组脊髓损伤局部可见坏死空洞,空洞周边胶质细胞增生明显。免疫印迹检测结果表明,移植组神经丝蛋白(NF-200)相对含量高于对照组;对照组胶质纤维酸性蛋白(GFAP)的相对含量高于实验组。以上结果提示:纤维蛋白支架移植可促进大鼠脊髓损伤后神经再生,并抑制胶质瘢痕形成。纤维蛋白(原)作为生物材料用于构建修复脊髓损伤的组织工程支架具有良好的应用前景。     

Cell type-specific markers for human glial and neuronal cells in culture

We have used cell type-specific markers to identify and study the major classes of neural cells in dissociated cell cultures of optic nerve, spinal cord, and dorsal root ganglion from 15- to 21-week-old human fetuses. Astrocytes were identified by the intracellular expression of glial fibrillary acidic protein, oligodendrocytes by the cell surface expression of galactocerebroside, dorsal root ganglion neurons by their ability to bind tetanus toxin at their surface, and macrophages (including microglia) by cell surface Fc receptors and their phagocytic properties. Oligodendrocytes continued to express galactocerebroside (and some of them, also myelin basic protein) for up to 1 week in culture in the absence of neurons. While some dorsal root ganglion Schwann cells initially expressed galactocerebroside on their surface, and myelin basic protein and the major peripheral myelin glycoprotein (PO) intracellularly, they no longer expressed detectable amounts of any of these myelin-specific molecules after several days in culture. The Thy-1-like glycoprotein was found on the surface of fibroblasts, dorsal root ganglion neurons, and some astrocytes but not oligodendrocytes or the majority of leptomeningeal cells. Fibronectin was only expressed by fibroblasts and leptomeningeal cells.     

无血清培养新生Wistar大鼠神经干细胞及免疫荧光鉴定

目的在无血清条件下,分离培养新生1d的Wistar大鼠神经干细胞,观察其生长及分化情况,并对其生物学特性进行鉴定。方法取新生1d的大鼠海马组织,机械分离法分离神经干细胞,加入含有表皮生长因子、碱性成纤维生长因子、B27的DMEM/F12无血清培养基中培养、增殖。倒置显微镜下观察神经干细胞的增殖分化情况,采用免疫荧光染色鉴定其生物特性。结果从新生大鼠海马组织分离培养的神经干细胞在无血清培养基中不断增殖,免疫荧光染色显示巢蛋白呈阳性表达。诱导分化后,免疫荧光染色可见高分子量神经丝蛋白、胶质纤维酸性蛋白和髓鞘碱性蛋白阳性表达细胞。结论无血清条件分离培养的神经干细胞具有自我更新和增殖能力,能分化为神经元、星形胶质细胞和少突胶质细胞。     

Development of immunologically identified brain cells in culture: Quantitative aspects

Mechanically dissociated brain cells of 14 and 18-day-old mouse embryos and of mouse neonates were cultured for 3 weeks. Neurons, oligodendrocytes and astrocytes were identified at the 7th, 14th and 21st day in vitro by staining the cultures using the indirect immunoperoxidase technique with antisera directed against neuron specific enolase, galactocerebroside, myelin basic protein and glial fibrillary acidic protein. The number of neurons and oligodendrocytes was higher in embryonic cultures than in neonate cultures. The expression of some antigens was also different in the two types of culture. Our results indicate that the development of brain cells in mechanically dissociated brain cell cultures depends on the age of the animal at the time of plating.     

Effects of erythropoietin on glial cell development; oligodendrocyte maturation and astrocyte proliferation

We investigated the effects of erythropoietin (Epo) in glial cell development, especially the maturation of late stage immature oligodendrocytes and the proliferation of astrocytes. Epo mRNA level in oligodendrocytes was much more prominent than those in neurons or astrocytes, which were the same as those in the young adult kidney, while Epo receptor (Epo-R) mRNA level were almost the same among neural cells, kidney and liver tissues. On immunohistochemical examination, Epo-R expression was also detected in O4-positive immature oligodendrocytes and glial fibrillary acidic protein positive astrocytes. These results suggested that types of both glial cells are responsive to Epo. The numbers of mature oligodendrocytes, which are characterized by myelin basic protein and process development, were increased by treatment with recombinant human Epo (rhEpo) (0.001-0.1 U/ml). The maturation of oligodendrocytes was also enhanced by coculture with astrocytes in vitro. However, when mixed cultured cells (oligodendrocytes+astrocytes) were treated with anti-Epo antibody and/or soluble Epo-R, the differentiation of oligodendrocytes was partially inhibited. Interestingly, high dose rhEpo (1, 3, 10 U/ml) markedly enhanced the proliferation of astrocytes. These results suggested that Epo not only promotes the differentiation and/or maturation in oligodendrocytes, but also enhances the proliferation of astrocytes. It is generally accepted that astrocytes produce Epo, and therefore Epo might act on astrocytes in an autocrine manner. The astrocytes stimulated with Epo may further accelerate the maturation of oligodendrocytes. These comprehensive effects of Epo might also affect the ability of oligodendrocyte lineage cells to promote myelin repair in the normal and damaged adult central nervous system.