脑出血大鼠脑内神经干细胞移植的研究

目的分离并克隆新生大鼠神经干细胞,研究其移植入脑出血大鼠脑内的生物学特征,了解神经干细胞移植治疗脑出血的可行性.方法用尾状核注射Ⅶ型胶原酶制作脑出血模型,从Wistar新生大鼠脑室下区分离并克隆神经干细胞,经Brdu(5-溴脱氧尿嘧啶)掺入标记后移植入脑出血同侧的侧脑室或脑出血对侧的尾状核中.经免疫组织化学鉴定了解移植细胞在大鼠脑内的生存、迁移及分化情况.结果将稳定培养的神经干细胞移植入脑出血大鼠脑内,发现移植后4 d移植细胞仍存在.侧脑室移植组中移植细胞多在侧脑室周边区存在,尾状核移植组可见移植细胞开始向对侧迁移.免疫荧光双标证实细胞大多分化成神经元,少部分分化成胶质细胞.结论神经干细胞移植入脑出血大鼠脑内后能够存活,并能有效地穿过室管膜和向脑出血部位迁移.移植细胞在脑内大部分分化成神经元,少部分分化成胶质细胞.     

神经干细胞移植对脑出血模型大鼠神经功能的影响☆

背景：外源性神经干细胞具有神经修复作用，可能对脑出血后的神经功能恢复起到一定的作用。 目的：观察胎鼠神经干细胞的体外生长、分化及移植到脑出血大鼠后的存活、迁徙、分化情况，探讨神经干细胞对脑出血模型大鼠受损神经功能的修复作用。 设计：完全随机分组设计，对照动物实验。 单位：复旦大学附属华山医院神经外科 材料：选用健康雄性成年SD大鼠18只为受体，体质量280~320 g，由中国科学院上海实验动物中心提供。实验用鼠抗BrdU为Neomarkers产品, 鼠抗胶质纤维酸性蛋白和兔抗微管相关蛋白2 为Chemicon产品。 方法：实验于2006-02/12在复旦大学附属上海医学院解剖组胚实验室完成。从胎龄14 d的胎鼠海马中分离、培养、鉴定神经干细胞。16只受体SD大鼠被随机分为3组：对照组，PBS组和移植神经干细胞组。均通过尾状核内注射自体动脉血制作大鼠脑出血模型。移植NSC组在造模后30 min在血肿腔周围四点分别移植浓度为2×1011 L-1神经干细胞悬液5μL；PBS组于相同时间点在脑内相同部位注射PBS；PBS和神经干细胞的移植方法同自体血的移植方法。对照组大鼠在造模后30 min只造成四点损伤，不注射任何物质。 主要观察指标：在造模后立即，1，3，5，14，21，28 d采用前肢评分和转身评分对大鼠神经功能进行评估。大鼠于造模后28 d麻醉后取脑，并通过双标胶质纤维酸性蛋白、微管相关蛋白2、BrdU免疫组化来检测移植入脑的神经干细胞在体内的分化情况。 结果：①神经功能评分：造模后5 d，各组差异无显著性意义（P > 0.05）。造模后14~28 d，干细胞移植组较其他3组明显改善（P < 0.05）。②脑组织切片双免疫组织学双标染色结果：干细胞移植组血肿周围凋亡细胞少于PBS组。受体大鼠脑组织切片显示有BrdU, 微管相关蛋白2,胶质纤维酸性蛋白阳性细胞，说明神经干细胞可以在宿主脑内存活、迁徙和分化，可以分化为神经元样细胞和神经胶质样细胞。 结论：神经干细胞移植可能通过分化为神经元样细胞和神经胶质细胞促进大鼠脑出血的神经功能恢复。     

人胚神经干细胞植入脑出血大鼠的存活和分化状态

背景: 研究证实外源性神经干细胞能修复神经,促进脑出血后神经功能障碍的恢复。然而,脑出血后局部内环境对移植神经干细胞存活分化的影响是一个复杂多变的过程。 目的：观察人胚神经干细胞植入脑出血大鼠脑内的存活和分化状态。 设计、时间及地点：免疫组织化学水平的开放性实验,于2007-05/2008-04在泸州医学院附属医院分子生物实验室完成。 材料：纳入40只SD雌性大鼠,由中国医学科学院实验动物研究所提供;8周龄流产胚胎大脑由德阳市人民医院医院妇产科提供。 方法：取8周龄流产人胚胎大脑皮质细胞,体外培养获得人胚神经干细胞。通过注射自体动脉血到尾状核制作大鼠脑出血模型,出血后2 d将标有5’-溴脱氧尿嘧啶的人胚神经干细胞悬液移植到血肿腔周围的4点,1,2周后处死大鼠,相邻脑组织切片行5’-溴脱氧尿嘧啶/微管相关蛋白2和5’-溴脱氧尿嘧啶/胶质纤维酸性蛋白免疫组织化学双染。 主要观察指标：应用免疫组织化学及免疫荧光染色方法观察移植入脑出血大鼠脑内的人胚神经干细胞存活、分化状态和迁徙情况。 结果：5’-溴脱氧尿嘧啶阳性细胞为椭圆形棕褐色,移植后1周及2 周均可见其存活并向周围迁移,且移植后2周迁移的范围广。移植后1周,脑组织切片见5’-溴脱氧尿嘧啶/微管相关蛋白2和5’-溴脱氧尿嘧啶/胶质纤维酸性蛋白双阳性细胞,且5’ -溴脱氧尿嘧啶/微管相关蛋白2双阳性细胞多于5’ -溴脱氧尿嘧啶/胶质纤维酸性蛋白双阳性细胞。移植后2周,5’-溴脱氧尿嘧啶阳性细胞数明显减少,在脉络丛和微血管中可见,且5’-溴脱氧尿嘧啶/胶质纤维酸性蛋白双阳性细胞多于5’ -溴脱氧尿嘧啶/微管相关蛋白2双阳性细胞。 结论：人胚神经干细胞移植入脑出血大鼠脑内能够存活,移植后逐渐分化为神经细胞和星形胶质细胞。     

星形胶质细胞条件培养液体外诱导胎鼠室管膜前下区神经干细胞向多巴胺能神经元的分化

背景：在神经干细胞移植治疗神经系统退行性变及修复神经系统功能损伤过程中,有效的神经干细胞体外增殖与多巴胺能神经元的定向诱导分化尤为关键。 目的：以星形胶质细胞条件培养液为诱导剂,观察胎鼠室管膜前下区神经干细胞体外向多巴胺能神经元的分化。 设计、时间及地点：细胞学体外对照观察,于2006-12/2007-08在解放军第三军医大学新桥医院实验中心完成。 材料：清洁级新生2 d龄KM小鼠15只,孕16 d Wistar大鼠40只,均由解放军第三军医大学实验动物中心提供。 方法：采用差速黏附法和振荡分离法纯化培养KM小鼠星形胶质细胞,收集传至第3代、培养5 d的星形胶质细胞条件培养液,-20 ℃冻存待用。体外分离Wistar胎鼠室管膜前下区神经干细胞,加入含B27和碱性成纤维细胞生长因子的DMEM/F12无血清培养基进行原代培养,传至第3代后按0.5×108 L-1密度接种,设立2组：对照组单纯加入含体积分数0.1为胎牛血清的DMEM/F12培养基予以自然分化,实验组加入已制备的星形胶质细胞条件培养液进行诱导分化。 主要观察指标：免疫细胞化学染色鉴定胎鼠室管膜前下区神经干细胞,流式细胞仪检测胎鼠室管膜前下区神经干细胞向多巴胺能神经元分化的阳性率。 结果：培养的神经球细胞表达巢蛋白,可分化为神经元特异性烯醇化酶、胶质纤维酸性蛋白阳性细胞。诱导分化7 d后,实验组可见酪氨酸羟化酶阳性细胞,胞体呈圆形或椭圆形,酪氨酸羟化酶位于胞质及突起中;对照组酪氨酸羟化酶阳性细胞在数量、细胞成熟形态上均未达到实验组水平。实验组酪氨酸羟化酶阳性细胞分化率明显高于对照组（t=35.296,P < 0.01）。 结论：在体外星形胶质细胞条件培养液可显著促进胎鼠室管膜前下区神经干细胞向多巴胺能神经元分化。     

半乳凝素1对脑损伤大鼠室管膜下区内源性神经干细胞原位增殖及迁移的影响

背景：半乳凝素1表达于室管膜下区的星形胶质细胞中并诱导其分化，分化的星形胶质细胞可明显提高脑源性神经生长因子的产生。 目的：观察侧脑室注入半乳凝素1对脑缺血损伤大鼠内源性神经干细胞增殖、迁移的影响。 设计、时间及地点：细胞学体内观察实验，于2007-03/11在佳木斯大学神经科学研究所完成。 材料：纯种清洁级成年Wistar大鼠48只，随机分为模型组、药物组，24只/组。半乳凝素1为北京晶美生物工程公司产品。 方法：两组大鼠均采用线栓法制作局灶性大脑中动脉闭塞模型。造模后24 h，药物组经右侧侧脑室注入10 μL浓度为0.2 g/L的半乳凝素1，模型组注射等量生理盐水。分别于缺血再灌注后3，7，14，28 d处死大鼠，取脑组织制作石蜡切片，处死前1 d腹腔注射BrdU。 主要观察指标：免疫组织化学染色检测大脑室管膜下区BrdU和巢蛋白阳性细胞的表达。 结果：模型组缺血再灌注3 d后大脑室管膜下区BrdU、巢蛋白阳性细胞开始增加，7 d增殖达高峰，14 d后表达开始下降，28 d后下降至最低。药物组缺血再灌注3 d后大脑室管膜下区两种阳性细胞均明显增加；7 d 增殖达高峰，半定量分析BrdU、巢蛋白阳性细胞数分别是模型组的2倍和1.5倍，且BrdU阳性细胞向腹外侧迁移，巢蛋白阳性细胞胞体变大，突起增长，并有向外侧迁移进入脑实质的迹象；14 d后开始下降；28 d降至最低，但仍明显多于模型组（P < 0.05）。 结论：经侧脑室注入半乳凝素1在大鼠脑缺血后可激活内源性神经干细胞原位增殖，并存在由增殖区向外周脑实质迁移的趋势。     

老年大鼠脑出血后海马齿状回神经干细胞的增殖与分化

目的 观察老年大鼠脑出血后海马齿状回神经干细胞(NSCs)的增殖与分化,探讨脑出血后NSCs的变化规律.方法 制作老年大鼠脑出血模型,5-溴脱氧尿核苷(BrdU)腹腔注射标记增殖细胞,用免疫组化法检测大鼠海马齿状回BrdU、神经元核抗原(NeuN)、胶质纤维酸性蛋白(GFAP)阳性细胞数的变化.结果 正常组和假手术组老年大鼠海马齿状回均有少量BrdU阳性细胞,脑出血后大鼠各时间段的BrdU阳性细胞数目均较正常组和假手术组明显增加,7d组达到峰值后逐渐下降,28d组仍高于正常组和假手术组.正常老年大鼠海马齿状回可见少量BrdU/NeuN和BrdU/GFAP双标阳性细胞,脑出血后双标阳性细胞数较正常组明显增加.结论 脑出血后老年大鼠海马齿状回NSCs增殖明显,且可以向神经元和神经胶质细胞分化.     

海马神经元条件培养液体外诱导大鼠骨髓间质干细胞向神经元样细胞和神经胶质样 细胞的分化：与含b-FGF培养基和无血清培养基的比较*☆

背景：目前关于骨髓间质干细胞能否向神经元方向分化的报道不多,且争论多集中在分化后的神经元是否仅具有神经元形态而不具有神经元功能。 目的：探讨海马神经元条件培养液诱导大鼠骨髓间质干细胞向神经元样细胞和神经胶质样细胞分化的可能性。 方法：将第5代大鼠骨髓间质干细胞分为4组：条件培养基组加入海马神经元和胶质细胞的培养液;b-FGF组加入含b-FGF的DMEM培养基;无血清培养组加入含Neurobasal和B27的无血清培养基;阴性对照组加入含胎牛血清的DMEM。各组诱导12,24 h后,应用免疫细胞化学染色行神经元特异性烯醇化酶、微管相关蛋白2、胶质纤维酸性蛋白的鉴定,Western-blot法检测细胞神经元特异性烯醇化酶、微管相关蛋白2和胶质纤维酸性蛋白的表达。 结果与结论：诱导12,24 h后,条件培养基组、b-FGF组、无血清培养组骨髓间质充干细胞微管相关蛋白2、胶质纤维酸性蛋白、神经元特异性烯醇化酶均呈阳性表达,阴性对照组未见表达。与阴性对照组比较,诱导后24 h,条件培养基组、b-FGF组、无血清培养组微管相关蛋白2表达均明显增强(P < 0.05),且条件培养基组增强幅度显著高于另两组(P < 0.05);条件培养基组、b-FGF组、无血清培养组神经元特异性烯醇化酶及胶质纤维酸性蛋白表达无明显差异。结果证实海马神经元条件培养液可体外诱导大鼠骨髓间质干细胞分化为神经元样细胞和神经胶质样细胞,与含b-FGF的培养基和无血清培养基相比,海马神经元条件培养基诱导的神经元和神经胶质细胞阳性率最高。     

三七总皂苷干预股骨头缺血性坏死兔成骨细胞护骨素基因的表达*

背景：前期研究已明确三七总皂苷能抑制乙醇诱导的兔骨髓基质干细胞成脂分化。 目的：进一步观察三七总皂苷对兔成骨细胞的增殖和分化以及骨保护素、细胞核因子κB受体活化因子配体mRNA表达的影响。 方法：白酒灌胃4周制备新西兰兔股骨头缺血坏死模型,随机分为生理盐水组、复方骨肽组和三七总皂苷组。通过组织块培养法提取各组兔成骨细胞,检测细胞的分化,骨保护素、细胞核因子κB受体活化因子配体基因表达情况,检测各目的基因杂交信号的强弱。 结果与结论：三七总皂苷组兔碱性磷酸酶活性、钙结节计数及骨保护素、细胞核因子κB受体活化因子配体的表达强度、骨保护素mRNA/细胞核因子κB受体活化因子配体mRNA均高于生理盐水组(P < 0.01);与复方骨肽组效果接近。证实三七总皂苷能够促进兔成骨细胞的增殖和分化,并能提高成骨细胞的骨保护素mRNA的相对表达量而对其细胞核因子κB受体活化因子配体mRNA有抑制作用。     

液压性脑损伤后室下区神经干细胞的分离培养与鉴定

目的分离液压性脑损伤室下区巢蛋白（nestin）和胶质酸性纤维蛋白（GFAP）阳性（nestin^＋／GFAP^＋）共存细胞进行培养和诱导分化，观察其分裂、增殖和分化能力，以阐明损伤反应性星形胶质细胞增生过程中nestin^＋／GFAP^＋共存细胞是否具有神经干细胞特性。方法用液压冲击法建立动物模型，分离损伤成年SD大鼠室下区nestin^＋／GFAP^＋共存细胞，制成单细胞悬液，培养和诱导分化，以免疫荧光化学方法对原代和传代培养形成的神经球以及原代和传代培养诱导分化的细胞进行鉴定。结果结果显示培养及传代的细胞不断分裂增殖，可以形成悬浮生长nestin阳性的神经球；神经球诱导分化后可以分化为少突胶质细胞、神经元和星形胶质细胞。结论成年大鼠液压性脑损伤后分离的室下区细胞具有自我更新能力和多向分化潜能，是中枢神经系统神经干细胞。     

多聚赖氨酸和明胶对神经干细胞增殖和分化的影响

摘要 背景：目前大鼠神经干细胞体外诱导分化的研究报道诸多,但其分化过程很难控制,很多实验的操作方法复杂,分化比率也很低。 目的：探索大鼠胚胎前脑神经干细胞体外原代及传代培养方法,并观察其分化规律。 方法：胎鼠在无菌条件下分离出前脑,制备单细胞悬液,以1×1011L-1接种于含N2的DMEM/F12培养基中培养,传代培养过程中加入BrdU,标记神经干细胞球。诱导分化实验分为多聚赖氨酸铺板组、明胶铺板组和无铺板组。采用体积分数20%胎牛血清刺激其分化。免疫细胞化学检测nestin、BrdU及在血清诱导条件下神经干细胞向神经细胞分化的能力。 结果与结论：细胞呈神经干细胞样生长,具有连续增殖能力,可以传代培养。传代神经球中的细胞均呈nestin阳性和BrdU阳性。多聚赖氨酸铺板组和明胶铺板组贴壁后分化为神经细胞能力强于无铺板组(P < 0.01)。多聚赖氨酸铺板组略强于明胶铺板组(P > 0.05)。神经谱系标记物神经胶质纤维酸性蛋白和微管相关蛋白2的免疫细胞化学结果均阳性。结果表明,大鼠胚胎前脑富含神经干细胞,其分化观察,多聚赖氨酸和明胶在诱导神经干细胞分化中作为细胞贴壁支持物提高分化细胞数量的作用,且多分化为星形胶质细胞。 关键词：多聚赖氨酸;神经干细胞;明胶;增殖分化;体外培养 doi:10.3969/j.issn.1673-8225.2010.47.004     

Asymmetry in the structural organization of the ganglion layer of the retina in the frog

Silver-impregnated retinal preparations were used to study the distribution density and topographic features of small and large ganglionic cells (GC) of Rana ridibunda and Rana temporaria. For both species the increased density of GC (a streak) stretched higher than the naso-temporal axis passing through the optic disk. Beyond the streak the density of small GC was maximal in the central zone of the retina and decreased towards its periphery. For the upper quadrants of the retina the density of small GC was higher than that for the lower ones by 26% on the average. On the contrary, the density of large GC was higher in the lower part of the retina as compared to the upper one, the difference being more pronounced for R. temporaria. The density of large GC was also asymmetric with respect to the dorso-ventral axis being higher in nasal quadrants than in temporal ones by 40-55%. The highest density of large GC was found in the middle zone of the retina. The found structural asymmetry in the retinal output raster may bear an adaptively ecological meaning and may condition the particularities of the formation of the visually guided prey-catching and avoidance reactions.     

Local nonuniformities in the syncytium of the horizontal cells of the retina in the turtle

Electrical coupling between horizontal cells of the turtle retina was investigated by means of two microelectrodes (current and recording ones) penetrating neighbouring cells at a fixed distance from each other. The morphological coupling was revealed by means of fluorescent dye Lucifer Yellow. The electrical coupling was confirmed between elements of similar type (L1--axonal terminals, or L2--cell bodies, or R/G type cells) and no coupling was found between elements of different types, though L1 and L2 are directly connected through thin axons. In the L1 syncytium the electrical coupling at small (less than or equal to 50 microns) but fixed distances between microelectrodes could differ several times depending on the minimal displacement of microelectrodes. This local nonuniformity of coupling can be explained on the basis of structural nonuniformities in the L1 (axon terminal) network. It is unlikely however that the structural nonuniformities can influence the functional properties of horizontal cell network when the retina is stimulated adequately (by light).     

Inhomogeneities in the propagation of the slow wave in the stomach

The propagation of the slow wave in the stomach and its role in inducing sweeping peristaltic contractions toward the pylorus, essential for a proper digestion and emptying, have been studied for many years. Irregularities in the timing or in the pattern of propagation of the slow wave have been known to induce various gastric malfunctions and, recently, several types of gastric dysrhythmias have been described which could lead to gastric contraction abnormalities. In this study, Du et al. have analyzed the disturbances caused by a simple transmural incision in a human stomach, performed to obtain a biopsy of the muscle, on the propagation pattern of the slow wave. In addition, they show that such an incision may by itself also induce new types of gastric dysrhythmias. These results are important in demonstrating that the function of the stomach can easily be disturbed by such procedures. This mini‐review describes several ways in which inhomogeneities in propagation may affect the conduction pattern of the slow wave, including the genesis of several dysrhythmias, and what is currently known about their impact on gastric contraction and digestion.     

Early stages in the formation of the cerebellum in the frog

The formation of the cerebellum was studied during the first 6 months of the tadpole stage of the bullfrog by using standard histological methods and reconstructions from serial horizontal sections. Three major developmental phases were noted in the formation of the cerebellum. (1) During the first 5 weeks of development, the neuroepithelium proliferated and the dorsal mesencephalic plates increased in size. (2) Starting in the sixth week, a patch of neuroepithelium began to differentiate and gave rise to a small population of Purkinje cells. In subsequent weeks, the area of differentiation continued to spread and a Purkinje cell layer became established along the dorsal margin of the cerebellar plate. (3) In the 12th week, the ventrolateral part of the cerebellar plate began to increase in size and generate two populations of small cells. The lateralmost part of the neuroepithelium in this area generated a group of cells that formed an external granular layer that was one cell deep. Cells of this external granular layer migrated inward into the primitive molecular layer, and by the 26th week only a remnant of an external granular layer remained in the cerebellum. The more medially situated part of the neuroepithelium gave rise to another population of small cells that formed a column, which appeared to be continuous with the Purkinje cells, but differed from them in size. It should be noted that full maturation of the cerebellum occurs during metamorphosis, which in this species remains some 2 years away.     

Gradients of histogenesis in the ependymal lining of the third ventricle in the rabbit

Tritiated thymidine autoradiography was used to analyze the site, time of origin, and developmental gradients of the specialized lining of the ependymal surface of the third ventricle. Cells destined to form the ependyma are generated between days 15 and 22 of embryogenesis (gestation: 30 +/- 2 days), the majority of the cells undergoing final division on the 18th day of gestation. Ependymal cells originate in an orderly fashion according to 3 gradients. Two gradients of opposite direction (ventrodorsal and dorsoventral) are found in the parasaggital plane. Both gradients start at the level of the hypothalamic sulcus, progressively departing from this anatomical landmark as histogenesis progresses. A third gradient occurs in the caudorostral axis, such that cells located in caudal regions originate earlier than those located in rostral sectors. Thus, an orderly relationship exists between the time of origin of ependymal cells and their final location within the lining of the ventricular wall. These findings indicate, once again, the topographic nature of the gradients of histogenesis. The histogenic gradients displayed by the ependymal lining of the third ventricle appear strongly related to those exhibited by other diencephalic derivatives. The latter suggests that common factors govern the developmental sequence of all diencephalic derivatives as a function of their relative topographic location, independently of their functional role in the adult.     

Challenges in the Management of the HIV Patient in the Third Decade of AIDS

The epidemic of AIDS has been increasingly recognized as a major health and socioeconomic problem, not only in the United States or Africa, but also the rest of the world. The face of the epidemic has changed. The role that mental health providers play has also significantly grown as the epidemic continues on. Prior to the introduction of Highly Active Anti-Retroviral Therapy (HAART) and other advances in HIV care, the patients faced issues that related to death and dying. These advances brought with them renewed hope and resurrected lives. The patients fought with issues related to living new lives with HIV no longer an imminent death threat. In the third decade of AIDS, the struggles of the post-HAART era continue but bring with it more challenges. Mental health providers need to familiarize themselves with these issues so that they can better help HIV patients cope with this devastating disease.     

Analysis of the habituation-like changes in transmission in the temporodentate pathway of the rat

Habituation-like decrements in extracellular measures of synaptic activation (population EPSP) and cell discharge (population spike) were analyzed in the dentate gyrus of the rat following repetitive low-frequency stimulation of the medial and lateral entorhinal cortex. Stimulation of either subdivision of the entorhinal projection system resulted in comparable habituation-like response decrements with similar stimulation regimens. However, habituating stimulation of one subdivision did not result in decreased responsiveness to stimulation of the other. Repetitive low-frequency stimulation or even a single pulse delivered to either subdivision did, however, result in a potentiation of granule cell discharge in response to stimulation of the other subdivision (a form of heterosynaptic potentiation). This heterosynaptic potentiation of granule cell discharge was not accompanied by any increase in the extracellular EPSP. Comparisons of the relationship between the population EPSP and population spike before and during habituating stimulation revealed changes in cell discharge in response to the habituating stimulus which could not be accounted for by changes in synaptic activation alone. The results suggest that repetitive activation of the temporodentate pathway alters granule cell output as a result of two processes, a habituation-like decrement in synaptic activation, and a potentiation of granule cell discharge as a consequence of prior activation.