培养脊髓神经元中微管相关蛋白5的表达

目的　探讨初代培养脊髓神经元中微管相关蛋白 (MAP5 )的表达特征及其调节因素。方法　应用荧光免疫细胞化学检测初代培养脊髓神经元中MAP5的表达。结果　MAP5在培养脊髓神经元中呈网状分布在胞浆及突起中。诺考达唑引起微管解聚及MAP5的结构破坏 ,荧光物质分布不均匀 ,突起呈串珠或断裂。PMA(phorbol 12 myristate 13 acetate)能诱发微管及MAP5聚合 ,胞浆中荧光物质呈疏松的网状结构。结论　MAP5在初代培养脊髓神经元胞浆及突起中明显表达 ,且表达受微管解聚剂诺考达唑及微管聚合剂PMA的调控。     

培养的大鼠脊髓神经元中微管相关蛋白5与溶酶体的相关性

目的 探讨微管相关蛋白5(MAP5)与溶酶体之间的相互关系。方法 神经细胞培养、免疫荧光细胞化学方法、激光共聚焦扫描显微镜。结果 在培养脊髓神经元中，MAP5及组织蛋白酶D均分布在胞质及突起中，融合图像中两者分布大致相似。分别使用Nocodazole及PMA处理后MAP5及组织蛋白酶D在培养脊髓神经元的变化具有一致性。结论 提示MAP5和溶酶体的形态及分布依赖于微管的完整性。     

免疫电镜观察培养脊髓神经元中微管相关蛋白-5的表达

目的　研究微管相关蛋白 5在培养脊髓神经元的表达变化。方法　使用ACLAR膜培养脊髓神经元 ,分为四组 ,即正常脊髓神经元组 ,加诺考达唑 (nocodazole)组 ,加乙酸佛波醇 (PMA)组 ,空白对照组。免疫电镜观察四组微管相关蛋白 5的分布。结果　正常脊髓神经元微管相关蛋白 5阳性颗粒在胞浆及突起中均匀分布 ,nocodazole引起微管相关蛋白 5阳性颗粒排列紊乱 ,PMA则使微管相关蛋白 5阳性颗粒在胞浆及突起中密集存在。结论　微管相关蛋白 5在脊髓神经元的胞浆及突起中均有表达 ,并受nocodazole及PMA的调节     

谷氨酸转运抑制剂诱导脊髓运动神经元选择性损伤

目的：观察谷氨酸转运体抑制剂苏-羟天冬氨酸(THA)对器官型培养的脊髓片的影响。方法：取出生后8d乳鼠的腰段脊髓组织切片做脊髓器官型培养,在培养液中加入THA,免疫组化染色进行神经元计数,测定培养液中谷氨酸的含量,电镜观察超微结构改变。结果：THA组随培养时间的延长谷氨酸含量增高,α运动神经元数目减少,超微结构损伤明显,而脊髓背角的中间神经元的数目无显著变化,超微结构损伤相对较轻。结论：谷氨酸转运异常可以诱导选择性运动神经元损伤。     

大鼠脊髓损伤后不同时间自噬相关蛋白beclin-1和神经元超微结构的变化

目的:观察大鼠脊髓腰段全横断损伤后beclin-1及脊髓神经元超微结构的变化。方法:雄性Wistar大鼠随机分成损伤后6 h,1、3、7、14 d和21 d及假手术组共7组。脊髓损伤组用手术刀片横切腰段。电镜观察脊髓损伤以下神经元内超微结构形态学变化,免疫印迹检测beclin-1蛋白的表达。结果:脊髓损伤早期出现自噬体和自噬溶酶体,神经元内细胞器以退行性变化为主,随着时间延长,神经元超微结构出现明显改变;脊髓损伤后6 h,1、3、7、14 d和21 d,与假手术组相比,大鼠脊髓组织中beclin-1蛋白明显增加,差异有统计学意义;脊髓损伤后3 d beclin-1蛋白表达最高,7 d开始降低,21 d降到最低。结论:脊髓腰段全横断损伤后神经元自噬活动增强,beclin-1可反映神经元自噬状态的变化。     

生川乌与不同比例生半夏配伍对大鼠肝细胞超微结构的影响

目的:观察生川乌与不同比例生半夏配伍对大鼠肝细胞超微结构的影响。方法:SD大鼠随机分为正常对照组、生川乌组、生川乌配生半夏组。电镜下观察肝细胞超微结构变化。结果:生川乌组肝细胞形态不规则,胞质溶解,粗面内质网明显减少,线粒体肿胀,溶酶体增多;生川乌配生半夏(1:0.25)组,肝细胞肿胀,核不规则,异染色质增多,胞质溶解减轻,粗面内质网较生川乌组增多,线粒体肿胀,溶酶体增多;生川乌配生半夏(1:0.5)组,肝细胞肿胀减轻,核异染色质较少、边集,粗面内质网增多,线粒体肿胀,次级溶酶体明显增多;生川乌配生半夏(1:1)组,肝细胞肿胀明显减轻,核接近正常,粗面内质网增多,线粒体肿胀减轻,糖原较少,溶酶体减少。结论:生川乌可明显损伤大鼠肝超微结构,与生半夏配伍后可减轻其损伤作用,配伍比例与损伤程度呈反比。     

红藻氨酸处理对星形胶质细胞超微结构影响的研究———原子力显微镜及透射电镜观察

为了观察体外培养的大鼠海马星形胶质细胞(ASTs)在红藻氨酸(KA)作用下的超微结构变化,我们体外培养大鼠乳鼠海马ASTs,在KA不同浓度(25μmol/L、250μmol/L)及时间(10min、100min)处理条件下,采用原子力显微镜(AFM)扫描细胞表面超微结构改变,透射电镜(TEM)观察细胞内部超微结构变化。结果表明,与对照组相比,低浓度KA(25μmol/L)处理组的细胞表面超微结构无明显改变,但胞内出现空泡样变,溶酶体增多;高浓度KA(250μmol/L)处理组的细胞表面有“孔洞”、“裂隙”样改变,直径在数百纳米,这些表现在KA长时间处理组(100min)更加明显;TEM下可见溶酶体增多、胞核扭曲,在KA长时间处理组甚至出现了自噬小体。以上结果说明培养的ASTs经KA处理后能发生一些损伤性的超微形态结构变化,且具有剂量及时间依赖性,而AFM观察到的KA处理使细胞膜出现“孔洞”、“裂隙”样变化可能是不可逆性兴奋性神经毒性损伤的结构基础。     

丹那唑对体外培养的原位与异位子宫内膜细胞生长的影响

应用胶原凝胶细胞培养和传代培养的方法培养原位与异位子宫内膜细胞，并经丹那唑处理在无丹那唑对照培养中，细胞生长良好，形态呈星形，以胞浆突起相互连接。而经两种浓度丹那唑处理后，细胞生长受抑，传代后２４ｈ细胞贴壁力受损，部分细胞呈致死性病理改变，丹那唑作用的超微结构变化包括胞浆内大量空泡与脂滴，溶酶体增多，线粒体肿胀与细胞核不规则，核膜内陷，提示胶凝胶培养系统为较佳的培养子宫内膜细胞的方法，丹那唑可直接     

脊髓半横断损伤后腹角神经元神经生长因子、脑源性神经营养因子、神经营养因子-3和神经营养因子-4表达的变化

目的:探讨大鼠脊髓半横断损伤(htSCI)后脑源性神经营养因子(BDNF)、神经生长因子(NGF)、神经营养因子(NT-3、NT-4)在脊髓腹角神经元表达的早期变化。方法:免疫组织化学ABC法分别染4种神经因子并作阳性细胞计数。结果:NGF主要分布于脊髓腹角神经元的胞核,BDNF、NT-4与NT-3主要分布于胞浆。htSCI前后它们在细胞内的分布范围没有变化。BDNF、NGF与NT-3的3 d在损伤尾侧段脊髓双侧腹角阳性神经元数与对照组相比显著减少。BDNF与NGF的14 d的双侧腹角阳性神经元数量均较正常组明显增多,NT-3与NT-4的14 d~21 d的双侧腹角阳性神经元数量均较正常组明显增多,BDNF7~21 d以及NGF14 d的健侧的阳性神经元数量均分别多于相应的伤侧。结论:内源性BDNF、NGF、NT-3、NT-4增加对脊髓损伤修复具有重要作用,BDNF和NGF在健侧表达的增加说明健侧代偿功能的活跃。     

挤压伤后脊髓神经元NGF表达的变化

本研究运用免疫组织化学 ABC法和灰度值测定探讨脊髓挤压伤后 NGF在脊髓腹角和背角神经元表达的早期变化。结果显示 :NGF主要分布于脊髓灰质神经元的胞核及胞浆 ;腹角阳性神经元数量在损伤后 2 1d组较正常组明显增多 (P<0 .0 1) ,而背角的阳性神经元在损伤后 2 4h、7d及 2 1d组均较正常组明显增多 (P<0 .0 1)。腹角和背角神经元的阳性灰度值在挤压伤后各组均较正常组显著降低 (P<0 .0 1)。提示 ,内源性 NGF增加对脊髓损伤修复具有重要作用     

A three-dimensional observation of acid phosphatase-positive structures in rat testis by high voltage electron microscopy.

Using a fixative of low concentrated glutaraldehyde in PIPES, we examined three-dimensional structures of acid phosphatase (ACPase) activity in the cells of testes of normal adult and 30 min heat-treated rats by high voltage electron microscopy. There were less ACPase-positive structures in germ cells, but more in Sertoli cells, particularly in residual bodies, and also in macrophages lying between seminiferous tubules. Reaction products of ACPase were distributed in the trans-most cisternae of Golgi apparatus, many spherical lysosomes, and acrosomes. The elongated thread-like lysosomes (nematolysosomes) were absent on the testicular cells. After heat-treatment of rat testis, though ACPase-positive lysosomes increased in number, nematolysosomes were never seen in Sertoli cells and most of the other testicular cells; a number of rod-like lysosomes, however, emerged in macrophages. The results indicate that (1) Sertoli cells usually have more ACPase-positive lysosomal contents than germ cells; (2) Sertoli cells are less affected by stress which may largely hurt germ cells; (3) nematolysosome is not a ubiquitous structure in all the types of cells; and (4) the function of nematolysosomes in macrophages may involve in the disposition of disintegrated cells in rat testis.     

豚鼠脊髓腹角神经元线状溶酶体酶细胞化学研究及其电子探针X射线能谱分析

为探讨豚鼠脊髓腹角神经元是否存在线状溶酶体及其酶细胞化学活性分布特点，用偏磷酸酶（Ｍｅｔａｐｈｏｓｐｈａｔａｓｅ，ＭＰａｓｅ）和酸性磷酸酶（Ａｃｉｄｐｈｏｓｐｈａｔａｓｅ，ＡＣＰａｓｅ）电镜细胞化学方法和电子探针Ｘ射线能谱分析技术，证实豚鼠脊髓腹角神经元存在线状溶酶体（Ｎｅｍａｔｏｌｙｓｏｓｏｍｅ，ＮＬＹ），同时于原位测定ＮＬＹ内的铅含量以反映酶活性强弱。ＭＰａｓｅ和ＡＣＰａｓｅ反应产物分布于圆形溶酶体和ＮＬＹ，同时在高尔基复合体的部分扁囊也有酶活性，表明该酶是在高尔基复合体上加工后输送至溶酶体。在神经元胞体、突起及突触前成分中均有呈ＭＰａｓｅ阳性和ＡＣＰａｓｅ阳性的ＮＬＹ的分布，并和线粒体紧密相贴，提示酶是由线粒体提供能量的ＮＬＹ从胞体输送到神经终末，可能参与神经递质的降解及神经元代谢物质的处理。电子探针Ｘ射线能谱分析测定结果，ＭＰａｓｅ活性强于ＡＣＰａｓｅ活性，提示ＭＰａｓｅ是比ＡＣ－Ｐａｓｅ更为敏感的溶酶体标志酶     

Localization of Alkaline Phosphatase and Cathepsin D during Cell Restoration after Colchicine Treatment in Primary Cultures of Fetal Rat Hepatocytes

Localization of alkaline phosphatase (ALP) and cathepsin D (CAPD) in primary cultures of fetal rat hepatocytes was examined using double immunofluorescent staining in order to investigate the relationship between lysosome movement and the fate of ALP during cell restoration after microtubule disruption by colchicine. At 3 hr and 24 hr after colchicine treatment, numerous coarse dots containing ALP were observed throughout the cytoplasm, and some of these showed colocalization with CAPD. At 48 hr and 72 hr after colchicine treatment, although most of the dots containing ALP in the cytoplasm disappeared, dots containing CAPD remained. The present results suggest that the denatured ALP proteins remaining in the cytoplasm of hepatocytes during cell restoration after colchicine treatment are digested by lysosomes.     

Morphological manifestations of secretory activity of the neutrophils in inflammation]

Neutropholic leukocytes of aseptic peritoneal exudate of rabbits differed from neutrophilic leukocytes of the blood in greater variability in the content of cationic proteins. Some lysosomes ceased to be stained on cationic proteins, which produced a false impression of degranulation of leukocytes. Ultramicroscopically, there was observed a "clearing" of the lysosomes content and its outflow into cytoplasm. Lysosomes acquired the shape of spherical "holed" bodies and were transformed into clear vacuoles with remnants of the electron-dense material. The rupture of lysosome membranes led to an "explosive" type of liberation of the lysosome content into the cytoplasm. Partly degranulated leukocytes remained vital and activity moved about in a homologous serum. Following contacts with histone F1 leukocytes lost cationic proteins. The liberation of cationic proteins from leukocytic took place without destruction of leukocytes and lysosomes themselves.     

Microtubule-associated protein 2 and tubulin are differently distributed in the dendrites of developing neurons

We have followed the appearance of two microtubule proteins, tubulin and microtubule-associated protein 2, in rat hippocampal neurons differentiating in cell culture. Double-label immunofluorescence staining showed that from day 1 in vitro onward tubulin appeared as filaments but that microtubule-associated protein 2 remained distributed throughout the cytoplasm. This difference persisted throughout development and was also detectable in cells that had reached morphological maturity. When cells were treated with the microtubule-depolymerizing agent nocodazole, the depolymerized tubulin became spread throughout the cytoplasm so that its distribution was then identical to microtubule associated protein 2. At the same time, multiple side branches began to emerge along the dendrites. When cells which had been exposed to nocodazole were allowed to recover before staining, the tubulin was again present as filaments but the microtubule-associated protein 2 remained distributed throughout the dendritic cytoplasm. Under these conditions the previously extended proximal side branches were resorbed into the main process. These results suggest that cellular microtubule-associated protein 2 is not necessarily exclusively associated with microtubules. Neuronal dendrites in particular appear to contain this protein at levels in excess of the capacity of microtubular microtubule-associated protein 2 binding sites. In view of the known effectiveness of microtubule-associated protein 2 as a promoter of tubulin polymerization, its abundance in dendrites suggests that it acts to ensure total polymerization of dendritic microtubules. In this way it would contribute both to the support of the growing process and the suppression of adventitious sidebranching.     

细胞自噬的形态学特征和功能意义

目的 自噬是细胞受到刺激后吞噬自身的细胞质或细胞器,最终将吞噬物在溶酶体内降解的过程.按吞噬物进入溶酶体的途径,自噬可分为巨自噬、微自噬和分子伴侣介导的自噬3类.在巨自噬,自噬前体包裹细胞质或细胞器后形成自噬体,继而自噬体与溶酶体结合形成自噬溶酶体,自噬体内容物被降解.在微自噬,溶酶体膜凹陷,直接吞噬细胞质、细胞器或细胞核,形成自噬体,然后被溶酶体酶降解.分子伴侣介导的自噬是通过溶酶体膜的受体将细胞质内的蛋白质转运入溶酶体.自噬从酵母至哺乳动物细胞均很保守,对于耐受饥饿和缺血,清除衰老细胞器,清除细菌和异物,维持细胞活性和延长寿命等起着重要作用.自噬活动受自噬基因的调控,自噬基因缺失或功能障碍时可导致某些疾病的发生.深入认识自噬过程以及由此产生的自噬体等结构及其功能有助于探讨自噬对于人体生理和病理作用的机制.本文综述了自噬的形态学特征及其功能意义.     

胶质细胞源性神经营养因子体外对脊髓运动神经元的作用

目的 :观察不同浓度的胶质细胞源性神经营养因子 (GDNF) ,对大鼠胚胎脊髓运动神经元生长活性的作用。方法 :取大鼠胚胎脊髓腹侧组织体外分离 ,进行原代细胞培养 ,应用抗神经微丝单克隆抗体 (mAb)SMI32进行运动神经元的免疫细胞化学染色 ,从细胞形态学及应用MTT比色法 ,研究GDNF对大鼠脊髓运动神经元的影响。结果 :GDNF能明显促进体外培养的大鼠脊髓运动神经元存活及突起的生长 (P <0 .0 5 ) ,且具有剂量依赖的趋势。结论 :不同浓度的GDNF对体外培养的大鼠胚胎脊髓运动神经元 ,有不同程度的促生长作用