肿瘤坏死因子α对体外培养视神经星形胶质细胞的促增殖作用

目的：观察肿瘤坏死因子α对体外培养的视神经星形胶质细胞的促增殖作用，为进一步分析星形胶质细胞的功能奠定实验基础。 方法：实验于2005-06／10在贵阳中医学院第二附属医院眼科和贵阳医学院解剖学教研室完成。将出生24h以内的新生小鼠断颈处死后，采用机械法分离视神经管内段的视神经，进行视神经星形胶质细胞的原代和传代培养及纯化，倒置相差显微镜观察培养细胞的生长过程和形态学变化。取第3，5，6代以后的细胞接种于24孔培养板中，贴壁后取出分别进行免疫荧光化学染色和免疫细胞化学染色，检测细胞胶质纤维酸性蛋白及S-100阳性产物情况。在第6代培养细胞中添加肿瘤坏死因子α，四唑盐比色法观察肿瘤坏死因子α对胶质细胞增殖的影响。 结果：①原代及传代培养的细胞形态光学倒置相差显微镜观察结果：经机械分离培养的原代细胞24h开始贴壁，贴壁细胞形态以梭形、不规则形为主。经消化传代去除悬浮的死亡细胞，培养至第4代以后细胞形态基本一致，细胞体积大，呈星形、梭形及不规则形，突起少，核相对较大，贴壁细胞呈片状生长。②培养的视神经星形胶质细胞胶质纤维酸性蛋白及S-100阳性产物免疫组织化学检测结果：免疫荧光染色及免疫细胞化学染色显示，胶质纤维酸性蛋白及S-100呈阳性，髓磷脂碱性蛋白免疫细胞化学染色阳性率小于1％，未加一抗的呈阴性。第6代以后细胞阳性率达97％以上。③细胞增殖活性四唑盐比色法检测结果：随着肿瘤坏死因子α的浓度加大，胶质细胞表现出增殖亢进现象，以30m表现最为显著，亢进效果明显强于未添加肿瘤坏死因子α的培养细胞（0．197&;#177;0．002，0．276&;#177;0．005，P〈0．001）。 结论：机械分离纯化新生小鼠视神经星形胶质细胞纯度高，肿瘤坏死因子α能够促进星形胶质细胞增殖，且呈现出明显的剂量依赖性效应，可在短期内获取大量的细胞，是进一步分析神经胶质细胞结构和功能的可靠材料。     

次声作用后大鼠海马星形胶质细胞的变化

目的 研究8Hz,90dB和130dB次声作用后不同时间点，大鼠海马中胶质原纤维酸性蛋白（GFAP）表达的变化。方法 Hz,90dB和130dB次声作用于大鼠，2h/d，分别作用于1，7，14，21，28d后采用免疫组织化学方法，观察大鼠海马星形胶质细胞GFAP表达的时程改变。结果 130dB组从作用1d开始大鼠海马中GFAP阳性星形胶质细胞数量开始增加，14d达到高潮，21d开始下降，但仍然维持较高水平90dB组大鼠各时间点GFAP反应均比130dB组弱。结论 8Hz,90dB和130dB次声作用可以激活海马大量星形胶质细胞，对神经元具有保护作用。     

大鼠高眼压后筛板区星形胶质细胞的免疫荧光组织化学变化

目的：观察大鼠青光眼和正常眼视乳头筛板区星形胶质细胞标志物神经胶质纤维酸性蛋白和Nogo-C的表达。方法：实验于2004—03／07—21在第三军医大学大坪医院野战外科研究所眼科和第三军医大学大坪医院野战外科研究所第三研究室完成。步骤：①用532-二极管激光对16只正常SD大鼠的右眼行跨越角膜缘小梁网激光光凝。②Tonopen眼压计测量双眼眼压变化。⑧应用免疫荧光组织化学-激光共聚焦显微镜观察星形胶质细胞标志物神经胶质纤维酸性蛋白和Nogo-C的表达变化。结果：大鼠模型组16眼和非模型组16眼均进入结果分析。①大鼠模型组、非模型组眼压情况：模型组激光光凝之后的眼压呈上升趋势，第3天显著高于非模型组[（3．12&;#177;0．01），（2．29&;#177;0．03）kPa，P〈0．01]，一直持续60d均显著高于非模型组[（3．29&;#177;0．03），（2．30&;#177;0．02）kPa，P〈0．01]。②大鼠模型组、非模型组眼球标本免疫荧光组织化学染色结果：大鼠高眼压后60d，视乳头筛板区星形胶质细胞标志物神经胶质纤维酸性蛋白和Nogo-C表达强、密度高，模型组星形胶质细胞标志物神经胶质纤维酸性蛋白红色荧光、Nogo-C绿色荧光以及红色和绿色叠加后的黄色荧光（灰度值）显著高于非模型组（19．13&;#177;0．12，17．82&;#177;0．23，18．30&;#177;0．14比3．11&;#177;0．15，6．52&;#177;0．16，4．16&;#177;0．20，P〈0．01）。结论：星形胶质细胞可能参与了青光眼神经纤维的进一步损害和抑制了神经纤维轴索再生。     

癫痫发作1h后延髓内脏带区域内反应神经元与星形胶质细胞功能单位的分布特征：激光共聚焦显微镜研究

背景：传统观点认为癫痫是大脑不同区域内神经元出现异常兴奋引起的复杂神经行为紊乱现象。而对星形胶质细胞在癫痫发病中的作用目前研究较少。目的：研究戊四氮诱导大鼠癫痫发作后延髓内脏带内神经元和星形胶质细胞的反应。设计：随机对照的实验研究。地点和材料：实验在南方医科大学珠江医院神经外科实验室和解放军第四军医大学全军神经科学研究所完成。成年健康SD大鼠14只，体质量180-220g，清洁级，由解放军第四军医大学实验动物中心提供。干预：用抗Fos蛋白、抗酪氨酸羟化酶和抗胶质原纤维酸性蛋白的三重免疫荧光组织化学方法结合激光共聚焦显微镜技术，显示癫痫发作1h后延髓内脏带内反应性神经元与星形胶质细胞的分布。主要观察指标：对延髓内脏带内Fos，胶质原纤维酸性蛋白和抗酪氨酸羟化酶阳性细胞的分布及胶质原纤维酸性蛋白阳性星形胶质细胞与神经元的关系进行观察。结果：延髓内脏带内的Fos阳性神经元和胶质原纤维酸性蛋白阳性星形胶质细胞明显增多，三重免疫组化方法显示反应性神经元(Fos阳性)与反应性星形胶质细胞(胶质原纤维酸性蛋白阳性)关系密切，发现3种不同标记的“神经元-星形胶质细胞复合体”：即抗酪氨酸羟化酶 ／Fos ／胶质原纤维酸性蛋白 三标记复合体、抗酪氨酸羟化酶 ／胶质原纤维酸性蛋白 ／Fos-和Fos ／胶质原纤维酸性蛋白 ／抗酪氨酸羟化酶-二标记复合体。结论：延髓内脏带内的神经元和星形胶质细胞在癫痫发作时反应强烈，可能以神经元一星形胶质细胞复合体作为功能单位参与癫痫发病的调节。     

糖尿病早期大鼠海马和皮质星形胶质细胞的变化

目的：观察链脲佐菌素(streplozotocin，STZ)诱导的糖尿病大鼠早期(6周)海马和皮质星形胶质细胞的变化，探讨星形胶质细胞在糖尿病脑病发病中的重要作用。方法：实验在解放军第四军医大学航空航天医学系病理学实验室完成。在建模前和建模后6周时检测糖尿病大鼠的血糖、尿糖和体质量，应用免疫组织化学方法观察糖尿病组和对照组大鼠海马和皮质星形胶质细胞胶质原纤维酸性蛋白(glial fibrillary acidic protein，GFAP)的表达情况。结果：对照组大鼠体质量随周龄增加而明显增加；糖尿病组大鼠体质量随周龄的增加不明显。对照组大鼠的血糖始终处于大鼠血糖值正常范围，尿糖检测始终都为阴性，而糖尿病组大鼠的血糖值处于模型要求的标准之上。尿糖自建模后都呈强阳性。GFAP免疫组化标记可见糖尿病大鼠海马和皮质星形胶质细胞明显增生，且增生细胞在CA1区和CA4区最为明显。结论：糖尿病大鼠早期星形胶质细胞增生反映了神经组织对机体糖代谢紊乱的适应性重塑过程，提示神经组织改变在糖尿病脑病的发病中可能起了重要作用。     

大鼠高眼压后筛板区星形胶质细胞的免疫荧光组织化学变化

目的:观察大鼠青光眼和正常眼视乳头筛板区星形胶质细胞标志物神经胶质纤维酸性蛋白和Nogo-C的表达。方法:实验于2004-03/07-21在第三军医大学大坪医院野战外科研究所眼科和第三军医大学大坪医院野战外科研究所第三研究室完成。步骤:①用532-二极管激光对16只正常SD大鼠的右眼行跨越角膜缘小梁网激光光凝。②Tonopen眼压计测量双眼眼压变化。③应用免疫荧光组织化学-激光共聚焦显微镜观察星形胶质细胞标志物神经胶质纤维酸性蛋白和Nogo-C的表达变化。结果:大鼠模型组16眼和非模型组16眼均进入结果分析。①大鼠模型组、非模型组眼压情况:模型组激光光凝之后的眼压呈上升趋势,第3天显著高于非模型组[(3.12±0.01),(2.29±0.03)kPa,P<0.01],一直持续60d均显著高于非模型组[(3.29±0.03),(2.30±0.02)kPa,P<0.01]。②大鼠模型组、非模型组眼球标本免疫荧光组织化学染色结果:大鼠高眼压后60d,视乳头筛板区星形胶质细胞标志物神经胶质纤维酸性蛋白和Nogo-C表达强、密度高,模型组星形胶质细胞标志物神经胶质纤维酸性蛋白红色荧光、Nogo-C绿色荧光以及红色和绿色叠加后的黄色荧光(灰度值)显著高于非模型组(19.13±0.12,17.82±0.23,18.30±0.14比3.11±0.15,6.52±0.16,4.16±0.20,P<0.01)。结论:星形胶质细胞可能参与了青光眼神经纤维的进一步损害和抑制了神经纤维轴索再生。     

大鼠脑皮质未成熟星形胶质细胞的分离、鉴定及原代培养

目的探讨大鼠脑皮质未成熟星形胶质细胞的分离、鉴定及原代培养技术。方法采用差速贴壁法清除成纤维细胞及酶消化去除少突胶质细胞污染,应用免疫荧光进行细胞鉴定。取不同时间点的细胞在倒置显微镜下观察细胞形态变化,双重免疫荧光染色观察细胞生长过程的分化状态。结果细胞形态观察提示:6~14 d细胞呈均匀扁平多角状,16 d后部分细胞肥大增生,呈成熟状态。免疫组化染色显示:GFAP(+)细胞达97%。免疫荧光双重标记发现:16 d后细胞表达vimentin和nestin这两种蛋白减弱。结论本实验技术可获得纯度高,形态和功能良好,表达稳定的大鼠脑皮质未成熟星形胶细胞。     

大鼠在空间学习记忆时脑海马区内星形胶质细胞纤维酸性蛋白的表达

目的：利用水迷宫试验观察大鼠内海马内星形胶质细胞胶质纤维酸性蛋白的表达在空间辨别性学习记忆时的变化。方法：实验于2003-12／2004-07在暨南大学医学院人体解剖学教研室实验室完成。雄性SD大鼠30只，随机分为对照组、模型5d组和模型15d组，每组10只。采用Morris水迷宫训练，对模型5d组和模型15d组大鼠以定位航行试验建立空间辨别性学习记忆模型，对照组大鼠在无平台的迷宫内自由游泳，2次／d，2min／次，持续5d。各组大鼠停止训练后12h，取脑，冠状连续切片。免疫组织化学S-P法染色以胶质纤维酸性蛋白标记星形胶质细胞。Tiger 2000图象分析系统对胶质纤维酸性蛋白的表达进行定量分析。结果：建立了空间辨别性学习记忆模型后，大鼠海马内胶质纤维酸性蛋白的表达模型5d组(CAl区：330．83&;#177;36．85；CA3区：333．3l&;#177;30．37；齿状回区：326、20&;#177;36．75)和模型15d组(CAl区：398．33&;#177;38．47；CA3区：404．99&;#177;48．9l；齿状回区：423．55&;#177;40．50)高于对照组(CAl区：297．69&;#177;27．22；CA3区：296．98&;#177;31．13；齿状回区：163．50&;#177;33．98)．差异有显著性意义(t=2．87．5．37，P均&;lt;0．01)。结论：星形胶质细胞参与空间辨别性学习记忆过程并与记忆的进一步巩固有关。     

次声作用对小鼠脑中胶质纤维酸性蛋白表达和分布的影响

目的　观察次声作用后小鼠脑中胶质纤维酸性蛋白（GFAP）的表达和分布。方法　将BALB/C小鼠暴露于16Hz、声压130dB次声,2h/次     

糖尿病早期大鼠海马和皮质星形胶质细胞的变化

目的:观察链脲佐菌素(streptozotocin,STZ)诱导的糖尿病大鼠早期(6周)海马和皮质星形胶质细胞的变化,探讨星形胶质细胞在糖尿病脑病发病中的重要作用。方法:实验在解放军第四军医大学航空航天医学系病理学实验室完成。在建模前和建模后6周时检测糖尿病大鼠的血糖、尿糖和体质量,应用免疫组织化学方法观察糖尿病组和对照组大鼠海马和皮质星形胶质细胞胶质原纤维酸性蛋白(glialfibrillaryacidicprotein,GFAP)的表达情况。结果:对照组大鼠体质量随周龄增加而明显增加;糖尿病组大鼠体质量随周龄的增加不明显。对照组大鼠的血糖始终处于大鼠血糖值正常范围,尿糖检测始终都为阴性,而糖尿病组大鼠的血糖值处于模型要求的标准之上。尿糖自建模后都呈强阳性。GFAP免疫组化标记可见糖尿病大鼠海马和皮质星形胶质细胞明显增生,且增生细胞在CA1区和CA4区最为明显。结论:糖尿病大鼠早期星形胶质细胞增生反映了神经组织对机体糖代谢紊乱的适应性重塑过程,提示神经组织改变在糖尿病脑病的发病中可能起了重要作用。     

Binding and biological effects of tumor necrosis factor alpha on cultured human neonatal foreskin keratinocytes

Tumor necrosis factor alpha (TNF alpha) localizes to the epidermis when injected in vivo, but its role in the skin has heretofore not been evaluated. As a first approach to assessing the role of TNF alpha in the skin, we evaluated the binding and biological effects of TNF alpha on human neonatal foreskin keratinocytes maintained in culture. We found that TNF alpha at 0.3-1.0 nM inhibited proliferation of keratinocytes in a reversible fashion as demonstrated by a reduction in total DNA content and clonal growth. The antiproliferative effects were most marked when TNF alpha was added in the preconfluent stages of cell growth. Accompanying this antiproliferative effect was a stimulation by TNF alpha of differentiation of keratinocytes as indicated by the stimulation of cornified envelope formation. Keratinocytes specifically bound TNF alpha, reaching maximal binding in 2 h at 34 degrees C or 8 h at 4 degrees C. Much of the apparent binding at 34 degrees C was due to internalization of the TNF alpha. At 4 degrees C the rate of internalization was much less. Confluent keratinocytes showed a single class of high-affinity receptors with 1,250 receptors/cell and a Kd of 0.28 nM. These data suggest a role for TNF alpha in the growth and differentiation of the epidermis.     

Tumor necrosis factor alpha induces adhesion molecule expression on human fetal astrocytes

Leukocyte adhesion molecules on endothelial cells of the blood-brain barrier may participate in the entry of leukocytes into the central nervous system. Because astrocytes are also a component of the blood-brain barrier and have been associated with inflammation, we studied the ability of astrocytes to express leukocyte adhesion molecules using Northern blot and immunocytochemical techniques. Astrocytes treated with the proinflammatory cytokine tumor necrosis factor alpha (TNF) expressed messenger RNA for the adhesion molecules E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1, as well as their corresponding proteins. In addition, TNF-treated astrocytes expressed a monocyte adhesion protein identified by our laboratory, recognized by the monoclonal antibody IG9. These results indicate that under inflammatory conditions in the central nervous system, such as multiple sclerosis and acquired immune deficiency syndrome, astrocyte expression of adhesion molecules may facilitate the migration of leukocytes and contribute to the disease process.     

Adeno-associated virus production of soluble tumor necrosis factor receptor neutralizes tumor necrosis factor alpha and reduces arthritis

The major limitation of adenovirus is its association with induction of an inflammatory response and relatively short-term production of the gene therapy transgene product. Adeno-associated virus (AAV) is a 4.68-kb single-strand DNA virus that contains ITRs for viral replication and a packaging signal, and also has been engineered to contain therapeutic genes up to 5 kb in length. Transduction of recombinant AAV (rAAV) results in low inflammatory response and long-term expression. We have cloned a low-immunogenic form of human sTNFRI (sTNFRI2.6D) into AAV (rAAVsTNFRI). This vector was analyzed for its ability to transfect and neutralize the effect of TNF-alpha on primary rheumatoid arthritis synovial fibroblast (RASFs). The rAAVsTNFRI was transduced into the cells at 1.8 x 10(1), 1.8 x 10(2), and 1.8 x 10(3) viral particles per cell. There was greater than 90% neutralization of TNF-alpha at 1.8 x 10(3) viral particles/cell. There was a significant decrease in the synovial cell hyperplasia and cartilage and bone destruction in human TNF-alpha transgenic mice treated intraarticularly with rAAVsTNFRI. These results indicate that the low-immunogenic and long-term expressing vector, rAAVsTNFRI, can be used to deliver the soluble TNF-alpha in vitro and in vivo and effectively reduce the severity of arthritis.     

Protein kinase regulates tumor necrosis factor mRNA stability in virus- stimulated astrocytes

Infection of astrocytes with Newcastle disease virus stimulated the production of 1,2-diacylglycerol, and resulted in the kinase-dependent expression of mRNAs encoding tumor necrosis factor (TNF), interferon alpha and beta, and interleukin 6. The half-life of TNF mRNA was significantly decreased in the presence of protein kinase inhibitors H-7 and staurosporine, but not in the presence of HA1004. In contrast to the decay of TNF mRNA, the half-lives of other cytokine mRNAs were only minimally affected by the kinase inhibitors. These data indicated that the stability of TNF mRNA was regulated through a novel, kinase-dependent pathway.     

Effect of tumor necrosis factor alpha on mitogen-activated human B cells

In this study we demonstrate that the monocyte/macrophage product, tumor necrosis factor alpha (TNF-alpha), has significant in vitro effects of B cell function. It costimulated with anti-mu in the induction of B cell DNA synthesis, and it prolonged the DNA synthesis initiated in B cell cultures stimulated with the human B cell mitogen, Staphylococcus aureus Cowan strain I (SAC). The addition of either IL-1 or IFN-gamma to TNF-alpha resulted in a substantial further increase in DNA synthesis. The addition of TNF-alpha to IL-2, a known inducer of SAC-activated B cell Ig secretion, resulted in a twofold enhancement in the amount of IL-2 stimulated B cell Ig secretion. Receptor binding studies with 125I-TNF-alpha demonstrate a marked increase in TNF-alpha binding sites after B cell activation (approximately 6,000 sites per cell, with an apparent Kd of 2.0 X 10(-10) M). Thus, TNF-alpha may be an important factor in human B cell function and is likely to interact with other T cell and monocyte derived cytokines in the regulation of human B cell proliferation and Ig production.     

Study on the correlation between tumor necrosis factor and nerve root pain

Objective:To study the correlation between TNF and nerve root pain,and try to find out its significance.Methods 45 cases who had cervical syndrome,lumbar disc protrusion and lumbar spine stenosis were studied.TNF in blood serum was determined with subsequence saturation liquid phase competitiver radio-immunity,and the results were further statistically processed.Results:TNF value in 45 cases was highr than normal value(P&lt;0.01).T-check-out was made between pain degree.we gained P&lt;0.01.Conclusion There is notable correlation between TNF and nerve root pain,which is benifit to guide clinial diagnosis and treatment.     

Lipopolysaccharide-induced selective priming effects on tumor necrosis factor alpha and nitric oxide production in mouse peritoneal macrophages

Preculture of thioglycollate-elicited C3HeB/FeJ mouse peritoneal macrophages in vitro with subthreshold stimulatory concentrations of lipopolysaccharide (LPS) can induce hyporesponsiveness (desensitization) to both tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) production when these cells are subsequently stimulated with 100 ng/ml of LPS. We have established, however, that the primary dose of LPS required for inducing downregulation of NO production is significantly lower than that required for inducing downregulation of TNF-alpha production. Further, when LPS-pretreated macrophages become refractory to subsequent LPS stimulation for NO production, the secondary LPS-stimulated TNF-alpha production is markedly enhanced, and vice versa. These results indicate that LPS- induced TNF-alpha and NO production by macrophages are differentially regulated, and that the observed desensitization process may not reflect a state in which macrophages are totally refractory to subsequent LPS stimulation. Rather, our data suggest that LPS- pretreated macrophages become selectively primed for differential responses to LPS. The LPS-induced selective priming effects are not restricted to LPS stimulation, but extend as well to stimuli such as zymosan, Staphylococcus aureus, and heat-killed Listeria monocytogenes.