轴突生长抑制性蛋白

轴突生长抑制性蛋白是阻止成年哺乳动物中枢神经再生的重要因素。近年来一些重要的抑制性蛋白陆续得到鉴定和克隆。本文概述轴突生长抑制性蛋白的发现过程,着重介绍勿动蛋白（Nogo）,髓磷脂相关性糖蛋白（MAG）以及崩溃蛋白（Collapsin）的结构和功能,同时介绍有关轴突生长抑制性蛋白的受体和信号转导等方面的研究进展。     

淫羊藿苷抑制中枢神经髓磷脂抑制因子的表达：在局灶性脑缺血大鼠中的研究

摘要 研究背景：研究表明,成年哺乳动物的中枢神经损伤后勿动蛋白、髓磷脂相关糖蛋白、少突胶质细胞髓磷脂糖蛋白等髓磷脂抑制因子的产生是轴突再生的主要障碍。淫羊藿苷是中草药淫羊藿的主要活性成分,对脑缺血有较好的治疗作用,但对髓磷脂抑制因子的影响还不清楚。 研究目的：观察淫羊藿苷对由大脑中动脉闭塞模型大鼠脑缺血区勿动蛋白、髓磷脂相关糖蛋白、少突胶质细胞髓磷脂糖蛋白表达的影响。 设计、时间、地点：本项目采用随机对照的神经化学研究,于2008年1月至6月在山东中医药大学大学药学院和中心实验室完成。 材料：淫羊藿苷是从中草药淫羊藿干燥的切片中提取的有效成分,由山东中医药大学药学院提供。Nogo-A、MAG购买于武汉博士德生物工程有限公司。Omgp购买于美国ACCURATE公司。高清晰图像分析系统（HPIAS-1000）购买于武汉千屏影像技术有限责任公司。 方法：30只SPF级雄性SD大鼠随机分为假手术组、模型组和药物组,每组10只。假手术组合模型组以10mL/Kg的剂量给予生理盐水;药物组以20 mg/kg的剂量给予淫羊藿苷。各组均于造模24小时后灌胃给药,每天1次,连续14天。 主要检测结果：在最后给药后进行大鼠神经功能评分,并用免疫组织化学方法分析勿动蛋白、髓磷脂相关糖蛋白、少突胶质细胞髓磷脂糖蛋白在缺血区脑组织中的表达。 结果：药物组大鼠神经功能评分较模型组明显降低。模型组中勿动蛋白、髓磷脂相关糖蛋白、少突胶质细胞髓磷脂糖蛋白在缺血区脑组织中的表达明显高于假手术组。药物组动蛋白、髓磷脂相关糖蛋白、少突胶质细胞髓磷脂糖蛋白在缺血区脑组织中的表达较模型组明显降低。 结论：淫羊藿苷能改善缺血性脑血管病的神经功能恢复,其可能的机制是通过抑制了髓磷脂相关抑制因子的表达,促进了轴突的发芽与再生。     

小儿急笥感染性多发性神经根炎抗髓磷脂碱蛋白抗体的测定…

作者测定了６４例急性多发性神经根炎患儿的血和脑脊液中髓磷脂碱蛋白抗体，并与５０例正常儿童进行了比较。结果表明：患儿的血和脑脊液中该抗体明显高于正常对照组；混合型病例不论是血中还中脑脊液中存在的该抗体明显高于脊神经型病例；患儿血中和脑脊液中存在的该抗体具有明显相关性。提示：该抗体在本病发病中可能占重要地位；抗体的存在与疾病的严重性有关；脑脊液中该抗体可能来源于外周血。由此进一步推测，本病脑脊液中增高     

骨髓间充质干细胞移植治疗对大鼠脑梗死后神经再生抑制因素的影响

目的探讨骨髓间充质干细胞(BMSCs)静脉移植对大鼠脑梗死后Nogo-A、少突胶质细胞髓磷脂糖蛋白(OMgp)和髓磷脂相关糖蛋白(MAG)蛋白的影响。方法实验动物分成假手术组、损伤对照组、溶剂对照组和移植组,各组再分为3d、7d、14d和21d组。全骨髓贴壁法分离培养大鼠BMSCs,线栓法制作大鼠脑梗死模型。移植组自大鼠尾静脉注射BMSCs悬液1ml,溶剂对照组注射磷酸盐缓冲液(PBS)。对各组动物进行神经功能缺损程度评分,免疫组化方法检测Nogo-A、OMgp和MAG的表达水平。结果移植组7、14和21d神经功能恢复优于对照组;移植组术后3、14和21d Nogo-A蛋白表达较对照组降低(P<0.05);移植组7、14和21d OMgp蛋白表达较对照组降低(P<0.05);移植组术后3、7、14和21d MAG蛋白表达较对照组降低(P<0.05)。结论 BMSCs移植可促进大鼠脑梗死后的神经功能恢复,其作用机制与下调Nogo-A、OMgp和MAG的表达有关。     

小儿急性感染性多发性神经根炎抗髓磷脂碱蛋白抗体的测定及意义

作者测定了６４例急性多发性神经根炎患儿的血和脑脊液中髓磷脂碱蛋白抗体，并与５０例正常儿童进行了比较。结果表明：患儿的血和脑脊液中该抗体明显高于正常对照组；混合型病例不论是血中还是脑脊液中存在的该抗体明显高于脊神经型病例；患儿血中和脑脊液中存在的该抗体具有明显相关性。提示：该抗体在本病发病中可能占重要地位；抗体的存在与疾病的严重性有关；脑脊液中该抗体可能来源于外周血。由此进一步推测。本病脑脊液中增高的蛋白质亦可能来源于外周血。     

髓磷脂碱性蛋白的提纯及其对PBMC产生TNF和IFN的影响

目的 探讨髓磷脂碱性蛋白（MBP）诱导EAE产生的可能原因。方法 通过酸抽提和CM－Sephadex50层析,分离提取牛MBP,观察其致AE活性,用生物活性法检测MBP刺激的PMBC上清中TNF和IFN的活性。结果 分离 纯化得到电泳1条主带的MBP,证明其具有致EAE活性,MBP刺激的PBMC可产生TNF和IFN。结论 MBP可通过在外周刺激PBMC产生炎性因子TNF和IFN诱导EAE的产生。     

口服髓磷脂碱性蛋白治疗实验性自身免疫性神经炎的临床及神经病理观察

目的 探讨口服髓磷脂碱性蛋白（MBP）汉字实验性免疫性神经炎（EAN）的效果。方法 应用牛周围神经BMP免疫豚鼠建立EAN动物模型。应用国际分级标准和临床评分对发病豚鼠进行临床评定；对EAN豚鼠坐骨神经进行常规病理、免疫组化、单薄切片的光镜及电镜观察；剥离单神经纤维，评定其脱髓复髓情况。结果 用BMP建立豚鼠EAN模型其发病率为87．5％，免疫诱导的12－16d出现典型瘫痪症状，高峰期在14－21d，以后进入恢复阶段。发病高峰期坐骨神经病理为小血管周围大量的淋巴细胞、巨噬细胞浸润，纤维肿胀、变性，髓鞘脱失；恢复期神经组织内浸润的细胞明显减少，神经纤维肿胀、脱髓的现象明显减轻，施万细胞明显增生。在发病高峰时开始治疗，口服BMP降腹腔注射静用丙种球蛋白（IVIG）治疗均促使豚鼠临床得分提前下降，坐骨神经病理在21d时表现为浸润的淋巴细胞减少，脱髓鞘纤维减少，复髓纤维增多。结论 BMP口服治疗EAN，明显地促进其临床和病理提前恢复，其机制可能是大剂量BMP口服诱导机体免疫耐受的结果。     

抑制神经再生的髓鞘蛋白研究进展

一、中枢神经系统 (centralnervoussystem ,CNS)再生的概述中枢神经系统损伤后的再生修复是十分复杂的病理生理过程 ,涉及从分子、细胞到整体的各个层次的变化。一个成功的再生可分为以下三个阶段 :第一 ,受损神经元及支持细胞的存活 ;第二 ,由存活神经元再生的轴突长过损伤区、到达合适的靶区 ,并重建有功能的突触联系 ;最后 ,新建立的神经环路成功补偿神经系统损伤后的功能缺失。这种轴突再生的能力只存在于周围神经系统及发育期的中枢神经系统 ,成年哺乳动物中枢神经系统损伤后不能成功再生 ,造成的功能缺失也是不可逆的。自从本世纪初…     

下调蛋白磷酸酯酶2A活性可抑制海马神经元轴突生长

目的探讨下调蛋白磷酸酯酶2A(PP2A)活性对胎鼠海马神经元轴突生成的影响。方法选取体外培养原代海马神经元为研究模型;采用PP2A抑制剂OA下调PP2A活性,最后采用荧光双标检测PP2A对神经元轴突的影响。结果对照组海马神经元培养至72h时神经元轴突已经形成,而OA处理后的神经元轴突生长明显受到抑制;OA处理后的神经元轴突长度分别为对照组(DMSO)的47%,单个神经元的平均轴突数目也从正常1.1/neuron下降到0.6/neuron。结论下调PP2A活性能抑制原代海马神经元轴突生成,提示PP2A在海马神经元轴突生成中起重要作用。     

嗅鞘细胞移植对脊髓损伤区勿动蛋白表达的影响

背景：近年来人们认为只要能抑制脊髓损伤后神经再生的不利因素就能促进损伤脊髓的再生，抑制因子包括髓鞘相关抑制分子和胶质瘢痕。其中髓鞘相关抑制分子主要有勿动蛋白、髓磷脂相关糖蛋白及少突起胶质细胞髓鞘相关糖蛋白。 目的：观察嗅鞘细胞移植前后脊髓损伤区勿动蛋白的动态变化。 设计、时间及地点：开放性实验，于2006-09/2007-05在西安交大医学院教育部环境与基因重点实验室完成。 材料：8周龄成年SD大鼠40只，体质量（2.50±0.25）kg，雌雄不拘，随机数字表法分为正常组、模型组、嗅鞘细胞组、DF12对照组，10只/组。另取30只健康成年雄性SD大鼠用于嗅鞘细胞的取材，体质量200~250 g。 方法：除正常组外，其余各组均建立全横切脊髓损伤模型。嗅鞘细胞组将原代培养12 d的嗅鞘细胞悬液调整为1×1011 L-1，在距损伤缘上下各1 mm处分4点应用微量注射器注射，深度1.0 mm，每处各注射1 μL。DF12对照组同法每点注射等量DF12培养液，模型组、正常组不进行任何处理。 主要观察指标：各组分别于移植后1，4，8周采用免疫组织化学技术动态检测脊髓损伤区勿动蛋白的变化。同时在移植后8周行嗜银染色检测组织形态学变化。 结果：①勿动蛋白的变化：正常组勿动蛋白吸光度值明显低于其余3组（P < 0.05）。移植后1，4，8周，嗅鞘细胞组脊髓损伤区勿动蛋白均明显低于模型组和DF12对照组（P < 0.01），而模型组和DF12对照组差异无显著性意义（P > 0.01）。②组织形态学变化：嗅鞘细胞移植8周后，除正常组外，其余各组均可见明显的神经纤维再生，但模型组与DF12对照组大部分纤维排列紊乱，再生纤维方向性较差；嗅鞘细胞组可见明显的新生轴突，且神经纤维跨越损伤部位修复脊髓损伤，无论在数量还是质量上均优于模型组及DF12对照组。 结论：嗅鞘细胞移植可能通过降低脊髓损伤区勿动蛋白水平促进损伤脊髓的修复。     

Neurite formation modulated by nerve growth factor, insulin, and tumor promoter receptors

Until recently, nerve growth factor could be considered the only neurotrophic factor with an established physiological role. We discuss the emerging evidence indicating that the insulinlike factors may constitute a family of related neurotrophic proteins, and the observations suggesting that the receptor for the phorbol ester tumor promoters is closely associated with neuronal differentiation. The emphasis of the discussion is placed on neurite formation under multiple modulation by insulinlike factors, nerve growth factor, and tumor promoter receptors in sensory, sympathetic and human neuroblastoma cells.     

Neurite growth modulation associated with astrocyte proteoglycans: influence of activators of inflammation.

Of the three classes of sulphated proteoglycans produced by type 1 astrocytes in vitro and released into conditioned medium, only heparan sulphate (HS) was associated with enhanced neurite growth by sensory neurons following pretreatment of a laminin substratum. Astrocyte-conditioned medium (ACM) produced in the presence of certain inflammatory mediators had reduced titers of neurite-promoting activity. The low activity ACM contained inhibitors of neurite growth. Heparan sulphate proteoglycans may modulate neurite growth when complexed to constituents of the extracellular milieu either directly or by interacting with other growth-promoting or growth-inhibitory factors.     

Neurite branching on deformable substrates

The mechanical properties of substrates underlying cells can have profound effects on cell structure and function. To examine the effect of substrate deformability on neuronal cell growth, protein-laminated polyacrylamide gels were prepared with differing amounts of bisacrylamide to generate substrates of varying deformability with elastic moduli ranging from 500 to 5500 dyne/cm. Mouse spinal cord primary neuronal cells were plated on the gels and allowed to grow and extend neurites for several weeks in culture. While neurons grew well on the gels, glia, which are normally co-cultured with the neurons, did not survive on these deformable substrates even though the chemical environment was permissive for their growth. Substrate flexibility also had a significant effect on neurite branching. Neurons grown on softer substrates formed more than three times as many branches as those grown on stiffer gels. These results show that mechanical properties of the substrate specifically direct the formation of neurite branches, which are critical for appropriate synaptic connections during development and regeneration.     

Neurite growth on different substrates: permissive versus instructive influences and the role of adhesive strength.

Growing axons use environmental cues to guide them to their targets. One class of cues is thought to be adhesion molecules on cells and in the extracellular matrix that axons interact with as they grow to their targets. In choosing between two possible pathways, the relative adhesiveness of the two substrates could be an important factor in controlling neurite growth. We conducted experiments in vitro to study how naturally occurring adhesion molecules influence neurite growth. Neurite growth rates, the degree of neurite fasciculation, the choices neurites make between two substrates, and the relative adhesiveness of different substrates were examined. We found that the relative adhesiveness of a substrate was a poor predictor of either axon growth rate or the degree of fasciculation. Furthermore, neurites showed little selectivity between three different naturally occurring substrates, L1, N-cadherin, and laminin. These results suggest that some adhesion molecules may serve as permissive substrates in that they can define axonal pathways but they do not provide information about which path to take at a choice point or about which direction to go along the path. Finally, these results suggest that substrates in vivo may not exert their effects on axon guidance principally via relative adhesiveness.     

Proteoglycans: Road Signs for Neurite Outgrowth

Proteoglycans in the central nervous system play integral roles as ＂traffic signals＂ for the direction of neurite outgrowth. This attribute of proteoglycans is a major factor in regeneration of the injured central nervous system. In this review, the structures of proteoglycans and the evidence suggesting their involvement in the response following spinal cord injury are presented. The review further describes the methods routinely used to determine the effect proteoglycans have on neurite outgrowth. The effects of proteoglycans on neurite outgrowth are not completely understood as there is disagreement on what component of the molecule is interacting with growing neurites and this ambiguity is chronicled in an historical context. Finally, the most recent findings suggesting possible receptors, interactions, and sulfation patterns that may be important in eliciting the effect of proteoglycans on neurite outgrowth are discussed. A greater understanding of the proteoglycan-neurite interaction is necessary for successfully promoting regeneration in the iniured central nervous system.     

Neurite outgrowth inhibition by chondroitin sulfate proteoglycan: stalling/stopping exceeds turning in human neuroblastoma growth cones

Chondroitin sulfate proteoglycan (CSPG) inhibits outgrowth from embryonic chick and rodent neurons in vivo and in vitro and is upregulated during development and following injury. The role of CSPG in outgrowth from human neurons has been largely untested, but is critical for our understanding of regeneration in humans following nervous system injury. Here we determined the effects of CSPG on platelet-derived growth factor (PDGF)-stimulated neurite outgrowth from SH-SY5Y human neuroblastoma cells, a well-accepted model of neuronal differentiation. Cells were plated on glass coverslips adsorbed with laminin (LN), CSPG, or a patterned substratum consisting of alternating stripes of the two molecules. Similar to other studies using chick or rodent neurons, SH-SY5Y cells extend neurites on LN, displaying a 15.2% increase in the total neurite length/cell as compared to cells plated on glass. Cells plated on CSPG alone exhibited reduced neurite outgrowth compared to cells plated on glass or LN. Interestingly, SH-SY5Y growth cones extending on LN and then encountering a CSPG border display more stopping/stalling (62.3%) than turning (27.9%) behaviors. Soluble CSPG inhibits neurite initiation from SH-SY5Y cells plated on glass, but not on LN. These data demonstrate that several CSPG-elicited responses of human neuron-like cells are similar to those from nonhuman neurons. However, approximately 70% of SH-SY5Y growth cones stop or stall at a CSPG border while over 80% of chick sensory neurons turn at a CSPG border. The experimental difference between these models may well indicate a functional difference between animal and human neuronal regeneration.