雪旺细胞无血清条件培养液中的神经营养蛋白活性

目的　进一步探测雪旺细胞无血清条件培养液中神经营养蛋白活性 .方法　收集成年大鼠坐骨神经雪旺细胞无血清条件培养液 (Schwanncellserum -freecondionedmedium ,SC -SFCM ) ,通过PM10超滤获取 >10Kd浓缩液 ,再经Disc -PAGE分离和Biotrap电洗脱 ,从SC -SFCM中分离出A、B、C、D四组蛋白带 ,选择其最佳活性浓度 ,四组蛋白带按照 7种不同组合加入脊髓前角神经元 (SAHN)培养液中 ,通过MTT法进行神经营养活性检测 .结果　含D蛋白带的各组 ,其OD值均显著高于对照组 p <0 .0 1) ,而不含D蛋白带的其它各组 ,其OD值与对照组比较无显著性差异 (p >0 .0 5 ) .结论　来自于SC -SFCM中的D蛋白带具有明显促进SAHN体外存活的神经营养活性     

雪旺细胞源神经营养因子对背根节感觉神经元的保护作用

目的：了解雪旺细胞源神经营养因子对周围神经高位损伤所致脊髓背根节感觉神经元死亡的保护作用。方法：选出生3周SD鼠高位切断L4、L5神经根，神经近侧断端应用雪旺细胞源神经营养因子或生理盐水，4周后观察损伤神经根背根节感觉神经元的存活率和形态学变化。结果：术后4周，营养因子组神经元的存活率是91．8％，生理盐水组是58．6％；生理盐水组存活神经元胞体明显萎缩。结论：雪旺细胞源神经营养因子对受损的背根节感觉神经元有明显的神经营养活性．     

神经营养因子与周围神经再生

周围神经损伤后 ,成功的神经再生取决于很多因素。这其中神经当时所处的微环境起着很重要的作用。大量的实验证明 ,该微环境中的神经营养因子对周围神经的维持和存活有着不可缺少的作用。本文拟对神经营养因子与周围神经再生的关系作一综述。1　神经营养因子　　神经营养因子 (ＮｅｕｒｏｔｒｏｐｈｉｃＦａｃｔｏｒｓ,ＮＴＦ)是一类在靶组织内合成 ,并逆行运输至效应神经元的能对中枢和周围神经发挥营养作用的小分子肽类物质和蛋白质。神经营养因子种类繁多 ,大致可分为生长因子类 (ＧｒｏｗｔｈＦａｃｔｏｒｓ,ＧＦ)和细胞因子类 (Ｎｅｕ…     

复合神经营养因子的神经导管研究进展

目前周围神经的组织工程研究热点之一就是研制具有生物活性的神经导管,主要方法是神经导管与雪旺细胞或者神经营养因子相结合来促进周围神经的再生。就复合神经营养因子的神经导管的相关研究进展作一综述。     

自体神经桥接陈旧性周围神经缺损后雪旺细胞的组织学观察

目的:探索长时间失神经损伤后的雪旺细胞的促神经再生功能。方法:切除成年雌性SD大鼠左侧坐骨神经4 mm,分别饲养3、6个月后,修剪近、远侧神经断端制成不同持续时间的大鼠坐骨神经10 mm陈旧性缺损模型。实验组切取对侧正常坐骨神经桥接缺损,对照组缺损模型制备完成后不予任何修复。各组动物术后再饲养3个月取材,标本进行神经三色染色、免疫荧光染色,电镜等组织学方法观察。结果:各组损伤近端神经结构无明显差异,实验组桥接物段可观察到粗细不等的有髓神经纤维。电镜下,实验组远侧断端皆可观察到髓鞘形成良好的有髓神经纤维和存活的雪旺细胞。结论:长时间失神经损伤的雪旺细胞仍能在一定时间内存活并维持其促神经再生功能。     

血清浓度对雪旺细胞诱导神经上皮干细胞分化的影响

目的:探讨血清对雪旺细胞诱导神经上皮干细胞分化的影响。方法:从新生鼠坐骨神经及臂丛神经分离纯化雪旺细胞,从孕11.5 d大鼠胚胎分离培养神经上皮干细胞,含0%、1%、5%、10%胎牛血清的培养液联合培养雪旺细胞与神经上皮干细胞,收集上清作为条件培养液诱导神经上皮干细胞分化,1周后MAP-2和GFAP细胞免疫化学染色,显微镜下观察统计神经上皮干细胞分化为神经元与星形胶质细胞的比例。结果:条件培养液促进神经上皮干细胞存活和分化,分化形成的神经元大体形态与成熟神经元相似,0%、1%、5%、10%血清条件培养液组神经元与星形胶质细胞比例分别为1.53:1、1.13:1、1.03:1、0.75:1。结论:血清影响雪旺细胞诱导神经上皮干细胞向神经元分化,血清浓度增加,神经上皮干细胞分化形成的神经元减少。     

神经营养因子促周围神经再生的研究进展

<正>神经营养因子是靶组织合成的蛋白质,在生理条件下经逆向运输至神经元胞体,为其提供营养支持,促进神经发育[1];而在损伤时神经营养因子的合成增加、活性增强,能够保护受损的神经元,促进周围神经的修复[2,3]。虽然文献报道外源性神经营养因子有促神经再生的作用,但是实际应用效果难以令人满意,一个主要的原因是多数研究选取的是单一的因子[4,5],而近来的研究表明,修复周围神经损伤多因子     

巨噬细胞条件培养基神经营养活性成分的分离,纯化和鉴定

用快速自动比色微量分析法检测巨噬细胞条件培养基(MΦCM)对体外培养的生后7d SD大鼠小脑皮质神经元的作用。结果表明,MΦCM内分子量大于30kD的组分(加入量40μl/孔)对神经元(细胞密度1×10~5/孔)具有明显的神经营养活性,与对照组相比有显著性差异(P<0.01)。盖片培养法证明,此组分还具有促神经元突起生长的作用。另外,把MΦCM进行Sephadex G-100凝胶层析及生物活性鉴定,获得具有神经营养活性的第一峰洗脱液。降此洗脱液及上述MΦCM分子量大于30kD的组分经SDS-聚丙烯酰胺梯度凝胶电泳分析,证明MΦCM中含有支持神经元生存和增强其活性作用的成分,是一种分子量为97.9kD的蛋白质。     

脑源性神经营养因子对雪旺细胞增殖和分泌的影响

目的:评价脑源性神经营养因子(BDNF)对雪旺细胞(SCs)增殖、分泌功能的影响。方法:取新生SD大鼠双侧臂丛神经,用双酶消化法体外培养SCs,并检测特异性标志蛋白S-100的表达。按培养基中BDNF终浓度的不同将所培养的SCs设立0、10、20、50、100 ng/mL组,共5组培养2周,每2 d用MTT法测细胞活力。并对不同浓度BDNF处理的SCs培养24 h后,取上清液,ELISA法测细胞分泌神经生长因子(NGF)、成纤维细胞生长因子(FGF)水平。结果:培养出的细胞呈S-100阳性,为SCs。与0 ng/mL BDNF组比较,终浓度为50、100 ng/mL BDNF组的SCs细胞活力明显高于0 ng/mL组(均P<0.05),而浓度为10、20 ng/mL时虽有差异但无统计学意义(均P>0.05)。SCs能分泌NGF和FGF;其中终浓度为50 ng/mL BDNF组的SCs分泌NGF和FGF水平最高(P<0.05)。结论:BDNF对SCs的增殖和分泌功能有促进作用,终浓度为50 ng/mL时,SCs的活力与分泌能力最佳。     

牙髓炎症时胶质源性神经营养因子的表达

目的　探讨胶质源性神经营养因子 (GDNF)在人炎症牙髓中的表达及意义。方法　应用免疫组织化学方法比较正常和炎症牙髓中GDNF的表达变化。结果　正常人牙髓组织GDNF染色呈阴性 ,炎症牙髓组织GDNF染色呈阳性 ,主要分布在神经纤维 ,特别是血管周围的神经纤维。结论　GDNF可能参与人牙髓感染后的修复性反应 ,对神经末梢再生发芽有促进作用     

适于杂交瘤细胞长期大量繁殖的SCI－8910培基的研制

单抗的免疫导向治疗已成为国内外肿瘤研究的热点之一。目前杂交瘤分泌单抗的来源一部分是小鼠腹水，另一部分培养于含血清的培养基内，这对单抗纯化带来困难，对免疫导向治疗也带来许多不利条件，如牛的ＩｇＧ、鼠的病毒等的污染。因此，我们着手研制一种能适应于杂交瘤长期培养的无血清培基，定名为ＳＣＩ－８９１０。现已进行了９株杂交瘤细胞的长期培养，同时用该培基在１．５Ｌ的Ｃｅｌｌｉｇｅｒ（ＮＢＳＵＳＡ）搅拌生物反应器中长期培养鼠－鼠杂交瘤细胞２Ｆ７（分泌抗人小细胞肺癌单抗）获得成功，经检测单抗纯度达到一定要求，产品在８ｍｇ／Ｌ以上。结果提示，该无血清培基制备的ＭＣＡｂ用于免疫导向治疗的可行性及大规模生产的可能性。     

施万细胞来源的Toll样受体在外周神经损伤及修复中的作用和机制

Toll样受体参与组成机体抵抗外来病原微生物入侵的第一道防线,在非感染性疾病中与内源性配体结合可触发免疫炎症反应。在外周神经损伤后,各种内源性配体释放,可与外周神经中表达的Toll样受体结合,通过信号级联传导,参与免疫炎症反应过程。本文将着重阐述施万细胞来源的Toll样受体在外周神经损伤后瓦勒变性及外周神经修复中的作用和机制。     

脑源性神经营养因子研究现状

近年来神经科学的研究表明,神经营养因子是选择性调节周围神经和中枢神经系统神经生长和存活的一类蛋白质。脑源性神经营养因子（brain derived neurotrophic factor,BDNF）是神经生长因子（nerve growth fac-tor,NGF)发现后约30年由德国神经生物学家Barde^[1]报告的另一神经营养因子,它对CNS多种类型神经元的生长、发育、分化、维持和损伤修复都具有重要作用,对神经系统疾病诸如早老性痴呆、帕金森氏病和肌萎缩侧索硬化等退变性疾病的治疗具有潜在应用前景。本文就脑源性神经营养因子的结构、功能及应用前景作一综述。     

Activity of Allergenic Proteins from Dermatophagoides pteronyssinus

Two purified allergens from Dermatophagoides pteronyssinus, Dp 42 (identical to P1) and Dp X were studied for their ability to induce histamine release from washed leukocytes and to bind to IgE antibodies from the serum of 27 mite-sensitive children. Almost all patients were demonstrated to be sensitive to both proteins by both assays. Dp 42 was found to have the highest allergenic activity, releasing histamine from leukocytes at a median concentration 10 times lower than for Dp X. There was a positive correlation between basophil sensitivity to both proteins and allergen specific serum IgE concentrations.     

Antigenicity of Recombinant Proteins Derived from Rhoptry-Associated Protein 1 of Plasmodium falciparum

Rhoptry-associated protein 1 (RAP1) of Plasmodium falciparum is a potential component of a malaria vaccine. We have expressed in Escherichia coli eight recombinant RAP1 proteins representing almost the entire sequence of the mature protein and assessed the antigenicity of the proteins by immunization of mice. Antisera to six of the recombinant proteins reacted specifically with parasite-derived RAP1 (PfRAP1), as determined by indirect immunofluorescence and by immunoblotting. These proteins were then used in enzyme-linked immunosorbent assays to evaluate human antibody responses to RAP1 during naturally transmitted infections in The Gambia. Immunoglobulin G (IgG) antibodies specifically reactive with the recombinant RAP1 proteins are directed mostly towards fragments containing the N-terminal sequences of mature PfRAP1. The most N-terminal segment (residues 23 to 175) contains only minor epitopes, while major epitopes are outside this region. Antibodies from malaria patients do not compete for a linear epitope recognized by an inhibitory anti-RAP1 monoclonal antibody. Analysis of IgG subclass distribution shows that human anti-RAP1 antibodies are predominantly IgG1.Rhoptries are club-shaped organelles located at the apical end of the merozoite, the extracellular form of malaria parasites responsible for invasion of erythrocytes. Antigens associated with these organelles are among targets for antimalaria vaccines (19). One such antigen of Plasmodium falciparum is a complex of two noncovalently linked proteins, rhoptry-associated protein 1 (RAP1) and RAP2 (4, 5, 7, 10, 18, 28, 30, 31).Unlike many other malaria antigens, RAP1 shows minimal genetic polymorphism, and its amino acid sequence is highly conserved between isolates. Full or partial amino acid sequences of the protein deduced from gene sequences obtained from seven distinct isolates of different geographical origins were more than 99% identical, with only nine amino acid substitutions identified (14–16, 28). This suggests that interstrain antigenic diversity may not be a problem for a RAP1-based vaccine.Experimental immunizations of Saimiri monkeys with affinity-purified RAP1-RAP2 complex conferred partial protection against P. falciparum infection (27). The epitopes responsible for this immunity were not determined. Monoclonal antibodies (MAbs) to conserved linear epitopes of RAP1 inhibit the development of P. falciparum in vitro (13, 31), suggesting that antibodies to this antigen may reduce the replication of the parasite. Since RAP1 is a component of an endotoxin-like exoantigen that stimulates in vitro production of tumor necrosis factor by human mononuclear cells (22), it was proposed that antibodies against RAP1 might protect against the disease by removing the toxin-like exoantigen from circulation.The knowledge of human immune recognition of RAP1 is inadequate. To date, only four studies of human immune responses to RAP1 during natural malaria infection have been reported. Jakobsen and colleagues (20) showed that lymphocytes from most of 21 Ghanaian donors proliferated in vitro in response to a recombinant protein representing the N-terminal third of RAP1 (amino acids [aa] 23 to 294), suggesting the presence of T-cell epitopes in this region. Sera from these donors also contained antibodies to the recombinant RAP1 (rRAP1). A larger study using the same rRAP1 and sera of 425 Tanzanians living in an area where malaria is holoendemic showed that the proportion of responders increased with age and, in addition, indicated an association between high levels of anti-RAP1 immunoglobulin G (IgG) antibodies and protection against high P. falciparum densities in children (21). A more recent study of 100 Papua New Guineans confirmed that the recognition of RAP1 correlated with age (35). Only one study compared the relative immunogenicities of different regions of RAP1 (15). Testing sera of 26 individuals by immunoblotting for antibodies to rRAP1 antigens and visual scoring of results, the study indicated that most antibodies detectable by this method were against epitopes within an N-terminal region (aa 1 to 122) (15).The work presented here describes the production and immunological characterization of a new set of rRAP1 proteins and their use in an enzyme-linked immunosorbent assay (ELISA) for evaluation of antibody responses in Gambian malaria patients. We show that although patients have IgG antibodies to an rRAP1 containing the N-terminal sequence from aa 23 to 175, more antibodies are targeted to major epitopes outside this region. The antibodies are mainly of the IgG1 subclass.     

hBDNF基因在成纤维细胞中的异位表达

将hBDNF全长编码序列插入人Ⅰ型胶原基因（Colia1）增强子－启动子调节序列之下,构建了含Colia1-BDNF微基因的重组pSCEPBFCAT,并转染人胚肌腱成纤维细胞。成功地利用了Colia1基因调控元件介导DBNF基因在成纤维细胞中异位表达,SDS－PAGE显示表达产物分子量为27kDa,免疫斑点杂交、Elisa和Western杂交证实该表达产物具有BDNF抗原活性。     

Induced Pluripotent Stem Cell Lines Derived from Equine Fibroblasts

The domesticated horse represents substantial value for the related sports and recreational fields, and holds enormous potential as a model for a range of medical conditions commonly found in humans. Most notable of these are injuries to muscles, tendons, ligaments and joints. Induced pluripotent stem (iPS) cells have sparked tremendous hopes for future regenerative therapies of conditions that today are not possible to cure. Equine iPS (EiPS) cells, in addition to bringing promises to the veterinary field, open up the opportunity to utilize horses for the validation of stem cell based therapies before moving into the human clinical setting. In this study, we report the generation of iPS cells from equine fibroblasts using a piggyBac (PB) transposon-based method to deliver transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, expressed in a temporally regulated fashion. The established iPS cell lines express hallmark pluripotency markers, display a stable karyotype even during long-term culture, and readily form complex teratomas containing all three embryonic germ layer derived tissues upon in vivo grafting into immunocompromised mice. Our EiPS cell lines hold the promise to enable the development of a whole new range of stem cell-based regenerative therapies in veterinary medicine, as well as aid the development of preclinical models for human applications. EiPS cell could also potentially be used to revive recently extinct or currently threatened equine species.     

Effect of Hypoxia/Reoxygenation on Cell Viability and Expression and Secretion of Neurotrophic Factors (NTFs) in Primary Cultured Schwann Cells

As the primary myelin‐forming cells of the peripheral nervous system, Schwann cells (SCs) play a key role in the regeneration of injured peripheral nerves. However, hypoxia causes injury of SCs, as observed in peripheral neuropathies, including those caused by diabetes. So we investigated the effect of hypoxia/reoxygenation (H/R) on SCs in this study. To do so, SCs were cultured in hypoxic condition in vitro and then in normal condition for 24 hr; The effects H/R on SCs were evaluated by MTT (3(4,5‐dimethylthiazol‐2‐yl)2,5‐diphenyltetrazolium bromide) assay, Hoechst staining, immunocytochemistry, western blotting, ELISA, and RT‐PCR. H/R resulted in a significant decrease in SCs survival and an increase in caspase‐3 activity. H/R also reduced the mRNA level of BDNF (brain derived neurotrophic factor) and its secretion, but NGF mRNA level was elevated in these cells. These observations showed that H/R induces death of primary cultured SCs, and different mechanisms responsible for regulating NGF and BDNF expression. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.