体外培养中少突胶质细胞在立体网架上的迁移

应用聚酯纤维在培养基上形成的立体网架,接种生后7天大鼠视神经,然后对少突胶质细胞在三维空间的迁移进行体外培养观察。以抗髓鞘碱性蛋白抗体标记少突胶质细胞,并用扫描电镜和透射电镜对在聚酯纤维上迁移的少突胶质细胞超微结构进行了研究。结果表明,聚酯纤维能引导少突胶质细胞迁移;在其表面,少突胶质细胞的突起相互交错并将其包绕;有些突起已在其表面融合,形成“类膜”结构,有些部位可见有2～3层这样的膜性结构。免疫细胞化学染色结果表明,少突胶质细胞突起在聚酯纤维表面形成的“类膜”结构为髓鞘碱性蛋白阳性。以上结果提示,在体外培养条件下,聚酯纤维能引导少突胶质细胞迁移,在无神经元存在时,少突胶质细胞仍可形成髓鞘碱性蛋白阳性的膜性结构。本文对中枢神经系统髓鞘形成模式进行了讨论。     

电针对脊髓损伤后少突胶质细胞再髓鞘化的影响

目的：观察电针治疗对脊髓损伤后少突胶质细胞增生及新生轴突髓鞘再形成的影响。方法：选用成年大鼠,制作中度脊髓损伤模型,应用督脉电针治疗。电镜观察各组髓鞘和少突胶质细胞的超微结构变化;原位杂交显示髓磷脂碱性蛋白（myelin basic protein,MBP）的基因表达。结果：脊髓损伤后髓鞘明显肿胀,部分崩解;少突胶质细胞坏死溶解。1～2周后出现少量增生少突胶质细胞和厚薄不等的髓鞘。电针组髓鞘肿胀较轻,少突胶质细胞坏死少。1周后可见较多增生的少突胶质细胞和完整髓鞘。原位杂交显示电针组MBP基因表达明显高于损伤组,3d组最低,1周达高峰,此后逐渐下降。结论：脊髓损伤后电针治疗可有效防止神经纤维的溃变,促进少突胶质细胞增生和再生神经纤维髓鞘再形成。     

神经元来源细胞外囊泡促进神经干细胞的神经生成

背景：已有研究表明，神经干细胞能够分化为神经元、星形胶质细胞和少突胶质细胞等。间充质干细胞来源细胞外囊泡被证实能够透过血脑屏障到达中枢神经损伤部位，促进神经修复。然而，神经元来源细胞外囊泡是否促进神经干细胞向有益于神经生成的方向分化，帮助神经修复，还不是很明确。目的：探究神经元来源细胞外囊泡是否有利于神经干细胞向神经生成的方向分化。方法：用胰酶消化法从新生SD大鼠大脑皮质中提取神经元和神经干细胞。收集培养神经元的细胞上清，提取神经元来源细胞外囊泡。将培养10 d的神经干细胞与神经元来源细胞外囊泡或PBS共培养7 d，采用免疫印迹、免疫荧光和RT-qPCR技术分别检测神经元、神经干细胞、少突胶质细胞和星形胶质细胞特异性标志蛋白的表达。结果与结论：神经干细胞与神经元来源细胞外囊泡共培养后，高表达神经元特异性蛋白β3微管蛋白、神经丝蛋白200和少突胶质细胞特异性蛋白髓鞘碱性蛋白，低表达星形胶质细胞特异性标志蛋白胶质纤维酸性蛋白。这些结果说明，神经元来源细胞外囊泡能够促进神经干细胞向神经元和少突胶质细胞分化，抑制其向星形胶质细胞分化。     

无血清培养新生Wistar大鼠神经干细胞及免疫荧光鉴定

目的在无血清条件下,分离培养新生1d的Wistar大鼠神经干细胞,观察其生长及分化情况,并对其生物学特性进行鉴定。方法取新生1d的大鼠海马组织,机械分离法分离神经干细胞,加入含有表皮生长因子、碱性成纤维生长因子、B27的DMEM/F12无血清培养基中培养、增殖。倒置显微镜下观察神经干细胞的增殖分化情况,采用免疫荧光染色鉴定其生物特性。结果从新生大鼠海马组织分离培养的神经干细胞在无血清培养基中不断增殖,免疫荧光染色显示巢蛋白呈阳性表达。诱导分化后,免疫荧光染色可见高分子量神经丝蛋白、胶质纤维酸性蛋白和髓鞘碱性蛋白阳性表达细胞。结论无血清条件分离培养的神经干细胞具有自我更新和增殖能力,能分化为神经元、星形胶质细胞和少突胶质细胞。     

丙戊酸钠对小鼠原代少突胶质细胞增殖和分化的影响

目的:观察丙戊酸钠(VPA)药物对原代培养的小鼠少突胶质细胞增殖和分化的影响。方法:体外培养并纯化小鼠少突胶质前体细胞,对增殖或诱导分化的细胞分为Control组和VPA组。利用CCK-8试剂检测VPA对少突胶质前体细胞活性的影响,5-溴脱氧尿嘧啶核苷(Brd U)标记检测增殖期细胞并计算所占比例,免疫细胞化学法检测在细胞不同分化阶段时2',3'-环核苷酸3'-磷酸二酯酶(CNP)和髓鞘碱性蛋白(MBP)、髓磷脂脂蛋白(PLP)和髓鞘相关糖蛋白(MAG)等标志性分子的表达情况real time RT-PCR和Western Blot法分别检测细胞增殖和分化时期相关分子的mRNA和蛋白质水平的表达情况。结果:VPA在1 mmol/L时对细胞活性促进作用最显著;免疫细胞化学显示:与Control组相比,VPA处理组Brd U阳性增殖细胞数量显著增加,CNP和MBP阳性细胞数量明显减少; RT-PCR法检测到与Control组相比,VPA处理后少突细胞中血小板衍化生长因子受体α(PDGFRα) mRNA表达水平增加,CNP、MBP和PLP等分化相关分子的mRNA表达水平降低;同时Western Blot方法检测到细胞分化阶段MBP蛋白水平表达显著降低。结论:VPA能够显著促进体外培养小鼠少突前体细胞的增殖,并抑制细胞分化。     

墨西哥钝口螈脊髓全切后胶质细胞的变化

背景：墨西哥钝口螈脊髓切断可以再生,再生过程伴随胶质细胞数目及分布的改变,研究墨西哥钝口螈脊髓全切后胶质细胞的变化,对进一步探讨其脊髓切断再生机制有重要意义。 目的：观察墨西哥钝口螈脊髓全切后小胶质细胞、星形胶质细胞及少突胶质细胞的变化。 方法：选用成年墨西哥钝口螈,分为脊髓全切组和对照组,利用免疫组织化学法观察脊髓全切后1,3和10 d的损伤脊髓及周围区cd11b标记的小胶质细胞、胶质细胞原纤维酸性蛋白标记的星形胶质细胞及髓鞘碱性蛋白标记的少突胶质细胞的变化。 结果与结论：脊髓全切后短期内cd11b染色阴性;脊髓损伤后胶质细胞原纤维酸性蛋白及髓鞘碱性蛋白阳性细胞染色强度,1 d组阳性细胞染色强度与对照组比较无显著差异,3及10 d组阳性细胞染色强度较对照组低。墨西哥钝口螈小胶质细胞染色阴性,可能存在不同于哺乳动物的标记蛋白;脊髓全切后3及10 d在损伤脊髓及周围区的胶质细胞原纤维酸性蛋白及髓鞘碱性蛋白阳性细胞染色强度较对照组低,提示钝口螈脊髓急性损伤早期未见星形胶质细胞及少突胶质细胞增生,无胶质瘢痕形成。     

人胚胎纹状体来源神经干细胞的体外培养

背景：目前神经干细胞多由动物获得,不适合人类临床移植治疗。 目的：探索体外环境下人胚胎纹状体来源神经干细胞的培养方法,同时观察其生物学特性。 方法：取经水囊引产的孕8-16周人胚胎纹状体,体外用无血清DMEM培养基进行培养,待细胞形成神经球后进行传代,并应用含体积分数10%胎牛血清的DMEM/ F12培养液进行诱导分化。 结果与结论：体外培养的人胚胎纹状体来源神经干细胞生长迅速,表达神经干细胞标志物nestin。克隆形成实验显示细胞克隆形成率为6.0%-7.0%;BrdU掺入实验显示细胞增殖率为37.9%。免疫荧光染色显示经诱导分化的细胞表达神经元标志物Ⅲ型β微管蛋白、星形胶质细胞标志物胶质纤维酸性蛋白及神经干细胞标志物nestin,但不表达少突胶质细胞标志物髓鞘碱性蛋白。可见人胚胎纹状体来源神经干细胞在体外无血清条件下可保持其生物学特点,具有自我更新能力,经胎牛血清诱导后可向神经元及星形胶质细胞分化。     

SOX蛋白与少突胶质细胞的发育

SOX蛋白是一类在动物中发现的转录因子,它们属于高移动组分(h igh mob ility group,HMG)超家族的DNA结合蛋白。它们参与性别决定、骨组织发育、神经系统发育及晶状体发育等多种胚胎发育过程。近年发现,在脊髓中形成髓鞘的少突胶质细胞表达SOX E家族的成员SOX8,SOX9和SOX10。这3个转录因子与少突胶质细胞的发育关系密切:SOX9参与少突胶质细胞的早期定向;SOX10主要影响少突胶质细胞的终末分化和髓鞘形成;SOX8与SOX9、SOX10有协同作用,既参与早期定向又参与终末分化,但作用较弱。     

Ara-C处理小脑与异种视神经的联合体外培养研究

采用细胞分裂抑制剂阿糖胞苷(Cytosine Arabinoside,Ara-C)抑制体外培养小鼠小脑组织中少突胶质细胞的增殖而造成小脑组织的脱髓鞘模型,以10μg/ml阿糖胞苷处理7天为最佳剂量。利用该模型与大鼠视神经组织联合培养2周后,少突胶质细胞自视神经迁至经Ara-C处理的小脑组织中,并在视神经附近形成髓鞘,表明异种动物之间神经组织联合培养能形成髓鞘。少突胶质细胞在形成髓鞘前先进行分裂增殖。本实验建立和采用的体外小脑组织脱髓鞘模型和联合培养系统对研究影响中枢神经髓鞘再生的因素是有效的。     

大鼠少突胶质细胞的纯化培养与鉴定

目的探讨新生2dSprague—Dawley（SD）大鼠脑少突胶质细胞的分离方法和生长条件，为少突胶质细胞损伤后髓鞘形成障碍或脱髓鞘疾病的研究奠定基础。方法根据星形胶质细胞和少突胶质细胞的生长时间差异、细胞生长方式及细胞对培养层粘附等特性的不同，采用两次恒温摇床振荡分离纯化法和条件限定培养基培养获取并鉴定高纯度的大鼠少突胶质细胞。结果培养出突起有如蜘蛛网状的成熟少突胶质细胞，半乳糖脑苷脂（Galactocerebroside，Gal）阳性。结论两次恒温摇床振荡分离纯化法和条件限定培养基培养可获取高纯度的大鼠少突胶质细胞。     

钾离子对体外培养中枢神经髓鞘形成的影响──一种新的体外培养中枢神经脱髓鞘模型的建立

本文应用组织培养和电镜等方法研究了ＫＣＩ对体外培养中枢神经系统髓鞘形成的影响。结果表明：体外培养Ｂ６Ｃ３小鼠小脑组织髓鞘形成的关键期是１０～１４ｄ，在１０～１２ｄ时体外培养的小脑组织对ＫＣＩ最为敏感；当培养液内ＫＣＩ浓度达到３０ｍｍｏｌ／Ｌ时，即可完全抑制髓鞘形成。本文对钾离子引起体外培养中枢神经组织脱髓鞘的机制进行了讨论。     

体外培养小鼠小脑的髓鞘形成及电生理研究

本文应用组织培养、免疫细胞化学和电生理方法研究了体外培养条件下小脑髓鞘形成及电生理表现。本文对体外培养条件下中枢神经系统髓鞘形成及小脑发育各阶段电生理表现进行了讨论。     

高钾处理小脑与同种及异种视神经体外联合培养的研究

应用上海科大学解剖教研室建立的以高浓高钾离子体外培养中枢神经系统脱髓鞘模型，移植正常同种及异种动物神经、进行联合培养并结合电镜和免疫细胞化学，对髓鞘再生过程作了直观的观察和研究。     

Construction of a bispecific antisense oligonucleotide containing multiple binding sites for the treatment of hormone insensitive prostate tumors

Antisense oligonucleotides (oligos) have demonstrated efficacy for the treatment of various cancers, infectious diseases and metabolic disorders. While most studies have utilized single oligos either administered alone, or more recently in combination therapy with other drugs, some investigators have administered more than one oligo in a combined administration or have designed oligos which target multiple proteins (those which share mRNA sequence homology). Antisense oligos inhibit mRNA translation through complementary base pair binding, often about the AUG initiation codon. This inhibition is further enhanced through destruction of the mRNA:oligo hybrid by RNAse H. Construction of an oligo with multiple binding sites located about the respective mRNA initiation codons could simultaneously block translation of more than one protein, even those unrelated in sequence. Such binding could produce a complex mRNA:oligo hybrid more prone to degradation and clearance. Furthermore, such a formulation would increase oligo specific activity and cellular uptake, reduce toxicity, and stabilize at 1:1 the ratio between multiple oligo active sites which otherwise must be comparably delivered (in amount) and individually targeted. The activity of these newly constructed oligos could be tested in both in vitro and in vivo prostate tumor models, utilizing the hormone sensitive LNCaP and the hormone insensitive PC-3 lines. In vitro testing would evaluate oligos administered either alone or in combination with other chemotherapeutics. In vivo testing would administer the oligos to tumors carried in athymic nude mice by either intratumoral inoculation or by using a diffusion pump. Antisense oligos which target proteins associated with growth factors or their receptors, could have a role in the treatment of human prostate cancers when administered with hormone deprivation therapy, or against tumors which have already become hormone insensitive. In the later case, such treatment could form the basis of a second tier of therapy based upon growth factor deprivation.     

Down-regulation of the axonal polysialic acid-neural cell adhesion molecule expression coincides with the onset of myelination in the human fetal forebrain

The polysialic acid (PSA) modification of neural cell adhesion molecule, which reduces neural cell adhesion molecule (NCAM) - mediated cell adhesion, is involved in several developmental processes, such as cell migration, axonal growth, path finding, and synaptic plasticity. It has been suggested that PSA-NCAM expression may inhibit myelination. To clarify the relationship between myelination and the expression of PSA-NCAM we systematically investigated its expression in the human forebrain from embryonic stage to midgestation (19-24 gestation weeks, gw). Immunofluorescence on cryosections showed that PSA-NCAM is expressed at the earliest stage studied (5.5 gw) in the primordial plexiform layer of the telencephalon, which mainly consists of neuronal processes. At midgestation, cortical axonal tracts in the emerging white matter were PSA-NCAM+, but they were not yet myelinated, based on the lack of myelin basic protein (MBP) immunoreaction. To follow the progression of myelination we developed organotypic slice cultures that included the subventricular and intermediate zones of the fetal forebrain. In freshly prepared slices, similar to cryosections, axonal tracts were PSA-NCAM+ but did not express MBP. After 5 days in culture there was a dramatic increase in MBP expression around the axons of the intermediate zone, which suggested the onset of myelination. Simultaneously with MBP up-regulation PSA-NCAM expression in axons was completely lost, as demonstrated both with immunofluorescence and Western blot analysis. These results support the idea that in the human fetal forebrain axonal PSA-NCAM expression is inversely related to primary myelination.     

Synthesis of branched antisense oligonucleotides having multiple specificities. Treatment of hormone insensitive prostate cancer

Antisense oligonucleotides (oligos) directed against transforming growth factor-alpha (TGF-alpha) and its binding site, the epidermal growth factor receptor (EGFR), have demonstrated in vitro and in vivo efficacy against both the PC-3 and LNCaP prostate tumor models. In an attempt to increase the efficiency of these oligos a new type of antisense compound called a bispecific oligo has been evaluated in vitro both alone and in combination with traditional chemotherapeutic agents. These bispecifics, which were first proposed in this journal in 2004, include binding sites for both TGF-alpha and EGFR along the same stretch of complementary DNA. Such bispecifics are able to deliver essentially two antisense activities in an equal molar ratio and can be directed against mRNA encoding proteins of different biochemical pathways. The first bispecifics were developed against two proteins regulating a single autocrine loop. Subsequent bispecifics have been developed which target both EGFR and the apoptosis regulating protein bcl-2. Bispecific activity of a single linear sequence oligo has already been shown to have efficacy. To further develop this multispecific approach, we now propose a branched antisense compound, again, having multiple binding site activities (to complementary sequenced mRNA). Active oligos would be attached to a fat soluble backbone which might enhance targeting and also intracellular entry, release and activity. Such a structure would also permit the customization of these branched forms to include oligos targeting specific proteins related to the growth of various tumor types. Problems associated with the development of antisense oligos have included both membrane solubility and specific targeting. By designing this branched form of antisense structure, multiple activities can be retained (added), solubility improved and delivery enhanced. Such a new formulation would include several antisense oligos covalently bound to and branching off from a lipid-like backbone. An elongated hydrocarbon chain would increase fat solubility and would permit oligo incorporation into nanoparticles or liposome derived delivery vehicles. Specific delivery of oligos could also be enhanced by the tendency of these nanoparticle or liposomal microbubbles to be disrupted under the influence of ultrasonic waves beamed at the targeted tissue.     

Soluble antigen therapy induces apoptosis of autoreactive T cells preferentially in the target organ rather than in the peripheral lymphoid organs

The administration of soluble myelin proteins is an effective way of down-regulating the inflammation in the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. To shed more light on the mechanism of this antigen-specific therapy, we determined the effect of the intraperitoneal (i.p.) injection of soluble myelin basic protein (MBP) on T cell apoptosis in the CNS and peripheral lymphoid organs of Lewis rats with EAE induced by inoculation with MBP and complete Freund's adjuvant. In particular we assessed the level of apoptosis of Vβ8.2⁺ T cells, which constitute the predominant encephalitogenic MBP-reactive T cell population in the Lewis rat. The daily i.p. injection of MBP for 3 days from the onset of neurological signs inhibited the further development of neurological signs of EAE. Using two-color flow cytometry we found that a single i.p. injection of MBP increased the level of apoptosis of the Vβ8.2⁺ T cell population in the CNS to 26.2 % compared to 7.4 % in saline-treated rats and 7.6 % in ovalbumin-treated rats. In contrast, treatment with MBP did not increase the level of apoptosis of the Vβ8.2⁺ population in the popliteal lymph node draining the inoculation site (1.4 %) or in the spleen (1.6 %) above that occurring in saline-treated rats (1.6 % and 1.1 %, respectively). Limiting dilution analysis revealed that the frequency of T cells reactive to the major encephalitogenic epitope, MBP_{72 – 89}, was decreased in the CNS but not in the popliteal lymphnode by this treatment. Three-color flow cytometry in MBP-treated rats demonstrated that CNS Vβ8.2⁺ T cells expressing Fas (CD95) and Fas ligand were highly vulnerable to apoptosis compared to Vβ8.2⁺ T cells not expressing these proteins. We conclude that the i.p. injection of MBP increases the spontaneously occurring Fas-mediated activation-induced apoptosis of auto reactive T cells in the CNS in EAE and that this contributes to the therapeutic effect of the injection.     

Selective blockade of gene expression in a single identified snail neuron

In the present study, the applicability of antisense morpholino oligos for loss-of-function experiments in neurobiology was investigated. The identified withdrawal interneurons of the parietal ganglia expressing helix command neuron-specific 2 (HCS2) gene were pressure injected with HCS2 antisense or control morpholino oligo solution at a final concentration 1-4 microM. No toxic or side effects for the neural functioning were noted immediately or several hours after injection. The changes in the concentration of HCS2-encoded protein in neurons after injection were monitored by two methods, Western blotting and immunostaining of the brain. The amount of the peptide immunoreactive with the HCS2 antibody started to decline in the injected cells at day 2 post-injection, decreased four- to five-fold at day 4, and stayed at this low level thereafter. Similar results obtained by both methods suggest significant selective blockade of production of the HCS2-encoded peptide. In contrast, no substantial decrease of the HCS2-encoded polypeptide was observed after injection with control oligos. Due to the high stability of the morpholino oligos in the cell, they represent a highly efficient tool for a specific long-term blockade of gene expression in molluscan neurons.     

Inhibition of myelin formation by HIV-1 gp120 in rat cerebral cortex culture

Summary To study the role of HIV-1 gp120 in loss of myelin in HIV encephalopathy, the binding of gp120 to various types of neural cells and its effects on myelination were examined in rat primary brain culture. Doublestaining of cultured cells with gp120 and specific antibodies for different neural cell types showed that gp120 bound to most of the galactocerebroside (GalC)-positive oligodendrocytes, a small population of type-2-like astrocytes and a few small neurons. Gp120 did not bind to type-1-like astrocytes, most neurons, or to macrophage/microglia. To assay myelination, cells were bathed in a myelination medium containing chick embryo extract and high glucose, with or without gp120. Seven days after the application, myelination in the culture was observed morphologically and by staining with anti-myelin basic protein (MBP) antibody, and was found to be significantly inhibited by the addition of gp120 (50–100 nM). The processes of oligodendrocytes were reduced in length and arborization relative to the control, but MBP production by oligodendrocytes was unaffected. These results show that gp120 can cause a functional disorder of oligodendrocytes and thus could underlie the diffuse loss of mylein sheaths of HIV encephalopathy.