犬骨髓基质干细胞体外定向分化为软骨细胞

背景：骨髓基质干细胞的多向分化性不利于向软骨细胞单一方向分化,植入体内后成骨系细胞分泌的骨形态发生蛋白作用于非定向分化的前体细胞,使其向成骨细胞分化,而已定向分化的细胞则不受骨形态发生蛋白的影响,形成相应的组织。 目的：体外诱导犬骨髓基质干细胞定向分化为软骨细胞,探讨体外诱导成软骨的方法和条件。 设计、时间及地点：单一样本观察,于2005-03/2006-01在中山大学组织工程实验室完成。 材料：选取4个月龄雄性家犬1只,骨髓基质干细胞取自犬肋骨。 方法：自犬肋骨取骨髓2.0~3.0 mL行体外原代骨髓基质干细胞分离培养。8~11 d细胞接近融合时,加入胰酶消化,用含体积分数10%胎牛血清的L-DMEM合成培养液终止消化,收集细胞悬液,离心后,用培养液悬浮细胞,按1∶3接种传代。取第3代细胞培养扩增,换液时加入10 μg/L碱性成纤维细胞生长因子2 mL,换液2次后,再加入 1 mg/L转化生长因ß1 2 mL诱导骨髓基质干细胞向软骨细胞分化。 主要观察指标：行甲苯胺蓝、阿新蓝染色检测软骨基质的分泌,免疫组织化学染色检测软骨特异性Ⅱ型胶原表达。 结果：骨髓基质干细胞体外行原代和传代培养至P4代生长良好,经碱性成纤维细胞生长因子和转化生长因ß1扩增诱导的细胞甲苯氨蓝异染性、阿新蓝染色阳性;II型胶原免疫组织化学检测阳性,显示被诱导的骨髓基质干细胞具备软骨细胞特性。 结论：应用碱性成纤维细胞生长因子和转化生长因ß1体外可以诱导犬骨髓基质干细胞分化为软骨细胞,诱导的软骨细胞可作为软骨组织工程较理想的种子细胞。     

骨关节炎软骨细胞的体外分离培养

背景：由于体外分离培养骨关节炎患者软骨细胞存在难度，同时软骨组织中因富含大量的基质, 且软骨细胞分布于其中的软骨陷窝中, 因此与其他细胞相比, 软骨细胞的分离更为困难。 目的：从骨关节炎行人工膝关节置换者的关节软骨中分离软骨细胞，并改良其酶消化法分离软骨组织，观察软骨细胞的生物学特性。 设计、时间及地点：对比观察，实验于2007-04/2008-03在解放军第三军医大学烧伤研究所实验室完成。 对象：标本取材于解放军第三军医大学西南医院关节外科中心同期收治的10例人工膝关节置换患者软骨组织（> 3 cm）。 方法：切取原发性骨关节炎患者关节软骨，将软骨剪成1 mm×1 mm×1 mm左右的碎块，以Ⅱ型胶原酶顺序消化联合胰蛋白酶消化分离关节软骨细胞,同期对比用Ⅱ型胶原酶加胰蛋白酶消化法消化软骨后的收获细胞数及细胞成活率，分离细胞进行原代和传代培养。 主要观察指标：以倒置相差显微镜观察体外培养软骨细胞形态；以甲苯胺蓝染色以及Ⅱ型胶原免疫组织化学染色进行软骨细胞鉴定；四甲基偶氮唑盐法测定骨关节炎细胞增殖情况。 结果：改良的Ⅱ型胶原酶顺序消化联合胰蛋白酶法，对原代关节软骨细胞的分离取得了优良而稳定的效果，与传统的胰蛋白酶消化法相比，收获细胞数为3.72×106，细胞存活率为97.5%，二者相比有显著性差异（P < 0.05）。体外培养观察软骨细胞贴壁和生长均较缓慢。原代和传代后细胞Ⅱ型胶原免疫组织化学和甲苯胺蓝异染反应均较弱。软骨细胞增殖和生长缓慢。 结论：Ⅱ型胶原酶顺序消化联合胰蛋白酶分离骨关节炎患者软骨细胞的技术简单且易操作, 分离培养的软骨细胞符合骨关节炎时软骨退变的表现, 可为骨关节炎的病因研究及软骨细胞移植治疗骨性关节炎提供实验基础。     

组织工程软骨细胞体外培养技术要点☆

通过组织工程技术，采用自身软骨细胞体外增殖构建软骨来修复关节软骨损伤，在临床上已成为一种有效的新方法。近年来，有关体外软骨细胞培养技术的研究较多。通过对入选文献进行分析、整理，对影响体外构建软骨的因素包括培养空间、应力、细胞密度和氧张力等方面进行分析，体外采用三维多条件联合培养软骨细胞可以改善构建软骨的生物品质。但是影响软骨细胞生长的因素众多复杂，在体外如何更好地构建软骨，有待进一步研究。     

结缔组织生长因子对体外培养的兔关节软骨细胞增殖和表型的影响

背景：在关节软骨损伤修复的过程中,由于目前实验室中常用的细胞因子存在半衰期短、价格昂贵等缺点,不能广泛应用于临床。 目的：假设结缔组织生长因子对体外培养的兔关节软骨细胞增殖和表型有一定的影响,为组织学方法修复关节软骨缺损寻找新的细胞因子。 设计、时间及地点：随机区组设计对照实验,于2007-08/2008-02在深圳龙岗中心医院完成。 材料：健康1月龄新西兰大白兔2只;结缔组织生长因子为Propetech公司产品。 方法：获取兔关节软骨组织,将其剪碎,Ⅱ型胶原酶消化法原代分离培养兔关节软骨细胞,待细胞铺满瓶底80%胰酶消化传代,取其第2代,细胞贴壁后随机分组,实验组分别加入30,50,100,150 μg/L的结缔组织生长因子,对照组仅加入DMEM培养液进行体外培养。 主要观察指标：采用倒置相差显微镜下观察细胞形态及数量改变,采用四唑盐(MTT)法检测不同浓度的结缔组织生长因子对软骨细胞增殖的影响,免疫组织化学法(SABC法)检测Ⅱ型胶原的表达情况。 结果：①结缔组织生长因子作用下的兔关节软骨细胞生长迅速,呈典型的软骨细胞形态,部分细胞聚集成团,形成软骨结节。②结缔组织生长因子能显著促进兔关节软骨细胞增殖,与对照组相比差异具有显著性意义(P < 0.01)。不同质量浓度的结缔组织生长因子均能促软骨细胞增殖,100 μg/L为最佳作用质量浓度(P < 0.01)。③结缔组织生长因子作用下的兔关节软骨细胞Ⅱ型胶原表达增多,且始终为阳性。 结论：结缔组织生长因子可以刺激体外培养的兔关节软骨细胞增殖,促进软骨特异性Ⅱ型胶原表达,维持软骨表型。结缔组织生长因子可能用来作为组织工程学修复软骨缺损,治疗骨关节炎的新的生长因子。     

碱性成纤维细胞生长因子对生长板软骨细胞增殖与分化的影响

背景: 体外培养软骨细胞经历多次传代后，其增殖能力将逐渐退化。实验表明，碱性成纤维细胞生长因子可促进软骨细胞增殖，并可能影响软骨细胞的表型与分化。 目的：观察碱性成纤维细胞生长因子对生长板软骨细胞增殖和分化的影响，并筛选其用于体外软骨细胞培养的最佳剂量。 设计、时间及地点：以细胞为观察对象的观察对比实验，于2004-10/2005-02在暨南大学医学院生化教研室组织工程实验室完成。 材料：选用12只新西兰白兔用于分离软骨细胞，碱性成纤维细胞生长因子由英国PeproTech公司生产。 方法：分离并在低血清条件下培养兔生长板软骨细胞。根据实验需要加入0，1，2.5，5，10，25，50及100 μg/L 8个剂量的碱性成纤维细胞生长因子，进行各项指标观察。 主要观察指标：应用改良四甲基偶氮唑蓝比色法检测细胞增殖倍数；应用羟脯氨酸法测定软骨细胞胶原产量；应用酶动力学方法测定碱性磷酸酶活性。 结果：①碱性成纤维细胞生长因子剂量在5~100μg/L范围内可以促进软骨细胞增殖，并以25 μg/L时刺激效果最为显著。②当碱性成纤维细胞生长因子剂量高于 25 μg/L时，软骨细胞的胶原合成被抑制。③当碱性成纤维细胞生长因子剂量高于 1 μg/L时，软骨细胞的碱性磷酸酶活性被抑制。 结论：碱性成纤维细胞生长因子可以刺激生长板软骨细胞增殖，并在剂量高于25 μg/L时抑制生长板软骨细胞的分化。     

海藻酸钠复合载体长期培养软骨细胞的生物学稳定性

摘要 背景：近年研究发现在海藻酸钠三维培养体系中软骨细胞存活时间延长,细胞外基质分泌旺盛。　 目的：观察海藻酸钠复合载体对体外长期培养软骨细胞生物学稳定性的影响。 方法：将兔软骨细胞接种至以海藻酸钠、透明质酸、壳聚糖、纤维连接蛋白、碱性成纤维细胞生长因子为主要成分的复合载体三维培养体系中,以平面培养作为对照。定期观察在两种不同培养体系中软骨细胞形态学改变,细胞生长曲线差异,细胞上清液糖胺多糖含量差异。 结果与结论：软骨细胞在海藻酸钠复合载体中生长良好,生长旺盛,并形成球状细胞团,细胞分裂增殖活跃;培养2 d时复合载体培养软骨细胞增殖稍高于平面培养软骨细胞;培养4 d时可见复合载体培养软骨细胞增殖明显加快;培养6~14 d时复合载体培养的仍保持较稳定增殖,而平面培养的软骨细胞增殖逐渐降低;复合载体细胞外液糖胺多糖含量明显高于平面培养软骨细胞。说明海藻酸钠复合载体可以长期培养软骨细胞并保持其生物学稳定性。 关键词：海藻酸钠;软骨细胞;培养;载体;生物学稳定性 doi:10.3969/j.issn.1673-8225.2011.16.005     

体外团状培养系统中Ad-hTGF-β1诱导骨髓间充质干细胞向软骨细胞的分化

背景：组织工程技术的发展给修复关节软骨缺损带来了希望,最近的比较研究证实骨髓间充质干细胞是修复全层软骨缺损的最佳细胞源。 目的：在体外团状培养系统中,以Ad-hTGF-β1诱导骨髓间充质干细胞向软骨细胞定向分化,并对分化后的细胞进行鉴定。 设计、时间及地点：以细胞为对象的重复观察测量实验,于2007-06/2008-01在解放军第三军医大学新桥医院中心实验室完成。 材料：2月龄日本大耳白兔由解放军第三军医大学实验动物中心提供。 方法：兔骨髓间充质干细胞体外分离培养扩增,Ad-hTGF-β1转染后,在团状培养系统中培养。通过组织学、免疫组化及反转录-聚合酶链反应方法检测其成软骨分化。 主要观察指标：组织学染色观察细胞形态改变,免疫组化方法及反转录-聚合酶链反应检测蛋白多糖和Ⅱ型胶原的表达。 结果：组织学染色显示,诱导后细胞呈软骨细胞样形态。免疫组化方法及反转录-聚合酶链反应结果显示,诱导后的细胞表达蛋白多糖和Ⅱ型胶原。 结论：骨髓间充质干细胞经Ad-hTGF-β1诱导后在团状培养系统中可分化为软骨细胞。     

反转录病毒载体介导转化生长因子β1基因体外转染兔膝关节软骨细胞的表达

背景：将功能基因片段整合到基因载体内,再将基因载体转染入靶细胞中或转染入关节腔内,通过转基因的靶细胞持续大量分泌功能基因产物,在一个较长的时期内保持局部治疗浓度,可修复关节软骨损伤。 目的：以重组反转录病毒载体介导转化生长因子β1体外转染兔膝关节软骨细胞,观察其表达情况及其对软骨细胞生物学性状的影响。 方法：采用胰酶消化法分离培养兔软骨细胞。载体经PLNCX2 Hind Ⅲ/Not Ⅰ双酶切、去磷酸化后,与载体pDsRed2双酶切得到大的部分多克隆位点和RFP基因连接,构建PLNCX2-RFP。转化生长因子β1基因从PGEMT-TGF中扩增,经双酶切后与PLNCX¬2-RFP连接,构建PLNCX2-TGFβ1-RFP。包装反转录病毒载体,并检测病毒上清滴度。将培养的兔膝关节软骨细胞分组转染：对照组(不做任何转染)、转染PLNCX2组、转染PLNCX2-TGFβ1-RFP组,持续筛选2周,观察细胞变化。收集稳定转染的细胞上清液,以NO检测试剂盒检测基因转染对软骨细胞的影响,以ELISA法检测细胞培养上清液中人转化生长因子β1表达。 结果与结论：重组基因PLNCX2-TGFβ1-RFP经酶切鉴定测序TGFβ1、RFP、序列均正确,表明构建成功预期的真核表达载体PLNCX2- TGFβ1-RFP。转染到包装细胞并筛选培养后,病毒滴度为1×106 CFU。稳定转染软骨细胞后可观察到红色荧光,证明转染成功。持续筛选2周,散在贴壁细胞形成阳性克隆,逐渐弥漫融合,并有细胞簇出现,双核多见,细胞增生活跃。转染PLNCX2-TGFβ1-RFP组NO浓度高于对照组、转染PLNCX2组(P < 0.05),对照组、转染PLNCX2组间差异无显著性意义。对照组与转染PLNCX2¬组均无转化生长因子β1表达,转染PLNCX2-TGFβ1-RFP组转化生长因子β1质量浓度为(28.08±3.73) ng/L。提示反转录病毒载体PLNCX2介导的人转化生长因子β1能有效转染到兔膝关节软骨细胞并获得稳定表达,同时转染后的软骨细胞增生活跃。     

转化生长因子β3诱导骨髓间充质干细胞分化为软骨细胞

背景：转化生长因子β是一族多肽类生长因子,胚胎形成期可诱导原始的间充质干细胞分化形成软骨组织,有研究报道转化生长因子β3和碱性成纤维细胞生长因子可促进软骨细胞的代谢和增殖。目的：验证转化生长因子β3体外诱导骨髓间充质干细胞分化为软骨细胞的可行性。方法：骨髓来源于髋关节手术时的松质骨碎片或于下肢骨开放性手术时收集,分离培养骨髓间充质干细胞,鉴定其表面抗原,用含体积分数10%胎牛血清以及10 μg/L转化生长因子β3的条件培养基诱导,诱导后细胞通过软骨细胞特征性染色,即甲苯氨蓝染色以及Ⅱ型胶原免疫组织化学染色鉴定。结果与结论：骨髓间充质干细胞表面抗原CD73,CD90,CD105阳性,CD14,CD34,CD45,CD106以及HLA-DR阴性。诱导后细胞形态明显改变,甲苯氨蓝以及Ⅱ型胶原染色结果阳性。提示骨髓间充质干细胞在转化生长因子β3作用下,体外可分化为软骨细胞,可以作为组织工程种子细胞的一种有效来源。     

体外定向诱导鹿茸间充质干细胞向软骨细胞的分化**☆

目的：间充质干细胞的诱导分化的研究多见于骨髓、脐血等组织,而在鹿茸组织中分离的间充质干细胞是否能诱导为软骨细胞目前尚不清楚。实验拟建立梅花鹿鹿茸生长中心间充质干细胞体外培养方法,观察转化生长因子β1体外诱导鹿茸间充质细胞分化为软骨细胞的可行性。 方法：实验于2006-03/2007-06在吉林大学畜牧兽医学院基础兽医研究室完成。①实验材料：4岁龄健康雄性梅花鹿由中国农科院左家特产研究所提供,实验过程中对动物处置符合动物伦理学标准。②实验方法：于生长早期锯取梅花鹿鹿茸,在解剖显微镜下定位和切取间质层（突起部）,即为鹿茸间充质干细胞所在的组织层。Ⅰ型胶原酶消化法原代分离培养鹿茸生长中心间充质干细胞,锥虫蓝染色显示细胞活性达90%以上,将活性确定后的细胞液氮冻存。取第3代细胞,用含10%胎牛血清以及10 μg/L转化生长因子β1的条件培养基诱导培养。③实验评估：培养12 d 后于倒置显微镜下观察细胞形态,采用细胞化学及免疫细胞化学鉴定细胞。 结果：①鹿茸生长中心间充质干细胞的分离与培养：Ⅰ型胶原酶消化法培养可以获得均一的间充质干细胞,贴壁的间充质干细胞形态均匀,呈长梭形,克隆样生长,增殖迅速。②转化生长因子β1体外定向诱导鹿茸间充质细胞分化为软骨细胞：转化生长因子β1诱导分化的鹿茸间充质细胞生长迅速,诱导后细胞形态明显改变,呈典型的软骨细胞形态,甲苯胺蓝染色阳性,Ⅱ型胶原表达阳性。 结论：采用酶消化法可以从鹿茸生长中心间充质层中分离出间充质干细胞,在体外转化生长因子β1具有促进鹿茸生长中心间充质干细胞分化为软骨细胞的能力。     

丝裂原在海马神经干细胞培养中的作用

目的 研究表皮生长因子(epidemal growth factor，EGF)和碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)在海马神经干细胞培养中的作用，确定适合海马神经干细胞体外培养的丝裂原。方法 应用EGF和bFGF刺激海马神经干细胞增殖，观察神经干细胞生长、增殖和分化等特性。结果 EGF和bFGF都能刺激神经干细胞扩增，bFGF反应性神经干细胞团增殖缓慢，细胞团中部分细胞迁出，迁出的细胞形成新的细胞团或分化成神经细胞或神经胶质细胞，而且bFGF反应性神经干细胞贴壁很紧，不易传代；相反，EGF反应性神经干细胞快速增殖，易于传代，较少迁移和分化。结论 EGF可促使海马神经干细胞快速增殖，是体外培养海马神经干细胞比较合适的丝裂原。     

Expression of Ciliary Neurotrophic Factor and the Neurotrophins-Nerve Growth Factor,Brain-Derived Neurotrophic Factor and Neurotrophin 3-in Cultured Rat Hippocampal Astrocytes

Cultured astrocytes are known to possess a range of neurotrophic activities in culture. In order to examine which factors may be responsible for these activities, we have examined the expression of the genes for four known neurotrophic factors – ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) – in purified astrocyte cultures derived from neonatal rat hippocampus. Hippocampal astrocytes were found to express mRNA for three neurotrophic factors – CNTF, NGF and NT3 – at significantly higher levels than other cultured cell types or cell lines examined. BDNF messenger RNA (mRNA), however, was undetectable in these astrocytes. The levels of CNTF, NGF and NT3 mRNA in astrocytes were largely unaffected by their degree of confluency, while serum removal caused only a transient decrease in mRNA levels, which returned to basal levels within 48 h. Astrocyte-derived CNTF was found to comigrate with recombinant rat CNTF at 23 kD on a Western blot. Immunocytochemical analysis revealed strong CNTF immunoreactivity in the cytoplasm of astrocytes, weak staining in the nucleus, but no CNTF at the cell surface. NGF and NT3 were undetectable immunocytochemically. CNTF-like activity, as assessed by bioassay on ciliary ganglion neurons, was found in the extract of cultured astrocytes but not in conditioned medium, whereas astrocyte-conditioned medium supported survival of dorsal root ganglion neurons but not ciliary or nodose ganglion neurons. This conditioned medium activity was neutralized with antibodies to NGF. Astrocyte extract also supported survival of dorsal root ganglion and nodose ganglion neurons, but these activities were not blocked by anti-NGF. Part, but not all, of the activity in astrocyte extracts which sustained nodose ganglion neurons could be attributed to CNTF.     

Fibroblast Growth Factors and Insulin Growth Factors Combine to Promote Survival of Rat Schwann Cell Precursors Without Induction of DNA Synthesis

In embryonic rat nerves, we recently identified an early cell in the Schwann cell lineage, the Schwann cell precursor. We found that when these cells were removed from contact with axons they underwent rapid apoptotic death, and that in a proportion of the cells this death could be prevented by basic fibroblast growth factor (bFGF, FGF-2). We now report that 100% of Schwann cell precursors isolated from peripheral nerves of 14-day-old-rat embryos can be rescued by a combination of insulin-like growth factor (IGF) 1 or 2 in combination with either acidic FGF (aFGF, FGF-1), bFGF or Kaposi's sarcoma FGF (K-FGF; FGF-4). The precursors display an absolute requirement for both an IGF and an FGF to achieve maximal survival. Elevation of intracellular levels of cAMP by forskolin does not result in a significant shift in the IGF/FGF dose-response curves. In contrast, the percentage of precursors rescued by FGF in the presence of insulin is dramatically increased by elevation of cAMP. These growth factor combinations did not stimulate DNA synthesis significantly in Schwann cell precursors. These findings show that cooperation between growth factors is required to suppress cell death in Schwann cell precursors, and suggest that survival and DNA synthesis are regulated by distinct growth factor combinations in these cells. The observations are consistent with the idea that survival regulation by FGFs and IGFs plays an important role in the development of glial cells in early embryonic nerves.     

分离扩增肋软骨细胞在三维支架上的种植与培养

摘要背景：外科长段气管切除后,需要应用气管替代物,理想的替代材料需具有相当的强度、可弯曲性以及内面覆盖的上皮。合适的种子细胞、性质优良的载体、良好的生长媒介是组织工程化气管构建中最重要的组成部分。目的：寻找分离培养肋软骨细胞的最佳方法,构建软骨-支架模型。方法：将取出的兔第5,6肋软骨切成组织片,经胰酶、蛋白酶消化或酶处理后贴壁,分离肋软骨细胞并扩增。传代2次后种植于聚乳酸羟基乙酸或涤纶支架上,继续载体外环境下培养。观察单层培养扩增中软骨细胞的形态、结构,及在支架上培养时的细胞生长状态、外分泌基质。结果与结论：酶消化法和组织块培养法所获细胞均为原代肋软骨细胞,单次传代时间为5 d,但组织块培养法分离细胞较慢,且存在成纤维细胞污染情况,提示酶消化法适合用于肋软骨细胞的分离。肋软骨细胞种植于支架后贴附良好,第2周时可见基质分泌,获得软骨-支架模型,可用于构建组织工程化气管。关键词：软骨;培养;支架;酶消化法;生物材料;组织块培养法;组织工程doi:10.3969/j.issn.1673-8225.2010.34.007     

Effect of Single Growth Factor and Growth Factor Combinations on Differentiation of Neural Stem Cells

Objective

The effects on neural proliferation and differentiation of neural stem cells (NSC) of basic fibroblast growth factor-2 (bFGF), insulin growth factor-I (IGF-I), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF) were assessed. Also, following combinations of various factors were investigated : bFGF+IGF-I, bFGF+BDNF, bFGF+NGF, IGF-I+BDNF, IGF-I+NGF, and BDNF+NGF.

Methods

Isolated NSC of Fisher 344 rats were cultured with individual growth factors, combinations of factors, and no growth factor (control) for 14 days. A proportion of neurons was analyzed using β-tubulin III and NeuN as neural markers.

Results

Neural differentiations in the presence of individual growth factors for β-tubulin III-positive cells were : BDNF, 35.3%; IGF-I, 30.9%; bFGF, 18.1%; and NGF, 15.1%, and for NeuN-positive cells was : BDNF, 34.3%; bFGF, 32.2%; IGF-1, 26.6%; and NGF, 24.9%. However, neural differentiations in the absence of growth factor was only 2.6% for β-tubulin III and 3.1% for NeuN. For β-tubulin III-positive cells, neural differentiations were evident for the growth factor combinations as follows : bFGF+IGF-I, 73.1%; bFGF+NGF, 65.4%; bFGF+BDNF, 58.7%; BDNF+IGF-I, 52.2%; NGF+IGF-I, 40.6%; and BDNF+NGF, 40.0%. For NeuN-positive cells : bFGF+IGF-I, 81.9%; bFGF+NGF, 63.5%; bFGF+BDNF, 62.8%; NGF+IGF-I, 62.3%; BDNF+NGF, 56.3%; and BDNF+IGF-I, 46.0%. Significant differences in neural differentiation were evident for single growth factor and combination of growth factors respectively (p<0.05).

Conclusion

Combinations of growth factors have an additive effect on neural differentiation. The most prominent neural differentiation results from growth factor combinations involving bFGF and IGF-I. These findings suggest that the combination of a mitogenic action of bFGF and postmitotic differentiation action of IGF-I synergistically affects neural proliferation and NSC differentiation.     

Regulation of Rat Schwann Cell Po Expression and DNA Synthesis by Insulin-like Growth Factors In Vitro

Myelination by Schwann cells is likely to be regulated in Vitro by positive and negative epigenetic factors. In Vitro , the positive regulation of myelin differentiation, in particular expression of the major myelin protein P_o, can be mimicked by cAMP elevating agents, while serum, transforming growth factor (TGF)βs, and fibroblast growth factor (FGF)2 have been shown to exert a negative effect on this differentiation. Growth factors which promote P_o induction have not, however, been identified previously. Using a forskolin concentration (0.4 μM) which alone produces little P_o mRNA or protein induction, we show that insulin-like growth factor (IGF)-I, IGF-II and high concentrations of insulin promote high levels of P_o induction, although in the absence of forskolin they have no effect. Another event related to Schwann cell differentiation, induction of galactocerebroside expression in response to cAMP analogues, is also potentiated by IGFs. In a different context, IGFs regulate Schwann cell DMA synthesis. We find that in defined medium forskolin plus FGF2, TGFβ or platelet-derived growth factor (PDGF) BB causes minimal DNA synthesis in the absence of IGFs and that IGFs act as potent mitogens under these conditions. IGFs also potentiate DNA synthesis induced by β isoforms of neu-differentiation factors (NDFs), although in this case considerable DNA synthesis occurs even in the absence of IGF. These results show that IGFs can act as powerful stimulators of both proliferation and differentiation in Schwann cells, and that the total growth factor input determines which of these pathways IGFs will promote.     

Cultured neurons release an inhibitor of astroglia proliferation (astrostatine)

Using in vitro techniques, we looked for a possible downregulation of rat astroglia proliferation by neuronal cells. We demonstrate that medium conditioned by 7-day-old rat cerebellar granule neurons or by 16-day-old rat embryo hippocampal neurons strongly inhibits the proliferation of cultured astroglial cells. Two neuronal cell lines, the PC12 rat pheocromocytoma and the neuro 2A (N2A) murine neuroblastoma also release such an activity. This release in N2A-conditioned medium (CM) occurs when the cells are at high density and show a low proliferation rate. This activity is present in media conditioned by neuronal cells, but not in media conditioned by normal astrocytes, by two glioma cell lines, or by one fibroblastic cell line. This proliferation inhibitor addresses normal astrocytes: the proliferation of two glioma cell lines, of a fibroblastic cell line, and of the two neuronal cell lines (PC12, N2A) is not inhibited by N2A CM. Moreover, this activity is directed against type 1 astrocytes, but not against type 2. Using three different assays, we demonstrate that DNA synthesis by astroglial cells is inhibited. N2A CM has no cytotoxic effect on astrocytes and does not modify their overall protein synthesis. Using affinity and gel filtration chromatography, we show that this activity is associated with a protein whose molecular weight ranges between 15 and 20 kDa. The possible relationship between this N2A cell-derived astroglia proliferation inhibitor and other types of potential glial proliferation inhibitors has been investigated. A brain glycoprotein immunologically related to epidermal growth factor receptor (EGFR) was reported to inhibit astroglial cell proliferation in vitro. Using polyclonal and monoclonal antibodies against EGFR, we were unable to immunoprecipitate the astrocyte proliferation inhibitor in N2A CM or to demonstrate by immunoblotting the presence of an EGFR-like immunoreactivity in the N2A CM or in the active chromatographic fractions of N2A CM. Transforming growth factor beta (TGF beta) is a well-known modulator of the proliferation of various cell types and was shown to be present in N2A CM. Using a polyclonal anti-TGF beta antibody that recognizes TGF beta on Western blots of N2A CM, we were unable to immunoprecipitate the astrocyte proliferation inhibitor of N2A CM. It seems thus far that the neuronal astroglia proliferation inhibitor is a new protein for which we propose the name astrostatine.     

Growth Factors as Mediators of Exercise Actions on the Brain

Physical exercise has long been recognized as highly beneficial for brain and body health. The molecular mechanisms responsible for translation of exercise stimuli in the brain have claimed attention due to mounting evidence for the neuroprotective actions of the exercise and its positive effects in preventing both ageing and neurodegenerative disease. These molecular mediators are currently under investigation with new tools able to yield deep insights into the neurobiology of exercise. In the present work we focus on the evidence pertaining to the mediation of exercise effects by insulin-like growth factor 1 (IGF1), as recent reports suggest that this growth factor shows brain area-specific, temporal rank-sensitive, and behavioural task-dependent features in response to exercise.