骨髓间充质干细胞与同种异体脱钙骨基质组合生长因子诱导向软骨细胞分化*

背景：用组织工程学方法高质量修复关节软骨缺损并达到很好的远期疗效目前尚无定论。鉴于此,课题组提出“同种异体骨髓间充质干细胞关节腔内定向培养组织工程软骨”的实验设技。 目的：同种异体脱钙骨基质组合转化生长因子β1和胰岛素样生长因子Ⅰ诱导骨髓间充质干细胞向软骨分化的能力,同时探索其在关节腔内培养促进向关节软骨定向分化的方法。 方法：分离兔骨髓间充质干细胞并行体外培养,分为两组：实验组DMEM培养液中加入转化生长因子β1和胰岛素样生长因子Ⅰ,对照组中未加入诱导因子。比较两组细胞的增殖情况和成软骨分化情况。制备同种异体脱钙骨基质支架材料,实验组骨髓间充质干细胞负载到同种异体脱钙骨基质中,构建组织工程软骨复合体,取另2只兔的腰背筋膜包裹后缝合固定到30只兔膝关节腔内行腔内培养。分别于植入后4,8,12周各取10只标本进行组织学切片观察及Ⅱ型胶原免疫组织化学观测,并对结果进行分析。 结果与结论：实验组中骨髓间充质干细胞集落形成效率明显高于对照组(u=3.326,P < 0.01)。实验组细胞爬片做Ⅱ型胶原免疫组化检测呈阳性,对照组未见阳性细胞。组织工程复合体在腔内培养12周后,苏木精-伊红染色见大量软骨细胞增生,胞核染色呈蓝色;甲苯胺蓝染色见软骨细胞成串排列,大量软骨陷窝形成,周围大量基质包绕;Ⅱ型胶原免疫组织化学反应见细胞外基质中出现大量棕黄色颗粒,Ⅱ型胶原染色强阳性。提示转化生长因子β1和胰岛素样生长因子Ⅰ可显著促进骨髓间充质干细胞增殖和成软骨分化,骨髓间充质干细胞与同种异体脱钙骨基质结合后可在关节腔内成功培养出组织工程软骨。同种异体脱钙骨基质符合组织工程软骨支架材料的基本要求。     

胰岛素样生长因子1、血小板源性生长因子、转化生长因子β1对

背景：生长因子可调整椎间盘细胞的增殖、分化及基质代谢,用于椎间盘退变的生物学治疗,既往的研究偏重于成骨性生长因子对髓核细胞的作用,对纤维环细胞的研究相对较少。 目的：观察体外培养条件下胰岛素样生长因子1、血小板源性生长因子、转化生长因子β1对人退变纤维环细胞生物学活性的影响。 设计、时间及地点：对照观察实验,于2007-11/2008-12在解放军南京军区福州总医院完成。 材料：腰椎间盘手术患者的纤维环组织。 方法：结合酶消化法和组织块培养法,体外单层培养人退变纤维环原代细胞。传代后通过免疫组织化学染色鉴定细胞。对传3代细胞分别采用不同生长因子干预,分为 100 μg/L胰岛素样生长因子1组、10 μg/L 血小板源性生长因子组、10 μg/L 转化生长因子β1组、100 μg/L 胰岛素样生长因子 1+ 10 μg/L 血小板源性生长因子组、100 μg/L 胰岛素样生长因子1+ 10 μg/L 转化生长因子β1组、10 μg/L 血小板源性生长因子+10 μg/L 转化生长因子β1组、100 μg/L 胰岛素样生长因子1+10 μg/L血小板源性生长因子+10 μg/L 转化生长因子β1组,以不加生长因子为对照组。 主要观察指标：免疫组织化学染色观察传3代细胞。干预3,6 d后,采用MTT法测定传3代细胞增殖,ELISA法测定细胞培养上清液中Ⅰ、Ⅱ型胶原和聚集蛋白聚糖质量浓度。 结果：传3代细胞Ⅰ、Ⅱ型胶原免疫组织化学染色阳性。胰岛素样生长因子1促进人退变纤维环细胞增殖,轻度抑制细胞合成Ⅰ型胶原,促进合成Ⅱ型胶原和聚集蛋白聚糖,促Ⅱ型胶原合成作用轻度强于转化生长因子β1。血小板源性生长因子促进细胞增殖,作用强于胰岛素样生长因子1,其抑制细胞合成Ⅰ型胶原,轻度促进合成Ⅱ型胶原,无促合成聚集蛋白聚糖作用。转化生长因子β1抑制细胞增殖,促进合成Ⅰ、Ⅱ型胶原及聚集蛋白聚糖,促聚集蛋白聚糖合成作用强于胰岛素样生长因子1。多种因子联合作用较单种未见明显优势,未能呈现协同效应。 结论：胰岛素样生长因子1、血小板源性生长因子、转化生长因子β1明显改变人退变纤维环细胞的生物学活性,可应用于对人类椎间盘退变的生物学治疗。     

胰岛素样生长因子1对人脂肪来源的间充质干细胞向软骨细胞定向诱导分化的作用☆

背景：近年研究者发现胰岛素样生长因子1还可以诱导骨髓来源间充质干细胞向软骨细胞方向分化,但目前尚未见胰岛素样生长因子1诱导脂肪来源间充质干细胞向软骨细胞方向分化及在此过程中与转化生长因子β1相互作用的报道。 目的：观察胰岛素样生长因子1诱导脂肪来源间充质干细胞向软骨细胞定向分化的可能性及在诱导分化中和转化生长因子β1的相互作用。 方法：获取脂肪来源间充质干细胞,以2×105 cells/cm2的密度接种于培养瓶,使用含胰岛素样生长因子1或(和)转化生长因子β1的无胰岛素软骨诱导剂诱导脂肪来源间充质干细胞。2周后获取细胞,制备细胞爬片,进行甲苯胺蓝染色和Ⅱ型胶原免疫组织化学染色,观察细胞内高硫酸化的蛋白聚糖和Ⅱ型胶原着色情况。RT-PCR检测Ⅱ型胶原蛋白、aggrecan及Sox9 mRNA的表达。 结果与结论：加入诱导剂后,甲苯胺蓝染色显示3个诱导组细胞呈多角形,胞浆及胞膜呈蓝色异染。Ⅱ型胶原免疫组织化学染色显示3个诱导组细胞胞浆及胞膜呈棕黄色着色。RT-PCR检测显示胰岛素样生长因子+转化生长因子组Ⅱ型胶原蛋白、aggrecan、Sox9 mRNA的表达均显著强于胰岛素样生长因子组和转化生长因子组,胰岛素样生长因子组和转化生长因子组显著强于对照组,而胰岛素样生长因子组与转化生长因子组差异无显著性意义。提示胰岛素样生长因子1可以单独诱导脂肪来源间充质干细胞向软骨细胞分化,表达软骨细胞特异性细胞表型,胰岛素样生长因子1与转化生长因子β1诱导脂肪来源间充质干细胞向软骨细胞分化有协同作用。     

威灵仙干预体外培养兔膝关节软骨细胞增殖及转化生长因子β1 mRNA基因的表达

背景：有研究显示,威灵仙能够维持和促进软骨细胞合成蛋白多糖与Ⅱ型胶原,并可能通过抑制白细胞介素1水平的增高保护关节软骨,延缓骨关节炎的发展。 目的：在以往研究基础上,观察中药威灵仙对体外培养兔膝关节软骨细胞增殖及转化生长因子β1 mRNA基因表达的影响,探讨其治疗骨关节炎的作用及可能机制。 方法：取新西兰白兔膝关节软骨,剪碎后,采用酶消化法原代分离培养软骨细胞,甲苯胺蓝染色鉴定后,取处于对数生长期第3代细胞随机分组,实验组分别加入0.01,0.05,0.1,0.5,1.0 g/L威灵仙培养基,对照组仅加入普通培养基进行培养。倒置显微镜下观察原代培养的软骨细胞形态变化及甲苯胺蓝染色鉴定, MTT法检测不同质量浓度威灵仙对软骨细胞增殖影响,RT-PCR法检测转化生长因子β1 mRNA表达。 结果与结论：原代软骨细胞呈圆球形,悬浮状态,24 h后大部分细胞贴壁;传代后软骨细胞生长速度加快,融合成片,外观呈典型“铺路石”状;传4代后逐渐出现长梭形软骨细胞,传代培养至6代时,呈成纤维细胞样外观,生长速度明显减慢,传代周期延长。软骨细胞经甲苯胺蓝染色后细胞外基质可见蓝色异染颗粒,细胞核染成深蓝色。不同质量浓度威灵仙均能促进软骨细胞增殖,尤以0.5 g/L增殖效果明显,且增殖高峰出现在威灵仙干预后的第3天。0.05,0.1,0.5,1.0 g/L威灵仙均能促进软骨细胞转化生长因子β1 mRNA 表达,4组组间比较差异无显著性意义(P > 0.05),但作用的高峰为0.5 g/L组。威灵仙能促进软骨细胞增殖及转化生长因子β1 mRNA的表达,提示这可能是其治疗骨关节炎的作用及可能机制之一。     

兔骨髓间充质干细胞体外培养定向诱导分化为软骨细胞

背景：骨髓间充质干细胞具有分化为骨、软骨、肌腱、脂肪等组织的多分化潜能。 目的：体外培养兔骨髓间充质干细胞并定向诱导分化为软骨细胞,探讨体外诱导分化为软骨的方法和条件。 方法：取兔股骨骨髓,全骨髓贴壁法分离培养骨髓间充质干细胞,碱性成纤维生长因子体外培养后,取第3代细胞在培养基中添加不同质量浓度转化生长因子β1的软骨分化诱导剂,2周后倒置显微镜观察细胞形态,甲苯胺蓝染色,RT-PCR技术检测Ⅱ型胶原和聚集蛋白聚糖的表达。 结果与结论：可以从兔骨髓中培养出骨髓间充质干细胞,碱性成纤维生长因子能明显的促进骨髓间充质干细胞增殖。经软骨诱导分化后骨髓间充质干细胞呈软骨细胞形态,甲苯胺蓝染色阳性,Ⅱ型胶原和聚集蛋白聚糖的表达阳性。10 μg/L的转化生长因子β1诱导成软骨分化能力最强。     

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。     

转化生长因子β1诱导骨髓间充质干细胞向软骨细胞分化*

背景：转化生长因子β1是关节软骨组织工程研究的首选生长因子，适当浓度能刺激关节软骨细胞增殖、分裂和分化。 目的：建立含有转化生长因子β1的特殊诱导体系培养条件下骨髓间充质干细胞分化为软骨细胞的能力，观察诱导后的细胞形态及表型变化。 方法：于兔胫骨结节内侧抽取骨髓，采用贴壁培养法分离纯化兔骨髓间充质干细胞，取第3代骨髓间充质干细胞行流式细胞仪检测鉴定其表面抗原，以含有转化生长因子β1的特殊软骨诱导体系的培养条件下对第3代骨髓间充质干细胞诱导培养21 d，诱导后与人鼻中隔软骨细胞进行比较。采用免疫组织化学法对Ⅱ型胶原进行定性检测。 结果与结论：贴壁培养法可分离并纯化兔骨髓间充质干细胞，所得第3代骨髓间充质干细胞表面抗原CD44 阳性，CD34、CD45 阴性。经诱导培养21 d后细胞形态变为不规则，Ⅱ型胶原免疫组织化学染色显示可见阳性细胞。提示含有转化生长因子β1的特殊软骨诱导体系的培养条件下，骨髓间充质干细胞可以转化为软骨细胞，且与正常软骨细胞无明显差异。     

壳聚糖和软骨细胞外基质复合组织工程骺软骨支架的性能观察***◇

背景：在组织工程骺软骨构建过程中,采用何种载体承载已经扩增了的软骨细胞越来越受到人们的关注。 目的：探讨软骨细胞外基质与壳聚糖复合构建组织工程骺软骨支架的可行性并对其性能进行初步观察。 设计、时间及地点：体外观察实验,于2007-12/2009-03在解放军总医院骨科研究所完成。 材料：壳聚糖(脱乙酰度90%,Mr10×105)由青岛海汇生物工程公司提供。市售猪关节软骨。 方法：将新鲜的猪关节软骨在液体中粉碎,梯度离心后制备成3%软骨微丝悬液,将其按1︰1的比例与2%的壳聚糖溶液混合,采用冷冻冻干法制备脱细胞软骨基质三维多孔支架。 主要观察指标：对梯度乙醇处理再次干燥后支架材料进行组织学及扫描电镜观察,测定支架孔径和孔隙率、吸水率,并采用MTT 法分析支架浸提液毒性。分离培养兔骨髓间充质干细胞,用转化生长因子β1 成软骨诱导后种植至支架,倒置显微镜、电镜观察细胞在支架上的生长、分化情况。 结果：支架内孔洞相互连通,孔大小(161±31) μm,孔隙率为(90.1±1.6)%,吸水率为(2 361±132)%。组织学观察显示,三维多孔支架甲苯胺蓝染色、番红O染色、Ⅱ型胶原免疫组织化学染色均呈阳性。MTT 法结果显示支架无细胞毒性。细胞在支架表面黏附良好,分布均匀,并有基质分泌。 结论：制备的软骨细胞外基质和壳聚糖复合三维多孔支架结合良好,含有软骨细胞外基质主要成分,无毒,具备合适的孔径和孔隙率,生物相容性良好。     

碱性成纤维细胞生长因子对离心管内培养骺板细胞生成软骨组织的作用**☆

背景：采用离心管技术体外培养骺板细胞的报道已很多。 目的：观察碱性成纤维细胞生长因子对离心管内培养的骺板细胞生成类骺板组织的影响。 方法：获取3周龄新西兰白兔股骨远端的骺板组织，利用组织块纱巾培养法获得骺板细胞，加入含有10 μg/L碱性成纤维细胞生长因子的DMEM培养液，连续培养4周。 结果与结论：离心管内聚集的骺板细胞在含有碱性成纤维细胞生长因子的DMEM培养液中形成类骺板样组织块。在组织块外周形成类似骺软骨的生发层，中心的骺板细增殖情况良好，向肥大细胞方向分化。类骺板样组织甲苯胺蓝及番红“O”染色阳性，Ⅱ型胶原免疫组织化学染色呈强阳性。说明碱性成纤维细胞生长因子能够促进离心管中的骺板细胞形成富含蛋白聚糖及Ⅱ型胶原等细胞外基质的类骺板样软骨组织。     

兔脂肪干细胞体外构建组织工程软骨：负载胰岛素样生长因子1基因的作用

背景：前期研究证实在二维培养条件下,转染胰岛素样生长因子1基因能够明显促进脂肪间充质干细胞的增殖。 目的：在前期研究基础上,利用壳聚糖明胶复合物为立体培养的载体,观察三维立体培养条件下胰岛素样生长因子1基因转染对兔脂肪间充质干细胞增殖能力的影响,体外初步构建组织工程软骨。 方法：分离、培养成年新西兰大耳白兔脂肪间充质干细胞,构建稳定表达pcDNA3.1-IGF-1的细胞株,鉴定后接种于壳聚糖三维支架材料上,分组培养：空白对照组接种未转染基因的脂肪间充质干细胞,空载体组接种胰岛素样生长因子1诱导条件下培养的脂肪间充质干细胞,基因转染组接种稳定转染胰岛素样生长因子1基因的脂肪间充质干细胞,培养1周。扫描电镜观察各组细胞形态,MTT法绘制细胞增殖曲线,CM-DiL荧光标记后观察细胞增殖比率及细胞分布,DMMB法测定糖胺聚糖含量。 结果与结论：成功构建稳定表达pcDNA3.1-IGF-1的细胞株,转染后的细胞株在mRNA水平上获得胰岛素样生长因子1的表达,并成功翻译为蛋白。扫描电镜示各组细胞在支架上贴附、伸展良好,基因转染组细胞生长最为旺盛。MTT 及GAG检测结果提示,基因转染组增殖能力最强、糖胺聚糖最高(P < 0.01),荧光标记显示细胞分布均匀,存活率高(P < 0.05)。提示胰岛素样生长因子1基因转染联合三维立体培养能够有效促进兔脂肪间充质干细胞的增殖和软骨细胞外基质糖胺聚糖的分泌。     

Characteristics of the nerve growth factor receptors and a nerve growth factor receptor--nerve growth factor covalent complex

Nerve growth factor is a polypeptide hormone that is required for the normal growth and development of the embryonic sensory and sympathetic nervous systems. On these cells, there are two different receptors for the nerve growth factor. Recently, these receptors have been isolated from three cell types and shown to have essentially the same binding characteristics. Molecular weights for receptors from two of these cell types, embryonic sensory and rat pheochromocytoma cells, have been determined. In addition, the formation of a covalent nerve growth factor, nerve growth factor receptor complex, has been investigated on embryonic sensory and sympathetic neurons. The formation of this covalent complex containing 125I-beta nerve growth factor is prevented by the addition of excess unlabeled nerve growth factor or by the addition of sodium fluoride and dinitrophenol. This complex forms at 4 degrees, 22 degrees, and 37 degrees, indicating that it is occurring on the cell surface. A disulfide bond is involved in the formation of this covalent complex.     

Macrophage colony-stimulating factor in nerve growth factor preparations

Following a report that nerve growth factor preparations have granulocyte-colony-stimulating activity, we investigated the presence of colony-stimulating factors in 7s mouse submaxillary nerve growth factor and its subunits. Macrophage colonies were formed in mouse bone marrow cultures after exposure to preparations of 7s nerve growth factor, the gamma subunit, and, to a small extent, the alpha subunit; the beta subunit, which is responsible for the nerve growth function, did not stimulate colony growth. Furthermore, the esteropeptidase activity of the gamma subunit was not detected in preparations of macrophage colony-stimulating factor purified from the giant cell tumor (GCT) cell line. Immunoprecipitation of radiolabeled gamma subunit with a polyclonal antibody to L-cell macrophage colony-stimulating factor showed a protein band that could represent the gamma subunit of nerve growth factor. Separation of the macrophage activity from the esteropeptidase activity of the gamma subunit was accomplished on the basis of molecular size. Thus, macrophage colony-stimulating factor was a contaminant of nerve growth factor produced by the mouse submaxillary gland and copurified with the gamma subunit.     

Nerve growth factor and the neurotrophic factor hypothesis

The discovery of nerve growth factor (NGF) over 40 years ago led to the formulation of the “Neurotrophic Factor Hypothesis”. This hypothesis states that developing neurons compete with each other for a limited supply of a neurotrophic factor (NTF) provided by the target tissue. Successful competitors survive; unsuccessful ones die. Subsequent research on NTFs has shown that NTF expression and actions are considerably more complex and diverse than initially predicted. Even for NGF, different regulatory patterns are seen for different neuronal populations. As would be predicted by the “Neurotrophic Factor Hypothesis”, NGF levels critically regulate basal forebrain cholinergic neuron size and neurochemical differentiation. In contrast, the level of trkA, the NGF receptor, regulates these properties in caudate-putamen cholinergic neurons. Understanding NTF regulation and actions on neurons has led to their use in clinical trials of human neurological diseases. NTFs may emerge as important therapies to prevent neuronal dysfunction and death.     

Epidermal growth factor influences the neurotrophic/differentiating action of nerve growth factor

Exposure of naive PC12 cells, sympathetic neurons from rat superior cervical ganglia, and brain-derived septal neurons to epidermal and nerve growth factors simultaneously resulted in some alteration of cellular events induced by nerve growth factor alone. A more pronounced decline of catecholamine content, no additional change in acetylcholinesterase activity, and additive stimulation of RNA and protein syntheses were found in PC12 cells. Earlier elevation of the enzyme activity was observed in sympathetic but not in septal neurons. Epidermal growth factor appeared to support independently the same level of acetylcholinesterase activity in septal neurons as revealed for nerve growth factor during the first week and cell survival throughout 2 weeks of observation. The data obtained indicate that epidermal growth factor augments temporarily some effects of nerve growth factor, thus supporting the idea of an important role of mitogenic growth factors in neural development as complementary and/or substitutive regulators of nerve cell differentiation and survival.     

Identification of basic fibroblast growth factor as a cholinergic growth factor from human muscle

Dissociated embryonic chick ciliary ganglion cells in culture were used as a bioassay to isolate a cholinergic growth-promoting protein from extracts of autopsied adult human muscle. An active protein was purified after acid and salt precipitation of extract, cation exchange, molecular sieving, heparin affinity chromatography, and in some cases, SDS-PAGE. This protein increased levels of choline acetyltransferase activity and ACh synthesis with time in culture. The protein was identified as basic FGF by several criteria. It shared the high affinity for heparin and was the same approximate molecular weight, 18 kD, as basic FGF. Activity was removed from solution by antibodies specific for basic FGF. Recombinant human basic FGF was equally effective in stimulating CAT activity, but was not additive with our purified protein at saturating concentrations. Basic FGF was also found in extracellular matrix and conditioned medium from cultured embryonic chick muscle. The activity could be released from extracellular matrix by treatment with heparinase or high salt extraction. Basic FGF stimulates neurite outgrowth as well as the capacity for transmitter synthesis. Thus, basic FGF is present in embryonic and adult muscle and capable of acting as a growth regulator for cholinergic neurons.     

S-180 cells secrete nerve growth factor protein similar to 7S-nerve growth factor

The nerve growth factor protein (NGF) has been identified by biological assay and rocket immunoelectrophoresis in media conditioned by monolayers of mouse S-180 sarcoma cells, a transformed cell line of salivary gland origin. By utilization of a purification procedure designed to enrich for acid-dissociable, high-molecular-weight complexes similar to the 7S-NGF isolated from male mouse submaxillary gland, it has been determined that a significant portion of mouse sarcoma NGF is initially present as a complex at neutral pH with a molecualr weight indistinguishable from that of 7S-NGF. At pH 4.0 the mouse sarcoma NGF complex dissociates, and the active subunit can be isolated as a species with a molecular weight of less than 40,000. The induced dissociation at pH 4.0 of the mouse sarcoma NGF high-molecular-weight complex, as well as the complex's behavior in isoelectric focusing and sucrose gradient sedimentation is consistent with the hypothesis that 7S-NGF is packaged as a subunit containing protein intracellularly prior to secretion into the extracellular space. Moreover, the stability of the mouse sarcoma NGF complex at dilute concentrations is similar to that reported for the purified 7S-NGF complex.     

Fibroblast growth factor and hydrocephalus

The immunohistochemical localization of basic fibroblast growth factor (bFGF) was studied in ventricular ependyma and choroid plexus of aged-matched normotensive and spontaneously hypertensive rats at different ages using a polyclonal antibody against bFGF. The bFGF-like immunoreactivity was observed in brain ependyma and choroid plexus of young and old normotensive rats. However, a progressive loss of immunoreactivity was observed with age in spontaneously hypertensive rats, that was associated with a progressive cerebroventricular dilation. These results show a new neuroendocrine anomaly to be added to the many others previously observed in this rat strain, when they develop hydrocephalus as they age.