细胞外基质2
四、层粘连蛋白 laminin，LN
1.分布：存在于各种动物胚胎及成体组织 的各种基膜中，主要存在基膜的透明层 紧贴细胞基底的表面。
2.结构：是由一条重链（A）和两条轻链 （B1、B2）借二硫键交联成不对称十字 形 分 子 ， 重 链 约 400000 ， 轻 链 约 21000物。
一、胶原(Collagen)
1．胶原的分布、结构及类型 （1）胶原的分布
存在于各种动物的组织、器官内，是细胞外基 质的骨架结构，约占人体总蛋白的30%；腱、 骨、软骨中丰富，刚性、抗张力强度大。
（2）组成：
由原胶原交联而成，原 胶原是三条α肽链形成的 超螺旋纤维状蛋白质，原 胶原是杆状分子，长 300nm，直径1.5nm； 每条α链一般由1000AA 组成；各类胶原蛋白的氨 基酸组成完全不同。
（3）影响细胞迁移
– 早期神经脊细胞的迁移；胚胎发育过程中有多种细胞 要发生迁移以便完成个体发育，FN在细胞的迁移中担 负有重要任务。
– 在创伤修复中，纤粘连蛋白促进巨噬细胞和其它免疫 细胞迁移到受损部位；
– 在血凝块形成中，纤粘连蛋白促进血小板附着于血管 受损部位；
– 癌细胞扩散中，切断FN或消化之，破坏FN形成的网络， 癌细胞通过血液或淋巴系统漂移出去迁移到其它部位。
2．胶原的生成及降解 P139-141
1）合成：主要由成纤维细胞（成软骨细胞、 成骨细胞）和其它一些细胞（如上皮细胞）分 泌。一般胶原的生成分为细胞内和细胞外两个 阶段。
胶原基因进行转录后需进行大量而精确的剪 接才能生成胶原肽链的mRNA，从mRNA翻译 出的肽链还必须通过复杂的修饰过程才能变成 功能完善的胶原分子。
（3）胶原的类型 p138
已知20种胶原，不同类型的胶原及同类型的 胶原的不同α链都分别由不同基因编码，这些基 因在进化上是相关的，结构上各具特点。
在不同组织或同一组织的不同分化阶段表达； 不同的胶原定位在特定组织，也有多种胶原存 在于同一种组织，但常以某种为主。
不同胶原具有不同的化学结构和免疫特性。
• 合成装配主要过程：前体肽链—前胶原— 胶原—胶原原纤维—胶原纤维
2）胶原的降解
胶原的三股超螺旋结构对一般蛋白水解酶有较 强的抵抗性，只有经胶原酶水解后，才能被蛋 白水解酶作用。
胚胎发育、组织分化以及创伤愈合过程中，发 生胶原类型的转换就是借助组织胶原酶清除原 有胶原，再由分化细胞产生新胶原。
胶原酶在组织中分布广泛常以无活性形式存在， 无活性的胶原酶究竟是酶原或是与抑制剂的复 合物尚无定论。
3.胶原的生物学功能
(1)参与形成结缔组织，为腱、角膜、骨、皮肤组 织中的主要蛋白，为细胞外基质提供水不溶性的 骨架结构，决定细胞外基质的机械性能（如刚性、 抗张力强度）。
(2)其它组分通过与胶原结合形成结构与功能的复 合体。
3.FN的生成和组装
• 来源：血浆FN主要来自肝实质细胞或血管内 皮细胞；细胞外基质及细胞表面FN主要来自 中胚层演变的细胞。
• FN的结构基因似乎只有一个，其mRNA的多 样性是由前体mRNA剪接上的差异所造成的。 此外翻译后修饰也是原因之一，结果形成不同 的FN亚单位。
• 与胶原不同，FN不能自发组装，而是通过 细胞表面受体指导进行，只存在于某些细胞 （如成纤维细胞）表面。
• 常以罗马数字表示不同的胶原类型。I、II、III型胶原含 量最丰富，形成类似的纤维结构，但并非所有胶原都形 成纤维结构。P138表4-3
I型胶原多分布在皮肤、肌腱、韧带和骨中，抗张强度 较大； II型胶原多分布在软骨中； III型胶原形成微细的原纤维网，广泛分布于伸展性的组 织，如疏松结缔组织； Ⅳ型胶原形成二维网格样结构，是基膜的主要成分及骨 架。
第二节 细胞外基质
extracellular matrix，ECM
• ECM是分布于细胞外空间，由细胞合成、分泌 的蛋白质和多糖组成的网络。
• 包括：①结构蛋白，如胶原和弹性蛋白，它们 赋 予 基 质 一 定 的 强 度 和 韧 性 ;② 黏 着 蛋 白 (adhesive proteins)，如纤维粘连蛋白和层粘 连蛋白，它们促使细胞同基质结合;③蛋白聚糖 (proteoglycan)。
2.弹性蛋白的结构
弹性蛋白的 结构模型
组织的弹性通过改变散布在弹性纤维中的胶原的数量来控制； 弹性蛋白的生物合成及加工了解不多，降解由弹性蛋白酶完成。 老年组织中弹性蛋白的生成减少，降解增强，组织失去弹性。
三、纤粘连蛋白 Fibronectin，FN
1.分布和类型：分布广泛，从海绵、海胆—哺乳类 血浆FN：V形二聚体亚基相似，可溶，血浆、体液 细胞FN：多聚体亚基来源不同，不溶，细胞表面 已鉴定的FN亚单位20种以上。由同一基因 编码，转录后剪接不同，形成不同的mRNA； 翻译后修饰也有差异，形成多种异型分子。
• ECM将细胞粘连在一起构成组织，同时提供一 个细胞外网架，主要起连接、支持作用。
• ECM在结缔组织中含量较高，如骨、软骨组 织、血管中的结缔组织。
• ECM的成分及组装形式由所产生的细胞决定， 并与组织的功能相适应。
• ECM影响细胞的形态和功能活性、存活、死 亡、增殖和分化以及信号转导和形态发生。
• FN降解依靠FN水解酶，该酶由正常细胞合 成，没有活性。
4.FN的生物学作用
（1）介导细胞粘着：FN可同其在细胞膜上的受体结合，或 将细胞连接到ECM。由于FN具有同时与细胞外基质各类成 分相结合的特点，并可促进细胞外基质的其它成分的沉积， 故认为FN是细胞外基质的组织者。
（2）控制细胞的生长与分化：FN作为生长 因子，可使细胞快速铺展，加速增殖；或 抑制某些细胞的分化与去分化。
(3)控制细胞的形态，促进细胞生长，诱导细胞分 化。促进细胞生长；胶原基质和提纯的胶原底物 具有维持并诱导细胞分化的作用。
成纤维细胞周围的胶原纤维
二、弹性蛋白 elastin
1.分布
• 在肺管壁和肺、心脏、韧带、皮肤、肌腱和 疏松结缔组织中含量多，是弹性纤维的主要 成分。
• 弹性蛋白常与胶原纤维共同存在，分别赋予 组织以弹性及抗张能力。弹性蛋白依靠伸张 与收缩，使得某些组织具有弹性。
08-4-胞外基质
11.4 细胞外基质同学好！大家很容易理解，在多细胞生物体内，每一个细胞作为细胞社会中的一个成员，彼此相互作用，需要一个和谐稳定的生活环境。
我们前几讲一起学习了细胞表面、细胞连接和黏附分子，依赖这些重要的结构和分子，细胞与细胞，细胞与环境之间发生着各种各样的细胞社会联系。
那么由近及远，细胞表面邻近的周边环境怎么样？都由哪些成分构成呢？这就是我们今天要讨论的主题：细胞外基质(extracellular matrix, ECM)。
它是由细胞分泌产生的多种蛋白质和多糖大分子构成的精密有序的结构网络，填充在细胞间隙并介导细胞社会联系，构筑起细胞赖以生存的外部微环境。
一、细胞外基质的主要成分细胞外基质主要是由一些蛋白质和多糖构成。
包括：（一）胶原蛋白(collagen)胶原是细胞外最重要的水不溶性纤维蛋白，是构成细胞外基质的骨架。
1、结构单位：胶原蛋白的基本结构单位是原胶原，原胶原肽链的一级结构具有(Gly-X-Y)n重复序列，其中X常为脯氨酸，Y常为羟脯氨酸或羟赖氨酸，羟赖氨酸可发生糖基化修饰，糖在胶原中约占10%。
2、结构模式：原胶原是由三条α肽链盘绕而成的三股螺旋纤维结构，长约300nm，直径仅为1.5nm左右，原胶原进一步交联成原纤维，继而可形成直径达数微米的胶原纤维，形同绳索，其中原胶原的三股螺旋是维系胶原稳定和坚韧的重要结构基础。
3、主要类型：目前已发现胶原为类型有10多种，最主要有是I、II、III、IV 型胶原。
其中前三种为纤维状，广泛分布于各种组织的细胞外基质中。
IV型胶原则不形成螺旋纤维，而以分子头对头相接的方式形成三聚体，再相互交联成网络层结构，是各种基膜的重要构造。
胶原与多种疾病的发生和病理过程有关。
例如：过去远洋航海船员因维生素C缺乏引起的坏血病，就与胶原异常有关。
现已证明，当机体缺乏维生素C时，原胶原中的脯氨酸、赖氨酸不能有效羟基化，难以形成稳定的螺旋结构，导致组织中胶原减少，严重时引起血管脆弱，表现渗血出血倾向，称为坏血病。
陈红丽-第五章 细胞连接、细胞黏着和细胞外基质(二)
第五章 细胞连接、细胞黏着 和细胞外基质
授课教师：陈红丽 副教授 E-mail：chenhl@
目
第五章
录
细胞连接、细胞黏着 和细胞外基质
第一节 细胞连接
第二节 细胞黏着和细胞外基质
医学细胞生物学
2
第二节 细胞外基质
细胞外基质(extracellular matrix,ECM)是个体 发育过程中，由细胞合成并分泌到细胞外的蛋白 质和多糖所构成的网络结构。在结缔组织中含量 最丰富，主要由成纤维细胞所分泌。 不同结缔组织中细胞外基质的分子类型和组织方 式不同，以适应不同的生理功能。
医学细胞生物学
33
思考题
1、名词解释：（extracellular matrix，ECM )，
糖萼 2、试述ECM的化学组成和功能。 3、简述胶原或非胶原糖蛋白与疾病的关系，胶原 在生物医学中的应用？
医学细胞生物学
34
参考文献及网站
1. 翟中和等主编. 细胞生物学，第3版,北京，高等教育出 版社，2007 2. 杨保胜，孙银平.基础医学概要(四).人民卫生出版社， 2009 3. （美）B.Alberts等著(张新跃，钱万强等译) ．细胞的分 子生物学[M]．北京：科学出版社，2008 4. 韩贻仁主编. 分子细胞生物学(第3版),高等教育出版 社,2007 5. 细胞生物学专业信息网 / 6. 新乡医学院精品课程 /yj.asp 7. 细胞生物学在线 /
动脉粥样硬化患者：血管内皮细胞表面硫酸皮肤素PG↑，导 致脂类沉积。 肝硬化和肝癌患者血中HA含量明显升高。
医学细胞生物学
20
四、非胶原糖蛋白
非胶原糖蛋白(粘着成分)使细胞与 ECM相互粘 着，同时介导细胞运动迁移，在细胞分化和创伤 修复中起重要作用。对细胞的存活、形状、粘着、 铺展、迁移、增殖、分化有直接影响。 这一大类非胶原糖蛋白已发现数十种，如纤 连蛋白(fibronectin, FN)和层粘连蛋白 (laminin, LN)，它们都是多功能大分子，具有 分别与细胞及细胞外其他成分结合的多个结构域， 是 ECM 成分的组织者。
细胞质基质2
三、高尔基体与细胞内的膜泡运输
高尔基体在细胞内膜泡蛋白运输中起重要的枢纽作用
膜泡运输的主要途径, 膜泡运输的主要途径,其中多数与高尔基体直接相关
第四节 溶酶体与过氧化物酶体
一、溶酶体的结构
溶酶体（lysosome）：是单层膜围绕、内含多种酸性水解酶 类的囊泡状细胞器，其主要功能是进行细胞内消化。 C. de Duve与B. Novikoff 1955年首次发现。几乎存在于所有 的动物细胞中。 具有异质性（heterogenous），形态大小及内含的水解酶种 类都可能有很大的不同。执行不同生理功能。 共同特征：含有大量酸性水解酶，酸性磷酸酶是标志酶。 膜有质子泵，将H+泵入溶酶体，使其PH值降低。 膜蛋白高度糖基化，可能有利于防止自身膜蛋白降解 内有多种载体蛋白可将水解的产物向外转运。
Figure 13-20 The plant cell vacuole. This electron micrograph of cells in a young tobacco leaf shows that the cytosol is confined by the enormous vacuole to a thin layer, containing chloroplasts, pressed against the cell wall. The membrane of the vacuole is called the tonoplast. (Courtesy of J. Burgess.)
3、残体（residual body） 残体（ body） 又称后溶酶体（post-lysosome）已失去酶活性，仅 留未消化的残渣，故名。残体可通过外排作用排出细胞， 也可能留在细胞内逐年增多，如表皮细胞的老年斑，肝细 胞的脂褐质。
14_细胞外基质-2008.5-I
(一)
胶 原（collagen）
分布（Distribution)与类型(Category) 分布（Distribution) 不同的结缔组织其结构和功能的不同， 主要是由于胶原含量、类型及排布方式不 同所决定的 产生的细胞：以间质型细胞为主，如成 纤维细胞、成骨细胞及成软骨细胞等
(一) 胶 原（collagen）
病理 创伤的修复、肿瘤转移、肾病、肝病、肺 病、胶原病、心血管病、骨关节病及糖尿病等。
ECM结构示意图
第一节 细胞外基质的分子组成
(一) 胶原 collagen (一) 胶原 collagen
(一) 非胶原糖蛋白 (二) 胶原 collagen non-collagenous glycoprotein
胶原生成的细胞内阶段
主要包括羟化和糖基化修饰。
羟化修饰
前链在内质网进行翻译中和翻译后的修饰，
体内缺乏维生素C,则正常体温下的前胶原的 羟化不足，因而不能形成稳定的三股超螺旋
膳食中缺乏维生素C所导致的坏血病出现血 管、肌腱、皮肤变脆、易出血等症状即与其 所致胶原结构的异常有关。
胶原生成的细胞内阶段
(三) 弹性蛋白 elastin
(四) 氨基聚糖与蛋白聚糖 glycosaminoglycan, GAG & proteoglycan, PG
(一) 胶
原
分布与类型(Category) 分子结构(Elementary structure) 胶原的生物合成过程(Biosynthesis) 胶原的降解过程(Degradation)
V
VI VII VIII IX X
含有I型胶原组织中的次要组 成成分
大多数结缔组织 表皮、小肠黏膜的锚定原纤 维 血管内皮和其他组织 含有Ⅱ型胶原的组织 增生软骨
细胞生物学 第十一章 细胞外基质及其与细胞
第十一章细胞外基质及其与细胞的相互作用细胞外基质（ECM）：是由细胞分泌到细胞外空间，由蛋白和多糖构成的精密有序的网络结构。
不仅对组织细胞起支持、保护、营养作用，而且还与细胞的增殖、分化、代谢、识别、黏着、迁移等基本生命活动密切相关。
糖胺聚糖（AGA）：是细胞外基质的主要成分，是由重复的二糖单位构成的直链多糖，过去称为黏多糖，其二糖单位之一是氨基己糖（N-乙酰氨基葡萄糖或N-乙酰氨基半乳糖），故又称氨基聚糖，另一个糖残基多为糖醛酸（葡萄糖醛酸或艾杜糖醛酸）糖胺聚糖可分为六种：透明质酸HA、硫酸软骨素CS、硫酸皮肤素DS、硫酸乙酰肝素HS、肝素、硫酸角质素KS蛋白聚糖（PG）：是由糖胺聚糖（除透明质酸外）与核心蛋白共价结合形成的高分子量复合物，是一种含糖量极高的糖蛋白。
黏多糖累积病：由于基因突变引起先天性缺乏降解糖胺聚糖的酶（如糖苷酶或硫酸酯酶）可导致糖胺聚糖或蛋白聚糖及其降解中间产物在体内一定部位堆积，造成黏多糖累积病如Hunter综合征。
胶原（collagen）：是细胞外基质中的骨架结构，动物体内高度特化的纤维蛋白家族，是人体内含量最丰富的蛋白质，遍布于体内各种器官和组织，在结缔组织中特别丰富，可由成纤维细胞、软骨细胞、成骨细胞以及某些上皮细胞合成并分泌到细胞外。
原胶原：典型的胶原分子呈纤维状，是由3条α多肽链盘绕而成的3股螺旋结构，称原胶原。
胶原合成与组装始于内质网，在高尔基体修饰，最后在细胞外组装成胶原纤维①前α链：在糙面内质网附着核糖体上合成，不仅含有内质网信号肽，而且在其N端和C端各含有一段不含Gly-X-Y序列的前肽。
②前胶原：胶原合成过程中带有前肽的3股螺旋胶原分子称为前胶原，其两端的前肽部分保持非螺旋卷曲。
③原胶原分子：在细胞外，前胶原在前胶原N-蛋白酶和前胶原C-蛋白酶的作用下，分别水解去除两端的前肽，在两端各保留一段非螺旋的端肽区形成原胶原分子。
④胶原原纤维：原胶原分子在细胞外基质中相互呈阶梯式有序排列并发生侧向交联，自组装成胶原原纤维。
细胞外基质
细胞外基质（Extracellular Matrix，ECM）是由成纤维细胞、间质细胞、上皮细胞等体内各类组织和细胞合成和分泌的一类散布和聚集在细胞表面和细胞间质的大分子物质所组成的复杂网络结构，故称细胞外基质（间质），是细胞和组织赖以生存、活动和调剂的外环境。
要紧作用：一方面为细胞和组织提供支持、联结、固定、保水、缓冲等物理性的爱惜作用，另一方面又是细胞与外环境进行物质互换、信息传递和聚集的中介。
它可通过各类信号传递系统，调剂细胞生长、增殖、迁移、分化、粘附、代谢、损伤修复、组织重构等各类生理功能。
被称为是人体细胞和组织内稳态的要紧调剂者（The Central Regulator of Cell and Tissue Homeostasis）。
细胞外基质的成份十分复杂，除各型胶原之外，还有各类粘连蛋白（FN）、层连蛋白（LN）、氨基聚糖（GAG）、蛋白聚糖（PG）、弹性蛋白（Elastin）、内动素（Cytotatin）、血栓结合素（Thrombospondin）、整合素（Integrin）、玻连蛋白（Vitronetin VN）、连结蛋白（Connexins）、钙粘素（Cadherins）、选择素（Selectin）、粘附素（细胞粘合素）、细胞粘合素（Cytotatin）等几十个类别。
每一种类别又有几种至十几种亚型。
细胞不同产生和分泌的细胞外基质成份亦不同；组织不同所含的细胞外基质的成份和比例亦不同；即便同一种细胞，同一种组织，在不同的生理、病理和反映条件下，细胞外基质的成份、结构和构型亦不同；结构和构型不同，细胞外基质的功能和作用亦不同。
随着基因和蛋白质组生物学的研究进展，新的细胞外基质分子还在不断诞生，其类型、构型、构像还有更多发觉，其功能亦在不断的扩展，组成了一个十分复杂的细胞外基质的网络家族和体系。
细胞外基质尽管来源、成份、分型和功能不同，各司其责，但在结构和功能上，它们又排列有序、疏密相间、彼此联结、彼此协同，在细胞间质、组织间隙和器官内，形成各类复杂的相对固定的形式和分层网状结构，形成许多不同的功能结构区域，如在血管，能够形成内膜表面的粘附爱惜层、内膜基层、基底膜层、内弹力层、外弹力层、血管中层和外层系膜结缔组织等等。
细胞外基质
羟赖氨酸 ；低糖
类 由二糖单位（D-葡萄糖醛酸和N-乙酰-葡萄糖胺）重复聚合而成。
[α1(Ⅲ)] 2α2（Ⅳ）
Ⅳ [α1(Ⅲ)] 基底层 原胶原共价交联后成为具有抗张强度的不溶性胶原。
③粘着成份，如纤连蛋白和层粘连蛋白等。
α—链中保留前肽； 基膜
2α2（Ⅳ） [α1（Ⅰ）] 2α2（Ⅰ）
2、弹性纤维
弹性纤维主要存在于脉管壁及肺，亦少 量存在于皮肤、肌腱及疏松结缔组织中。
弹性纤维与胶原纤维共同存在，分别赋 予组织以弹性及抗张性。
弹性蛋白(elastin)是弹性纤维的主要成分。
弹性蛋白
是一种非糖基化的蛋白质，包含830个氨基酸 残基，富含脯氨酸和甘氨酸，但很少羟化。肽 链中不含胶原特有的 Gly-X-Y重复顺序，形成 不规则螺旋结构，并互相交错成网络。
第六节 细胞外基质
组成细胞外基质成份种类 繁多、结构复杂，一般 可分为三类：
①氨基聚糖和蛋白聚糖； ②纤维蛋白，主要包括胶
原和弹性蛋白； ③粘着成份，如纤连蛋白
和层粘连蛋白等。 不同组织的细胞外基质成
份的结构存在着差异
一、氨基聚糖和蛋白聚糖
氨基聚糖（glycosaminoglycan, GAG） 是由重复的二糖单位聚合成的无分支长 链多糖，因二糖单位中一个常为氨基糖 （N-乙酰氨基葡萄糖或N-乙酰氨基半乳 糖）而得名。
眼睛玻璃体
几种主要胶原及其特征
②纤维蛋白，主要包括胶原和弹性蛋白；
Ⅲ [α1 原纤维 皮肤、血管 、 高羟脯氨酸 ；低 氨基聚糖（glycosaminoglycan, GAG）是由重复的二糖单位聚合成的无分支长链多糖，因二糖单位中一个常为氨基糖（N-乙酰氨基葡萄
11细胞外基质
A Proteoglycan Complex
蛋白聚糖
蛋白聚糖多聚体
氨基聚糖
蛋白聚糖
软骨中蛋白聚糖电镜图片
2.蛋白聚糖在内质网中合成并装配 • 核心蛋白在内质网相连核糖体中合成 • 核心蛋白质的Ser残基在内质网中装配上 GAG链。 • 首先合成由四糖组成的连接桥（Xyl-GalGal-GlcUA）连接到Ser残基上，然后在糖 基转移酶的作用下延长糖链。 • 硫酸化、异构化修饰 • 除HA及肝素外，其他GAG均不游离存在。
• 交联：由侧向相邻的lys或hyl残基氧化后所 产生的两个醛基间进行缩合而成。交联后
后形成不溶性纤维，原胶原呈阶梯状排列，
电镜下可见间隔67nm的横纹。
– 新生儿的胶原交联程度低而易于抽提。 – 年龄增加，胶原交联程度高，皮肤血管组织僵 硬老化。
Structures of the collagen
Collagen fibrils around a fibroblast
1.胶原分子结构 胶原是三条α多肽链形成的 三股螺旋，长300nm,直径 1.5nm,富含Gly,Pro和Lys 胶原的每条链由重复的GlyX-Y序列构成。（X=pro， Y=hyp或hyl） Gly-X-Y序列使α链卷曲为 左手螺旋。三股链再绕成右 手超螺旋。
2．蛋白聚糖 (proteoglycan)
1.结构： 由氨基聚糖（除HA）与核心蛋白(core protein)的 丝氨酸残基共价连接形成的巨分子。 若干蛋白聚糖单体借连接蛋白以非共价键与透明质 酸结合形成多聚体巨分子。 显著特点是多态性：不同的核心蛋白, 不同的氨基 聚糖； 软骨中的蛋白聚糖是最大巨分子之一, 赋予软骨以 凝胶样特性和抗变形能力； 蛋白聚糖可视为细胞外的激素富集与储存库，可与 多种生长因子结合，完成信号的传导。
细胞外基质的生物学功能和应用
细胞外基质的生物学功能和应用细胞外基质（extracellular matrix，ECM）是由细胞合成并分泌到周围环境中的一组复杂的蛋白质、多糖和小分子组成的结构。
细胞外基质的主要功能包括提供对细胞的支持、促进信号传递、调节细胞分化和增殖、参与细胞外基质重塑等。
在本文中，我们将详细介绍细胞外基质的生物学功能以及其在生物技术和医学应用中的价值。
细胞外基质的生物学功能1.提供对细胞的支持和结构细胞外基质是细胞及组织之间的接口，它将细胞连接成组织和器官。
过去，人们通常将细胞外基质视为静态的结构，但现在已经发现，它是一种动态的结构，具有调节细胞行为的重要作用。
细胞外基质中最重要的成分是胶原蛋白，它是一种纤维性蛋白质，质地坚韧。
除了机械支持之外，胶原蛋白还可以通过与细胞外基质中其他成分的相互作用，调节胞外基质的物理性质和化学性质，从而影响细胞的生理过程。
此外，细胞外基质中还有积累在地面物质（ground substance）中的大量的葡萄糖胺聚糖（glycosaminoglycans）。
这些多糖具有负电张和胶冻状态的特性，可以在形成透明质酸之前，提供一种有效的滑润层。
这可以保护细胞免受机械性损伤，并协助它们在经历的现实环境变化时维持其形态和生理功能。
2.促进信号传递除了为细胞提供支持之外，细胞外基质还具有信号传递的作用。
细胞外基质上存在着许多导向细胞行为和增殖的细胞外生信号分子，包括肽、糖和脂质等。
这些信号分子作用于细胞表面的受体，从而触发一系列的细胞信号级联反应，包括细胞增殖、分化和转移等。
此外，细胞外基质本身也可以促进信号传递。
例如，细胞外基质中的蛋白质可以与调节蛋白相互作用，从而改变它们的空间位置和功能，影响细胞的行为。
3.调节细胞分化和增殖细胞外基质可以通过许多不同的方式调节细胞的行为，其中增殖和分化是最为重要的两个方面。
细胞外基质可以激活或抑制某些信号通路，进而影响细胞的生长和偏向性。
另外，在某些时候，细胞外基质可以促进细胞分化，例如在骨骼生长相和软骨分化中。
细胞外基质
A new technique to expand human mesenchymal stem cellsusing basement membrane extracellular matrixTakehiro Matsubara,a,c,f Shinichi Tsutsumi,b Haiou Pan,a Hisatada Hiraoka,c Ryo Oda,d Masahiro Nishimura,e Hiroshi Kawaguchi,c Kouzou Nakamura,c and Yukio Kato a,f,*aJapan Science and Technology Corporation (JST),Chiyoda-ku,Tokyo 102-8666,JapanbDepartment of Orthopedic Surgery,School of Medicine,Gunma University,Maebashi 371-8511,Japan cDepartment of Orthopedic Surgery,Graduate School of Medicine,University of Tokyo,Tokyo 113-8655,JapandDepartment of Operative Dentistry,Graduate School of Biomedical Science,Hiroshima University,Hiroshima 734-8553,Japan eDepartment of Prosthetic Dentistry,Graduate School of Biomedical Science,Hiroshima University,Hiroshima 734-8553,JapanfDepartment of Dental and Medical Biochemistry,Graduate School of Biomedical Science,Hiroshima University,Hiroshima 734-8553,JapanReceived 12November 2003Abstractshort proliferative life span and readily lose the differentiation potential in culture.span of the stem cells markedly increased using tissue culture dishes coated with a which was produced by PYS-2cells or primary endothelial cells.Furthermore,the stem cells expanded on the extracellular matrix,but not those on plastic tissue culture dishes,retained the osteogenic,chondrogenic,and adipogenic potential throughout many mitotic divisions.The extracellular matrix had greater effects on the proliferation of MSC and the maintenance of the multi-lineage differentiation potential than basic fibroblast growth factor.Mesenchymal stem cells expanded on the extracellular matrix should be useful for regeneration of large tissue defects and repeated cell therapies,which require a large number of stem or progenitor cells.Ó2003Elsevier Inc.All rights reserved.Keywords:Mesenchymal stem cell;Basement membrane;Extracellular matrix;Regeneration;DifferentiationMesenchymal stem cells (MSC)can be induced to differentiate into a variety of tissues including bone,cartilage,tendon,fat,heart,muscle,and brain,in vitro and in vivo [1,2].Autologous MSC have advantages over ES cells:there is no teratocarcinoma formation,no immune rejection,and there are no ethical problems.However,compared with ES cells,which have an un-limited proliferative life span (period before the cells reach growth arrest in culture)and consistently high telomerase activity,MSC have very poor replicative capacity and short proliferative longevity [3,4].Thus,an important challenge in regenerative medicine is to im-prove the replicative capacity of MSC,thereby to obtain a number of MSC sufficient to repair large defects.Forced expression of telomerase in MSC markedlyincreases their proliferative life span and MSC with a high telomerase activity showed osteogenic potential [5].However,it is unknown whether these cells can maintain the chondrogenic and adipogenic potential or whether these cells have a risk of transformation.We report here that the growth rate and the proliferative life span of MSC markedly increased using tissue culture dishes coated with a basement membrane-like extracellular matrix (“bmECM”).Furthermore,MSC that expanded 106-fold on bmECM retained its osteogenic,chondro-genic,and adipogenic potential.Materials and methodsPreparation of bmECM-coated dishes .PYS-2cells,which produce laminin,type IV collagen,and heparan sulfate proteoglycans [6–8],were supplied by Dr.Atsumi (Riken,Wako,Japan),and bovine corneal endothelial cells were isolated and maintained as described [9].*Corresponding author.Fax:+81-82-257-5629.E-mail address:ykato@hiroshima-u.ac.jp (Y.Kato).0006-291X/$-see front matter Ó2003Elsevier Inc.All rights reserved.doi:10.1016/j.bbrc.2003.11.143Biochemical and Biophysical Research Communications 313(2004)503–508BBRC/locate/ybbrcBmECM-coated dishes were prepared according to the method of Dr. Gospodarowicz[10].The cells were seeded at2Â104cells/cm2on60-mm tissue culture dishes(Corning,Corning,NY)and maintained in 4ml of Dulbecco’s modified Eagle’s medium(DMEM)-Ham’s F12 medium(1:1)(Sigma,St.Louis,MO)in the presence of10%fetal bo-vine serum(Hyclone,Logan,Utah)and antibiotics(100U/ml penicillin G and100l g/ml streptomycin)(medium-A).Medium was changed every other day.Once the cultures became confluent,the media were renewed by4ml of medium-A supplemented with5%dextran (200,000Da,Wako,Osaka,Japan)and the cultures were further in-cubated for7days.Treatment of the cultures with20mM NH3resulted in cell lysis,exposing the extracellular matrix adhering to the substrata of tissue culture dishes.The substratum was washedfive times with PBS.Previous studies have shown that bmECM is composed of lami-nin,heparan sulfate,entactin,and type IV collagen[11,12].Preparation of laminin-coated dishes,type IV collagen-coated dishes, and ECM gel-coated dishes.Three milliliters of10mM NaHCO3 containing30l g/ml type IV collagen or30l g/ml laminin(Koken, Tokyo,Japan)was incubated in60-mm plastic tissue culture dishes at 4°C for12h.Two hundred micrograms per milliliter ECM gel solution (Sigma)was made by diluting with DMEM-high glucose.Three mil-liliters of the ECM gel solution was incubated in60-mm tissue culture dishes at4°C for12h.These concentrations of laminin,type IV col-lagen,and ECM gel were optimal for proliferation of MSC(data not shown).MSC culture.Human MSC were obtained from the ilium or the alveolar bone according to a protocol approved by ethical authorities at Hiroshima University.Cells in marrow aspirates(1ml/100-mm dish) were seeded on plastic tissue culture dishes.Passages were performed when cells were approaching confluence.Unless otherwise specified, MSC obtained from the primary cultures were seeded at1Â103cells/ cm2on60-mm of laminin-,type IV collagen-or ECM gel-coated dishes,on bmECM-coated dishes or on plastic tissue culture dishes, and cells were fed with DMEM-low glucose supplemented with10% fetal bovine serum and antibiotics(medium-B)every3days.Sub-sequent passages were performed on the appropriate substrata.In these studies,we seeded MSC at a low density(1Â103cells/cm2)to avoid frequent passages and the risk of contamination considering clinical application,although MSC showed a higher growth rate and a longer proliferative life span at a high seeding cell density(5Â103cells/ cm2)(data not shown).Differentiation.Chondrogenic,osteogenic or adipogenic conversion of MSC was determined according to the procedures reported by Pittenger et al.[1],with some modifications.For chondrogenic differ-entiation,cells were seeded at2.5Â105cells per15ml plastic centrifuge tube and maintained in0.5ml of serum-free a-MEM supplemented with3500mg/ml glucose, 6.25l g/ml insulin, 6.25l g/ml transferrin, 6.25ng/ml selenite,5.33l g/ml linolate,1.25mg/ml bovine serum al-bumin,10ng/ml transforming growth factor-b3,100nM dexametha-sone,and50l g/ml ascorbic acid-2-phosphate.The cultures were fed with0.5ml of the medium until3days after seeding.Thereafter,the cultures were fed with1ml of the medium every other day.Cells were cultured under the chondrogenic status for28days.For osteogenic differentiation,cells were seeded at4Â104cells per16-mm dish and maintained for21–28days in DMEM-low glucose supplemented with 10l g/ml insulin,10mM b-glycerophosphate,100nM dexamethasone, and50l g/ml ascorbic acid-2-phosphate.For adipogenic differentia-tion,cells were seeded at2Â105cells per35-mm dish and grown to confluence in medium-B.Thereafter,adipogenic differentiation was induced by subjecting confluent monolayers to3–4rounds of adipo-genic treatments.Each round had two steps;incubation with adipo-genic medium(DMEM-high glucose,10%fetal bovine serum,0.2mM indomethacin,1l M dexamethasone,0.5mM methyl-isobutylxanthine, and10l g/ml insulin)for72–96h and incubation with maintenance medium(DMEM-high glucose,10%fetal bovine serum,and10l g/ml insulin)for72–96h.Cells were cultured under the adipogenic status for 28days.Glycosaminoglycan content,alkaline phosphatase activity,calcium level,glycerol-3-phosphate dehydrogenase activity,and DNA content. The glycosaminoglycan(GAG)content was determined using a sul-fated GAG assay kit(Biocolor,Newtownabbey,UK)[13].The alka-line phosphatase(ALP)activity was determined by the method of Bessey[14].The calcium level was determined by the method of Git-elman[15].The glycerol-3-phosphate dehydrogenase activity was de-termined using an assay kit(Hokudo,Sapporo,Japan)[16].The DNA content was determined using afluorescent DNA quantification kit (Bio-Rad,Chicago,IL).RT-PCR.Total RNA was extracted using Isogen(Nippon Gene, Tokyo,Japan).Thefirst-strand cDNA was synthesized from1l g of total RNA using the SUPERSCRIPT II RNase HÀreverse trans-criptase(Life Technologies,Rockville,MD).Using the cDNAs as a template,PCR was carried out under the following conditions:dena-turation at94°C for30s and primer extension at65°C for1.5min in 27cycles.Pairs of nucleotides,50-TGGTGGAGCAGCAAGAGCAA-30and50-TGCCCAGTTCAGGTCTCTTA-30for type II collagen,50-CCCAACACCAAGACACAGTT-30and50C-ATCACCTTTGATG CCTGGCT-30for type X collagen,and50-GTCAAGGCCGAGAAT GGGAA-30and50-GCTTCACCACCTTCTTGATG-30for GAPDH, 50-CATTTTGGGAATGGCCTGTG-30and50-ATTGTCTCCTCCG CTGCTGC-30for bone sialoprotein,50-CTAGGCATCACCTGTGC CATACC-30and50-CAGTG ACCAGTTCATCAGATTCATC-30for osteopontin,50-CCACCGAGACACCATGAGAG-30and50-CCATA GGGCTGGGAGGTCAG-30for osteocalcin,and50-CATTCTGGC CCACCAACTT-30and50-CCTTGCA TCCTTCACAAGCA-30for PPAR-c2were used as primers for RT-PCR.Obtained PCR products were separated on1%agarose gels and stained with ethidium bromide.Statistical analysis.Student’s t test was used.ResultsThe extracellular matrix produced by PYS-2cells or endothelial cells adhered to the substratum of plastic tissue culture dishes and could be easily cut with a needle and turned over like a sheet of paper(Fig.1A).When cells in marrow aspirates were seeded on plastic culture dishes,adherent cells—MSC—proliferated in the pres-ence of10%fetal bovine serum at a high growth rate in primary cultures[3],but their growth rate rapidly de-creased in secondary and tertiary cultures(Figs.1B–D). In cultures on plastic tissue culture dishes,non-adherent cells were removed completely by thefirst passage. However,when cells in marrow aspirates were seeded directly on bmECM,both MSC and many other cells adhered to the substratum and these adherent cells were not removed by changing the medium.Accordingly,we harvested MSC when the cells were approaching con-fluence in primary cultures on plastic tissue culture dishes,and seeded the isolated MSC on bmECM or plastic tissue culture dishes without the matrix (“plastic”)to examine the effects of bmECM on the proliferation of MSC.The growth rate of human MSC isolated from the ilium(Fig.1B)or the alveolar bone(Fig.1C)on bmECM was much higher than that on plastic,and thus the cumulative cell number in the cultures on bmECM was105-fold greater than that on plastic on day50. After MSC obtained from primary cultures were seeded504T.Matsubara et al./Biochemical and Biophysical Research Communications313(2004)503–508on bmECM or plastic,the proliferative life span of MSC on bmECM(50.3Æ1.5days)was also significantly (p<0:0001)longer than that of MSC on plastic (29.2Æ4.4days)(Figs.1B and C).The effect of bmECM produced by PYS-2was similar to that of endothelial cell bmECM(Fig.1).MSC seeded at a low density and grown on plastic lost their spindle-like appearance,be-comingflat with an increase in the passage number (Fig.1E).Theflat appearance is characteristic of se-nescent cells.However,most MSC grown on bmECM maintained the spindle-like appearance until the5th passage culture on day45(Fig.1F),suggesting that bmECM suppressed cell senescence.Laminin and type IV collagen are the major com-ponents of bmECM,but MSC on laminin-or type IV collagen-coated dishes showed lower growth rates than on bmECM(Fig.1F).The ECM gel isolated from Engelbreth–Holm–Swarm murine sarcoma also showed less growth stimulation than bmECM(Fig.1F),sug-gesting a loss of active substances during isolation of the extracellular matrix components or the necessity of an intact structure for growth stimulation.The chondrogenic potential of MSC was examined as a function of the passage number.MSC obtained from the2nd and the5th passage cultures on day15and day 51(Fig.1B)were maintained in pellet cultures for28 days(Fig.2A).The amount of cartilage proteoglycan stained with toluidine blue was greater in the pellets obtained from the5th passage cultures grown on bmECM than in the pellets from the2nd and the5th passage cultures grown on plastic.The expressions of type II collagen and type X collagen mRNAs were higher in pellets from the5th passage cultures on bmECM than in pellets from the5th passage cultures on plastic(Fig.2B).GAG content and ALP activity in the pellets decreased with the increase in the passage num-ber,irrespective of the presence or absence of bmECM. However,at each passage number,the GAG content (Fig.2C)and ALP activity(Fig.2D)were higher with MSC from cultures on bmECM than with MSC from cultures on plastic.Next,MSC from the2nd and the5th passage cultures on bmECM or plastic were incubated under the osteo-genic status on plastic tissue culture dishes.DuringT.Matsubara et al./Biochemical and Biophysical Research Communications313(2004)503–508505osteogenesis,we did not use bmECM to discriminate the effect of the extracellular matrix on proliferation from its direct effect on differentiation.MSC from cultures on bmECM became stained with alizarin red more in-tensely than MSC from cultures on plastic on day 21(Fig.2E).The expressions of bone sialoprotein,osteo-pontin,and osteocalcin mRNAs on day 28were also higher in cultures of MSC from cultures on bmECM than in cultures of MSC from cultures on plastic at the 5th passage (Fig.2F).ALP activity and calcium level on day 28decreased with the increase in the passage num-ber.However,MSC from cultures on bmECM showed a higher ALP activity (Fig.2G)and a higher calcium level (Fig.2H)than MSC from cultures on plastic at the 2nd and the 5th passages.To examine the adipogenic potential,MSC from the 2nd and the 5th passage cultures on bmECM or plastic were incubated under the adipogenic status on plastic tissue culture dishes for 28days.MSC from cultures on bmECM showed higher adipogenic differentiation,which was indicated by more intense staining with oil-red O (Fig.2I),higher PPAR-c 2mRNA expression (Fig.2J),and higher glycerol-3-phosphate dehydroge-nase activity (Fig.2K).Next,human MSC were grown on bmECM or plastic with 10%human serum,since human serum may be safer than fetal bovine serum for clinical use.Under these conditions,MSC proliferated more rapidly and showed a longer proliferative life span on bmECM than on plastic (Fig.3A).Furthermore,this effect of bmECM was greater than that of basic fibroblast growth factor (FGF).The MSC grown on bmECM for 66days de-veloped into a cartilage-like tissue (Fig.3B),even though these MSC on bmECM had lost proliferation capability.In contrast,scarcely any cartilage-like tissue was formed with MSC obtained from 66-day-old cul-tures on plastic (Fig.3C).DiscussionThe extracellular matrix (ECM)plays a vital role in organ morphogenesis,maintenance,and reconstruction following injury,and actions of ECM can be attributed to its effect on proliferation of stem cells,since stem cells reside on the basement membrane in the epithelium and some other tissues [17].In muscle,satellite cells (stem cells)—which can be induced to differentiate intomuscle,Fig.2.Retention of chondrogenic,osteogenic,and adipogenic potential on bmECM.The MSC obtained from the 2nd and the 5th passage cultures on plastic or bmECM produced by PYS-2cells (Fig.1B)were transferred into the chondrogenic status in pellet cultures for 28days (A–D)and stained with toluidine blue (A).(B)The mRNA levels of type II collagen (type II)and type X collagen (type X)were analyzed by RT-PCR.GAG content (C)and ALP activity (D)were determined on day 28.The MSC from the 2nd and the 5th passage cultures on plastic or bmECM were transferred into the osteogenic status (E–H).(E)The cell layers were stained with alizarin red on day 21.(F)The mRNA levels of bone sialoprotein (BSP),osteopontin (OP),and osteocalcin (OC)were analyzed by RT-PCR on day 28.The ALP activity (G)and calcium level (H)of the cell-matrix layers were determined on day 28.The MSC obtained from the 2nd and the 5th passage cultures on plastic or bmECM were cultured under the adipogenic status for 28days (I–K).(I)The cell layers were stained with oil-red O.(J)The mRNA level of PPAR-c 2was analyzed by RT-PCR.(K)Glycerol-3-phosphate dehydrogenase activity was determined.(C,D,G,H,and K)“)”and “+”represent MSC from culture on plastic and bmECM,respectively.Values are averages þ=ÀSD for four cultures.*p <0:05,**p <0:01,and ***p <0:001vs plastic.506T.Matsubara et al./Biochemical and Biophysical Research Communications 313(2004)503–508bone,cartilage,and fat —are also in close contact with the basement membrane;satellite cells became myo-blasts after detachment from the basal membrane [18].These observations suggest that the basement membrane and/or some other ECMs play a crucial role in the proliferation of stem cells and the maintenance of their undifferentiated state.In this study,we showed that bmECM markedly increased both the growth rate and the proliferative life span of MSC.Furthermore,MSC that had expanded 106-fold on bmECM maintained its multi-lineage differentiation potential.The mechanism by which bmECM stimulates MSC proliferation and maintains their differentiation potential is not known,but even MSC transfected with telomerase gradually decreased its osteogenic potential with the increase in the passage number [5],although their replicativecapacity was maintained.In contrast,MSC on bmECM maintained the differentiation potential even after they lost their replicative capacity.Thus,the extracellular matrix and telomerase may have complementary effects on the proliferation of MSC and their differentiation potential.In any case,the remarkable effects of bmECM in MSC cultures demonstrated here meet the expecta-tions of doctors eager to expand MSC extensively in vitro from a small volume of marrow aspirates before transplantation.Henceforth,bmECM will be prepared using human ES or human cell lines,and in the near future bmECM-coated dishes will probably have a great use in MSC studies and regeneration medicine.References[1]M.F.Pittenger, A.M.Mackay,S.C.Beck,R.K.Jaiswal,R.Douglas,J.D.Mosca,M.A.Moorman,D.W.Simonetti,S.Craig,D.R.Marshak,Multilineage potential of adult human mesenchy-mal stem cells,Science 284(1999)143–147.[2]R.J.Deans,A.B.Moseley,Mesenchymal stem cells:biology and potential clinical uses,Exp.Hematol.28(2000)875–884.[3]S.Tsutsumi,A.Shimazu,K.Miyazaki,H.Pan,C.Koike,E.Yoshida,K.Takagishi,Y.Kato,Retention of multilineage 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