Current research and application prospect of mesechymal stem cells in tissue-engineered trachea

doi:10.3969/j.issn.2095-4344.2013.10.027

Abstract

Abstract:

BACKGROUND: Mesenchymal stem cells (MSCs) have become an ideal source of seed cells used for tissue-engineered trachea because they have good biological characteristics.
OBJECTIVE: To summarize the current research progress, existing problems and clinical prospects of mesenchymal stem cells used for reconstruction of tissue-engineered trachea.
METHODS: A computer-based online retrieval of Pubmed and CNKI databases was performed to search papers describing tissue-engineered trachea published during 1979-2012 using the key words “mesenchymal stem cells, tissue-engineered trachea, tracheal chondrocytes, tracheal epithelial cells, tracheal vascular endothelial cells” in English and Chinese. In the same research field, papers published recently or in high impact factor journals were selected. Fifty-one papers were included in the final analysis.
RESULTS AND CONCLUSION: Seed cells used for tissue-engineered trachea primarily include chrondrocytes, epithelial cells and vascular endothelial cells. Studies have demonstrated that mesenchymal stem cells can be differentiated into chondrocytes and cytokines play a key role in induced chondrogenic differentiation of mesenchymal stem cells. At present, there have been no proper factors that can be used to induce mesenchymal stem cells to differentiate into tracheal epithelial cells. Mesenchymal stem cells have been shown to exhibit the potential to differentiate into epithelial cells. Mesenchymal stem cells can be induced to differentiate into vascular endothelial cells under in vivo or in vitro special conditions, which provides experimental evidence for reconstruction of tissue-engineered trachea by vascular endothelial cells differentiated from mesenchymal stem cells。

Key words: stem cells, stem cell review, mesenchymal stem cells, tissue-engineered trachea, tracheal chondrocytes, tracheal epithelial cells, tracheal vascular endothelial cells, National Natural Science Foundation of China

CLC Number:

R318

Tan Rong-bang, Shi Hong-can. Current research and application prospect of mesechymal stem cells in tissue-engineered trachea[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(10): 1884-1890.

Figures/Tables 1

2.1 间充质干细胞的生物学特性 1976年Friedenstein[5]首先发现间充质干细胞是一类易贴附于塑料的成纤维样细胞。并最先由Fridenshtein鉴定[6]。骨髓间充质干细胞具有多向分化潜能，静脉输注体内后可定向分化相应的新细胞替代损伤的组织细胞[7-9]，目前已应用于缺血性脑病、神经退行性疾病、心肌梗死和糖尿病治疗中，并取得良好的治疗效果[10-13]。间充质干细胞因为抑制T细胞的活性，所以具有良好的免疫调节功能[14]，移植间充质干细胞后宿主体内可产生免疫耐受，无移植排斥反应。 2.1.1 间充质干细胞的来源、分离纯化间充质干细胞来源广泛，多分布于成人骨髓、骨松质、骨膜、真皮组织、骨骼肌、脂肪组织、外周血、滑膜组织中，也可从胎儿外周血及胎儿肝脏等组织中分离出了间充质干细胞[15]。骨髓是其主要来源，但骨髓中间充质干细胞的数量有限，所占的比例非常小，一般十万至百万个骨髓有核细胞中仅有1个间充质干细胞[16]。正是因为间充质干细胞含量少，所以体外分离、纯化间充质干细胞并使其高效率快速增殖是间充质干细胞应用于组织工程化气管的前提。目前间充质干细胞分离纯化方法有：贴壁培养分离法、密度梯度分离法、流式细胞分离法、免疫磁性分离法等[17]。以上4种方法各有优缺点。由于尚未发现间充质干细胞特异性表面标志物，且检测费用高，检测过程可能损伤细胞，因此很少应用流式细胞分离法和免疫磁珠分离法。目前实验常用的是贴壁培养分离法、密度梯度分离法，两种方法分离出来的间充质干细胞都不同程度混杂有其他细胞，如单核细胞、造血细胞和红细胞等。有学者认为混杂的单核细胞和破骨细胞前体细胞因缺乏1，25-(OH)2- D3或集落刺激因子而不能生长，所以一般在原代培养末期杂质细胞基本消除，即使有残留也会随换液、传代而去除。一般认为贴壁培养分离法通过定期换液、消化传代后纯化间充质干细胞效果同密度梯度离心法。 2.1.2 间充质干细胞的鉴定方法间充质干细胞的抗原表型无特异性，同时表达间充质干细胞、内皮细胞和肌细胞的特性[18]，而不表达造血细胞的标志，所以间充质干细胞无特异性表面标志物[19]。由于目前尚未发现间充质干细胞特异性的标记分子，缺乏特异的检测技术，不同实验室的数据之间的可比性较差，且尚未建立鉴定间充质干细胞的统一标准，所以一般间充质干细胞鉴定多采取一系列的表型和形态结构功能特性来综合鉴定[20]。形态学鉴定：间充质干细胞培养24 h后首次换液可见部分细胞贴壁生长，刚贴壁的细胞大部分呈圆形，有少数伸出突起。培养72 h，贴壁细胞体积增大，分裂增殖伸出突起，呈梭形、三角形和多角形。第5-7天可见细胞以集落方式生长，呈梭形，有粗大突起伸出。第10天后细胞进一步增殖，许多细胞彼此通过突起相连，呈大的长梭形，紧密排列呈漩涡状、网状、辐射状。培养约2周以后，细胞铺满瓶底，呈梭形，两极朝向不规则，细胞排向混乱。约3周以后出现致密的贴壁细胞层，细胞两极开始规律排列呈束状。表型鉴定：即流式细胞仪检测：用胰酶消化生长状态良好的间充质干细胞，加入相应的抗体孵育，流式细胞仪检测。目前发现间充质干细胞表达的一系列表面抗原有SH2、 SH3、SH4、OCT-4 [21]、CD29、CD44、CD13、CD71、CD90、CD105、CD106、CD166、CD146、Sca-1，不表达造血细胞表面抗原如造血前体细胞标志抗原CD34、白细胞标志抗原CD45、成熟造血细胞标志抗原CD38、单核细胞/巨噬细胞表面抗原CD14[22-24]、STRO-1[25]，且不同来源的间充质干细胞所表达的抗原有所不同，表达量也有所不同，如CD44，成人的骨髓间充质干细胞内CD44正常表达[21]，而新生胎儿的中胚层早期细胞CD44表达量很少[26]。功能鉴定：即分化能力，间充质干细胞诱导分化各种相应细胞，同时也证明了所诱导的细胞为间充质干细胞。体外和体内特定的环境下加入特定的生长因子，间充质干细胞可分化为成骨、软骨、骨骼肌、肌腱、韧带、真皮、脂肪和骨髓基质，也可分化成神经元和神经胶质细胞[27-28]。分化出的细胞可通过形态学、组织学和免疫组织化学等相关的检测技术来鉴定，如软骨细胞通过甲苯胺蓝染色、免疫荧光染色和免疫细胞化学染色检测软骨细胞特异性细胞外基质成分如Ⅱ型胶原来鉴定。 2.2 间充质干细胞诱导分化组织工程化气管细胞气管由软骨环、弹性纤维、结缔组织、平滑肌及富含腺体的黏膜、血管构成，其中软骨环具有支撑作用，维持气管的形态，保持通气。气管黏膜由假复层纤毛柱状上皮细胞组成，呼吸上皮细胞具有分泌功能，纤毛摆动将分泌物排出，清洁呼吸道。血管内皮细胞构成的血管运输氧和营养物质，排出代谢产物。体外培养出软骨细胞、上皮细胞和血管内皮细胞，遵循组织工程学原理，才能构建出组织工程化气管。所以组织工程化气管种子细胞研究主要包括软骨细胞、上皮细胞和血管内皮细胞。一般认为体内间充质干细胞的分化主要取决于机体发育的需要和其所处的微环境，如在梗死的脑部可分化为神经元和神经胶质细胞。在体外的分化则是由加入诱导物而定，加入不同的诱导剂，可分化为不同的组织细胞，如加入转化生长因子β1，间充质干细胞可分化为软骨细胞。 2.2.1 间充质干细胞诱导分化气管软骨细胞目前大量实验已证实，间充质干细胞可定向分化为软骨细胞[29]。Fuchs等[30]将羊间充质干细胞种植于可降解的生物材料上，用转化生长因子β1诱导，然后植入羊的胎儿气管中，出生后发现由间充质干细胞诱导分化产生的组织工程化气管与其自身的气管组织相比较，无明显差异。Adelaide等[31]将间充质干细胞和气管上皮细胞种植在一旋转式双层反应器中，应用转化生长因子β3、胰岛素-转铁蛋白-硒、地塞米松和抗坏血酸-2-磷酸盐联合诱导间充质干细胞分化软骨细胞，特定酶联合免疫吸附检测证实分化的细胞含有软骨细胞特有的Ⅰ、Ⅱ型胶原。最终制成一气管替代物，并植入患者身上，1年后患者仍健康，呼吸功能正常。Hiroshi等[32]将间充质干细胞培养于一环状三维水凝胶气管模型，用β-甘油磷酸盐、维生素C和地塞米松联合诱导间充质干细胞分化为软骨细胞，分化出的软骨细胞起支撑气管作用，植入大鼠后，大鼠能自主呼吸。晏杰等[33]以L-抗坏血酸-2-磷酸盐、胰岛素、转铁蛋白溶液及地塞米松为基础诱导液，加入转化生长因子β1和胰岛素样生长因子1的不同组合诱导间充质干细胞分化为软骨细胞，发现联合应用转化生长因子β1和胰岛素样生长因子1时，间充质干细胞表达的Ⅱ型胶原量显著提高。Liu等[34]用乳酸乙醇酸聚合物包裹间充质干细胞团块制成管状结构，用转化生长因子β诱导间充质干细胞分化软骨细胞，经过4周孵育，乳酸乙醇酸聚合物降解，最终形成组织工程气管样替代物。周广东等[35]不采用具有诱导作用的细胞因子，直接将软骨细胞与间充质干细胞混合培养在体内非软骨形成部位，最终间充质干细胞向软骨细胞分化，形成软骨组织。由此可以设想间充质干细胞向软骨细胞分化机制可能与软骨细胞自身分泌有关。该实验为间充质干细胞向软骨细胞分化带来了新思路。一般认为，细胞因子在诱导间充质干细胞分化为软骨过程中起着关键作用。目前使用较多的细胞因子主要有转化生长因子β系列、骨形态发生蛋白及胰岛素样生长因1等。转化生长因子β系列是一种多肽，主要是通过旁分泌机制发挥作用，所以适用于体外刺激诱导分化细胞。具体机制是产生一种细胞信号，与细胞间内因子结合，调节细胞分化，促进细胞增殖并分泌细胞外基质。胰岛素样生长因子1能刺激细胞分泌细胞外基质，维持蛋白聚糖的代谢，调节软骨细胞新陈代谢，并且在转录水平调节Ⅱ型胶原的表达。骨形态发生蛋白与Ⅰ型和Ⅱ型丝氨酸、苏氨酸受体结合后，通过Smad蛋白信号传导通路诱导间充质干细胞分化为软骨细胞和成骨细胞，所以使用骨形态发生蛋白时应注意其分化成骨细胞作用。同时联合应用两种细胞因子诱导作用比单独使用一种因子作用明显提高很多，比如转化生长因子β1和胰岛素样生长因子1联合应用时，间充质干细胞表达的Ⅱ型胶原量比单独使用转化生长因子β1或胰岛素样生长因子1时有显著提高。地塞米松可抑制间充质干细胞增殖，与间充质干细胞的糖皮质激素受体结合，激活细胞表面受体，促进间充质干细胞向软骨细胞分化。维生素C可合成软骨胶原类基质从而刺激软骨分化。而胰岛素则为体外培养细胞的成活、有丝分裂、脂肪酸和糖原合成所必须。地塞米松、维生素C、胰岛素与细胞因子联合使用，起辅助作用。 2.2.2 间充质干细胞诱导分化气管上皮细胞目前尚未发现合适的诱导因子能特异性诱导间充质干细胞定向分化气管上皮细胞。有研究发现间充质干细胞具有向上皮细胞分化的潜能。Stefanidakis[36]、Paupert等[37]将间充质干细胞在植入受损伤的肺组织后发现间充质干细胞可以分化为肺泡上皮细胞和支气管上皮细胞。Sun等[38]发现大鼠气管上皮细胞分泌物质可诱导间充质干细胞分化气管上皮细胞，通过酶联免疫吸附试验(ILISA)检测出气管上皮细胞分泌物有7种蛋白，并推测其中的4种蛋白包括血管内皮生长因子、脑神经营养因子、转化生长因子β1和激活蛋白A等在诱导骨形态发生蛋白分化气管上皮细胞时起关键作用。王亚菁等[39]建立一气液界面培养模型，模拟气管上皮细胞生长环境，将骨髓间充质干细胞和兔气管上皮细胞以一定比例混匀共培养，成功将骨髓间充质干细胞诱导分化为上皮样细胞。Wang等[40]将间充质干细胞与大鼠气管上皮细胞共培养，发现间充质干细胞分化后表达数个上皮细胞标志物，下调Wnt/β-连环蛋白信号可促进间充质干细胞诱导分化为上皮细胞，表明大鼠气管上皮细胞可促进间充质干细胞诱导分化为上皮细胞，阻断Wnt/β-连环蛋白信号转导可促进间充质干细胞诱导分化为上皮细胞。Klause 等[41]把小鼠全骨髓种植到经致死性射线处死的雌性小鼠中，发现它们分化为细支气管上皮细胞和2型肺泡上皮细胞。Redondo-Muñoz 等[42]发现干细胞在损伤的肺的急性炎症期进行移植才能分化为上皮细胞。另外MadcPherson[43]、Wang等[44]发现用热休克损伤间充质干细胞，并将其与气管上皮细胞共培养，可以诱导骨髓间充质干细胞向气管上皮细胞分化。Spees等[45]将间充质干细胞和热休克小气道上皮细胞在体外组织修复模型中一起培养，发现间充质干细胞分化出的细胞表现正常小气道上皮细胞的表型。Peter 等[46]认为将间充质干细胞植入受损组织，间充质干细胞可分化为受损组织的细胞或是产生一种微环境以促进组织细胞的再生。目前考虑其机制是间充质干细胞与共培养的细胞之间发生细胞融合，或是损伤信号促进间充质干细胞分化为与之共培养的细胞。目前尚未发现合适的上皮细胞诱导因子，寻求能特异性诱导间充质干细胞向气管上皮细胞分化的细胞因子和方法是组织工程化气管研究的一项重大的任务。 2.2.3 间充质干细胞诱导分化气管血管内皮细胞新生的血管为气管移植物运输氧、营养物质，排出代谢产物，同时参与各种生物活动。体外构建的组织工程化气管替代物植入体内后同样需要足够的血供，因此血管化是组织工程化气管研究重要的组成部分之一。间充质干细胞在体内或体外特殊条件下可诱导分化为血管内皮细胞。Tremain等[47]发现间充质干细胞的2 353种基因中有分化为内皮细胞基因，说明间充质干细胞具有分化为血管内皮细胞潜能。研究表明间充质干细胞可作为血管内皮细胞来源的理想的种子细胞。目前国内外研究多是使用血管内皮细胞生长因子、内皮细胞生长添加剂、碱性成纤维细胞生长因子、胰岛素样生长因子1、转化生长因子β等生长因子诱导间充质干细胞向血管内皮细胞分化。Zhu等[48]将标志BrdU的间充质干细胞经耳缘静脉注入新西兰大耳兔体内，发现肺内有标志BrdU的间充质干细胞，并分化为肺泡上皮细胞和血管内皮细胞。Oswald等[49]使用50 μg/L血管内皮生长因子成功诱导人骨髓间充质干细胞向内皮细胞分化。Zhen等[50]从经木瓜酶处理过的肺提取细胞，然后与间充质干细胞联合共培养，发现间充质干细胞可促进血管内皮细胞生长因子A表达，并抑制肺细胞凋亡，抗肿瘤坏死因子α可促进间充质干细胞分泌血管内皮细胞生长因子A。韩云等[51]用深低温冻储2周和6周的气管进行Wistar大鼠同种异体气管移植后，用PKH-26标记的3-5代骨髓间充质干细胞经鼠尾静脉移植入受体内。发现术后1周的气管标本中，免疫组织化学测定骨髓间充质干细胞移植组的气管上皮内的血管内皮生长因子蛋白表达呈阳性，从而说明骨髓间充质干细胞能够促进移植物周围新生血管的增加，从而促进气管损伤的修复。由此可见目前间充质干细胞成功诱导分化血管内皮细胞，可为间充质干细胞作为种子细胞分化血管内皮细胞应用于组织工程化气管起到借鉴作用，为组织工程化气管血管化奠定了坚实的基础。"

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