【作者】 张秀英；

【导师】 李玉林；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2004， 博士

【摘要】 骨髓中的间充质干细胞（mesenchymal stem cells, MSCs）在不同的诱导条件下，具有向中胚层和神经外胚层组织细胞分化的能力，已成为组织工程的种子细胞。目前，许多文献报道了关于MSCs的分离、鉴定、生物学性状及定向诱导分化.。随着组织细胞工程学和基因工程学的发展，利用MSCs的多向分化潜能，在组织工程、基因治疗以及器官移植等方面的应用研究已成为研究热点。然而MSCs在血管组织工程中的作用及其与血管内皮细胞之间的关系的研究尚少。 MSCs具有内皮细胞的某些表型，因此研究和分析MSCs生长、发育、分化和表型特征，不仅对研究血管内皮细胞相关疾病的治疗有着重要的指导意义，而且给血管组织工程学带来了一次革命性转变。MSCs作为血管组织工程的种子细胞，具有其他细胞所不可比拟的优越性：①取材方便，损伤轻微，不必通过手术从病人身上得到自身成熟细胞，避免对病人造成不必要的二次创伤；②从患者本人骨髓血培养扩增的MSCs诱导为血管内皮细胞，不存在组织相容性问题；③体内成熟内皮细胞其分裂增殖能力有限，且还存在去分化形成肿瘤的倾向。MSCs具有旺盛的增殖分化潜能，无诱导剂情况下可保持25代内无分化趋势，并且MSCs在体内具有免疫调节作用，至今未<WP=112>见体内发生肿瘤的报导；④人工合成材料的可降解性材料聚乙二醇酸PGA、聚丙醇酸PLA、聚乙醇酸PLLA，与MSCs相结合使用，将会给血管组织工程学带来不可估量的使用前景。殖分化潜能，无诱导剂情况下，可保持25代内无分化趋势。细胞分化程序的启动和进行是多个基因有次序的开启/关闭或表达增强/降低的结果，受细胞与细胞（包括间质细胞如成纤维细胞、肥大细胞等）、细胞与细胞外基质间相互作用的影响。细胞因子在传递细胞与胞外基质之间、细胞与细胞之间的信息中起重要作用。 Flt-1/VEGF，Flk-1/VEGF系统在内皮细胞增殖分化、内皮细胞游走及管腔形成过程中起重要作用并且大量实验表明VEGF在内皮细胞诱导分化过程中起着关键的作用。细胞外基质成分的改变也影响干细胞的分化，胞外某些信息通过细胞外基质传递给干细胞，以触发跨膜信号传导，调控细胞的基因表达。这一过程不仅改变干细胞的分裂方式，而且也激活干细胞的多潜能性，使干细胞产生一种或多种定向祖细胞，以适应组织修复的需要。本研究拟通过免疫荧光、全细胞膜片钳技术、激光共聚焦显微镜实时监控技术及三维图象重组技术、Bodyen 小室技术、体外血管形成实验，从形态学、电生理等多角度逐渐深入地研究VEGF在MSCs内皮细胞诱导分化过程中所起的作用；并利用抑制性消减杂交技术从分子水平上寻找内皮细胞分化的机理，探讨MSCs 作为血管组织工程种子细胞的可能性。结果：1．本实验在贴壁筛选与密度梯度离心法相结合的基础上，传代过程中与筛网过滤法结合，除去少量已分化或老化的体积较大而扁平的细胞（mMSCs），使细胞纯度达95%，平均为93.26±2.48％。与以往部分研究不同，CD29阴性，CD44阳性率达95%，说明分离的细胞群纯度较高。 <WP=113>2．MSCs的细胞生物学特征及电生理特征研究甚少。本实验发现，MSCs中S+G2 期的细胞约占18%,而G1期的细胞为 82 % (即静止期细胞 ) ,只有少数细胞处于活跃的增殖期。在对原代、冻存后复苏及传代培养至P12的MSCs的染色体核型分析中发现其保持稳定的二倍体核型且核型正常，因此，从细胞生物学角度进一步证实MSCs有望成为稳定培养的细胞系，作为细胞治疗的种子细胞。3．MSCs细胞上存在电压依赖型钙通道（VOCCs）。其峰值电流(ICa-Peak)在+30 - +40mV为（97.83±8.98，pA，n=8），此电流可被10uM的Nicardipine阻断从而证实为细胞的钙通道电流。4．MSCs具备内皮分化的细胞免疫学基础。表达早期内皮细胞分化标志和EPC(内皮前体细胞)标志之一Flk-1，表达Flt-1，与早期内皮细胞发育有关。5．全细胞膜片钳技术观察VEGF作用下MSCs细胞内Ca2+ 峰值电流升高，（实验组与对照组分别为110.00±7.62、97.50±7.01 (p<0.05),pA,n=9）。与对照组相比VEGF组有统计学意义(P<0.05)，说明MSCs能接受VEGF刺激，引起胞内钙离子浓度发生相应变化。6．给予VEGF165刺激，细胞内Ca2+开始上升，经12秒趋于平稳，持续10秒后达到高峰，后开始下降，VEGF使细胞内Ca2+波动后维持在比加药前稍高的细胞内Ca2+水平上（P<0.001），提示VEGF作用于MSCs后，细胞内Ca2+经各种信号分子调节而波动后，细胞可重新建立新的钙平衡，即在无细胞钙毒性前提下提高了细胞内静息钙水平，而这种变化对于MSCs分化具有重要意义。7．利用LSCM实时动态监测了MSCs在Matrigel/VEGF和Matrigel/内皮细胞条件诱导液构成的内皮细胞生长微环境中的运动迁移情况，发现内皮细胞条件诱导液促进MSCs的迁移，在Matrigel内迁移的深度及迁移至聚碳酸脂<WP=114>膜下的细胞均明显多于对照组（p〈0.05），而VEGF组迁移的深度虽高于对照组（p〈0.05），但迁移至聚碳酸脂膜下的细胞与对照组相比，并无统计学意义（p〉0.05），说明MSCs在细胞外基质的微环境迁移过程中，VEGF确实发挥一定作用，但还需其它各种复杂成分的协同参与。8．VEGF组能够形成管腔样结构，内皮细胞条件培养液组形成管腔样?更多还原

【Abstract】 Human mesenchymal stem cells(MSCs) are thought to be multipotent cells, which are present in adult bone marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. The isolation, expansion, and characterization of MSCs have been reported recently. With the development of histocell engineering and genetic engineering, many research has focused on the appliance of multi-differentiating potential of MSCs in histoengineering, gene therpy and organ transplanting. However, little research has been done about the function of MSCs in blood vesselengineering and the relationship between MSCs and endothelial cells.MSCs have phenotype of endothelial cells in some extent, so it is important to characterize MSCs in culture,expandition and differentiation, which will give advantage to some disease related to endothelial cells, but also bring great changes to blood vesselengineering. MSCs have some characteristics and advantages as seeding cells in blood vesselengineering: ①It was convenient to draw the materials and lightly injure, which avoided of unnecessary second trauma on the patients; ②The autologous expanded MSCs had no histocompatibility when induced to ECs; ③The proliferation of mature EC was <WP=116>limited and had the tumorigenic tendency of de-differentiation. MSCs had the highly ability of proliferation and maintained un-differentiation on 25 passages on the condition of no inducer. Meanwhile, MSCs had immunoregulation and no occurrence of tumor in Vivo; ④The degraded artificial synthetic materials such as PGA, PLA, PLLA binding with MSCs, which bring about the surprising prospect of application for the vessel-tissue engineering. Therefore, MSCs became a novel seeding cells, exploring the mechanism of EC differentiation possessed the important theory sense.The initiation and process of cell differentiation procedures is the result of orderly opening and shut-off of multi-genes or regulation of gene expression, which is also influenced by the interaction between cell and cell as well as cell and extracellar matrix. Cytokines play an important role in signal transduction between cell and cell as well as cell and extracellar matrix. Flt-1/VEGF and Flk-1/VEGF systems are important in endothelial cells proliferation, differentiation，migration and tube formation. Changes of extracellar matrix may also affects MSCs, differentiation because certain message exocell will be convested to stem cell by extracellar matrix, which will trigger transmembrane signal conduction and control some gene expression. Such process may not only alter stem cell,s cleavage manier, but also activate stem cell,s multipotent to differentiate to committed progenitor. To examine roles of VEGF on MSCs differentiation to endothelial cells, immunofluorescence, whole cell patch clamp technology, Bodyen chamber technology, tube formation in vitro, confocal laser scanning microscopy real-time monitoring and three-dimension figures recombination were used to <WP=117>study the actions in morphology, cell biology and electric physiology. To investigate MSCs differentiation mechanisms and identify differentially expressed genes between MSCs and endothelial cells, especially specified genes expressed in endothelial cells, a cDNA subtractive library of endothelial cells was constructed using suppression subtractive hybridization (SSH) technique.Results:1.Cells that have the characteristics of human mesenchymal stem cells were isolated from bone marrow aspirates of volunteer donors using density gradient and adherence to plastic dishes method combined with sieve filtering technique, which eliminate unwanted big and flat cells and keep undifferentiation state to highly homologous population. Different from obvious research, the mesenchymal cells were negative for CD29 .MSCs comprised a single phenotypic population (93.26±2.48%) by flow cytometric analysis of expressed surface antigens CD44 indicating更多还原