【作者】 赵自平；

【导师】 周江南；

【作者基本信息】 中南大学 ， 外科学， 2003， 博士

【摘要】 在最近十余年，组织工程发展迅猛，已在许多方面取得了重大突破，其中，骨组织工程最有可能首先获得成功并应用于临床。在我国，组织工程学研究虽然起步较晚，但在很多领域已接近或达到国际研究水平，某些项目还处于领先地位。例如，我国学者已在裸鼠和兔身上成功构建了人耳廓形状的软骨支架。尽管取得了如此大的发展与突破，但目前组织工程研究仍处于实验阶段，离临床应用还有相当的距离。其中，理想种子细胞的获取和基质支架材料的制备与改良是骨组织工程研究的关键与重点。 在骨组织工程研究中发现来源于骨、骨膜、骨髓、滑膜、肌肉等组织的成体间质干细胞，其发育分化接近终末状态，经过一段时间的体外培养后，容易衰老凋亡，丧失分泌骨基质的成骨能力，基于这些原因，本研究试图从早期胚胎组织中获取更原始的间质干细胞，用于骨组织工程研究。卵黄囊为胎膜的一部分，其间质层的主要细胞成分为中胚层来源的间质干细胞。与来源于骨髓等组织的成体间质干细胞相比，来自于早期胚胎的卵黄囊间质干细胞更原始，具有更大的增殖潜能和可塑性，并且不表达主要组织相容性复合物(major histocompatibility complex,MHC)相关抗原。本研究对小鼠卵黄囊间质干细胞进行分离、纯化和传代培养，观察其多向分化的潜能，以期为组织工程与基因治疗提供新的理想细胞来源。 中南大学湘雅医院 博士学位论文 构建组织工程骨不仅需要具有成骨潜能的理想种子细胞，还需要适宜于种子细胞生长的三维基质支架材料，羟基磷灰石与1型胶原分别是骨骼中最主要的无机成分与有机成分，由这两类物质复合所制备的生物材料，其成分与结构更接近正常骨组织，将有利于组织工程骨的构建。本研究用1型胶原对自制的磷酸钙骨水泥… phosphate cement，CPC）进行表面改性，观察改性的磷酸钙骨水泥对小鼠卵黄囊间质干细胞粘附生长的支持作用，探讨其作为骨组织工程基质支架材料的应用前景。 主要结果如下：一、小鼠卵黄囊间质干细胞的分离、纯化与鉴定 本研究取妊娠第8．5大的小鼠卵黄囊，经胶原酶消化、低温离心获取小鼠卵黄囊细胞，利用贴壁性从卵黄囊细胞中分离出间质于细胞，原代培养的卵黄囊间质干细胞贴壁生长，具有集落形成能力，组成集落的细胞呈梭形，形态一致，大小均一，核浆比例大，随着培养时间延长，集落相互融合，呈旋涡状生长，易于传代与纯化，经2～3次传代后即可获得纯化的卵黄囊间质干细胞。传代培养细胞与原代细胞相似。经流式细胞仪检测分析，纯化的卵黄囊间质干细胞表达整合素家族成员 CD29、粘附分于 CD44、CD 66及 CD 05，不表达造血干／祖细胞标志抗原CD34、白细胞共同抗原CD45和共刺激分子CD86，以上结果与文献报导的间质干细胞表型一致，证明所分高纯化的细胞为间质干细胞。原代和传代卵黄囊间质干细胞＊KP弱阳性，表达1型胶原，符合间质干细胞的生物学特征。在卵黄囊间质干细胞的传代培养过程中观察到随着传代次数的增加，卵黄囊间质于细胞的增殖能力无明显改变。以上结果提示：卵黄囊间质干细胞易于分离纯化，是一种具有较大增殖和自我更新能力的间质干细胞，有望成为组织丁程研究的理想种子细胞和基因治疗的理想靶细胞。二、小鼠卵黄囊间质干细胞多向分化潜能的研究 将传代纯化的第 4代小鼠卵黄囊间质干细胞，分别加入含 10“‘mol／L地塞米松、10 mmol／L D一甘油磷酸钠和 50 u g／ml维生素 C的 DMEM继续培养，进行成骨诱导；加入合 3ng／ml TGF小的无血清 DMEM继续培养，进行成软骨诱导；加入含 10 u g／ml胰岛素。10’mol／L消炎痛、10”‘mol／L地塞米松的 DMEM继续 一3－ 中南大学湘雅医院 博士学位论文培养，进行成脂诱导。观察诱导前后细胞的生长情况和细胞的形态学特征；原代、传代细胞及加入成骨、成软骨诱导剂培养7天的细胞进行AKP活性检测；成骨诱导培养8周进行Vo uK。ssa’s染色，检测矿化情况：成软骨诱导3周进行＊1d。blue染色检测酸性粘多糖；成脂诱导二周后进行油红O染色，检测成脂情况；免疫组化染色检测1、11型胶原的表达；RT－PCR检测BMP－l、BMP－2的表达。结果显示：成骨诱导3～5天后，部分间质干细胞的体积明显增大，核浆比例减小，细胞形态由校形向星形转化，＊KP的活性增高由弱阳性变为阳性：1型胶原染色呈阳性。当星形细胞连接成网形成类骨结节样结构时，AKP染色形成粗大的阳性颗粒、增多的阳性颗粒可相互融合，AKP活性增高至强阳性。成骨诱导7天，培养的卵黄囊间质干细胞逐渐形成类骨结节样结构，随着诱导时间延长，类骨结节样结构逐渐增大，至第 8周时经 VOn Kossa七染色可见细胞外基质中有钙盐沉积形成矿化区。成软骨诱导5～7天，部分间质干细胞积明显增大，核浆比例减小，细胞形态由梭形向多角形转化，并进一步发育为椭圆形或圆形细胞，AKP阳性，11型胶原染色阳性，瑞氏染色可见细胞分泌细胞?更多还原

【Abstract】 In recent decades, great advancements have achieved in the research of tissue engineering. Bone being no more complex than internal organ such as the kidney, liver and lung which consist of numerous cell types that must be arranged in the proper three-dimensional structure, the research of bone tissue engineering might be succeeded firstly, though it is still in laboratory stage at present. How to obtain the ideal seeding cells and how to improve the properties of scaffold biomaterials are of the most importance in the research of bone tissue engineering.Adult mesenchymal stem cells (MSCs) derived from bone, periosteum, bone marrow, synovium and muscle could be used in bone tissue engineering. As adult MSCs have limited potential in both proliferation and differentiation, the aims of this study were to investigate the Multilineage Potential of yolk sac Mesenchymal Stem Cells (YS-MSCs) derived from the early stage of embryonic development, the potential of YS-MSCs applied in tissue engineering research were investigated too. Yolk sac being a part of caul, YS-MSCs exist in the mesenchymal layer of yolk sac, Compared with adult MSC, YS-MSCs from the early stage of embryonic development are more primitive, have more potential in both proliferation and differentiation and do not express major histocompatibility complex (MHC) related antigen. In order to obtain ideal cell source for tissue engineering and gene therapy, murine YS-MSCs were isolated and purified, their multilineage potential was also investigated in this study.In addition, matrix scaffold biomaterial with three-dimensional structure is also needed for construction of tissue engineered bone. Hydroxyapatite (HA) and collagen type one are the dominating elements of inorganic and organic phases in bone respectively. The components and structure of HA/collagen composite being similar to normal bone tissue, this biomaterial should be suitable for construction of tissue engineered bone. Calcium phosphate cement (CPC) was surface-modified with collagen type one, its effects on the adhesion, proliferation and differentiation of YS-MSCs were also investegated in this study.The main results are as follows:1. Isolation and purification of murine yolk sac mesenchymal stemcellsThe murine yolk sacs were harvested on day 8.5 postcoitus (pc) and digested by 0.1% collagenase. YS-MSCs were existed in the adherent cells. The primary YS-MSCs were adherent cells of spindle shape, uniform in size, having colony forming ability. Purified YS-MSCs could be obtained at passages 2 or 3. Flow cytometric analysis shows the phenotype of purified YS-MSCs is uniformly positive for CD29, CD44, CD 105, and CD 166, and negative for reactivity to antigens CD34, CD45, or CD86. Both the primary and passaged YS-MSCs express collagen type one and weakly but clearly positive in AKP. The biological characteristics of YS-MSCs are coincident with the phenotype of MSCs reported by others. The proliferation ability of primary and passaged YS-MSCs is similar. The results suggest that as primitive MSCs with strong ability in proliferation and self-renewal, YS-MSCs are candidates for bone tissue engineering’s ideal seeding cells.2. Multilineage Potential of murine yolk sac mesenchymal stem cellsTo investigate the differentiation potential of purified murine YS-MSCs, the cells were cultured under conditions that were favorable for osteogenic, chondrogenic, or adipogenic differentiation. Osteogenic differentiation was appeared whenYS-MSCs at passage 4th were treated with 10~8mol/L dexamethasone^ 10 mmol/L 3 -glycerophosphate and 50 u g/ml vitamin C. YS-MSCs which were of spindle shape, uniform in size could be induced to pleomorphism osteoblast-like cells which were positive in collagen type I and expressed high level of AKP. Aggregates or nodules were formed at day 7 and calcium accumulation was detected by Von Kossa’s stain at week 8. BMP-2 mRNA expression of YS-MSCs was detected by RT-PCR (reverse transcriptase polymerase chain reaction) during osteogenic induction.Five更多还原