【作者】 张展；

【导师】 尹维田；

【作者基本信息】 吉林大学 ， 外科学， 2011， 博士

【摘要】 外伤或慢性骨关节疾病导致的关节软骨损伤和缺损在临床上十分常见,关节软骨由于无血管、淋巴管及神经组织,同时软骨细胞增殖能力有限,不能迁移,因此其自我修复能力非常有限。目前常用的治疗方法均不能真正恢复软骨的组织结构及生物力学特点。组织工程学是应用细胞生物学和工程学的原理,研究开发生物替代物来修复损伤组织形态、恢复损伤组织功能的一门科学。它的提出、建立和发展为治疗组织、器官缺损和功能障碍提供了一种新的方法。作为组织工程软骨的首要因素,种子细胞的选择和获得对组织的构建至关重要。自体软骨细胞是目前临床唯一得到应用的组织工程软骨种子细胞。但由于其供区有限,分离培养困难,体外增殖能力差,因此应用受到限制。胚胎干细胞、诱导的多潜能干细胞由于伦理、安全性等问题距离临床应用还有很远距离。成体干细胞在体内广泛存在,特别是脂肪来源的间充质干细胞,其来源广泛、获取简单,是理想的组织工程种子细胞。但如何在体外快速扩增出大量干细胞,并将其更有效的向软骨细胞方向诱导分化依然是目前亟待解决的问题。当前常用的促进干细胞增殖、分化的添加物是各种细胞因子,然而这些细胞因子均十分昂贵,并且单纯应用一种或几种细胞因子也很难模拟体内的多因子环境。由此我们想到了鹿茸。鹿茸是哺乳动物唯一可以完全再生的器官。在其快速生长期,生长速度可达每天1-2cm,并且鹿茸在3个月的时间内将完成生长、软骨化、骨化的全过程。研究表明鹿茸中含有大量多肽类生长因子,是天然的细胞因子库,这种因子的组合是天然的最佳组合。并且许多因子无种属特异性。因此我们选择鹿茸来研究其是否含有能促进脂肪干细胞增殖及分化的有效成分。首先本实验建立了方便有效的从皮下脂肪组织中分离、纯化hADMSCs的方法：即I型胶原酶消化、贴壁培养法。应用本方法可以从临床废弃的皮下脂肪组织中分离得到大量形态均一、贴壁生长的间充质干细胞。细胞呈典型的漩涡状、鱼群样生长,细胞增殖速度快，群体倍增时间47.8小时。流式细胞术检测所获得的细胞表达CD29、CD105、CD73和CD90抗原,不表达CD45、CD34、CD14、CD79α和HLA-DR。多向分化潜能是鉴定间充质干细胞的重要标志,本实验通过各种不同的诱导剂分别将所获细胞向脂肪细胞、成骨细胞、软骨细胞方向诱导分化,证实其具有多向分化潜能,符合间充质干细胞的鉴定标准。随后选取新鲜鹿茸,经缓冲溶液及乙醇提取天然鹿茸肽组分,并应用超滤分离、凝胶层析、高效液相色谱等技术对其进行进一步分离纯化。并应用CCK-8实验,对鹿茸肽组分的促脂肪干细胞增殖活性进行研究,结果显示鹿茸肽粗提物能促进hADMSCs的增殖,特别是其寡肽组分,具有更强的促增殖活性。应用质谱法对该寡肽组分进行分子量检测,显示其由分子量位于500-600Da之间的两个寡肽组成。最后,我们应用细胞微团培养模式,研究鹿茸肽对脂肪间充质干细胞成软骨分化的作用,结果表明单独应用鹿茸肽成软骨诱导分化作用微弱,但其与TGF-β1联合应用可起到协同作用,可以促进细胞内PGC-1α、SOX9、Co12al基因的表达,促进细胞外基质糖胺聚糖和二型胶原的聚集。从而促进脂肪间充质干细胞向软骨细胞分化。其作用呈浓度依赖性,以12.5ug/ml-25.0ug/ml最为适宜。其机制可能与鹿茸肽含多种天然来源的优化组合的细胞因子有关,同时鹿茸肽中的寡肽组分也可能通过促进细胞间及细胞与细胞外基质间相互作用来促进脂肪干细胞向软骨细胞分化。总之,本实验建立了简单、有效的分离高纯度人脂肪来源间充质干细胞的方法,并从新鲜鹿茸中提取、分离出具有促进hADMSCs增殖活性的组分,证实其为分子量位于500-600Da的两个寡肽成分组成。本实验发现单独应用酸醇法提取的鹿茸肽不能有效使hADMSCs向软骨细胞分化,但其与TGF-β1的成软骨诱导分化作用有明显的协同作用。并且这种作用有一定的浓度依赖性,以12.5ug/ml-25.0ug/ml最为适宜。更多还原

【Abstract】 Articular cartilage injury and defect caused by trauma and chronic osteoarthritis are common in clinic. The capacity of intrinsic repair of cartilage is limited due to its avascularity and the limited proliferation and migration ability of chondrocyte. The treatment methods commonly used today can not really restore the organizational structure and biomechanical property of cartilage. Tissue engineering is the development of the alternates to replace or support the function of defective or injured body parts using the principle of cell biology and engineering.It provides a new method to deal with tissue defect and functional disability.As the primary factor in cartilage tissue engineering, the selection and acquisition of seed cells is most important. Autologous chondrocyte is the only seed cell which has been used in clinic, but its application is limited because of the donor site morbility and difficulty in isolation and cultivation. Embryonic stem cells. induced pluripotent stem cells are far from clinical application because of ethic and security problems. Adult stem cells are widespread in the body.especially the adipose derived stem cells. It is an ideal seed cell for tissue engineering because of its accessibility and abundant resource.How to acquire a large number of stem cells in vitro in a short time,and promote their chondrogenesis differentiate is still problems to be solved. Various cytokines have been used to promote stem cell proliferation and differentiation now. However, they are extremely expensive, and a single cytokine or several cytokines can not minic the in vivo multi-factor environment. So we remind the deer velvet antler which is the only organ that can regenerate every year. In the rapid growth period, the growth rate can be 1-2cm per day. and during the 3 months, the velvet antler will reach a hight of 1 meter, and complete the process of growth, chondrogenesis and ossification. Some studies have revealed that velvet contains a large number of polypeptide growth factor. It is a natural repository of cytokines. The combination of these cytokines is a natural status. Some study revealed many factors have no species specificity. So we choose the velvet antler as an object to explore its effects on proliferation and differentiation of human adipose derived mesenchymal stem cells.First, this study established a convenient and effective method to isolate hADMSCs from subcutaneous adipose tissue:collagenaseⅠdigestion, and adherent culture. We can obtain a large number of uniform hADMSCs from the discarded subcutaneous adipose tissue. Cells showed the typical swirling, fish-like growth. The cells can proliferate at a high speed,the population doubling time is 47.8 hours. Flow cytometry examination reveals that the surface antigen CD105, CD73 and CD90 are positive, and the CD45, CD34, CD14, CD79αand HLA-DR are negetive. Multipotent differentiation is a important feature of mesenchymal stem cells. In this experiment, we differentiate the acquired cells to adipocytes, osteoblasts. and chondrocytes using different inducers to confirm their multipotent ability. The cells used in this experiment are coincidence with the minimal criteria for defining multipotent mesenchymal stromal cells proposed by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Celluar Therapy.Then we extract the natural peptides from new velvet antler using buffer solution and ethanol. isolate and purify its component using ultrafiltration. gel chromatography. high performance liquid chromatography technology. We test the component’s activity of promoting adipose stem cells proliferation using cell counting kit-8. We discover that the velvet antler peptides can promote adipose stem cells proliferation, and the oligopeptides have a more better activiey. Mass spectrometry examination reveals its molecular weight is between 500-600Da.Finally, we explore the effect of velvet antler peptides in chondrogenesis of adipose derived mesenchymal stem cells. The velvet antler peptides has little effect in chondrogenesis when used lonely, but it has a synergistic effect with TGF-β1. It can promote the expression of PGC-1α、SOX9、Col2a1 genes, and accumulation of glycosaminoglycans and collagen typeⅡ. So it can promote chondrogenesis of TGF-β1, and the effect is dose-dependent, the suitable concentration is 25ug/ml-50ug/ml. The possible mechanism of this effect may be related to the natural combination of cytokines from velvet antler, and the oligopeptides may be effective by promoting the interaction between cells and extracellular matrix.In short, we establish a simple and effective method to isolate adipose derived mesenchymal stem cells with high homogenous, and we extract and purified a component of oligopeptides from velvet antler, and confirm its promotion effect of proliferation in adipose derived mesenchymal stem cells. In this experiment, we find that the velvet antler peptides extracted by acid and ethanol can not induce chondrogenesis. but it has a synergistic effect with TGF-pM. This effect is dose-dependent in a limited concentration, the suitable concentration is 12.5ug/ml-25.0ug/ml.更多还原