骨桥蛋白体外诱导大鼠骨髓基质干细胞向成骨细胞分化中的作用

发布时间：2018-07-01 16:30

本文选题：骨桥蛋白 + 骨髓基质干细胞　；参考：《山西医科大学》2010年硕士论文

【摘要】： 目的骨创伤后的再生能力提示有能够自我复制并能多向分化的干细胞存在。一般认为,干细胞存在于骨、骨膜、骨髓及其他由中胚层发育来的组织中,在出生后仍然有少量干细胞继续存在,在机体损伤时参与组织的修复。组织工程的基础是有功能的细胞,骨组织工程需要大量的成骨细胞。十多年来,国内外学者和临床工作者对各种组织的细胞成骨能力进行了大量的研究,提出了定向性骨祖细胞、诱导性骨祖细胞和基质干细胞的概念。目前,在研究中使用的作为种子细胞的成骨细胞来源有以下几种骨、软骨、骨外膜、骨髓和骨外组织。越来越多的证据表明,骨髓基质干细胞有多分化潜能,移植人体内后可分化为骨、软骨、肌肉、脂肪、血管内皮、肝脏、神经等多种细胞。能够作为组织工程中不同细胞的来源在体内正常循环中能够分布于多种组织和器官,可以用于细胞和基因治疗。而且取材、分离培养、扩增以及导入外源基因也相对方便。通过抽取自体骨髓得到,对病人造成创伤较少,且无免疫排斥反应。因此,在实际应用时将具有潜在的优势。骨桥蛋白在骨形成与骨改建中扮演着重要角色,实验观察其是否能诱导体外培养的大鼠骨髓基质干细胞向成骨细胞分化,探讨其在骨折愈合过程中的作用机制,为其以后在骨折愈合方面的研究提供确切的理论依据。方法冲洗法获取Weistar大鼠骨髓,用贴壁法分离提纯骨髓基质干细胞,传至第3代细胞后分组,体外培养的BMSCs分为实验组和对照组,实验组添加含0.2nmol/L骨桥蛋白的DMEM条件培养基；对照组为不含骨桥蛋白的DMEM培养基,于不同时间点收集细胞。通过光镜观察培养的细胞形态,以细胞计数法测定生长曲线,应用流式细胞仪分析细胞周期,碱性磷酸酶(ALP)染色和活性测定,骨钙素(OCN)含量测定。结果实验组细胞具有成骨细胞的形态学特征；骨桥蛋白组引起细胞生长曲线右移,细胞Go／G。期比例平均增加13.17%(P0.01),S期比例减少74.93%(P0.01)；实验组ALP染色呈阳性,且ALP活性,OCN含量均较对照组明显提高,差异具有统计学意义。结论1.贴壁筛选法是一种简单有效的骨髓基质细胞的分离纯化方法。2.骨髓基质细胞在体外可诱导分化为成骨细胞,可作为骨组织工程中种子细胞的来源。3.OPN对BMSCs有促进成骨分化的作用。
[Abstract]:Objective the ability of regeneration after bone trauma suggests the existence of self-replicating and multi-differentiated stem cells. It is generally believed that stem cells exist in bone, periosteum, bone marrow and other tissues developed from mesoderm. A small number of stem cells continue to exist after birth and participate in the repair of tissues when the body is damaged. Tissue engineering is based on functional cells, and bone tissue engineering requires a large number of osteoblasts. For more than ten years, scholars and clinicians at home and abroad have done a lot of research on the osteogenic ability of various tissues, and put forward the concepts of directional bone progenitor cells, inducible bone progenitor cells and stromal stem cells. At present, the osteoblasts used as seed cells in the study are the following kinds of bone, cartilage, periosteal, bone marrow and extraosseous tissue. More and more evidence shows that bone marrow stromal cells can differentiate into bone, cartilage, muscle, fat, vascular endothelium, liver, nerve and so on. It can be used as a source of different cells in tissue engineering and can be distributed in various tissues and organs in normal circulation in vivo, and can be used in cell and gene therapy. It is also relatively convenient to select materials, isolate, culture, amplify and import foreign genes. Through extraction of autologous bone marrow, there is less trauma and no immune rejection. Therefore, there will be potential advantages in practical application. Osteopontin plays an important role in bone formation and bone remodeling. Whether osteopontin can induce the differentiation of rat bone marrow stromal cells into osteoblasts in vitro was observed and the mechanism of osteopontin in the process of fracture healing was discussed. It provides a theoretical basis for further research on fracture healing. Methods the bone marrow of Weistar rats was obtained by washing method. Bone marrow stromal cells were isolated and purified by adherent method. BMSCs cultured in vitro were divided into two groups: experimental group and control group. The experimental group was supplemented with DMEM conditioned medium containing 0.2nmol / L osteopontin. The control group was cultured in DMEM medium without osteopontin and the cells were collected at different time points. The morphology of cultured cells was observed under light microscope. The growth curve was measured by cell count method. Cell cycle was analyzed by flow cytometry, alkaline phosphatase (ALP) staining and activity were determined. Osteocalcin (OCN) content was measured by osteocalcin (OCN) content. Results the cells in the experimental group had the morphological characteristics of osteoblasts, and the osteopontin group caused the cell growth curve to shift to the right and the cells to go / G. The proportion of ALP in the experimental group increased by 13.17% (P0.01) and decreased by 74.93% (P0.01), and the ALP activity and OCN content in the experimental group were significantly higher than those in the control group (P < 0.05). Conclusion 1. Adherent screening is a simple and effective method for the isolation and purification of bone marrow stromal cells. Bone marrow stromal cells can be induced to differentiate into osteoblasts in vitro and can be used as a source of seed cells in bone tissue engineering. 3. OPN can promote osteogenic differentiation of BMSCs.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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