SDF-1修饰人骨髓间充质干细胞体外培养鉴定的研究

发布时间：2018-06-29 19:44

本文选题：人骨髓间充质干细胞 + 基质细胞衍生因子-1　；参考：《第三军医大学》2006年硕士论文

【摘要】： 目的: 造血微环境损伤是引起某些疾病及造血干细胞移植时造血重建不良的重要原因。骨髓基质细胞是造血微环境的主要成分,对造血干/祖细胞的粘附作用是促使造血干/祖细胞回髓定植和进一步增殖分化的基础。而骨髓间充质干细胞( bone marrow mesenchymal stem cells,BMSCs)是存在于骨髓中除造血干细胞(hemopoietic stem cells,HSCs)以外的另一种干细胞,不仅直接参与生成造血微环境支持造血,在适宜的条件下还可发育、分化为具有支持造血功能的多种骨髓基质细胞。因其具有高度自我更新能力和多向分化潜能等,是细胞替代和基因治疗理想的细胞来源。尤其在造血干细胞移植过程中,可以纠正或改善造血微环境的损伤,从而促进移植后造血和免疫功能的重建。基质细胞衍生因子-1(stromal cell derived factor-1,SDF-1)主要由骨髓基质细胞合成和分泌,同时,BMSCs也可合成、分泌SDF-1,是骨髓造血微环境的重要组成部分。人SDF-1基因定位于10号染色体,根据其编码氨基酸序列归属于趋化因子CXC亚家族,与其表达于造血干细胞表面的唯一受体CXCR4结合参与造血的调控。在造血干细胞动员、归巢、定植和增殖中发挥重要作用,并可单独或协同G-CSF、TPO等细胞因子通过细胞信号转导系统维持造血干细胞的正常功能。本实验在成功分离、培养、鉴定人骨髓间充质干细胞(hBMSCs)和构建SDF-1-pIRES2-EGFP真核表达载体的基础上,将重组质粒传染入hBMSCs,并检测其瞬时转染效率,为进一步的体内外实验研究SDF-1修饰hBMSCs对造血的支持作用奠定基础。方法: 一、共采集25例骨髓标本,均取自第三军医大学附属新桥医院血液科15岁至40岁正常、缺铁性贫血及特发性血小板减少性紫癜患者;携带SDF-1cDNA的质粒pCMV-SPORT6载体购自ATCC公司,Number:47612;pIRES2-EGFP真核表达载体为本实验室保存。二、通过密度为1.073kg/L的Percoll液,利用密度梯度离心法分离出人骨髓单个
[Abstract]:Objective: hematopoietic microenvironment injury is an important cause of hematopoietic reconstitution in some diseases and hematopoietic stem cell transplantation. Bone marrow stromal cells are the main components of hematopoietic microenvironment. The adhesion of hematopoietic stem / progenitor cells to hematopoietic stem / progenitor cells is the basis for promoting hematopoietic stem / progenitor cells to be replanted and further proliferated and differentiated. Bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells) are other stem cells in bone marrow besides hematopoietic stem cells (hemopoietic stem cells). They are not only involved in the generation of hematopoietic microenvironment to support hematopoiesis, but also can be developed under suitable conditions. Differentiation into a variety of bone marrow stromal cells with hematopoietic support. It is an ideal cell source for cell substitution and gene therapy because of its high self-renewal and multi-differentiation potential. Especially in the process of hematopoietic stem cell transplantation, the damage of hematopoietic microenvironment can be corrected or improved, thus promoting the reconstruction of hematopoietic and immune function after transplantation. Stromal cell derived factor 1 (stromal cell derived factor-1 (SDF-1) is mainly synthesized and secreted by bone marrow stromal cells (BMSCs), and BMSCs secrete SDF-1, which is an important part of bone marrow hematopoietic microenvironment. Human SDF-1 gene is located on chromosome 10 and belongs to the chemokine CXC subfamily according to its coding amino acid sequence. The SDF-1 gene binds to CXCR4, the only receptor expressed on the surface of hematopoietic stem cells, involved in the regulation of hematopoiesis. It plays an important role in the mobilization, homing, colonization and proliferation of hematopoietic stem cells, and can maintain the normal function of hematopoietic stem cells by signal transduction system. Based on the successful isolation, culture and identification of human bone marrow mesenchymal stem cells (hBMSCs) and the construction of SDF-1-pIRES2-EGFP eukaryotic expression vector, the recombinant plasmid was infused into hBMSCs and its transient transfection efficiency was measured. It provides a basis for further in vitro and in vivo study on the hematopoietic support of hBMSCs modified with SDF-1. Methods: 1. A total of 25 bone marrow specimens were collected from the Department of Hematology, Xinqiao Hospital affiliated to the third military Medical University. The plasmids pCMV-SPORT6 carrying SDF-1 cDNA were purchased from ATCC company, Number1 47612 pIRES2-EGFP eukaryotic expression vector for preservation in our laboratory, and the eukaryotic expression vector pCMV-SPORT6 containing SDF-1 cDNA was obtained from patients with iron deficiency anemia and idiopathic thrombocytopenic purpura. Secondly, using Percoll solution with density of 1.073 kg / L, a single human bone marrow was isolated by density gradient centrifugation.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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