VCAM-1基因修饰人脐血源基质细胞移植重建造血微环境功能
发布时间：2019-04-03 19:44
【摘要】： 骨髓造血微环境(hematopoietic inductive microenvironment,HIM)是造血干/祖细胞生长发育的“土壤”,基质细胞作为造血微环境中的重要成分,不仅与造血干/祖细胞的归巢定位、增殖分化和自我更新密切相关,而且在某些血液系统疾病的发生、发展和转归过程中具有非常重要的作用,探讨基质细胞在造血调控中的作用需继续深入。 人脐血中的细胞成分丰富且较骨髓和外周血更原始;脐血细胞具有来源广泛、采集方便、GVHD轻和高增殖的特点,临床应用前景广阔。目前对造血基质细胞的研究主要侧重于骨髓基质细胞,而对于脐血中是否存在基质细胞,脐血源基质细胞的生物学特点如何,能否作为一种新型的造血基质细胞来源等问题尚缺乏系统深入的研究;血管细胞粘附分子(vascular cell adhsion molecule-1,VCAM-1)是骨髓基质细胞参与造血调控不可缺少的粘附分子,它与受体整联蛋白(Integrin)家族中的α4β1特异性结合能起到固定造血干细胞于骨髓基质的作用,对造血干/祖细胞的增殖、分化、迁移中发挥重要调控作用。 鉴于此,本课题将人脐血CD34+细胞,采用Dexter贴壁细胞培养体系行脐血源基质细胞培养,探讨人脐血细胞体外扩增基质细胞的可行性和有效性;研究人脐血源基质细胞微环境的造血支持作用;构建荧光蛋白标记的VCAM-1腺病毒载体并转染人脐血源基质细胞,移植给造血微环境辐射损伤裸鼠,探讨其对重建造血微环境功能及促进造血损伤修复的作用,为平战条件下造血功能损伤的救治提供新的辅助治疗措施。 1.研究内容及方法: 1.1分离人脐血CD34+细胞,采用Dexter贴壁细胞培养体系行脐血源基质细胞培养、传代扩增,采用光镜,电镜、组织细胞化学、免疫细胞化学和流式细胞仪等技术对脐血源基质细胞进行鉴定; 1.2采用DNA重组技术,将目的基因VCAM-1克隆至含有报告基因EGFP的穿梭质粒;在BJ5183细胞中与腺病毒基因组进行同源重组,产生重组腺病毒;在脂质体介导下,将重组的腺病毒表达质粒转染293细胞,使腺病毒在293细胞中包装复制,并对转染后的293细胞进行荧光观测,检测培养上清目的蛋白的表达;用高效转染载
[Abstract]:Bone marrow hematopoietic microenvironment (hematopoietic inductive microenvironment,HIM) is the "soil" for the growth and development of hematopoietic stem / progenitor cells. As an important component of hematopoietic microenvironment, stromal cells are not only associated with homing and localization of hematopoietic stem / progenitor cells. Proliferation, differentiation and self-renewal are closely related to each other, and play an important role in the occurrence, development and prognosis of some hematological diseases. It is necessary to explore the role of stromal cells in hematopoiesis regulation. Human umbilical cord blood is rich in cellular components and more primitive than bone marrow and peripheral blood. Umbilical cord blood cells have a wide range of sources, convenient collection, light and high proliferation of GVHD, and have broad clinical application prospects. At present, the research on hematopoietic stromal cells mainly focuses on bone marrow stromal cells, but for whether there are stromal cells in umbilical cord blood, what are the biological characteristics of umbilical cord blood derived stromal cells? Whether it can be used as a new source of hematopoietic stromal cells and other issues is still lack of systematic and in-depth research; Vascular cell adhesion molecule (vascular cell adhsion molecule-1,VCAM-1) is an indispensable adhesion molecule for bone marrow stromal cells to participate in hematopoiesis regulation. Its specific binding to 伪 4 尾 1 in the receptor integrin (Integrin) family plays a role in fixing hematopoietic stem cells in bone marrow stroma and plays an important role in regulating the proliferation, differentiation and migration of hematopoietic stem / progenitor cells. In view of this, human umbilical cord blood CD34 cells were cultured with Dexter adherent cell culture system to explore the feasibility and effectiveness of expansion of stromal cells in vitro by human umbilical cord blood cells. To study the hematopoietic supporting effect of microenvironment of human umbilical cord blood-derived stromal cells (UCB). To construct VCAM- 1 adenovirus vector labeled with fluorescent protein and transfect human umbilical cord blood-derived stromal cells into nude mice with radiation-induced hematopoietic microenvironment injury, and to explore the effects of adenovirus vector labeled with fluorescent protein on the reconstruction of hematopoietic microenvironment and the repair of hematopoietic injury. To provide a new adjuvant therapy for the treatment of hematopoiesis injury in peacetime and war. 1. Content and methods: 1.1.1Human umbilical cord blood CD34 cells were isolated and cultured with Dexter adherent cell culture system. The cells were subcultured and amplified by light microscope, electron microscope, histochemistry, and the results were as follows: (1) Human umbilical cord blood derived stromal cells were cultured in vitro and cultured in vitro. Immunocytochemistry and flow cytometry were used to identify umbilical cord blood derived stromal cells. The target gene VCAM-1 was cloned into shuttle plasmid containing reporter gene EGFP by DNA recombination technique, and the recombinant adenovirus was generated by homologous recombination with adenovirus genome in BJ5183 cells. The recombinant adenovirus expression plasmid was transfected into 293 cells by lipofectamine, and the adenovirus was packaged and duplicated in 293 cells, and the transfected 293 cells were observed by fluorescence to detect the expression of the protein in the culture supernatant, and the recombinant adenovirus was transfected into 293 cells by high efficiency transfection.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329
本文编号：2453507
[Abstract]:Bone marrow hematopoietic microenvironment (hematopoietic inductive microenvironment,HIM) is the "soil" for the growth and development of hematopoietic stem / progenitor cells. As an important component of hematopoietic microenvironment, stromal cells are not only associated with homing and localization of hematopoietic stem / progenitor cells. Proliferation, differentiation and self-renewal are closely related to each other, and play an important role in the occurrence, development and prognosis of some hematological diseases. It is necessary to explore the role of stromal cells in hematopoiesis regulation. Human umbilical cord blood is rich in cellular components and more primitive than bone marrow and peripheral blood. Umbilical cord blood cells have a wide range of sources, convenient collection, light and high proliferation of GVHD, and have broad clinical application prospects. At present, the research on hematopoietic stromal cells mainly focuses on bone marrow stromal cells, but for whether there are stromal cells in umbilical cord blood, what are the biological characteristics of umbilical cord blood derived stromal cells? Whether it can be used as a new source of hematopoietic stromal cells and other issues is still lack of systematic and in-depth research; Vascular cell adhesion molecule (vascular cell adhsion molecule-1,VCAM-1) is an indispensable adhesion molecule for bone marrow stromal cells to participate in hematopoiesis regulation. Its specific binding to 伪 4 尾 1 in the receptor integrin (Integrin) family plays a role in fixing hematopoietic stem cells in bone marrow stroma and plays an important role in regulating the proliferation, differentiation and migration of hematopoietic stem / progenitor cells. In view of this, human umbilical cord blood CD34 cells were cultured with Dexter adherent cell culture system to explore the feasibility and effectiveness of expansion of stromal cells in vitro by human umbilical cord blood cells. To study the hematopoietic supporting effect of microenvironment of human umbilical cord blood-derived stromal cells (UCB). To construct VCAM- 1 adenovirus vector labeled with fluorescent protein and transfect human umbilical cord blood-derived stromal cells into nude mice with radiation-induced hematopoietic microenvironment injury, and to explore the effects of adenovirus vector labeled with fluorescent protein on the reconstruction of hematopoietic microenvironment and the repair of hematopoietic injury. To provide a new adjuvant therapy for the treatment of hematopoiesis injury in peacetime and war. 1. Content and methods: 1.1.1Human umbilical cord blood CD34 cells were isolated and cultured with Dexter adherent cell culture system. The cells were subcultured and amplified by light microscope, electron microscope, histochemistry, and the results were as follows: (1) Human umbilical cord blood derived stromal cells were cultured in vitro and cultured in vitro. Immunocytochemistry and flow cytometry were used to identify umbilical cord blood derived stromal cells. The target gene VCAM-1 was cloned into shuttle plasmid containing reporter gene EGFP by DNA recombination technique, and the recombinant adenovirus was generated by homologous recombination with adenovirus genome in BJ5183 cells. The recombinant adenovirus expression plasmid was transfected into 293 cells by lipofectamine, and the adenovirus was packaged and duplicated in 293 cells, and the transfected 293 cells were observed by fluorescence to detect the expression of the protein in the culture supernatant, and the recombinant adenovirus was transfected into 293 cells by high efficiency transfection.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329
【引证文献】
相关博士学位论文 前1条
1 冯一梅;高表达SDF-1人脐血源基质细胞经PECAM-1介导调控巨核细胞增殖迁移的机制研究[D];第三军医大学;2011年
，本文编号：2453507
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