脑源性神经营养因子（BDNF）基因克隆及其基因工程细胞对体外培养脑片生长的影响
发布时间：2019-04-01 10:44
【摘要】：帕金森氏病(PD)是神经系统锥体外系常见的慢性退行性疾病。主要病理变化是位于中脑黑质的多巴胺(DA)能神经元变性和消失,导致纹状体的DA能神经纤维终末变性消失,这种黑质-纹状体神经传导路的损害使得DA水平下降,由此产生了PD的典型症状。目前,临床上对PD治疗的研究主要集中在两个方面:一是以补充多巴胺的不足为目的药物治疗:如给病人定期服用左旋多巴,美多巴,息宁,安坦等,通过药物治疗病人的大多数临床症状改善,但并不能阻止病情的发展,即不能治愈。另一种方法是以抢救幸存神经元为目的的治疗:如导入神经营养因子的基因治疗。 脑源性神经营养因子(brain-derived neurotrophie factor,BDNF)作为神经生长因子家族成员,与神经系统发育有着密切关系,不仅对中枢神经元的发育、增殖分化、存活起重要作用,而且对损伤后神经元的修复与功能重塑也起了重要作用。近年来神经生长因子家族一些成员纷纷被克隆并应用于中枢神经系统退行性疾病的防治。 针对上述研究趋势,本研究采用脑皮质-纹状体-黑质三组织联合培养技术,选用脑源性神经营养因子(BDNF)通过RT-PCR及载体克隆的方法,构建BDNF真核表达载体pEGFP-N1-BDNF,以重组质粒pEGFP-N1-BDNF作为外源性基因,与脂质体一起转染骨髓基质干细胞(BMSCs),通过荧光显微镜观察,并结合BDNF免疫细胞化学染色等形态学方法鉴定稳定表达BDNF蛋白的基因工程细胞,检测其对体外联合培养脑片生长的影响。 材料与方法 (一) 大鼠骨髓基质干细胞(BMSCs)体外培养及向神经元诱导分化研
[Abstract]:Parkinson's disease (PD) is a common chronic degenerative disease in extrapyramidal nervous system. The main pathological changes were the degeneration and disappearance of dopaminergic (DA) neurons located in the substantia nigra, which led to the disappearance of the terminal degeneration of the striatum DA-denergic nerve fibers. The damage of the substantia nigra-striatal nerve conduction pathway reduced the level of DA. This results in typical symptoms of PD. At present, the clinical research on PD mainly focuses on two aspects: one is to supplement the deficiency of dopamine for the purpose of drug therapy: for example, to give patients regular use of levodopa, Medopa, Xining, Antan, and so on. Most of the patients' clinical symptoms improved by medication, but did not stop the development of the disease, that is, can not be cured. Another approach is treatment aimed at rescuing surviving neurons: gene therapy for the introduction of neurotrophic factors. As a member of nerve growth factor family, brain-derived neurotrophic factor (brain-derived neurotrophie factor,BDNF) is closely related to the development of nervous system. It not only plays an important role in the development, proliferation, differentiation and survival of central neurons, but also plays an important role in the development of central neurons. It also plays an important role in the repair and functional remodeling of neurons after injury. In recent years, some members of the nerve growth factor family have been cloned and applied to the prevention and treatment of degenerative diseases of the central nervous system. In view of the above research trends, the eukaryotic expression vector pEGFP-N1-BDNF, of BDNF was constructed by using the technique of co-culture of cerebral cortex, striatum and substantia nigra, and using the method of RT-PCR and vector cloning of brain derived neurotrophic factor (BDNF) to construct the eukaryotic expression vector pEGFP-N1-BDNF,. Recombinant plasmid pEGFP-N1-BDNF was used as exogenous gene and transfected with liposome into bone marrow stromal stem cells (BMSCs) (BMSCs), by fluorescence microscope. The gene engineering cells stably expressing BDNF protein were identified by BDNF immunocytochemical staining and the effects of these cells on the growth of co-cultured brain slices in vitro were detected. Materials and methods (1) Rat bone marrow stromal stem cells (BMSCs) were cultured in vitro and differentiated into neurons.
【学位授予单位】：中国医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329
本文编号：2451494
[Abstract]:Parkinson's disease (PD) is a common chronic degenerative disease in extrapyramidal nervous system. The main pathological changes were the degeneration and disappearance of dopaminergic (DA) neurons located in the substantia nigra, which led to the disappearance of the terminal degeneration of the striatum DA-denergic nerve fibers. The damage of the substantia nigra-striatal nerve conduction pathway reduced the level of DA. This results in typical symptoms of PD. At present, the clinical research on PD mainly focuses on two aspects: one is to supplement the deficiency of dopamine for the purpose of drug therapy: for example, to give patients regular use of levodopa, Medopa, Xining, Antan, and so on. Most of the patients' clinical symptoms improved by medication, but did not stop the development of the disease, that is, can not be cured. Another approach is treatment aimed at rescuing surviving neurons: gene therapy for the introduction of neurotrophic factors. As a member of nerve growth factor family, brain-derived neurotrophic factor (brain-derived neurotrophie factor,BDNF) is closely related to the development of nervous system. It not only plays an important role in the development, proliferation, differentiation and survival of central neurons, but also plays an important role in the development of central neurons. It also plays an important role in the repair and functional remodeling of neurons after injury. In recent years, some members of the nerve growth factor family have been cloned and applied to the prevention and treatment of degenerative diseases of the central nervous system. In view of the above research trends, the eukaryotic expression vector pEGFP-N1-BDNF, of BDNF was constructed by using the technique of co-culture of cerebral cortex, striatum and substantia nigra, and using the method of RT-PCR and vector cloning of brain derived neurotrophic factor (BDNF) to construct the eukaryotic expression vector pEGFP-N1-BDNF,. Recombinant plasmid pEGFP-N1-BDNF was used as exogenous gene and transfected with liposome into bone marrow stromal stem cells (BMSCs) (BMSCs), by fluorescence microscope. The gene engineering cells stably expressing BDNF protein were identified by BDNF immunocytochemical staining and the effects of these cells on the growth of co-cultured brain slices in vitro were detected. Materials and methods (1) Rat bone marrow stromal stem cells (BMSCs) were cultured in vitro and differentiated into neurons.
【学位授予单位】：中国医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329
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