腦源性神經(jīng)營養(yǎng)因子（BDNF）基因克隆及其基因工程細(xì)胞對體外培養(yǎng)腦片生長的影響
發(fā)布時間：2019-04-01 10:44
【摘要】：帕金森氏病(PD)是神經(jīng)系統(tǒng)錐體外系常見的慢性退行性疾病。主要病理變化是位于中腦黑質(zhì)的多巴胺(DA)能神經(jīng)元變性和消失,導(dǎo)致紋狀體的DA能神經(jīng)纖維終末變性消失,這種黑質(zhì)-紋狀體神經(jīng)傳導(dǎo)路的損害使得DA水平下降,由此產(chǎn)生了PD的典型癥狀。目前,臨床上對PD治療的研究主要集中在兩個方面:一是以補充多巴胺的不足為目的藥物治療:如給病人定期服用左旋多巴,美多巴,息寧,安坦等,通過藥物治療病人的大多數(shù)臨床癥狀改善,但并不能阻止病情的發(fā)展,即不能治愈。另一種方法是以搶救幸存神經(jīng)元為目的的治療:如導(dǎo)入神經(jīng)營養(yǎng)因子的基因治療。 腦源性神經(jīng)營養(yǎng)因子(brain-derived neurotrophie factor,BDNF)作為神經(jīng)生長因子家族成員,與神經(jīng)系統(tǒng)發(fā)育有著密切關(guān)系,不僅對中樞神經(jīng)元的發(fā)育、增殖分化、存活起重要作用,而且對損傷后神經(jīng)元的修復(fù)與功能重塑也起了重要作用。近年來神經(jīng)生長因子家族一些成員紛紛被克隆并應(yīng)用于中樞神經(jīng)系統(tǒng)退行性疾病的防治。 針對上述研究趨勢,本研究采用腦皮質(zhì)-紋狀體-黑質(zhì)三組織聯(lián)合培養(yǎng)技術(shù),選用腦源性神經(jīng)營養(yǎng)因子(BDNF)通過RT-PCR及載體克隆的方法,構(gòu)建BDNF真核表達(dá)載體pEGFP-N1-BDNF,以重組質(zhì)粒pEGFP-N1-BDNF作為外源性基因,與脂質(zhì)體一起轉(zhuǎn)染骨髓基質(zhì)干細(xì)胞(BMSCs),通過熒光顯微鏡觀察,并結(jié)合BDNF免疫細(xì)胞化學(xué)染色等形態(tài)學(xué)方法鑒定穩(wěn)定表達(dá)BDNF蛋白的基因工程細(xì)胞,檢測其對體外聯(lián)合培養(yǎng)腦片生長的影響。 材料與方法 (一) 大鼠骨髓基質(zhì)干細(xì)胞(BMSCs)體外培養(yǎng)及向神經(jīng)元誘導(dǎo)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.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R329
本文編號：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.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R329
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