| Hypoxic ischemic encephalopathy refers to the clinical and neuropathologic findings in infants who have experienced a significant episode of perinatal asphyxia. Despite major advances in obstetric and perinatal .medicine, there has been little change in the incidence of hypoxic-ischemic encephalopathy, which has been estimated to occur in approximately 2-4 neonates per 1 000 live-born at term; the incidence is higher in small preterm neonates. Depending on the gestational age and the severity of the insult, between 10 % and 60 % of 'asphyxiated neonates who have hypoxic-ischemic encephalopathy expire during the neonatal period, and 25% or more of the survivors develop permanent neuropsychological deficits such as cerebral palsy, mental retardation,learning disability, and epilepsy. As there is no available clinical means to restore the function of neurons damaged by hypoxia-ischemia (HI), treatment is typically supportive and designed to minimize brain injury. Grafting of neural tissue or cells was found to establish functional neuronal interconnections with the host brain and to replace neurons damaged by ischemia. Although some reports have addressed this possibility using adult hosts with ischemic brain damage, few reports has grafted of NSCs into the neonatal brain damaged by HI. To provide the theoretical bases of transplantation of NSCs in neonatal clinical application, we employed measures of sensorimotor performance and long-lasting behavioral alterations to assess whether NSCs grafts placed into the sensorimotor cortical or hippocampus region following ischemic-hypoxic injury in neonates can promote functional recovery. In addition, we have studied the optimization of Lipofectamine2000 mediated reporter gene transfection of NSCs to prelabling NSCs for transplant study, and provided experimental evidence for using NSCs as a resource of vehicle cells for gene therapy.Part I Isolation, Cultiva.tion and Identification of Neural Stem Cells from Embryonic RatObjective: To establish the method of isolation and cultivation of neural stem cells and to observe the characteristic of growth proliferation, and induced differentiation of neural stem cells. Methods: the embryonicrat cerebral cortices were dissociated mechanically and enzymatically. The primary and passage suspension culture without serum technique were performed to acquire cell clones. Indirect immunofluorescence cytochemistry was used to identify the neural stem cells and their differentiated offspring. Results: Cell clones could from the forebrain cortices of embryonic rat after primary and passage cultivation. The clones were nestin-positive and possessed the ability of proliferation and self-renewal. The differentiated cells expressed neuron, astrocytes specific antigen. Conclusion: The neural stem cells isolated and cultured by using the present methods have the ability of self-renewal and differentiation.Part II Transfection of Neural Stem Cells with Green Fluorescent Protein GeneObjective: To study the optimization of Lipofectamine2000 mediated reporter gene transfection of neural stem cells; To provide easy tracing and observing neural stem cells for transplant study, and provide experimental evidence for using NSCs as a resource of vehicle cells for gene therapy. Methods: We separated and cultured the neural stem cells from embryonic rats. Cotransfection of neural stem cells was conducted using fluorescent plasmid as a reporter gene with Lipofectamine 2000. For establishing optimal transfection conditions, we designed the techniques of determining the expression level of a reporter protein byvarying parameters, such as different transfection tin^ different expression time and different ratio of lipofectamine/DNA, and observed the expression of enhanced green fluorescent protein(EGFP); Screening cell clones which stable expressing GFP, observing their property of growth, Stable transfectants were stereotactically implanted into the lateral ventricle of SD neonatal rats, observed the expression of enhanced green f...
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