| PART ONEStudy of the Cultivation and Differentiation of Rat Embryonic Neural stem cellsNeural stem cells (NSCs) are defined as those multipotent undifferentiated cells within the central nervous system, which have the capacities of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes. It is already confirmed that neural stem cells exist extensively in the central nervous system of both embryonic and adult mammals. In this study, we have cultured neural stem cells which are derived from the subventricular zone and hippocampus of E14d rat.Material and Methods1. Animal: pregnant Sprague-Dawley Rat (E14).2. Reagents: 0.25% Trypsin , B27 serum-free supplement, DMEM/F12 medium, Basic Fibroblast Growth Factor (bFGF), Epidermal Growth Factor (EGF), Anti-Nestin IgG, Anti-GFAP IgG, Anti-NSE IgG, IHC Kit.3. Primary Culture and Passage of NSCs: The pregnant Sprague-Dawley Rat is killed in order to pick out the embryonic rats. Then dissect the embryonic brains, separate the subventricular zone and hippocampus. These tissues are incubated with Trypsin and EDTA, and dissociated mechanically with a pipette. Cells are seeded in dishes and cultured with DMEM/F12 media containing B27 supplement, EGF and bFGF. Fresh media are supplied every two or three days. After seven days of primary culture, cellular clusters are dissociated and passaged.4. Authenticating neural stem cells: After the cells attached to floor, they are stained by indirect immunocytochemistry for nestin. Nestin positive cells were considered as neural stem cells.5. Differentiation of NSCs: Neural stem cells are seeded on poly-lysine coated cover glass. They are cultured with DMEM/F12 media containing 10% FBS for 5 days. Then they are stained by immunocytochemistry for NSE and GFAP. NSE positive cells are considered as neurons. GFAP positive cells are considered as astrocytes.Results1. At the first day of primary culture, most cells attached to the floor. All of them were small and round in shape, without large projection. After 4-5 days, a lot of attached cells died; the surviving floating cells began to proliferate and form a round shape (Neurosphere) . At about the seven day of primary culture, neurospheres contained several hundreds of cells. By immunocytochemisty, we found most cells in neurospheres were nestin positive.2. After being cultured in DMEM/F12 media containing FBS, neural stem cells were stained by immunocytochemistry for NSE and GFAP. We found that most cells havedifferentiated into GFAP positive cells. The remaining cells differentiated into NSE positive cells.ConclusionsWe have established a routine to culture embryonic neural stem cells in vitro. This procedure provides a platform on which we can make further progress on neural stem cells research.We found that most neural stem cells differentiate into GFAP positive cells in vitro, if they are not treated with any special cytokines.Severe craniocerebral injury is a critical case in neurosurgery, having a high fatality rate. Even the survivors often suffer from such sequeii as paralysis, amentia, anepia, and epilepsy. Even though its fatality rate has been decreasing owing to the progress in microneurosurgery and intensive care technology, the rate of sequeii does not decrease according, mostly because of the limited reparing capacity of adult central nervous system. Moreover, current rehabilitation therapy cannot help much. Since neural stem cells have the capacity of giving rise to new neurons and glias, it is sensible to re-build damaged brain tissue with neural stem cells. We deduce that transplanting neural stem cells into severely traumatic brain will become a practical solution to reduce neuronal function deficiency.In this study, we will transplant embryonic neural stem cells into the severely injured brain. Then the animals' behavioral and morphologic properties will be studied, in order to establish a firm foundation for future clinical application of neural stem cell therapy.Material and Methods1. Animal: 150 Sprague-Dawley rats.2. Neural stem cells labeling: O... |
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