| dhe treatment of CNS axonal injury is a major problem in medicine and neurobiology. It was generally accepted that neurons and axons lose the ability to regenerate in adult mammalian CNS. CNS axon repair includes the regeneration of neurons and that of axons. Cell transplantation is an experimental approach to repair CNS injury, and it is also represents a possible strategy to restore brain function in neurodegenerative disorders such as Parkinson's and Huntington's disease (Gross CG, 2000; Sanberg, PR, 1999; Saporta, S 1999). Various cell types are under investigation in trial studies , of which the most promising tissue sources for neuronal transplantation are immature neurons or their progenitors, including fetal tissue, cell lines, and stem cells (Lindvall OP.1990; Date I, 2000; Flax JD, 1998; Lundberg C, 1997; Park KI, 1999; Ono T, 1997) . Neural plasticity is now widely accepted as a fundamental principle. According to modern scientific viewpoints, CNS plasticity not only means rearrangement of neurons and their interconnections, but also the formation of new neural cells in humans and animals during their whole life span (Gross CG, 2000). So it is possible for transplanted neurons to integrate into neuronal networks composed by host neurons, repairing the lost function caused by neurodegeneration or CNS injury. While a great deal of interest has been focused on the long-term survival and differentiation of transplanted neurons, less attention has been paid to the extent to which implanted neurons can give rise to mature neurons and develop essential properties, especially synapses connected withhost neurons.PC12 cells, a rat pheochromocytoma cell line, have been used extensively as a model for studying the function of neurotrophic factors and neuronal differentiation (Greene LA, 1976; Pang L, 1995) due to their ability to alter their phenotype to a neuron-like cell and be induced morphological changes featured by neurite-like processes and synapse-like structures with a differentiation-inducing reagent such as NGF (Takafumi N, 1999; Greene LA, 1976). Studies have revealed that the PC12 cells survived even 12 months after transplantation (Ono T, 1997) . But it is not clear whether implanted PC12 cells can develop synapses, especially synapses connected with host neurons. Here we established an in vitro co-culture system to investigate neuron-like cells derived from PC12 cells. Our findings demonstrate that neuron-like cells derived from PC12 cells can develop functional synapses with neurons from neonatal cortex.Part One: Co-culture system of primary neurons and PC12 cells Isolation of Neurons from the Cortexes of Newborn Rats: Using the chemical digestion method, we dispersed the newborn rat brain tissue into single cells, and cultured them in DMEM supplemented with 10% horse serum and NS. 4 days later, 0.2uM Ara-c was added into the medium to kill the dividing cells. Following the cells cultured for 6 days, many neurons had a clear nucleus and nucleolus, and their processes became very thick, forming a dense network. Preparation of PC12 Cells and transfection with EGFP: Rat pheochromocytoma PC12 cells were maintained in plastic flasks in medium containing DMEM supplemented with 10% horse serum and5% fetal bovine serum. EGFP-N1 vector was transfected into the PC12cells using HpofectAMINE 2000 reagent. Under the pressure of G418, stable transfectants of PC12 cells bearing the EGFP expression subclones were isolatedCo-culture system : Prepared PC12 cells were washed and harvested, then suspension of 5+104 cells was added into the disk of neurons from the neonatal cortexes. After 4 days, 0.2uM Ara-c was added into the medium to kill non-mitotic neurons.Part Two: induced neural differentiation of PC12 cells with NGF and synapse formationInduced neural differentiation of PC12 cells: 25 ng/ml NGF was added into medium after implantation to induce the differentiation of PC12 cells. One day later, neurites outgrowed from PC12 cell bodies, the neu... |
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