| Unlike peripheral nervous system (PNS) axons, severed central nervous system (CNS) axons are unable to regenerate. Scientists have tried many means to promote CNS regeneration, such as applying of neural trophic factors, deleting glia scars, transplanting stem cells and so on. In addition, gene therapy is also a valid provision of promoting the regeneration after CNS injury. Some new genes have been discovered during this process, of which Nephroblastoma Overexpression gene (nov) is an important one. As a proto-oncogene, nov was first discovered in 1991. NOV protein, which is encoded by nov gene, is a insulin-like growth factor (IGF) binding protein (IGFBP), belonging to the IGF family. Data indicated that nov gene might participate in the processes of regeneration and repair after CNS injury. In recent years, scientists have shown great interest in a kind of glial cell in the research of CNS injury. Interestingly, some results suggested that olfactory ensheathing cell (OEC), a kind of glia cells with the character between that of the Schwann cell and the astroglial cell, could promote axons' elongation. Like Schwann cells, OECs are also able to form myelin sheath and can induce myelin generation after the nervous regression lesion and the spinal cord injury. Since OECs' new function being discovered, it has been a hot point to be associated with CNS regeneration. But there are few reports about the research of transplanting OECs as a receptor cell expressing objective gene. Gene modification of OECs transplanting therapy conjugates the advantages of OECs and nov gene.In order to make it clear that the effect of nov on the proliferation and differentiation of OECs and that of nov genetic modified OECs on CNS repair and regeneration, we combine the promoting effect of nov gene on the CNS regeneration with the repairing effect of OECs on spinal cord injury, so that a new method to repair the CNS injury could be found. We have done the following work: (1) Drew the OECs from animals, cultured in vitro, investigated the cell growth property, proliferation, and activity in vitro(2) Observed the action of nov on the proliferation and differentiation of OECs by the amplification, transfection and expression of nov plasmid, (3) Analyzed the result of two-dimensional gel electrophoresis between,before and after nov transfection, to find differences and prepare for the evaluating of the discrepant proteins later. We expected that after transfection OECs could steadly express nov, and with the compound actions of OECs and nov, it might provide a more suitable microenvironment for repair and regeneration of CNS. Here are the main results:1. Under invert microscope, after inoculated 24 hours, the most OECs were ball shape, and there wer cloud and mist material distributed around. After cultured for three days, the OECs were shuttle shape with two poles and three-poles mainly, and with the obvious web shaped growth cone structure. Within 2~14 days, the purity of cells could reach 95% or above, and the purity did not decline obviously with the elongation of culture time.2. Cultured for 2 days after purification, most of the cells were two-poles and three-poles, tending towards the same directory, and shaping like Schwann cells. Eight days later, the cells were the same directory.3. OECs Immunocytochemical stain and purity detection: cultured for 2 days before purification, P75NGFr immunologic reaction showed that the cell body was stained deeper than apophysis, The cells tended toward for different directions. Cultured for 2 days after purification, cells were almost all positive. The cell membrane was deep stained. OECs were trigone or shuttle shapeed, tending toward the same direction. The background was clear.4. OECs kept in hypothermia grew well after revivfication. The cell form was shuttle shaped, and the activity was the same as that before frozen. The amount of live cells was over 80%.5. Plasmid amplification of pcDNA3.1-nov was done, and the nucleotide sequences of the cDNA were analyzed. Tran...
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