| The spinal cord is the major conduit through which sensory and motor signals pass between brain and body and its injuries always lead to a disruption of these pathways, resulting in dramatic and long-lasting functional losses. It is now widely accepted that axon regeneration depends on the interplay between extrinsic cues and intrinsic properties of the damaged neuron, encouraging various new therapeutic approaches to manipulate the regenerative potentials of lesion neuron and to modify the non-permissive environment in SCI.Following traumatic SCI, there is chronic demyelination of axons, shedding light on the importance of remyelination of injured axons. Transplantation of cells with myelinating potentials (Schwann cells, olfactory ensheathing glia, neural stem cells, et al) are used for this purpose but with unsatisfactory results. O2A progenitor cells have shown the capability to differentiate into oligodendrocytes and to remyelinate the demyelinated axons in the CNS, but the functional extent of remyelination is limited. All these lead to the idea that to enhance the migration of grafted O2A progenitor cells with genetic modification. It has been demonstrated in vivo that the migration of Schwann cells, which mediated by p75 neurotrophin receptor, contributed to the regenerative process of the axotomized axons in peripheral nervous system. It is especially promising that promotion of functional repair of SCI may be accomplished by engraftment of O2A progenitor cells modified by adenoviral vector-mediated p75NTR gene transfer.In the present study, O2A progenitor cells were purified by modified different- attachment, identified by immunochemistry and induced-differentiation, and then amplified by improved medium. Cre/loxP-mediated intracellullar homologous recombination method was employed to construct the p75NTR and EFGP adenoviral vector containing different promotor. Then, the modified O2A progenitor cells transfected by theses vectors were transplanted into the damaged spinal cord to investigate its effect on the functional repair of injured spinal cord.The main results and conclusions are as follows:1. In the culture system of primary mixed glial cell from newborn rat cortex, the slightly modified different-attachment method was used to isolate and purify the O2A progenitor cells (OP). The cells then were immunocytochemistrically identified by A2B5 marker and subsequently confirmed by the induced-differentiation in which OP divided into astrocyte and oligodendrocyte by serum. Also, OP could be induced into oligodendrocytes by T3. All of these results could indicate that the purified cells were oligodendrocytes-type-2-astrocyte progenitors.2. OP could survive and proliferate in the astrocyte-conditioned medium, which indicating that the medium contained mitogen of OP.3. Under culture condition, it was found by immunocytochemistry that there was a constitutive expression level of p75NTR in oligodendrocytes, while almost no p75NTR positive-immunoreactivities in OP was observed.4. Shuttle plasmids containing CMV or MBP promotor, pCMV-p75-shuttle, pCMV-EGFP-shuttle, PMB-p75-shuttle, pMBP-EGFP-shuttle, were constructed by molecular colony technique. The plasmids containing adenovirus genome were purified by CsCl method and identified by enzyme incision.5. Cre/loxp-induced intracellullar homologous recombinant method combined modern virology techniques were employed to construct high-titer recombinant-deficit adenoviral vector AdCMV-p75, AdCMV-EGFP, AdMBP-p75 and AdMBP-EGFP. The vectors were then identified by PCR at DNA level,by RT-PCR at RNA level and by immunocytochemisty / indirect immunofluorescence method at protein level in vitro. The vectors were also transfected into mixed gilal cells and injected into spinal cord, from which we concluded that the expression of transgene delivered by AdMBP-p75 and AdMBP-EGFP was only found in oligodendrocytes, and that the MBP promotor...
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