| Part I Experimental studies on effect of recombinant adenovirus-mediated hepatocyte growth factor gene on glial scar of rat spinal cord and exploration ofcorrelative mechanismObject: Spinal cord injury is a kind of trauma that affects seriously the health and life quality of mankind. There is yet no satisfactory medical therapy for the disease to date. It has been known that axonal regeneration, induction of remyelination, replacement of dead neurons, are all inevitably taking place in a glial scar environment. However, there is evidence that glial scar can inhibit both axon growth and myelination, and astrocytes is the principal component of glial scar. Some experimental strategies have been employed to inhibit astrocytes proliferation so as to promote axonal regeneration. There are reports that hepatocyte growth factor (HGF) can inhibit scar formation. In this study, we transfected recombinant adenovirus-mediated HGF to astrocytes in vitro, and scratching cultured astrocytes to simulate in vivo injury. Through observing the effect of HGF on proliferation of astrocytes, we exploring a novel strategy to inhibit glial scar formation.Methods and results: (1)We found that the transfection rate of adenoviral vector to the primary astrocytes cells of newborn rat was high through observing green fluorescence protein by fluorescence microscopy, and HGF can be detected in the culture medium through ELISA essay. To prepare a mechanical injury model, astrocytes cultured on 35-mm Petri plates were injured by making sharp crossing line using Rainin RT-20 pipette tip, and interval of lines was 3mm. By detecting the contents of glial fibrillary acidic protein and proportion of S phase, we investigated astrocytes proliferation at different time points after Ad-HGF transfection, and found that Ad-HGF can inhibit proliferation of astrocytes obviously on the first day after scratching injury; (2)Toinvestigate effect of HGF on astrocytes without scratching injury by 3H-TdR methods, we found that HGF had no effect on astrocytes in normal conditions; (3)By observation of two signal pathways relative to cell proliferation, mitogen activated protein kinase(MAPK) and sphingosine l-phosphate(SPP), we found that the activity of MAPK was high in astrocytes, so it may play an important role in physiologic function of astrocytes, but its activity was not enhanced by HGF. The expression of sphingosine kinase(SPK), the key enzyme of SPP increased at low dosage HGF but decreased at high dosage. Therefore, HGF mighe have double affection on astrocytes; (4)Using different doses of HGF protein to post-injury astrocytes, we could further elucidate effect of HGF on astrocytes.Conclusion: HGF has no effect on astrocytes in normal condition, but it has double action on proliferation of injured actrocytes, HGF promotes astrocyte proliferation at low dosage while inhibits it at high dosage. SPP pathway plays a role in the function of HGF.Part II Experimental studies of effect of hepatocyte growth factor onspinal cord axonal regenerationObject: The key strategy in treatment of spinal cord injury is to promote axonal regeneration. Though we don't yet know the mechanisms of axonal regeneration thoroughly, many studies showed that suitable microenviroment can promote axonal regeneration in the central nervous system. The strategy of gene therapy is to provide a suitable enviroment by transgene methods, and providing neurotrophic factors at injured region is the main approach at present. Nerve growth factor, brain-derived neurotrophic factor, ciliary neurotrophic factor, neurotrophic-3, neurotrophic-4/5 were usually used candidate genes. HGF extensively exists in the nervous system, and the level of HGF and c-met recptor expression are varied during development of the rat cerebral cortex. It is suggestd that HGF is involved in the development and maintenance of cortical neurons during differentiation, motogenesis, neuritogenesis and neuronal survival. HGF does have nourishing and protective effects on cortical axons in vitro. However, there...
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