Recombinant Adeno-associated Virus Aav Carrying The Human Gdnf Gene Damage The Protective Effect And Mechanism Of The Rat Spinal Cord Neurons

It is known that neuronal cell degeneration is a critical component of many neurologic diseases. There is evidence that both neurotrophic factor deprivation and oxidative stress are major potential players as pathological processes in neuronal degeneration.Neurotrophic factors are naturally-occurring functional proteins or peptides which are essential for the neuronal survial, maintainance, and differentiation. Glial cell line-derived neurotrophic factor (GDNF) was cloned as secreted protein from the glial cell line B49 based on its ability to promote dopamine uptake in embryonic mesencephalic cultures by Lin and his colleagues. Recent experiments provide the evidence that GDNF appears to have more potency to promote survival of motoneurons and inhibit the neuron retrograde degeneration when they were directly injected into the local tissues. However, the effect of neuronal protection is transient, lasting only 2-3 weeks. In order to achieve more potent effect to protect the motoneurons from death induced by glutamate and peripheral nerve injury, we utilize the adeno-associated virus to deliver the GDNF gene, which can stably express the GDNF during long term.1. The expression of GDNF in the mammalian cells and investigation of the bioactivty inducing the differentiation of PC12 cell lineIn our experiments, we obtained GDNF cDNA which contains the coding sequences for signal peptide and mature protein from human fatal brain tissues by RT-PCR. The mammalian expression vectors pTARGET/ hGDNF(±) were constructed, and identification of pTARGET/ hGDNF(±) were confirmed by endonuclease digestion and DNA sequence analysis, the results show that the sequence is similar to human GDNF cDNA sequences from the reports of Genebank (L19036 , L15306). The expression vector pTARGET/hGDNF (+) was constructed and transfected into BHK-21 cells mediated by Lipofectamine. The supernatant of BHK-hGDNF cells contains the GDNF about 2ng/ml detected by ELISA.The PC12 cell can be induced to differentiate to neuronal...