The Mechanism Research On Neurotrophin-3 Protection Against Neurocyte Apoptosis

Neurocyte apoptosis correlates closely with nosogenesis and therapeutics of orthopedics diseases such as trauma, tumour etc. It is important to explore the mechanism of action about neuronal apoptosis protection to provide information for the therapy of these diseases.Neurotrophin-3(NT-3) belongs to nerve growth factor family, and exists extensively in central nervous system and periphery tissue. NT-3 is an important regulator of neuronal survival, development, function, and neuronal differentiation, and has become the focus of intense interest because of its association with various neurological disorders. NT-3 was previously found to transiently protect immature oligodendrocytes from glutamate-mediated apoptosis, NT-3 has also exhibited anti-apoptotic effects on cultured cortical neurons, while the mechanism in them is not elucidated completely. Reports excluding oligodendrocytes and cortical neurons are rare. Therefore, It is significant to explore the mechanism of action about neuronal apoptosis protection to establish new strategies of gene therapy for these diseases.Firstly, We amplified target gene by RT-PCR and cloned it into the pMD-18T vector, then analyzed its sequence and compared it with the sequence in GenBank. We got a 777 bp gene segment by RT-PCR. The DNA sequence was identical to rat NT-3 gene sequence in GenBank. It suggested that the target gene was correctly inserted into the vector. We subcloned it into pRSET-A vector and introduced it into Escherichia coli BL21. The expression was induced by IPTG, and identified by SDS-PAGE. The fusion protein was purified by niccolum purify kit. A new protein band of about 34 kDa appeared on SDS-PAGE after induction of IPTG. We immuned rabbits with immunological adjuvant for specificity antibody preparation. A specific high titer antibody of 1:64000 was gained by immuning rabbits with immunological adjuvant. All this make it possible to do further investigation.Secondly, we treated PC12 cells with 6-hydroxydopamine (6-OHDA) which is a widely used neurotoxic agent that can be used to selectively damage dopaminergic neurons in vivo and in vitro. This treatment resulted in activation of caspases-3 and subsequent apoptosis, as detected by TUNEL staining. In addition, Akt phosphorylation was decreased. Pretreatment with NT-3 reduced the percentage of apoptotic cells and caspase-3 activity induced by 6-OHDA and suppressed the cleavage of caspase-3 and PARP with a significant decrease in cell viability. Moreover, Akt phosphorylation was enhanced and 6-OHDA-induced chromatin condensation was suppressed by NT-3. Such NT-3-evoked supression in chromatin condensation was reversed by anti-TrkA antibody receptor blockade. Further study revealed that LY294002, an inhibitor of PI3-kinase (a molecule upstream of Akt), enhanced 6-OHDA-induced apoptosis. These data indicate that NT-3 prevents 6-OHDA-induced apoptosis in PC12 cells via activation of PI3-kinase/Akt pathway.Finally, we also observed the protective effect of recombinant adenovirus-mediated NT-3 gene(Adeno-NT3) on the survival and regeneration of motoneurons after sciatic nerve crush. In the experimental group, Adeno-NT3 was injected directly into the spinal cord parenchyma, while in the control group, isotonic Na chloride was injected instead. Nissl staining and CTB retrograde were carried out to label the survival and regeneration motoneurons respectively, and the numbers of the motoneurons of survival and regeneration were counted as well. We found that numbers of motoneurons of both survival and regeneration were significantly higher in the experimental group than that of the control group, and the ones of regeneration were more than the ones of survival in experimental group. So we made a conclusion that Adeno-NT3 can promote the survival and regeneration ability of motoneuron after sciatic nerve crush, the mechanism in it still needs further investigation.To sum up, in this study we amplified target gene by RT-PCR and inserted it correctly into the pMD-18T vector and pRSET-A vector, and introduced it into Escherichia coli BL21. A new protein band of about 34 kDa appeared on SDS-PAGE after induction of IPTG. We used several methods to investigate the effect of NT-3 on 6-OHDA-induced apoptosis in PC12 cells . We found that NT-3 prevents apoptosis via activation of PI3-kinase/Akt pathway. We also observed the animal experiment and made a conclusion that Adeno-NT-3 can promote the survival and regeneration ability of motoneuron after sciatic nerve crush. Therefore, this study may bring a new strategy of gene therapy for orthopedics diseases such as trauma, tumour and other diseases.