The Research On The Correlation Between The Axon Regeneration And E3B1 Gene Expression Regulation

BackgroundThe CNS injury is an injury with severe consequence and a great burden to the patients and their families as well as the society due to its frequent occurrence in prime of life and poor effectiveness in treatment. Therefore how to improve the repair and regeneration and reconstruct function after CNS injury is one of the hot studies in orthopedics.The existance of axon growth inhibitors like myelin leads to the retraction and collapse of neuron axon and inhabit the regeneration of axon and repair after CNS injury.The mechamism of axon regenertion failure caused by axon growth inhibitors eventually lies in the neuronal cytoskeleton actin-depolymerization, stress fiber formation and inhabition in the cytoskeleton reconstrction. Previous studies have proved that to stimulate the neuronal cytoskeleton actin-depolymerization and reconstruct the cytoskeleton could enhance the axon growth,thus,to find a way of stimulating the neuronal cytoskeleton actin-depolymerization and reconstructing the cytoskeleton so as to release the inhibition is the key to CNS injury repair. The present studies believe that the neuronal cytoskeleton actin-depolymerization and reconstruction of the microfilament, microtubule of cytoskeleton could disinhibit axon regenertion due to the inhibitors and help the oriented axon growth in a prossess of multi-phases and multi-genes,while the existance of some essencial genes could not identified. It is the existance of such genes that make the CNS injury repair feasible for the their function of releasing of inhibitiin in the axon regeneration and enhancing the oriented growth.E3B1(also called Abi1)was separated and identified in 1995 as a binding-protein regulating the cells growth and proliferating the Abl Non-Receptor Tyrosine Kinase(also called Eps8).The homologous protein family mainly includes Abi2,Agbp and NESH. The further study showed that the E3B1 plays most important part in the cytoskeleton reconstrction and the neurite outgrowth and its over-expression could result in the actin-depolymerization and the inhabition in the cytoskeleton reconstrction, thus E3B1 could be a critical gene regulating actin-depolymerization and cytoskeleton reconstrction. Therefore in this study, we explored the changes of E3B1 protein expression prior to and after the neurite growth inhibition by using bio-chemical technique and finished the designing, constructing, screening and transforming siRNA to inhibit the E3B1 protein expression in the neurons and enhance the regeneration of axon by using the RNA interference technology, which provided a novel idea of treating the inhibition of the anox regeneration and a new way of regenearation and repair after the CNS injury.Methods and Materials1. The study on the changes of E3B1 expression in the acute spinal cord injury tissue of rats.A model of acute spinal cord injury tissue of rats was set up and the changes of E3B1 protein and E3B1mRNA in the tissues before and after spinal cord injury were observed in a dynamic way and its significance of the changes of E3B1 protein in the acute spinal cord injury was analyzed by way of immunohistochemistry, RT-PCR,Western blot.2. The separation, culture, and identification of the rat cortex neurons & the extraction and identification of CNS myelolysisThe primary isolation and culture of the rat cortex tissue were made and its growth and morphology was observed. The quantity and shape of the neutrons were labeled by with fluorescence dye of GFAP and NF200. The CNS myelolysis was extracted and its effect on the axonal growth in different concentration was observed. Then the most effective concentration was used to make component identification by way of Western blot.3. The identification of changes of the neuronal cells E3B1 gene expression1) The expression distribution of the E3B1 in the neuronal cells culture was observed by means of immunohistochemistry. The E3B1 protein and E3B1mRNA expression in the neuronal cells culture was identified by using RT-PCR,Western blot.2) The axon growth inhibitor, inhiCNS myelolysis extracted, was added into the neuronal cells culture and the E3B1 protein and E3B1mRNA expression during the inhibition of neuronal axon by using RT-PCR,Western blot.3) The changes of E3B1 and ?Ш-tubulin expression in the neuronal cells were observed by comparing the fluorescence labeling before and after the CNS myelolysis was added. The correlation between the changes of the E3B1 gene expression and inhibition of axon growth.4. The effect of regulation of E3B1 genes expression on the regeneration of axon growth. in the neuronal cells culture.1) The construction of the siRNA expressing plasmid targeting the E3B1: three RNAi target sites (named as Abi11,Abi12,Abi13) targeting the E3B1(NCBI:NM-024397)a identified target sites (negative control, named as HK) and positive control GAPDH-A were selected. The pGenesil-1 eukaryotic expression vectors with the neoR mark amd GFP green fluorescent mark were selected to construct E3B1 RNAi plasmid. The resistance screening, restriction enzyme and sequencing were made.2) The study on the efect of RNA interference on the E3B1 expression: the neuronal cells were transfected by the RNAi plasmid constructed by way of Lipofectamine 2000. The effects of RNAi targeting various sites on E3B1 genes expression were evaluated by testing the transfection efficiency with fluorescence microscope, RT-PCR and Western blot. The most effective siRNA in inhibiting E3B1 genes were screened.3) The effect of E3B1 regulation on the axon growth: The most effective siRNA in inhibiting E3B1 genes screened were used to transfect the neuronal cells. After the resistance screening was made and the inhibitor of axon growth was added, the axon growth inhibited by E3B1 genes expression was observed and compared by use of the ?Ш-tubulin immunofluorescence and Western blot.Results1. The study on the changes of E3B1 genes in the acute spinal cord injury tissue of rats.There only was little E3B1 mRNA and protein expression in normal spinal cord tissue that mainly lies in spinal cord matter. The E3B1 mRNA and protein expression increased significantly 6 hours after of the acute spinal cord injury and reach the peak at 24-48 hours after injury, then decreased gradually.2. The separation, culture, and identification of the rat cortex neurons & the extraction and identification of CNS myelolysisThere were cells adhering 3 hours after inoculation and becoming stable in quantity and shape at 7-10 hours. The outgrowth of neuritis became longer and net. No positive cells with both GFA and NF200 marks, while the positive cells with NF200 mark were 89.1% , the positive cells with GFAP 6.7% and the rest were 4.2% which were the other types of cells with neither GFA and NF200 mark. The Nogo-A,MAG,CSPG inhibiting protein was identified in the CNS myelolysis by method of Western which had significant inhibiting effect on the axon growth at concentration of 200μg/ml without obvious cytotoxicity.3. The identification of changes of the neuronal cells E3B1 gene expressionThere was little E3B1 mRNA and protein expression in normal neuronal cells culture hat mainly lies in the neuronal somas and the neurite. The ?Ш-tubulin expression decreased significantly while the E3B1mRNA and protein expression increased when the axon growth was inhibited.4. The effect of regulation of E3B1 genes expression on the regeneration of axon growth in the neuronal cells culture.1) The siRNA expressing plasmids targeting the E3B1: Abi11,Abi12,Abi13,HK and GAPDH were constructed. The construction of regrouping RNAi plasmid was proved conforming to the design and the sequence after resistance screening, restriction enzyme and sequencing2) The transfecting rates of neurons in each plasmid were 34.2%(HK),36.3%(Abi11),33.5%(Abi12)和35.7%(Abi13)respectively and no significant difference existing. There was no effect of HK on the E3B1mRNA and protein expression in control group while the E3B1mRNA and protein expression decreased and showed a significant inhibition effect in the experimental groups of Abi11, Abi12, Abi13. The decrease of E3B1mRNA and protein expression reached about 61.26%.3) The co- culture of siRNA- Abi13 transfecting neurons with most effective inhibiting effect on E3B1 with the axon inhibitors showed a well growth of the neurons. The ?-Шmicrotubule showed a high expression and a significant difference was showed comparing with the control group.Conclusion1. The changes of E3B1 gene expression before and after spinal cord injury were identified in a dynamic way. The close correlation between the increase of E3B1 gene expression for a long period time after spinal cord injury and the change of acute spinal cord injury stage indicates that it plays critical part in the spinal cord injury and repair, which has provided an evidence for in vitro experiment in correlation between the E3B1 and axon regeneration.2. The separation, culture, and identification of the rat cortex neurons were made and the extraction and identification of CNS myelolysis were performed successfully. It is identified that 200μg/ml of CNS cortex neuron is most effective concentration in experiment that has provided a base for experiment for correlation between the E3B1 and axon regeneration.3. The E3B1mRNA and protein expression increased significantly when the axon growth was inhibited, indicating that the E3B1 had take part in regulating the axon growth and there existing a close correlation between the its over expression and growth inhibition, which has given a solid foundation for further regulating E3B1 expression and axon growth.4. The siRNA expressing plasmid targeting the E3B1 was constructed successfully. The transfected neurons can surely deceased E3B1mRNA and protein expression. The co-culture of siRNA- Abi13 transfecting neurons with most effective inhibiting effect on E3B1 with the axon inhibitors showed a well growth of the neurons at the presence of CNS myelin and a significant difference with that in the control group, indicating that the over-expression of E3B1 gene is the critical factor in inhibiting the axon growth and can achieve the expected effects on enhancing the axon growth by way of E3B1 gene expression regulation.