| Traumatic Brain Injury(TBI) is the most dangerous injury in the Systemic injury because of the high mortality and disability rate. The reasons to this is the seriously damaged central nervous tissue and disorders of nervous functions, to cure the TBI need to reconstruction the neural function and research on the major basic subject of nerve regeneration.A success nerve regeneration in the adult CNS requires a multistep process. First, the injured neuron must survive, and then the damaged axon must extend its cut processes to its original neuronal targets. Once contact is made, the axon needs to be remyelinated and functional synapses need to form on the surface of the targeted neurons. Traditional views believed that nerve regeneration is only occurred in peripheral nervous system and the central nervous.in infancy. As reported by Ramony Cajal in1928, Tello showed in1911that adult CNS neurons could regrow if they were provided access to the permissive environment of a conditioned sciatic nerve. While in1980Aguayo and colleagues replicated these studies with new methods, they transplant the peripheral nerve tissues into the CNS, then they found the axonal from the CNS elongated into the transplantations, but was inhibited when run into the CNS again. regeneration was occurs in definitively confirmed that adult CNS neurons have regenerative capabilities which was inhibited by its glial Environment. it is generally believed that the differences regenerative capabilities between the CNS and the PNS is caused by their glial Environment, the gliocytes of CNS is oligodendrocytes, astroglias and microglia, while in PNS is Schwann Cell.Damage to the central nervous system (CNS) results in a glial reaction, leading eventually to the formation of a glial scar. In this environment, axon regeneration fails, and remyelination may also be unsuccessful. The glial reaction to injury recruits microglia, oligodendrocyte precursors, meningeal cells, astrocytes and stem cells. Damaged CNS also contains oligodendrocytes and myelin debris. Most of these cell types produce extracellular matrix(ECM) that have been shown to be inhibitory to axon regeneration. Chondroitin sulphate proteoglycans(CSPGs) and Tenascin-R(TN-R) are the major composition of the inhibitory ECM.Studies have found that chondroitinase ABC can promote the axonal regeneration after injured by decompose the CSPGs piled up in the damaged areas, while the treatments on TN-R have never be reported so far. Some researchers found that open-field locomotion in TNR-deficient mice recovered better that in wild-type littermates after compression of the thoracic spinal cord, and other researchers use Facial nerve injury model found that number of Tentacles jitter in TNR-deficient mice is more than in wild-type littermates. These researches illustrate that the treatment on tTN-R may promote the regenerative capabilities in CNS after injured. But they also found that the TNR-deficient mice have a lower motor coordination and environmental suitability ability, which was easily to be infuriated. These side effect suggested that treatment on TN-R must target on its specific domain.Sequence analyses of in chicken, rat and, later on, in human have demonstrated the structural relatedness of these molecules to each other and to TN-C. The predicted sequences of chicken and rat reveal a homology of more than80%, and that of human TN-R shows a homology to chicken and rat TN-R of75%and93%respectively. The TN-R sequence is thus phylogenetically highly conserved and displays an overall structural architecture identical to TN-C:a cysteine-rich N-terminal region is followed by4.5EGF-like domains,8FN Ⅲ-like repeats, and a C-terminal region homologous to the FG Ca2+binding sequence. The research express the protein of EGFL function region of human TN-R in prokaryocytes and to prepare rabbit anti-EGFL serum, study the effect on the cortical neuron of rat by TN-R combine with anti-EGFL serum in vitro, analyze the feasibility of anti-EGFL serum for treatment of central nervous system injury. The serum is rich in polyclonal antibody (pAb) which can be acquired by classical and easily process, While monoclonal antibody(mAb) has higher specificity, but was more difficult to gain, so it is not used in this research.Part1The preparation and identification of anti-EGFL serum Objectiveto prepare and identificate the rabbit-derived anti-EGFL antiserum, establishes the foundation of the research on its function in vitro.MethodsTo transform PGEX-4T-1-TN-R aal99-323recombinant plasmid into Rosetta feel-state bacteria, pick out the positive monoclonal colonies by PCR identification, inoculate in kanamycin-resistant LB medium. Add different final concentration of IPTG for induction, when the OD is between0.6-0.8. Collect samples at different time-points for SDS-PAGE electrophoresis, then staining by Coomassie brilliant blue for view the protein bands, and determine the optimal induction condition. Centrifuge the bacilli which contain highest amount of protein expression and re-suspended sedimentation by split buffer, get the Supernatant and sedimentation by ice bath ultrasonic lysis, then perform SDS-PAGE and western blot to verify the expression form of fusion protein. Cultivate1000mL bacilli in optimized expression conditions, get sedimentation by Centrifuge the bacilli and re-suspending by split buffer, harvest incision bodies by ice bath ultrasonic lysis and centrifugalization. resolve incision bodies in urea and recycle the EGFL fusion protein by chelating resin chromatography. The concentration of purified protein was determined by BCA protein assay. For higher purity protein antigen, all the fusion protein was loaded on the SDS-PAGE electrophoresis. The acrylamide piece with antigen were cut out and preserved at-80℃. After frozen overnight, glue block grind with Freund’s adjuvant adequately. After full emulsification, multi-point injection was done at rabbits back. strengthen the immune response for every7days. Harvest sera before immunization for negative control and a small amount of boold10days after3rd immunization. antiserum titer Was detected by ELISA. When the titer achieved the desired level, collect the blood for antiserum preparation and.preserve at-80℃. the total protein of Rosetta expressing the fusion protein and brain membrane protein of rat were perform by SDS-PAGE electrophoresis, then the prepared antiserum for the first antibody to verify its specialty by Western blot. The ELISA plates were coated overnight at4℃with EGFL fusion protein. While sera before immunization for negative control, the antiserum titer was detected by ELISA.ResultsBacteria Rosetta transformed recombinant plasmid, identified by PCR, Was inducted by IPTG at different conditions. Collect the total Bacterial protein for SDS-PAGE electrophoresis. The result indicated the fusion protein have highest expression at37℃,1mM IPTG induction for4h. Then the fusion protein Was be confirmed by western blotting with anti-His tag mAb for the first antibody. Bacterial precipitation broken by ultrasonic, the suprenant and sediment were collected for SDS-PAGE electrophoresis. The result confirmed that the fusion protein was in the form of inclusion body. Expanse culture system according to the optimal condition of step three, harvest inclusion bodys, then recycle the fusion protein after purification. The concentration of protein was0.65mg/L determining by BCA assay. Control sera was harvested before immunization by repeated blood sample method. Rabbits have Normal feeding, be in good health and no significant adverse reactions in whole immune process. serum titers were higher than1:512000after3rd immunization. The volume of blood harvested was32ml, and the volume of prepared antiserum Was10ml. The control sera and antiserum were filtered by0.18μm filter membrane, preserved at-80℃.ConclusionsThe prepared anti-EGFL antiserum has good specificity and high titer, and has laid an important foundation to further study on its function of TN-R protein in vitro.Part2The primary culture and identify of cortical neurons of SD RatObjectsTo improve previous method of primary rat cortical neuron culture to get stably and more long-lasting cells for the next researchMethodsSingle-celled levitation liquid method and microexplant method were used in this research. The brain were rapidly dissected from Neonatal Rat aged from2to3days, then the meninges and cerebral cortical vessels were removed under a microscope.next, the cortex was detached from white matte and rapidly butted to Slices of3～5mm thickness with microsurgery scissors. Slices were transferred with a wide borepipet to another tube at37℃containing0.05%pancreatic enzymes and lOug/ml DNAse I, after incubated for8～10min min in a37℃water bath with a platform rotating at170rpm to keep the slices suspended. Slices were transferred to a15ml tube containing1ml FBS. After1min at room temperature, slices were triturated10times (in about30s) with a l ml blue polypropylene pipet tip with an opening of0.9mm D (inferior). The suspending media was filtered through a200mesh and the filtrate centrifugalize5min,1000rpm at4℃, the sediment was potched twice with2ml DMEM/F12and then suspended in4ml DMEM/F12with10%FBS, the cell suspension was planted on perforated plate with a density of1～3×105/cm2. Four to six hours (4～6h) post-plating, all plating media was removed from cultures and replaced with Neurobasal medium supplemented with2%B27. Half of the culture medium was changed every3～5d. The microexplant used Slices of cortex from the previous experiment, the slices were triturated on a stainless steel filters of50mesh, and the filtrate centrifugalize3min,800rpm at4℃, the sediment was potched twice with2ml DMEM/F12, then the microtissues with0.3～0.5mm D were plant on perforated plate,30to60min later,0.2ml Neurobasal medium supplemented with2%B27was added into each holes softly,20hours later,0.3ml same medium was added into each hole. Half of the culture medium was changed every3～5d. The morphological changes of neuron cells were observed by light microscope Double immune-staining of β-Ⅲ-tubulin.ResultsThe improved method could get about2～16×10cells from each SD rat, most of what was neurons identified under fluorescence microscope, the primary culture was characterized by high uniformity, purity, normal synapse formation and longtime livability. The microexplants could survival on the perforated plate, and neurons migrated from the plants and formed normal synapse.Conclusions The improved culture method can provide quantity of neurons with high purity which can be used in the next experiment.Part3The effect on the cortical neuron of rat by Tenascin-R and anti-EGFL antiserum in vitroObjectiveTo study the effect on the cortical neuron of rat by TN-R combine with anti-EGFL serum in vitro, analyze the feasibility of anti-EGFL serum for treatment of central nervous system injury.MethodsThe anti-EGFL serum was obtained from the rabbits immunized with the protein of EGFL, then combine with TN-R coat petri dish to prepare various culture substrate. The effect on adhere, migrate and neurite outgrowth of neuron by differ culture substrate was observed.ResultsHigh concentration of antiserum against protein of EGFL obtained from the experiment of part1, and it has no effect on the neuron when coated on petri dish as culture substrate with PLL in vitro, but can add the adhesive to the neuron, partly neutralize the inhibitory on the neuron neurite of TN-R. The antiserum can also allow the neuron with its neurite to migrate to the area coated with TN-R.ConclusionThe anti-EGFL serum can weaken the inhibitory on the neuron of TN-R in vitro, the function in vivo need further research. |
