| Spinal cord injury (SCI), one most serious disease in the world, results in axonal degeneration and neuronal necrosis. The microenvironment after SCI in adults is much less receptive to regrowth, thus hindering the regeneration of the damaged nerve. However, accumulating evidence these years has demonstrated that the central nerves in adults can regenerate in a supportive environment following injuries such as SCI. So we are looking forward to the patients, such as Sang Lan, standing up again. Because of the multifaceted nature of SCI, the therapeutic efficacy of current treatment strategies is not so satisfactory till now. It is believed nowadays that combinatorial measures, including neurotrophic factors and biomaterials, have been investigated in potential protocols for SCI repair. Exogenous neuretrophic factor is extensively used as they supply nutrition and guidance cues for axonal regrowth, and application of the neurotrophic factors is required as they are insufficient after SCI. But researches at present indicate that the extensive application of neurotrophic factors is limited. Transplanting genetically-modified cells may result in immunologic rejection. Adenoviral vector may have safety problems. Mini-osmotic pump could cause infection. Therefore, a more feasible and effective way for NT3 delivery is required. NT3, a member of neurotrophin family, is a most crucial factor in promoting central nerve regeneration. It is possible that when NT3 is gene-modified, it may tightly bind to the injury site or certain biomaterial to have more effective therapeutic efficiency. In this study, the NT3, the collagen-binding NT3 and the laminin-biding NT3 are recombinated. After that, the binding assay and the biological activity of DRG cells are conducted. The polymer Poly (lactic-co-glycolic acid) (PLGA) and the collagen scaffold are two suitable bioscaffolds. PLGA is approved by the U.S. Food and Drug Administration. The multi-channel PLGA nerve conduits with porous architectures in this study were recently manufactured and proved useful for SCI reconstruction. We are to use this scaffold and the recombinant NT3 to save SCI. Collagen material could serve as a physical bridge to connect the rostral and caudal of the injured spinal cord and a vehicle to deliver NT3. As collagen could be easily obtained and have good plasticity and biocompatibility, it has been extensively used in SCI repair. We are exploring a kind of fibrous collagen which is fit to the scientific researches according to the previous ordered collagen manufacture. The retained mechanical strength and fiber arrangements make it possible for the cells and axons growing and extending. Furthermore, the neurotrophic factors can bind to the collagen scaffold to construct an ideal system for NT3 delivery. In the present research, the combination of the new fibrous collagen scaffold and the collagen-binding NT3 was used for SCI repair.Chapter One:The construction and harvest of the recombinant NT3, collagen-targeting NT3 and laminin-targeting NT3 and their function studiesObject:To recombine NT3, reconstruct collagen-targeting NT3 and laminin-targeting NT3 by fusing collagen binding domain and laminin binding domain, respectively. Obtain the three NT3 by protein expression, renaturation and purification for the future studies.Methods:Obtain the genome cDNA from the human brain tissue, amplify the coded sequence of NT3 mature peptide (nt3) using its upstream and downstream primers, and recombinate pET28a-nt3 with nt3 and his-tagged plasmid pET28a. Construct pET28a-CBD-nt3 and pET28a-LBD-nt3 by adding collagen binding domain (CBD) and laminin binding domain (LBD) to the part between his-tag and nt3, respectively. After the expression of NT3, collagen-targeting NT3 and laminin-targeting NT3 in E.coli, the E.coli was collected, ultrasonicated, washed and dissolved. The three kinds of protein were harvested in the inclusion body, followed by renaturing and purifying. The collagen binding ability and the laminin binding ability was tested, and indicated in the curve diagram. After isolated the rat embryos from E15 rats, the dorsal root ganglion (DRG) was obtained carefully. As NT3 could maintain neuron survival and promote neurite outgrowth of DRG, the bioactivity qualitation and quantitation assays of the three NT3 were tested with DRG and DRG cells. Finally, we investigated the biological activities of the collagen-targeting NT3 and laminin-targeting NT3 on collagen and laminin in vitro, respectively. The surviving neurons and the neurite outgrowth was quantificated.Results:The collagen binding domain and the laminin binding domain was fused with the correct gene sequence of NT3 (nt3). The recombination of nt3 was obtained, and the nt3 with collagen binding domain or laminin binding domain was constructed. The proteins were expressed at 37℃with IPTG induction. It showed that the three bonds were about 15.49KD,17.69KD和31.88KD. After renaturation and purification, the three purified proteins were in the corresponding place. Western Blotting results furtherly demonstrated the exact proteins. It was showed in the binding ability curve that the collagen binding ability of collagen-targeting NT3 was significantly higher than NT3, and the laminin binding ability of laminin-targeting NT3 was significantly higher than NT3. The bioactivity qualitation and quantitation assays indicated that the biological activities of the collagen-targeting NT3 and laminin-targeting NT3 were not decreased. It was also demonstrated in the functional study in vitro that collagen may bind more collagen-targeting NT3, and laminin may bind more lanminin-targeting NT3. In this way, these two proteins could maintain neuron surviving and promote neurite outgrowth of DRG cells more effectively.Conclusion:The method of protein expression, renaturation and purification was feasible and convenient. The bioactivity of either reconstructed collagen-targeting NT3 or reconstructed laminin-targeting NT3 was not decreased. However, the binding abilities of the two proteins were significantly higher than that of NT3. The results of the functional study in vitro demonstrated that the collagen-targeting and laminin-targeting NT3 could have more effective biofunctions as they have significantly stronger binding ability to collagen and laminin, respectively.Chapter Two:The combination of the PLGA and recombinant NT3 on spinal cord injury repairObject:To repair rats' completely transected spinal cord injury with the combination of the newly manufactured PLGA and the recombinant NT3. To verify the feasibility and the effect of the combinated treatment measures, and provide information for the future treatment strategies.Methods:Completely transect T8-T10 to make rat spinal cord injury models, and implant the PLGA and NT3 immediately into the injury site. The injured rats were given considered care, and perfused after 16 weeks post surgery. During the whole period, the locomotor functional recovery was monitored and compared through BBB rating scale and % right steps in the grid walk test.18 days before the animals sacrified, every 5 rats in each group were randomly chosen, and microinjected with anterograde tracer into the sensorimotor cortex (within 2mm according to the midline and Bregma).10 days before the animals died, every 5 rats in each group were randomly chosen, and microinjected with retrograde tracer into 10mm below the injury site. The cortical somatosensory evoked potential was tested immediately before the animals perfused, with stimulating the sciatic nerve, and recording in the sensorimotor cortex. After the rats were perfused, the spinal cord was carefully dissected and processed. Through the staining of the neurofilament and the glial fibrillary acidic protein, the neural fiber growing across the glial scar and the transplantation was analysed, and its positive area was quantified. The anterograde tracer labeled axons were visualized with horseradish peroxidase, and its migration distance and % positive area was counted. The spinal cord tissue with retrograde tracing was observed and analysed immediately, following the measurement of the migration distance and % positive area of the retrograde tracer labeling fibers.Results:In the PLGA+NT3 group of rats, BBB score was greately improved, especially in the first 7 weeks post surgery. During the later period, BBB score was slowly going up or even plateaued. BBB rating scores continued rising gradually in the groups of non-treated, NT3 alone and PLGA alone. Animals in the sham group, scoring 21, had normal performances after 1 week post surgery. At 7th,12th and 16th week after injury, BBB scores of the PLGA+NT3 were significantly higher than those of non-treated, NT3 alone and PLGA alone groups. The % right steps in the grid walk test was measured at 32d,60d,150d, showing that the performance of the PLGA+NT3 animals in the grid walk test was significantly better compared with that of the non-treated, NT3 alone and PLGA alone animals. Functional reconstruction was indicated in the BBB rating scale and the grid walk test, meanwhile, the tissue and fiber structural connection was demonstrated in the morphology of immunohistochemisty, anterograde tracing and retrograde tracing results. The immunohistochemisty results also showed that the scarring was weaker in the PLGA+NT3 group, permiting more neural fibers growing into the transplantation; the scarring was much more intensive in the non-treated group; and there were fewer fibers in the lesion in NT3 alone and PLGA alone groups. The anterograde and retrograde tracing results demonstrated that the tracers could go across the transplantation in PLGA+NT3 group, and there was significance in the migration distance and % positive area between this group and the others. However, CSEP results indicated the limited neural regeneration, as there was no induced evoked potential.Conclusion:To treat spinal cord injury, the combination of the PLGA and the NT3 could not only supply physical support, but also provide attractive microenvioronment for axonal regrowth. With the combination of PLGA and NT3 treatment, there was partial functional recovery in the injured rats. This manufactured PLGA was characterized as high porosity and good interconnection, thus easy for the neurotrophins entering. Therefore, the PLGA could have synergistic effect with neurotrphins, and enhance spinal cord injury repair.Chapter Three:The collagen-targeting NT3 promote spinal cord injury repair with binding to collagen scaffolds through the collagen binding domainObject:To manufacture new suitable ordered collagen scaffolds for spinal cord injury repair. To investigate the effect of the system of the scaffolds binding with collagen-targeting NT3 on spinal cord injury repair. Provide new strategies for spinal cord repair, or supply directions for the future treatment measures.Methods:Completely transect T8-T10 to make rat spinal cord injury models, and implant the ordered collagen scaffolds and collagen targeting NT3 immediately into the injury site. The injured rats were given considered care, and perfused after 16 weeks post surgery. During the whole period, the locomotor functional recovery was monitored and compared through BBB rating scale and % right steps in the grid walk test.18 days before the animals sacrified, every 5 rats in each group were randomly chosen, and microinjected with anterograde tracer into the sensorimotor cortex (within 2mm according to the midline and Bregma). After the rats were perfused, the spinal cord was carefully dissected and processed. With the staining of the neurofilament and the glial fibrillary acidic protein, the neural fiber growing across the glial scar and the transplantation was analysed, and its positive area was quantified. The % positive area of 5-hydroxytryptamine (5HT) was also measured. The anterograde tracer labelling axons were visualized with horseradish peroxidase, and the positive area within 5mm caudal to the injury was counted.Results:In the ordered collagen scaffold+collagen targeting NT3 group of animals, BBB rating scores continued rising in the 16 weeks after surgery. The functional recovery was so speedy that there was significance comparing with the transected-only group, collagen-targeting NT3 group, ordered collagen+PBS group and ordered collagen+NT3 group. The BBB scores of the transected animals hardly rised, and those of the collagen-targeting NT3 animals, ordered collagen+PBS animals and ordered collagen+NT3 animals gradually improved. The % right steps in the grid walk test was measured at 32-150d post surgery, showing that the performance of the ordered collagen+collagen targeting NT3 animals in the grid walk test was significantly better compared with that of the transected-only, collagen-targeting NT3 alone, ordered collagen+PBS and ordered collagen+NT3 animals. These two investigations indicated that①ordered collagen benefited spinal cord injury repair②functional recovery in the ordered collagen+collagen-targeting NT3 group of animals was significantly better than that in the collagen+NT3 animals. It indicated that collagen-targeting NT3 could strongly bind to the ordered collagen and have better therapeutical effect. The staining of neurofilament, BDA-labeled fibers and 5HT fibers and their positive area showed that the system of ordered collagen and collagen-targeting NT3 could repair spinal cord injury more effectively. Furthermore, these regenerating axons consisted with the functional recovery, indicated that the regenerating neural fibers could promote locomotor functional recovery.Conclusions:The system of the ordered collagen scaffold and collagen-targeting NT3 could effectively deliver NT3 to treat spinal cord injury. This therepy could enhance functional recovery and promote neural fiber regenerating. The neurofilament,5HT fiber and BDA-labeled corticospinal tract regeneration was consistent with the functional improvement. |
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