| Retinal ganglial cells (RGCs) regenerate difficulty after injury, which is the main reason for glaucoma or optical nerve injury causing blind. The inability of RGCs to regenerate following injury may be due, in part, to myelin. Inhibitive factor Nogo would appear to be the most relevant pertaining to neuronal regeneration amongst these myelin-associated proteins. Nogo express little in normal central nervous system (CNS), while express much more when the central nervous system damaged. It causes neuron death by repressing nerve fiber regenerating and starting an apoptosis process of neuron. Nogo has a 66 amino acid extracellular region (Nogo66), which to be an active region, inhibits CNS neuron outgrowth. Currently researches suggested that, active or passive immunization with myelin-associated peptides induced a protective autoimmune response, which promoted functional recovery by reducing degeneration. But its mechanism is still not unclear.At preliminary study, we prokaryotic expressed and purified Nogo66 protein, got the Nogo66-Freund's adjuvant vaccine. The Nogo66 protein vaccine can induce high titer IgG antibody, activate and proliferate T lymphoid cell increase neuroregeneration, decrease nerve fiber further injuring and the vaccine approach may represent a promising repair strategy to promote functional recovery following optical nerve injury.However, Freund's adjuvant is not referring to human, peripheric vaccination has possibility of complications. Therefore, it is necessary to change adjuvant and path of vaccination to be safer.Eyelids conjunctiva has intensive blood vessel and abundant blood supply. There are two lymphoid system, shallow layer and deep. There is importance mucosa local lymphoid tissue (CALT) in the lower lid conjunctiva reductus. Researches confirmed that the CALT can ingest antigens on mucosa surface, identify them and respond. From last century 60's, the conjunctiva vaccinate path already applicated in the animals.Chitosan (CS) existence extensively in the natural world,have the properties of non- toxicity, great biocompatibility, low immunological rejection. And itself has immunostimulatory activity, and can strengthen the medicine permeate and absorption. It is a kind of mucosa immunity slow release material and immunity adjuvant with a very great potential. Our study investigated the immunoprotection mechanism of Nogo66 protein vaccine to retinal or optical nerve injury, improved the adjuvant and vaccinate path of Nogo66 protein vaccine, and made a Nogo66 protein vaccine for eye with chitosan as adjuvant, and investigated its chemistry property, immunity property and nerve protection function mechanism.The study was divided into the following 7 parts: 1. to study immunoresponse of SD rats with different dose Nogo66 vaccine and the condition of rat. Prepared Nogo-66 peptide vaccine at different density and immunized SD rats and splenocyte T cells were tested with MTT. Complication of experimental autoimmune encephalomyelitis (EAE) on experimental animals was evaluated on all experimental animals. 2. To approach the mechanism of immunological effect, SD rats were immunized by Nogo66 vaccine with the optimal dose and killed to get spleen to measure level of immunoglobulins IL-2, IFN-γand IL-4 were detected by ELISA. 3. Models of the retinal vessel permeate of chronic ocular hypertension and optical nerve injury was established in SD rats and investigated the integrity of the blood-retinal barrier (BRB). 4. Nogo66-CS vaccine was prepared. Then their shape, PH, osmotic pressure, viscosity, content and stability were tested. 5. According to eye irritative experiment standard, score the experimental animal eye stimulative grade point, examinated the pathology of the main organ of the experimental animal, investigated the safety of the Nogo66-CS vaccine. 6. Detected the influence Nogo66-CS vaccine to the CD11b expression of the microglia cultured in vitro. After the Nogo66-CS vaccine conjunctiva inoculate, SD rats were examinatedthe IgG antibody title in tears liquid and serum, the antigen particularity IgG type plasma cell and lymphoid cell in conjunctiva organization, examinated CD11b, MHC-Ⅱ, iNOS, TNF-αexpression in retina with immunohistochemice, and investigated the immunogenicity of the Nogo66-CS vaccine. 7. The protecting function and mechanism for Nogo66-CS vaccine to chronic ocular hypertension and optical nerve injury SD rats promoting regeneration were marked the RGCs with the fluorescence gold treacherously, detected retina GAP43, CD3, BDNF, GDNF expression with immunofluorescence, immunohistochemice, and western blot,. Main result and conclusion is as follows:1. It can induce high titer IgG antibody and proliferate T lymphoid cell after inoculating different density of Nogo66 vaccine.The IgG antibody title and T cell propagating of the group of 200μg/300μl were higher than those of other groups (P<0.05). None of experiment rats discovered any similar behavior of experimental autoimmune encephalomyelitis, and none organ pathology examin had seen obvious abnormality. It suggests that 200μg/300μl is the best inoculate density.2. The splenocyte IL-2,IFN-γ,IL-4 level of all Nogo66 protein vaccine groups are higher than those of the control(P<0.01). Among them, the range of the Th1 class cell factor IL-2, IFN-γincreased higher than the Th2 class cell factor IL-4 (P<0.01). It suggests that Nogo66 protein vaccine induce mainly of Th1 immunity reaction, named T lymphocyte immunity effect.3. IOP of rats with chronic ocular hypertension begin to rise at 3 days after modeling, then kept on increasing slowly and reach at 21.5±3.7mmHg, there was significant difference compared to the control group) The toppest level 24.8±3.2mmHg reached at 4th week, there is no obvious decrease even at 12th week. It still has significant difference compared to the control group (P<0.05) maintenanced in a lower level, the tallest at 24.8±3.2mmHg. Retina had never been seen EB obviously on fluorescence microscope after lasered 1, 4 and 8 weeks. Retina permeability increment was slightly higher than those of the control group and didn't show significant deviation. Related analysis the chronic ocular hypertension rats'retina permeability and IOP showed a positive of straight line related. Optical nerve crashing rats'retina showed obvious of blue fluorescence at the next day of crashing, and weakened down slowly, which retina permeability higher than the control group (P<0.05). It suggested that chronic ocular hypertension still can't break rats BRB. Optical nerve crushing break rats BRB and increased retina permeability at earlier period, and this condition will keep on at least a month.4. The self-made Nogo66-CS vaccine was detected for characteristics, pH, osmotic pressure, viscosity, content and stability. The results showed it consistent with the medicine standard to an ocustilla prepn. It suggests that the Nogo protein vaccine has good physico-chemical property when using chitosan as an adjuvant.5. In the eye stimulative experiment, the irritative response score of the Nogo66-CS vaccine group and the control both<3 and it should be considered that Nogo66-CS vaccine and Sodium Chloride either no stimulative according to eye irritative response standard. Retina, heart, liver, kidney, lung, brain, testicle pathology check has never seen obvious abnormality. It suggests that Nogo66-CS vaccine have no stimulative and is safe for body.6. Microglia culture in vitro expressed great CD11b and cells activated after stimulated by Nogo66-CS or Nogo66, expressed less CD11b and cells quiescence after stimulated by CS, While in control rats, CD11b was not detected. Conjunctiva of Nogo66-CS vaccine group rats have a great deal of lymphoid cell infiltrated with IgG type plasma cell increase. CS group rats'conjunctiva have a little amount lymphoid cell infiltrated with no IgG type plasma cell. After vaccinate the Nogo66-CS group rats'tears liquid IgG antibody title kept on increase since the 20th day and had a significant deviation. With the control group (P<0.05), and the serum IgG antibody title started lower range increase at the 34th day. IgG antibody in Nogo66 group and CS group rats'tears liquid and serum didn't show significant variety. Each layer of retinal microglia of the Nogo66-CS group activated, expressed CD11b, MHC-Ⅱ, but no iNOS and TNF-α. It suggests that Nogo66-CS vaccine can activate to conjunctiva immunity and system immunity, and induce retina microglia to activate, it has much stronger immunogenicity.7. The RGCs of the chronic ocular hypertension rats or optical nerve crashing are more than the CS group or non-innoculation group. There are more GAP43, GDNF, BNDF, and CD3 expression in the Nogo66-CS vaccine group rats'retina, little in the CS group. It indicate that Nogo66-CS vaccine could increase the retina neurotrophic factor expression , promoting the damaged neuron regeneration , decreasing the RGCs death after the retinal optical nerve damage, protecting the retinal optical nerve.
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