The Immnue Neuroprotective Effect And Mechanism Of Recombinant Nogo66-cs Eye Vaccine To Retinitis Pigmentosa Degeneration Rats

Retinitis pigmentosa， RP is a progressive retinal degenerative disease with thecharacter of functional obstacle in photoreceptor cell and retinal pigment epithelium, theinitiate change of retinitis pigmentosa is the degeneration and apoptosis of photoreceptorcell, the second and third grade neuro of retinal are all changed pathologyly with thedegeneration of RP. Now in clinic there is no effective treatment of RP, the gene treatment,retina transplantation, microenvironment regulate (neurotrophic supply and the clear oftoxicity and inhibite factor), prosthesis implantation are all on research.The retina is one important part of CNS, the repair difficult of nerve injuried is alwaysthe problem of medical research. It is observed that a quantity of T lymphocyte aggreagateat the site of injuried nerve after injuried, appearing transient and weak neuroprotectiveeffect. This is the result of autoantigen activate the protective immunity mediated by Tlymphocyte. According to this phenomenon, in2000Schwartz firstly suggests the conceptof autoimmune protection mediated by T lymphocyte. And In2005and2009, he elaboratethat autoantigen active immunity may induce and enhance the autoimmune neuroprotectiveresponse by initiative and passive immunization., the research suggest that it have theeffective of promoting the regenerate repaire of injuried nerve, the main mechanism isinhibiting the secondary injury, to ease and prevent the advancement of injury, protect theunmarred nerve, to help the boundary nerve in the injury. The animal experiment furthersuggests that the injury of nerve would be more graveness at the absence of T lymphocyte.It is confimed that meyline inhibit protein is the main inhibiting factor which leading therepair difficult of injuried optic nerve after the hypertension and clamping. The Nogo is one ofthe myeline proteins. In fact during the degeneration of retinitis pigmentos, the RGC and PRcell of retina are all express the special acceptor of Nogo, Ng-R and P75NTR，which have thedirect relation with the progressive degeneration of RP. Observing the express tendency ofNogo in RP retina would supply the rationale basement for treating RP by the autoimmuneneuroprotection, realying the pathological interference to RP and supply neuroprotective effect. After the nerve injuried, the release and expression of Nogo will play inhibitiveeffection to nerve. At the mean time, it will switch the apoptosis of nerve and lead the celldeath. In general, the N-terminal and C-terminal of Nogo are all located in entramembrane,and the66-amino form the outer of membrane named as the Nogo-66. Nogo-66may lay itsinhibiting effective by binding the special acceptor NgR. Our groups early prokaryoticexpress the Nogo-66protein from the plasmid of pET-46EK/LIC-Nogo-66, its relativemolecular mass is7.34KD, and sequence analysis suggests that eight epitope of Tlymphocyte of Nogo-66could activate microglia in vitro and in vivo. RecombinantNogo66-cs eye vaccine could induce the autoimmune neuroprotective respose and play theprotective effect by local mucous immune and system immune.The royal college of surgery rat is the classic animal model of RP, having thesemblable pathological change and functional change. In our experiment, RCS served as themain experiment animal, at the first we test the express of Nogo-A/B protein in retina, thenimmune rat with the recombinant Nogo66-cs eye vaccine through the CALT(conjunctiva-associated lymphatic system); Research the immunation of recombinant Nogo66-cseye vaccine by detecting the express of special antibody IgG. To study the autoimmuneneuroprotective effect of Recombinant Nogo66-cs eye vaccine by observing thehistopathology and apoptosis of retinal cell during the pathogenesis of RP. At last, explorethe relative mechanism of autoimmune neuroprotect by detecting the express of CNTF andbFGF in the retina of RP. We hope further consummate the immuno strategy ofRecombinant Nogo66-cs eye vaccine and expanding its serviceable range.Our experimental contents include the following three parts：1. Observe the expression of myelin inhibiting protein Nogo-A/B in retinal of RCS：According to retinal degeneration status, RCS-p+(RCS rats with inherited retinaldystrophy) were divided into4groups: P15d、P30d、P60d、P90d, respectively thecorresponding period of the RCS-rdy+p+(RCS rat without retinal degeneration) served asthe control groups,5rats each groups. Immunohistochemical techniques and immuneimprinting (Western blotting, WB) were used to detect the expression of Nogo-A/B proteinin retinal tissue of RCS rats (RCS-p+/RCS-rdy+p+).The experiment animal supplied bythe animal center of the third military medical university.2. Definiting the best immnuo methods of recombinant Nogo66-cs eye vaccine： P20d RCS-P+were served as the experiment animal and were randomly derived intothe Nogo66-cs protein vaccine groups and the chitosan groups, nine rats each group.According immuno programe each group were divided into three teams: The first team:once booster immunization were carried out following the primary immunization (one timea week), dissect at the seventh day after the last immnue.; The second team: twice boosterimmunization were carried out following the primary immunization (one time a week),dissect at the seventh day after the last immune; The third team: thrice boosterimmunization were carried out following the primary immunization (one time a week),dissect at the seventh day after the last immnue; The control group vaccined with chitosan,the method was same with the vaccine groups. The IPP(image pro plus) image analysistechnology served to analysis the TUENL and the thinkness of ONL、INL of RCS; IPPwould be used to detect the apoptotic cells number of RP and the thickness of the retina inorder to comparing different immune protective effect of vaccine to retinal cells. WB wouldbe used to test the expression of IgG, to sure the local immune response induced byrecombinant Nogo66-cs eye vaccine through mucosal.3. The immnuoprotective effect of recombinant Nogo66-cs eye vaccine to RPP30d RCS-P+were randomly divided into two groups: the Nogo66-cs eye vaccinegroup and the chitosan group five rats each gruops. Twice booster immunization werecarried out following the primary immunization (once a week), dissect at the seventh dayafter the last immnue. The control groups vaccined with chitosan, the method was samewith the anterior. The IPP(image pro plus) image analysis technology served to analysis theTUENL cell and the thinkness of ONL、INL of RCS, to explore the immunoprotectiveeffect of recombinant Nogo66-cs eye vaccine to RP4. The immnuoprotective mechanism of recombinant Nogo66-cs eye vaccine to RPThe animal experiment grouping was samed as the former. Immunohistochemistry andwestern blotting technology would be used to detect the express of CNTF and bFGF in RCSrats retinal, to define the neuroprotective mechanism of recombinant Nogo66-cs eyevaccine to RCS.The main results and conclusions are as follows:1. The express of NogoA/B in retina of RCS(1) Pathology result compared with the control, at P15d and P30d, retinal cell in RCS-p+retinal have not any obvious pathological change, only organization of INL celldisorders sightly; At P60d and P90d, there was obvious disorders in ONL and INL ofRCS-P+, obvious reduced cells numbers appear in ONL and RGC layer. It suggests thatduring the degeneration of RCS, the inner and outer are all change.(2) Immunohistochemical and western-blot result of Nogo-A/B It showed thatexpression of Nogo-A/B protein were all positive in retina of RCS-p+rat at each time, itmainly located in INL and RGC layer. The positive expression of Nogo-A/B protein wasweak in retina of RCS-rdy+p+rats. At P15d, P30d, P60d and P90d, the grey value ofNogo-A protein in retinal of RCS-P+rats are respectively：0.82737±0.21292,1.11019±0.08999,1.31552±0.02857,1.26881±0.08042, the differences between groupsare statistically significant (P <0.05). All demonstrated said, Nogo-A/B protein is dynamicchange during the degeneration process in RP, further indicated that Nogo-A protein may beinvolved in the retinal pigment degeneration.2. Immnuoprotective effect and methods of Recombinant Nogo66-cs eye vaccine(1) Analysis of bosster immnue one、two and three booster immunization after theprimary, compare with the control, in vaccine immnue group, the thinkness of retinal INLlayer are respectively:12.4581±2.64716（P＜0.05）、11.0671±2.38886（P＜0.05）、8.94238±0.82968（P＞0.05）. compared with the control, in vaccine immnue groups, thethinkness of retinal ONL layer were respectively:11.6328±1.77681（P＞0.05）、15.4117±4.66376（P＜0.05）、11.3383±4.61539（P＞0.05）。TUNEL tested the apopotosis of retina cell, one、two and three booster immunizationafter the primary, compared with the control, in vaccine immnue groups, the IODSUM/Area ratio of apoptosis is respectively:0.060365219±0.060365（P＞0.01）、0.03565282±0.019462（P＜0.01）、0.107844636±0.107845（P＞0.01）. The differencesbetween groups are statistically significant;(2) Detecting the express of IgG in retina of RP After recombinant Nogo66-cs eyevaccine immune RCS by twice booster immune, the expression quantity of IgG antibody incase and control groups was respectly:1.15435±0.25090、0.43957±0.13643. the differencesbetween groups are statistically significant (P <0.01). It is indicated that by mucosallymphoal immune, recombinant Nogo66-cs eye vaccine could effectively induce specialimmune response in local retina. 3. The neuroprotection effect of recombinant Nogo66-cs eye vaccine to RCS(1) The apotosis of retina cell After Recombinant Nogo66-cs eye vaccine immuneRCS, compare with the control, in vaccine immnue groups, the IOD SUM/Area ratio ofapoptosis was0.0576±0.0038(P <0.05﹡) the differences are statistically significant; thecase groups.(2) The thinkness of INL and ONL After recombinant Nogo66-cs eye vaccineimmune RCS, compare with the control, in vaccine immnue group,the thinkness of INLwas:13.4905±0.6211（P＜0.01）；the thinkness of ONL is:4.8293±0.5943（P＜0.05） thedifferences between groups were statistically significant. It indicated that recombinantNogo66-cs eye vaccine could effectively inhibit apoptosis of retina cell, and delaying thethinner of retina layer, in which the change of INL is obvious.4. The express of CNTF and bFGF(1) The result of IH Recombinant Nogo66-cs eye vaccine could induce effectivelythe positive expression of CNTF and bFGF in retinal of RCS, CNTF mainly express in INLand RGC layer, bFGF mainly express in RGC layer;(2) The result of WB After RCS immune, recombinant Nogo66-cs eye vaccine couldinduce express quantity of bFGF,it is respectively：0.82572±0.02803,0.60233±0.04789(P<0.01)，the differences between groups were statistically significant. The express quantityof CNTF was respectively：0.91272±0.19833、0.60759±0.09207（P＜0.05），the differencesbetween groups were statistically significant. It is indicated that one mechanism ofrecombinant Nogo66-cs eye vaccine exerting its neuroprotective effect to RP is mediated byendogenous nutrition support of bFGF and CNTF.Conclusion:1、 Nogo-A protein may be involved in the retinal pigment degeneration.2、 Recombinant Nogo66-cs eye vaccine could effectively induce special immuneresponse in local retina.3、 One of mechanism that Recombinant Nogo66-cs eye vaccine exerting its neuroprotectiveeffect to RP is mediated by endogenous nutrition support of bFGF and CNTF.