本文選題：Nogo66 + 眼用疫苗　； 參考：《第三軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：原發(fā)性視網(wǎng)膜色素變性（retinitis pigmentosa，RP）是一類以感光細(xì)胞和色素上皮層功能障礙為特征的遺傳性視網(wǎng)膜退變疾病， RP以感光細(xì)胞（Photoreceptor cell，PRC）的變性及凋亡為初始病變，隨病程發(fā)展視網(wǎng)膜各層結(jié)構(gòu)、功能均受到嚴(yán)重影響，視網(wǎng)膜二、三級神經(jīng)元的變性凋亡是RP的重要病理變化。目前臨床上對RP尚無特效治療，基因治療、視網(wǎng)膜移植治療、微環(huán)境調(diào)節(jié)（營養(yǎng)因子供給、中和或消除毒性及抑制性物質(zhì)）、視覺假體植入等均在實驗研究中。 視網(wǎng)膜是中樞神經(jīng)系統(tǒng)（Central nervous system，CNS）的一部分，CNS損傷后再生困難一直是神經(jīng)醫(yī)學(xué)研究的難題。Cohen[1]觀察到CNS損傷后在受損神經(jīng)病灶周圍有大量T淋巴細(xì)胞的聚集并出現(xiàn)短暫、微弱的神經(jīng)保護效應(yīng)，這是因損傷部位自身抗原暴露，刺激T淋巴細(xì)胞活化而產(chǎn)生的保護性免疫反應(yīng)。根據(jù)這一現(xiàn)象Schwartz等在2000[2]年首次提出：“生理性T細(xì)胞介導(dǎo)的自身免疫性神經(jīng)保護效應(yīng)”的概念，并于2005年[3]和2009年[4]在Neuroscience中詳細(xì)闡述了用自身抗原髓磷脂相關(guān)蛋白，經(jīng)主動免疫和被動免疫可誘導(dǎo)并加強這種生理性免疫應(yīng)答的方法，證明生理性免疫應(yīng)答可促進神經(jīng)損傷后的再生修復(fù)。其機制在于通過啟動保護性自身免疫反應(yīng)抑制損傷神經(jīng)元的繼發(fā)損害，減輕或者防止神經(jīng)損害進展，保護未損傷的神經(jīng)元，幫助修復(fù)處于“損傷邊緣期”的神經(jīng)細(xì)胞。動物試驗還證實，上述反應(yīng)缺失時損傷后果更加嚴(yán)重。 已經(jīng)證實高眼壓[5]和視神經(jīng)鉗夾傷后[6]視網(wǎng)膜和視神經(jīng)中存在導(dǎo)致神經(jīng)再生修復(fù)困難的內(nèi)源性抑制因子—髓磷脂抑制蛋白，Nogo蛋白是其中之一。有研究指出RP視網(wǎng)膜可表達(dá)Nogo受體Ng-R[7-8]、P75NTR并與視網(wǎng)膜色素變性存在相關(guān)關(guān)系[9][10]。研究髓磷脂抑制因子Nogo是否參與了視網(wǎng)膜色素變性的病理過程，將為我們利用中樞神經(jīng)系統(tǒng)中存在的生理性自身免疫應(yīng)答機制，實現(xiàn)對RP病理過程的干預(yù)或者提供神經(jīng)保護。 中樞神經(jīng)損傷后Nogo釋放增加、表達(dá)增強，發(fā)揮對損傷神經(jīng)的再生抑制作用，同時啟動神經(jīng)元凋亡過程，導(dǎo)致神經(jīng)元死亡。Nogo蛋白的碳端和氮端在細(xì)胞膜內(nèi)，而胞膜外由66個氨基酸殘基形成拓?fù)浣Y(jié)構(gòu)，又稱Nogo-66。 Nogo-66通過與細(xì)胞表面受體Ng-R的配體受體式結(jié)合，介導(dǎo)了Nogo的中樞神經(jīng)抑制活性。本課題組前期利用基因重組技術(shù)[11]，從質(zhì)粒pET-46EK/LIC-Nogo-66原核表達(dá)得到純化的Nogo-66蛋白，分子量7.34Kd，經(jīng)序列分析Nogo-66蛋白存在多處能夠激活T淋巴細(xì)胞的抗原提呈表位肽段，在體外可直接激活視網(wǎng)膜小膠質(zhì)細(xì)胞。并且應(yīng)用重組Nogo66-cs疫苗經(jīng)全身免疫和局部粘膜淋巴免疫，有效的啟動了青光眼和視神經(jīng)損傷大鼠的生理性自身免疫反應(yīng)，并證實有神經(jīng)保護作用[12]。 皇家外科學(xué)院大鼠（royal college of surgery rat, RCS）是研究RP的經(jīng)典模型，具有和人類RP相似的病理變化及功能特征。本實驗以RCS大鼠為研究對象，首先觀察RCS大鼠自然病程中髓磷脂抑制蛋白Nogo-A/B的表達(dá)情況，然后給予重組Nogo66-cs眼用疫苗進行局部粘膜淋巴免疫。通過檢測免疫大鼠局部視網(wǎng)膜特異性抗體的表達(dá)研究疫苗的免疫反應(yīng)；觀察比較RCS大鼠自然病程中免疫組和對照組視網(wǎng)膜的組織病理學(xué)和視網(wǎng)膜神經(jīng)細(xì)胞凋亡情況，研究重組Nogo66-cs眼用疫苗的神經(jīng)保護作用。同時檢測免疫組大鼠和對照組視網(wǎng)膜中CNTF和bFGF的蛋白表達(dá)情況，探討重組Nogo66-cs眼用疫苗的神經(jīng)保護機制。以期進一步完善重組Nogo66-cs眼用疫苗滴眼液的免疫策略，拓展其使用范圍。 一、主要研究內(nèi)容 1.髓磷脂抑制蛋白Nogo-A/B在RCS視網(wǎng)膜自然病程中的表達(dá)取出生后15d、30d、60d和90d四個時間點的視網(wǎng)膜含色素變性大鼠（RCS-p+)各5只為實驗組，相同時間點視網(wǎng)膜含色素正常大鼠（RCS-rdy+p+)各5只為正常對照組。利用免疫組化和免疫印跡分別定性和定量檢測視網(wǎng)膜中Nogo-A/B的表達(dá)。實驗動物由第三軍醫(yī)大學(xué)大坪醫(yī)院野戰(zhàn)外科研究所實驗動物中心提供。 2.重組Nogo66-cs眼用疫苗經(jīng)粘膜淋巴免疫誘導(dǎo)保護性免疫應(yīng)答效應(yīng)和方式取出生后20天RCS-p+大鼠為研究對象，隨機分為Nogo66-cs疫苗組和CS組，各9只。免疫接種分3組：第一組，初次免疫后追加免疫1次（1次/周），末次免疫后7d取材；第二組，初次免疫后追加免疫2次（1次/周），末次免疫后7d取材；第三組，初次免疫后追加免疫3次（1次/周），末次免疫后7d取材；對照組以相同策略給予CS點眼。采用TUNEL原位末端凋亡法檢測視網(wǎng)膜凋亡陽性表達(dá)，IPP分析視網(wǎng)膜厚度變化，以比較追加免疫對視網(wǎng)膜結(jié)構(gòu)的影響。采用免疫印跡檢測視網(wǎng)膜IgG表達(dá)，以明確重組Nogo66-cs眼用疫苗是否可經(jīng)粘膜淋巴免疫誘導(dǎo)視網(wǎng)膜局部免疫反應(yīng)。 3.重組Nogo66-cs眼用疫苗對RCS視網(wǎng)膜免疫神經(jīng)保護作用研究取出生后30天RCS-p+為實驗對象，隨機分為重組Nogo66-cs眼用疫苗和CS組，各5只。雌雄不限。實驗組初次免疫后追加免疫2次（1次/周），末次免疫后7d取材，對照組采取相同策略。采用IPP檢測視網(wǎng)膜厚度及TUNEL凋亡陽性表達(dá)；探討重組Nogo66-cs眼用疫苗對RP視網(wǎng)膜細(xì)胞的免疫神經(jīng)保護作用。 4.重組Nogo66-cs眼用蛋白疫苗對RCS大鼠視網(wǎng)膜的免疫性神經(jīng)保護機制研究實驗分組及免疫策略同前。采用免疫組化法、免疫印跡法檢測兩組視網(wǎng)膜睫狀神經(jīng)生長因子（Ciliary neurotrophic factor，CNTF）、堿性成纖維生長因子（Basic fibroblast growthfactor，bFGF）蛋白表達(dá)。探討重組Nogo66-cs眼用疫苗對RP的免疫性神經(jīng)保護機制。 二、主要結(jié)果 （一）Nogo-A/B在RCS大鼠視網(wǎng)膜中的表達(dá) 1.組織病理學(xué)改變與對照組相比，P15d和P30d，RCS-p+大鼠視網(wǎng)膜外層細(xì)胞結(jié)構(gòu)和數(shù)目未出現(xiàn)明顯變化，P30d內(nèi)核層(Inner nuclear layer，INL)層細(xì)胞排列出現(xiàn)輕微紊亂；P60d、P90d大鼠視網(wǎng)膜外核層(Outer nuclear layer，ONL)和INL結(jié)構(gòu)出現(xiàn)明顯紊亂，細(xì)胞數(shù)目減少以O(shè)NL和神經(jīng)節(jié)細(xì)胞層(Ganglion cell layer，RGC)層較為明顯。提示RCS大鼠視網(wǎng)膜色素變性發(fā)展過程中，視網(wǎng)膜外層和視網(wǎng)膜內(nèi)層結(jié)構(gòu)均出現(xiàn)改變。 2. Nogo-A/B的免疫組化和免疫印跡顯示Nogo-A/B蛋白在RCS-p+實驗大鼠各時間段視網(wǎng)膜表達(dá)均為陽性，主要位于視網(wǎng)膜INL和RGC層。Nogo-A/B蛋白在RCS-rdy+p+對照大鼠各時間段視網(wǎng)膜表達(dá)為弱陽性表達(dá)。 WB免疫印記檢測RCS-P+大鼠P15d、P30d、P60d和P90d Nogo-A蛋白表達(dá)量分別為：0.82737±0.21292、1.11019±0.08999、1.31552±0.02857、1.26881±0.08042，，組間比較存在顯著差異(P＜0.05)，各時間點組內(nèi)比較存在統(tǒng)計學(xué)差異（P＜0.05）。提示RCS大鼠的RP變性過程中存在內(nèi)源性髓磷脂抑制蛋白Nogo-A/B的動態(tài)表達(dá)變化，表明Nogo-A蛋白參與RP病理過程。 （二）追加免疫后視網(wǎng)膜結(jié)構(gòu)變化和局部免疫效應(yīng) 1.追加免疫效應(yīng)分析初次免疫后追加免疫1次、2次和3次，與對照組相比，實驗組視網(wǎng)膜INL厚度分別為：12.4581±2.64716（P＜0.05）、11.0671±2.38886（P＜0.05）、8.94238±0.82968（P＞0.05）。與對照組相比，實驗組視網(wǎng)膜ONL厚度分別為： 11.6328±1.77681（P＜0.05）、15.4117±4.66376（P＜0.05）、11.3383±4.61539（P＞0.05）。 TUNEL分析視網(wǎng)膜凋亡陽性表達(dá)，初次免疫后追加免疫1、2、3次，實驗組單位面積內(nèi)凋亡陽性表達(dá)IOD sum/Area比值分別為：0.060365219±0.060365（P＞0.01）、0.03565282±0.019462（P＜0.01）、0.107844636±0.107845（P＞0.01）。與對照組相比差異具有統(tǒng)計學(xué)意義。提示2次追加免疫可延緩RP所致視網(wǎng)膜厚度變薄和視網(wǎng)膜細(xì)胞的進行性凋亡。 2.視網(wǎng)膜中IgG抗體檢測結(jié)果經(jīng)重組Nogo66-cs眼用疫苗2次追加免疫RCS大鼠， WB檢測實驗、對照組視網(wǎng)膜IgG抗體表達(dá)量分別為：1.15435±0.25090、0.43957±0.13643，兩組比較差異具有顯著統(tǒng)計學(xué)意義（P＜0.01）。提示重組Nogo66-cs眼用疫苗可經(jīng)粘膜淋巴免疫誘導(dǎo)視網(wǎng)膜局部特異性免疫反應(yīng)。 （三）重組Nogo66-cs眼用疫苗免疫后視網(wǎng)膜TUNEL和厚度變化 1.視網(wǎng)膜凋亡細(xì)胞計數(shù)IPP半定量分析視網(wǎng)膜TUNLEL凋亡陽性表達(dá)，與對照組相比，實驗組IOD SUM/Area為：0.0576±0.0038（P＜0.05），兩組間比較差異具有統(tǒng)計學(xué)意義 2.視網(wǎng)膜厚度IPP分析視網(wǎng)膜各層厚度，與對照組相比，實驗組INL層厚度為：13.4905±0.6211（P＜0.01）；ONL層厚度為：4.8293±0.5943（P＜0.05）。表明重組Nogo66-cs眼用疫苗免疫接種后可有效抑制RP所致視網(wǎng)膜神經(jīng)細(xì)胞進行性凋亡，并延緩視網(wǎng)膜各層厚度變薄，以INL明顯。 （四）CNTF和bFGF免疫組化和免疫印跡 1.免疫組化重組Nogo66-cs眼用疫苗免疫接種RCS大鼠后可誘導(dǎo)CNTF和bFGF在RCS視網(wǎng)膜上的陽性表達(dá)，CNTF表達(dá)以INL和RGC層為主，bFGF主要表達(dá)在RGC層； 2.免疫印跡WB檢測實驗、對照組視網(wǎng)膜bFGF表達(dá)分別為：0.82572±0.02803、0.60233±0.04789，組間比較差異具有顯著統(tǒng)計學(xué)意義（P＜0.01）；WB檢測實驗、對照組視網(wǎng)膜CNTF表達(dá)分別為：0.91272±0.19833、0.60759±0.09207，組間比較差異具有統(tǒng)計學(xué)意義（P＜0.05）；bFGF表達(dá)兩組比較差異相對較高。研究表明重組Nogo66-cs眼用疫苗延緩RP視網(wǎng)膜變性的機制之一是內(nèi)源性bFGF、CNTF的營養(yǎng)支持。 結(jié)論： 1. Nogo-A蛋白參與RP變性過程。 2.重組Nogo66-cs眼用疫苗經(jīng)粘膜淋巴免疫可有效誘導(dǎo)視網(wǎng)膜局部特異性免疫反應(yīng)。 3.重組Nogo66-cs眼用疫苗對RP視網(wǎng)膜具有免疫性神經(jīng)保護效應(yīng)，其機制之一是內(nèi)源性bFGF、CNTF的營養(yǎng)支持。
[Abstract]:Primary retinal pigment degeneration ( RP ) is a kind of hereditary retinal degeneration disease characterized by photoreceptor cell and pigment epithelium dysfunction . The degeneration and apoptosis of photoreceptor cell ( PRC ) are the primary pathological changes . The degeneration and apoptosis of retina II and tertiary neurons are the important pathological changes of RP . At present , there are no special effects on RP , gene therapy , retinal transplantation , micro - environmental regulation ( nutrient factor supply , neutralization or elimination of toxicity and inhibitory substance ) , visual prosthesis implantation and so on .

The results of this study indicate that physiologic immune responses can promote the regeneration and repair of nerve injury . The mechanism consists in inhibiting the secondary damage of injured neurons by starting protective autoimmune response , reducing or preventing the progression of nerve damage , protecting uninjured neurons , and helping to repair the nerve cells in the " damaged edge period " .

It has been proved that the endogenous inhibitory factor - myelin - inhibiting protein ( Nogo ) , which is one of the endogenous inhibitors of nerve regeneration in the retina and optic nerve after high intraocular pressure , has been proved to exist in the retina and optic nerve . It is pointed out that the RP - retina can express Nogo receptor Ng - R - 7 - 8 , P75NTR and related to the degeneration of retinal pigment . Whether the myelin inhibitory factor Nogo is involved in the pathological process of retinal pigment degeneration will provide us with the physiological self - immune response mechanism in the central nervous system to intervene in the RP - pathological process or provide neuroprotection .

Nogo ' s release is increased after central nervous system injury , and the expression of Nogo protein plays an important role in the regeneration inhibition of injured nerve . At the same time , the process of neuronal apoptosis is initiated , leading to neuronal death . The carbon and nitrogen ends of Nogo protein are in the cell membrane and 66 amino acid residues outside the membrane form the topological structure , also known as Nogo - 66 . Nogo - 66 ( Nogo - 66 ) mediated Nogo ' s central nervous inhibitory activity by binding to ligand receptors of the cell surface receptor Ng - R .

In this experiment , we observed the expression of myelin - inhibiting protein Nogo - A / B in the natural course of RCS rats , and then given recombinant Nogo66 - cs eye vaccine for local mucosal lymphoimmunity .
In order to further improve the immune strategy of recombinant Nogo66 - cs eye vaccine , the protective mechanism of recombinant Nogo66 - cs eye vaccine was investigated .

( 3 ) Changes of TUNEL and thickness of retina after immunization with recombinant Nogo66 - cs vaccine

1 . The expression of Nogo - A / B of myelin - inhibiting protein Nogo - A / B at four time points of RCS - p + were detected by immunohistochemistry and Western blot .

2 . The recombinant Nogo66 - cs vaccine was randomly divided into Nogo66 - cs vaccine group and CS group , and the RCS - p + rats were randomly divided into Nogo66 - cs vaccine group and CS group , and 9 were randomly divided into Nogo66 - cs vaccine group and CS group .
the second group , after the first immunization , the second time ( 1 time per week ) was added , and the material was obtained on the 7th day after the last immunization ;
The third group was immunized three times ( 1 time per week ) after the first immunization , and the material was obtained on the 7th day after the last immunization ;
The changes of retinal thickness were detected by TUNEL in - situ end - of - situ apoptosis assay . The changes of retinal thickness were analyzed in order to compare the effect of additional immunity on the retinal structure . Western blot was used to detect the expression of retinal IgG , so as to determine whether the recombinant Nogo66 - cs vaccine could induce local immune response in the retina via mucosal lymphatic immunity .

3 . The RCS - p + of the RCS - p + cells were randomly divided into two groups : the recombinant Nogo66 - cs eye vaccine and the CS group , 5 . Female and male were not limited . After the first immunization in the experimental group , two ( 1 time / week ) immunization was added and the same strategy was adopted in the control group .
To investigate the protective effect of recombinant Nogo66 - cs vaccine on the immune protection of RP retinal cells .

4 . Recombinant Nogo66 - cs eye protein vaccine was used to study the immune neural protection mechanism of RCS rats . Immunohistochemical method was used to detect the expression of ciliary neurotrophic factor ( CNTF ) and basic fibroblast growth factor ( bFGF ) in two groups of ciliary neurotrophic factor ( CNTF ) .

II . Main results

Expression of Nogo - A / B in the retina of RCS rats

1 . After P15d and P30d , the structure and number of outer layer cells of RCS - p + rats did not change significantly as compared with control group .
In the P60 and P90d rats , the structure of outer nuclear layer ( ONL ) was significantly decreased and the number of cells decreased with ONL and Ganglion cell layer ( RGC ) .

2 . Nogo - A / B immunohistochemistry and Western blot showed that Nogo - A / B protein was positive in RCS - p + experimental rats for all periods of time , mainly in the retina and RGC . Nogo - A / B protein was expressed weakly positive in RCS - rdy + p + control rats . The expression of Nogo - A protein in RCS - P + rats was 0.82737 鹵 0.21292 , 1.11019 鹵 0.08999 , 1.315 52 鹵 0.02857 and 1.26881 鹵 0.08042 , respectively .

( 2 ) Changes of retina structure after immunization and local immune effect

1 . The thickness of retina in experimental group was 12.4581 鹵 2.64716 ( P < 0.05 ) , 11.0671 鹵 2.38886 ( P < 0.05 ) , 8.94238 鹵 0.829 68 ( P > 0.05 ) .

11.6328鹵1.77681(P錛

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