重组Nogo66-cs眼用疫苗对RCS视网膜色素变性大鼠的免疫性神经保护和机制研究

发布时间：2018-07-03 02:08

  本文选题：Nogo66 + 眼用疫苗　； 参考：《第三军医大学》2012年硕士论文

【摘要】：原发性视网膜色素变性（retinitis pigmentosa，RP）是一类以感光细胞和色素上皮层功能障碍为特征的遗传性视网膜退变疾病， RP以感光细胞（Photoreceptor cell，PRC）的变性及凋亡为初始病变，随病程发展视网膜各层结构、功能均受到严重影响，视网膜二、三级神经元的变性凋亡是RP的重要病理变化。目前临床上对RP尚无特效治疗，基因治疗、视网膜移植治疗、微环境调节（营养因子供给、中和或消除毒性及抑制性物质）、视觉假体植入等均在实验研究中。 视网膜是中枢神经系统（Central nervous system，CNS）的一部分，CNS损伤后再生困难一直是神经医学研究的难题。Cohen[1]观察到CNS损伤后在受损神经病灶周围有大量T淋巴细胞的聚集并出现短暂、微弱的神经保护效应，这是因损伤部位自身抗原暴露，刺激T淋巴细胞活化而产生的保护性免疫反应。根据这一现象Schwartz等在2000[2]年首次提出：“生理性T细胞介导的自身免疫性神经保护效应”的概念，并于2005年[3]和2009年[4]在Neuroscience中详细阐述了用自身抗原髓磷脂相关蛋白，经主动免疫和被动免疫可诱导并加强这种生理性免疫应答的方法，证明生理性免疫应答可促进神经损伤后的再生修复。其机制在于通过启动保护性自身免疫反应抑制损伤神经元的继发损害，减轻或者防止神经损害进展，保护未损伤的神经元，帮助修复处于“损伤边缘期”的神经细胞。动物试验还证实，上述反应缺失时损伤后果更加严重。 已经证实高眼压[5]和视神经钳夹伤后[6]视网膜和视神经中存在导致神经再生修复困难的内源性抑制因子—髓磷脂抑制蛋白，Nogo蛋白是其中之一。有研究指出RP视网膜可表达Nogo受体Ng-R[7-8]、P75NTR并与视网膜色素变性存在相关关系[9][10]。研究髓磷脂抑制因子Nogo是否参与了视网膜色素变性的病理过程，将为我们利用中枢神经系统中存在的生理性自身免疫应答机制，实现对RP病理过程的干预或者提供神经保护。 中枢神经损伤后Nogo释放增加、表达增强，发挥对损伤神经的再生抑制作用，同时启动神经元凋亡过程，导致神经元死亡。Nogo蛋白的碳端和氮端在细胞膜内，而胞膜外由66个氨基酸残基形成拓扑结构，又称Nogo-66。 Nogo-66通过与细胞表面受体Ng-R的配体受体式结合，介导了Nogo的中枢神经抑制活性。本课题组前期利用基因重组技术[11]，从质粒pET-46EK/LIC-Nogo-66原核表达得到纯化的Nogo-66蛋白，分子量7.34Kd，经序列分析Nogo-66蛋白存在多处能够激活T淋巴细胞的抗原提呈表位肽段，在体外可直接激活视网膜小胶质细胞。并且应用重组Nogo66-cs疫苗经全身免疫和局部粘膜淋巴免疫，有效的启动了青光眼和视神经损伤大鼠的生理性自身免疫反应，并证实有神经保护作用[12]。 皇家外科学院大鼠（royal college of surgery rat, RCS）是研究RP的经典模型，具有和人类RP相似的病理变化及功能特征。本实验以RCS大鼠为研究对象，首先观察RCS大鼠自然病程中髓磷脂抑制蛋白Nogo-A/B的表达情况，然后给予重组Nogo66-cs眼用疫苗进行局部粘膜淋巴免疫。通过检测免疫大鼠局部视网膜特异性抗体的表达研究疫苗的免疫反应；观察比较RCS大鼠自然病程中免疫组和对照组视网膜的组织病理学和视网膜神经细胞凋亡情况，研究重组Nogo66-cs眼用疫苗的神经保护作用。同时检测免疫组大鼠和对照组视网膜中CNTF和bFGF的蛋白表达情况，探讨重组Nogo66-cs眼用疫苗的神经保护机制。以期进一步完善重组Nogo66-cs眼用疫苗滴眼液的免疫策略，拓展其使用范围。 一、主要研究内容 1.髓磷脂抑制蛋白Nogo-A/B在RCS视网膜自然病程中的表达取出生后15d、30d、60d和90d四个时间点的视网膜含色素变性大鼠（RCS-p+)各5只为实验组，相同时间点视网膜含色素正常大鼠（RCS-rdy+p+)各5只为正常对照组。利用免疫组化和免疫印迹分别定性和定量检测视网膜中Nogo-A/B的表达。实验动物由第三军医大学大坪医院野战外科研究所实验动物中心提供。 2.重组Nogo66-cs眼用疫苗经粘膜淋巴免疫诱导保护性免疫应答效应和方式取出生后20天RCS-p+大鼠为研究对象，随机分为Nogo66-cs疫苗组和CS组，各9只。免疫接种分3组：第一组，初次免疫后追加免疫1次（1次/周），末次免疫后7d取材；第二组，初次免疫后追加免疫2次（1次/周），末次免疫后7d取材；第三组，初次免疫后追加免疫3次（1次/周），末次免疫后7d取材；对照组以相同策略给予CS点眼。采用TUNEL原位末端凋亡法检测视网膜凋亡阳性表达，IPP分析视网膜厚度变化，以比较追加免疫对视网膜结构的影响。采用免疫印迹检测视网膜IgG表达，以明确重组Nogo66-cs眼用疫苗是否可经粘膜淋巴免疫诱导视网膜局部免疫反应。 3.重组Nogo66-cs眼用疫苗对RCS视网膜免疫神经保护作用研究取出生后30天RCS-p+为实验对象，随机分为重组Nogo66-cs眼用疫苗和CS组，各5只。雌雄不限。实验组初次免疫后追加免疫2次（1次/周），末次免疫后7d取材，对照组采取相同策略。采用IPP检测视网膜厚度及TUNEL凋亡阳性表达；探讨重组Nogo66-cs眼用疫苗对RP视网膜细胞的免疫神经保护作用。 4.重组Nogo66-cs眼用蛋白疫苗对RCS大鼠视网膜的免疫性神经保护机制研究实验分组及免疫策略同前。采用免疫组化法、免疫印迹法检测两组视网膜睫状神经生长因子（Ciliary neurotrophic factor，CNTF）、碱性成纤维生长因子（Basic fibroblast growthfactor，bFGF）蛋白表达。探讨重组Nogo66-cs眼用疫苗对RP的免疫性神经保护机制。 二、主要结果 （一）Nogo-A/B在RCS大鼠视网膜中的表达 1.组织病理学改变与对照组相比，P15d和P30d，RCS-p+大鼠视网膜外层细胞结构和数目未出现明显变化，P30d内核层(Inner nuclear layer，INL)层细胞排列出现轻微紊乱；P60d、P90d大鼠视网膜外核层(Outer nuclear layer，ONL)和INL结构出现明显紊乱，细胞数目减少以ONL和神经节细胞层(Ganglion cell layer，RGC)层较为明显。提示RCS大鼠视网膜色素变性发展过程中，视网膜外层和视网膜内层结构均出现改变。 2. Nogo-A/B的免疫组化和免疫印迹显示Nogo-A/B蛋白在RCS-p+实验大鼠各时间段视网膜表达均为阳性，主要位于视网膜INL和RGC层。Nogo-A/B蛋白在RCS-rdy+p+对照大鼠各时间段视网膜表达为弱阳性表达。 WB免疫印记检测RCS-P+大鼠P15d、P30d、P60d和P90d Nogo-A蛋白表达量分别为：0.82737±0.21292、1.11019±0.08999、1.31552±0.02857、1.26881±0.08042，，组间比较存在显著差异(P＜0.05)，各时间点组内比较存在统计学差异（P＜0.05）。提示RCS大鼠的RP变性过程中存在内源性髓磷脂抑制蛋白Nogo-A/B的动态表达变化，表明Nogo-A蛋白参与RP病理过程。 （二）追加免疫后视网膜结构变化和局部免疫效应 1.追加免疫效应分析初次免疫后追加免疫1次、2次和3次，与对照组相比，实验组视网膜INL厚度分别为：12.4581±2.64716（P＜0.05）、11.0671±2.38886（P＜0.05）、8.94238±0.82968（P＞0.05）。与对照组相比，实验组视网膜ONL厚度分别为： 11.6328±1.77681（P＜0.05）、15.4117±4.66376（P＜0.05）、11.3383±4.61539（P＞0.05）。 TUNEL分析视网膜凋亡阳性表达，初次免疫后追加免疫1、2、3次，实验组单位面积内凋亡阳性表达IOD sum/Area比值分别为：0.060365219±0.060365（P＞0.01）、0.03565282±0.019462（P＜0.01）、0.107844636±0.107845（P＞0.01）。与对照组相比差异具有统计学意义。提示2次追加免疫可延缓RP所致视网膜厚度变薄和视网膜细胞的进行性凋亡。 2.视网膜中IgG抗体检测结果经重组Nogo66-cs眼用疫苗2次追加免疫RCS大鼠， WB检测实验、对照组视网膜IgG抗体表达量分别为：1.15435±0.25090、0.43957±0.13643，两组比较差异具有显著统计学意义（P＜0.01）。提示重组Nogo66-cs眼用疫苗可经粘膜淋巴免疫诱导视网膜局部特异性免疫反应。 （三）重组Nogo66-cs眼用疫苗免疫后视网膜TUNEL和厚度变化 1.视网膜凋亡细胞计数IPP半定量分析视网膜TUNLEL凋亡阳性表达，与对照组相比，实验组IOD SUM/Area为：0.0576±0.0038（P＜0.05），两组间比较差异具有统计学意义 2.视网膜厚度IPP分析视网膜各层厚度，与对照组相比，实验组INL层厚度为：13.4905±0.6211（P＜0.01）；ONL层厚度为：4.8293±0.5943（P＜0.05）。表明重组Nogo66-cs眼用疫苗免疫接种后可有效抑制RP所致视网膜神经细胞进行性凋亡，并延缓视网膜各层厚度变薄，以INL明显。 （四）CNTF和bFGF免疫组化和免疫印迹 1.免疫组化重组Nogo66-cs眼用疫苗免疫接种RCS大鼠后可诱导CNTF和bFGF在RCS视网膜上的阳性表达，CNTF表达以INL和RGC层为主，bFGF主要表达在RGC层； 2.免疫印迹WB检测实验、对照组视网膜bFGF表达分别为：0.82572±0.02803、0.60233±0.04789，组间比较差异具有显著统计学意义（P＜0.01）；WB检测实验、对照组视网膜CNTF表达分别为：0.91272±0.19833、0.60759±0.09207，组间比较差异具有统计学意义（P＜0.05）；bFGF表达两组比较差异相对较高。研究表明重组Nogo66-cs眼用疫苗延缓RP视网膜变性的机制之一是内源性bFGF、CNTF的营养支持。 结论： 1. Nogo-A蛋白参与RP变性过程。 2.重组Nogo66-cs眼用疫苗经粘膜淋巴免疫可有效诱导视网膜局部特异性免疫反应。 3.重组Nogo66-cs眼用疫苗对RP视网膜具有免疫性神经保护效应，其机制之一是内源性bFGF、CNTF的营养支持。
[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 卤 0.21292 , 1.11019 卤 0.08999 , 1.315 52 卤 0.02857 and 1.26881 卤 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 卤 2.64716 ( P < 0.05 ) , 11.0671 卤 2.38886 ( P < 0.05 ) , 8.94238 卤 0.829 68 ( P > 0.05 ) .

11.6328卤1.77681(P锛

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