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]:Retinitis pigmentosa (RP) is a kind of hereditary retinal degeneration disease characterized by the dysfunction of photosensitive cells and pigment epithelium. RP is the initial lesion of the degeneration and apoptosis of the Photoreceptor cell (PRC), and the structure of the retina is developed with the course of the disease. The function of the retina is seriously affected. The degeneration and apoptosis of the two and three stage neurons of the membrane is an important pathological change of RP. There is no special treatment for RP at present. Gene therapy, retina transplantation, microenvironmental regulation (nutrient supply, neutralization or elimination of toxic and inhibitory substances), and visual prosthesis implantation are all in the experimental study.
The retina is part of the central nervous system (Central nervous system, CNS). The difficulty of regeneration after CNS injury has been a difficult problem in neurology..Cohen[1] observed the accumulation of a large number of T lymphocytes around the damaged nerve lesion after CNS injury and a transient, weak neuroprotective effect, which is due to the self antigen storm of the injured site. A protective immune response to the activation of T lymphocytes. According to this phenomenon, the concept of "physiological T cells mediated autoimmune neuroprotective effects" was first proposed in 2000[2], according to this phenomenon, and in [3] and [4] in 2009, in 2005, the myelin related protein of the autoantigen was explained in Neuroscience, and the main body of the protein was expressed in Neuroscience. Dynamic and passive immunity can induce and strengthen this physiological response. It is proved that the physiological immune response can promote regeneration and repair after nerve injury. The mechanism is to inhibit the secondary damage of injured neurons by activating the protective autoimmune reaction, reduce or prevent the progression of nerve damage, and protect the undamaged nerves. It also helped to repair nerve cells at the "edge of injury". Animal tests also confirmed that the loss of these reactions was more serious.
The endogenous inhibitory factor, myelin suppressor protein, which has been found in the [6] retina and optic nerve, is one of the endogenous inhibitors of myelin suppressor, which has been found in the retina and optic nerve of the retina and optic nerve after [5] and optic nerve clamp injury. There is a study that the RP retina can express the Nogo receptor Ng-R[7-8], P75NTR and the relationship with retinal pigment degeneration [9][10]. The study of whether the myelin suppressor factor (Nogo) is involved in the pathological process of retinitis pigmentosa will provide us with the mechanism of physiological autoimmune response in the central nervous system to intervene in the pathological process of RP or to provide neuroprotection.
After the central nerve injury, the release of Nogo increases, the expression is enhanced, and the regeneration of the injured nerve is inhibited, and the process of neuronal apoptosis is initiated, which leads to the neuronal death of the carbon end and nitrogen end of the.Nogo protein in the cell membrane, and the extracellular matrix is formed by 66 amino acid residues, which is also called Nogo-66. Nogo-66 through the cell surface receptor Ng. The ligand receptor binding of -R mediates the central nervous inhibitory activity of Nogo. In our group, we used gene recombination technology [11] to express the purified Nogo-66 protein and molecular weight 7.34Kd from the plasmid pET-46EK/LIC-Nogo-66 prokaryotic. The sequence analysis of Nogo-66 protein has several antigen presenting epitopes capable of activating the T lymphocyte. In vitro, the retinal microglia can be activated directly. And the recombinant Nogo66-cs vaccine is used to activate the physiological autoimmune reaction of the rats with glaucoma and optic nerve injury through systemic and local mucosal lymphoid immunization, and the neuroprotective effect of [12]. is confirmed.
The Royal surgery College (Royal College of surgery rat, RCS) is the classic model of the study of RP, with the pathological changes and functional features similar to human RP. The experiment was conducted to observe the expression of the Nogo-A/B of myelin suppressor protein in the natural course of RCS rats, and then the recombinant Nogo66-cs eye vaccine was given. Local mucosal lymphoid immunity was carried out. The immunoreaction of the vaccine was studied by detecting the expression of local retina specific antibody in the immune rats. The histopathology of the retina and the retinal nerve cell apoptosis in the immune and control groups of the RCS rats were observed and compared. The neuroprotective effect of the recombinant Nogo66-cs eye vaccine was studied. At the same time, the protein expression of CNTF and bFGF in the retina of the immune group and the control group was detected, and the neuroprotective mechanism of the recombinant Nogo66-cs eye vaccine was explored in order to further improve the immunization strategy of the recombinant Nogo66-cs eye drops and expand its scope of use.
First, the main research content
1. the expression of myelin suppressor protein Nogo-A/B in the natural course of RCS retina takes 5 retinal pigment degeneration rats (RCS-p+) at the four time points of 15d, 30d, 60d and 90d, each of the experimental group, and 5 of the retinal pigment normal rats (RCS-rdy+p+) at the same time point as the normal control group. Using immunohistochemistry and immunoblotting, respectively. The expression of Nogo-A/B in the retina was qualitatively and quantitatively examined. The experimental animals were provided by the experimental animal center of the Field Surgery Institute of Daping Hospital, Third Military Medical University.
2. the RCS-p+ rats of the recombinant Nogo66-cs eye vaccine were divided into Nogo66-cs vaccine group and CS group, each of which were randomly divided into 3 groups: the first group, the first group, 1 times after the first immunization (1 times / week), and the 7d after the last immunization; the second group, the first group. After immunization, 2 times (1 times / week) and 7d were taken after the last immunization; the third groups were immunized with 3 times (1 times / weeks) after the first immunization, 7d was obtained after the last immunization, and the control group was given CS points with the same strategy. The positive expression of retinal apoptosis was detected by TUNEL in situ end apoptosis method, and IPP was used to analyze the retinal thickness changes in order to compare additional immunity. The effects of pestilence on retinal structure were detected by immunoblotting to detect the expression of retina IgG in order to determine whether the recombinant Nogo66-cs eye vaccine could induce local retinal immune response through mucosal lymphatic immunization.
3. the study of the protective effect of recombinant Nogo66-cs eye vaccine on RCS retina immuno neuroprotection took RCS-p+ 30 days after birth as the experimental object, randomly divided into the recombinant Nogo66-cs eye vaccine and the CS group, each 5. The experimental group was immunized with 2 times (1 times / week) after the first immunization, the same strategy was taken after the last immunization, and the control group adopted the same strategy. IPP examination was adopted. Retinal thickness and positive expression of TUNEL apoptosis were measured. The immuno neuroprotective effect of recombinant Nogo66-cs ophthalmic vaccine on RP retinal cells was investigated.
4. the experimental group and immunization strategy of recombinant Nogo66-cs eye protein vaccine on RCS rat retina were divided into two groups: immunohistochemistry and Western blotting to detect the two groups of Ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (Basic fibroblast growthfacto). R (bFGF) protein expression. To explore the immune neuroprotective mechanism of recombinant Nogo66-cs ophthalmic vaccine on RP.
Two, the main results
(1) expression of Nogo-A/B in the retina of RCS rats
1. compared with the control group, there was no obvious change in the structure and number of cells in the outer retina of P15d and P30d rats, and a slight disorder in the P30d kernel layer (Inner nuclear layer, INL) layer cells, and there were obvious disorders in the outer retinal nucleus (Outer nuclear) and the structure of the P90d rat, and the number of cells. The decrease of the layer of ONL and the ganglion cell layer (Ganglion cell layer, RGC) is more obvious. It is suggested that the outer layer of retina and the inner layer of the retina change in the development of retinal pigment degeneration in RCS rats.
2. Nogo-A/B immunoblotting and immunoblotting showed that the expression of Nogo-A/B protein was positive in all time segments of RCS-p+ experimental rats, mainly located in the retina INL and RGC layer.Nogo-A/B protein in the RCS-rdy+p+ control rats, and the expression of retina was weak positive. WB immunoimprint was used to detect P15d, P30d, P60d and the.Nogo-A/B in RCS-P+ rats. The expression of o-A protein was 0.82737 + 0.21292,1.11019 + 0.08999,1.31552 + 0.02857,1.26881 + 0.08042, and there was a significant difference between the groups (P < 0.05), and there was a statistical difference in each time point group (P < 0.05). It suggested that the dynamic expression of the endogenous myelin suppressor protein Nogo-A/B in the RP denaturation process of RCS rats The Nogo-A protein is involved in the pathological process of RP.
(two) retinal structural changes and local immune effects after supplemental immunization
1. the additional immunization effect was analyzed for 1 times, 2 times and 3 times after primary immunization. Compared with the control group, the retinal INL thickness was 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 group, the retinal ONL thickness was respectively:
11.6328 + 1.77681 (P < 0.05), 15.4117 + 4.66376 (P < 0.05), 11.3383 + 4.61539 (P > 0.05).
TUNEL analyzed the positive expression of retinal apoptosis and added immunization 1,2,3 times after the first immunization. The positive expression of IOD sum/Area in the experimental group was 0.060365219 + 0.060365 (P > 0.01), 0.03565282 + 0.019462 (P < 0.01), 0.107844636 + 0.107845 (P > 0.01). The difference was statistically significant compared with the control group. 2 times. Supplemental immunization can delay retinal thinning and progressive apoptosis of retinal cells induced by RP.
2. the results of IgG antibody detection in the retina were added to the recombinant Nogo66-cs eye vaccine for 2 additional immunization of RCS rats and WB test. The expression of IgG antibody in the retina of the control group was 1.15435 + 0.25090,0.43957 + 0.13643 respectively. The two groups had significant statistical significance (P < 0.01). The pestilence induced local specific immunoreaction of the retina.
(three) changes in TUNEL and thickness of retina after immunization with recombinant Nogo66-cs ophthalmic vaccine.
1. retinal apoptotic cells count IPP semi quantitative analysis of retinal TUNLEL apoptosis positive expression, compared with the control group, IOD SUM/Area in the experimental group was 0.0576 + 0.0038 (P < 0.05). The difference of the two groups was statistically significant.
2. retinal thickness IPP was used to analyze the thickness of each layer of the retina. Compared with the control group, the thickness of the INL layer in the experimental group was 13.4905 + 0.6211 (P < 0.01), and the thickness of the ONL layer was 4.8293 + 0.5943 (P < 0.05). It indicated that the recombinant Nogo66-cs eye vaccine could effectively inhibit the sexual apoptosis of the retinal membrane neurons caused by RP and postpone the retina layers after the immunization of the recombinant Nogo66-cs eye. The thickness is thinner, with INL obvious.
(four) immunoblotting and immunoblotting of CNTF and bFGF
1. immunized recombinant Nogo66-cs eye vaccine inoculated RCS rats could induce positive expression of CNTF and bFGF on RCS retina. The expression of CNTF was mainly in INL and RGC layer, and bFGF was mainly expressed in the RGC layer.
2. immunoblotting WB test showed that the expression of bFGF in the retina of the control group was 0.82572 + 0.02803,0.60233 + 0.04789, and the difference between the groups was statistically significant (P < 0.01). The CNTF expression in the retina of the control group was 0.91272 + 0.19833,0.60759 + 0.09207 respectively, and the difference between the groups was statistically significant (P < 0.05). The comparison of bFGF expression between two groups was relatively high. The study showed that one of the mechanisms of recombinant Nogo66-cs eye vaccine to delay RP retinal degeneration was endogenous bFGF and CNTF nutritional support.
Conclusion:
1. Nogo-A protein participates in the RP denaturation process.
2. recombinant Nogo66-cs ophthalmic vaccine can effectively induce the local specific immune response of the retina through mucosal lymphatic immunity.
3. recombinant Nogo66-cs ophthalmic vaccine has an immunological neuroprotective effect on RP retina, one of which is endogenous bFGF and CNTF nutritional support.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.1

【参考文献】