Nogo66蛋白眼用疫苗对大鼠高眼压和视神经损伤的免疫保护作用及机制研究

发布时间：2018-05-19 06:49

本文选题：Nogo66蛋白疫苗 + 高眼压　；参考：《第三军医大学》2009年博士论文

【摘要】： 视网膜神经节细胞(retinal ganglial cells, RGCs)损伤后再生困难,这是青光眼、视神经损伤等眼部疾病致盲的主要原因。目前认为,RGCs受损后存活或再生困难的一个主要原因是由于髓磷脂蛋白的存在,抑制因子Nogo是其中重要成员之一。Nogo在正常中枢神经系统(central nervous system, CNS)有少量表达,中枢神经受损后Nogo因子释放增加、表达增强,不仅抑制神经纤维再生,而且可启动神经元凋亡过程,导致神经元死亡。Nogo有66个氨基酸残基在胞膜外形成的拓扑结构,即Nogo66,是Nogo的活性区域,介导了Nogo的中枢神经再生抑制活性。目前多项研究表明,用主动免疫或被动免疫阻遏髓磷脂或Nogo均可诱导CNS损伤动物产生自身免疫保护反应,减少损伤后变性,促进CNS神经元再生。其机制尚不明确。在前期工作中,我们原核表达纯化了Nogo66蛋白,制备了Nogo66蛋白福氏佐剂疫苗,发现Nogo66蛋白疫苗可诱导动物机体产生较高滴度抗原特异性IgG抗体,刺激脾T淋巴细胞活化增殖,并可促进慢性高眼压大鼠视网膜神经节细胞(RGCs)存活、损伤后的再生修复,减少视神经纤维进一步损害、使神经功能得到一定保护。证实了Nogo66蛋白疫苗具有免疫原性和视神经损伤后的保护和修复作用。然而,青光眼和视神经损伤是需要长期治疗的疾病,Nogo66蛋白疫苗接种后产生的免疫效应持续时间较短,需要不断加强免疫才能维持一定的抗体滴度和活化T细胞数量,长期高频率地通过外周皮下注射接种疫苗的方式显然很难保证患者的依从性,治疗效果可想而知;另外,Nogo66是一个分子量为7.53461kD的小分子蛋白,小剂量皮下接种很难激活机体免疫系统,要获得较好的免疫效应,需要增加剂量,则有致实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis, EAE)的风险。因此,必须改变接种途径,减少疫苗中蛋白的使用剂量,减少接种频率,使这种神经免疫保护的治疗方法得以实现。眼睑结膜血管分布密集,血液供应非常丰富,有浅层和深层2个网状淋巴系统,在下眼睑结膜皱褶中存在重要的局部淋巴组织,研究证实结膜的淋巴组织具有摄取粘膜表面多种抗原进行识别,并作出反应的功能,是一个理想的疫苗接种途径。从上世纪60年代开始,经结膜接种疫苗途径就已经在家养动物中广泛应用。壳聚糖(chitosan, CS)广泛存在于自然界,具有无毒、良好的生物相容性、低免疫排斥反应等一系列特殊的化学和生物特性。且其本身具有免疫刺激活性,并可增强药物渗透和吸收,是一种具有很大潜力的粘膜免疫缓释材料和免疫佐剂。本课题在探讨Nogo66蛋白疫苗对视网膜视神经损伤的免疫保护作用机制的基础上,改进Nogo66蛋白疫苗的佐剂和接种途径,制备一种滴眼液形式的Nogo66蛋白疫苗,并研究其理化性质、免疫性质和神经保护作用机制。主要研究内容包括以下7部分: 1.不同浓度Nogo66蛋白疫苗对大鼠免疫系统的影响和大鼠免疫状况评估。在正常SD大鼠接种不同浓度的Nogo66蛋白疫苗,检测接种后不同时间点血清IgG抗体滴度和脾T细胞增殖率,主要脏器病理检查并进行EAE状态评价。 2. Nogo66蛋白疫苗诱导大鼠机体产生的免疫效应。以最佳疫苗接种剂量接种正常SD大鼠后,检测大鼠血清IgG抗体滴度和脾分泌Th1类细胞因子IL-2、IFN-γ和Th2类细胞因子IL-4的情况。 3.建立慢性高眼压大鼠模型和视神经钳夹伤大鼠模型,通过检测眼压、荧光金逆行染色视网膜神经节细胞计数,和视网膜血管渗透率检测对两种视网膜视神经损伤模型进行鉴定。 4.制备Nogo66蛋白眼用疫苗并进行质量控制。以壳聚糖为佐剂制备滴眼液形式的Nogo66蛋白疫苗,检测其性状、pH值、渗透压、黏度值、含量和稳定性。 5.检测Nogo66蛋白眼用疫苗的安全性。按照眼刺激性试验标准,进行Nogo66-CS疫苗眼刺激性评分,HE染色法检测Nogo66-CS疫苗对大鼠视网膜、心、脑、睾丸等主要器官的病理损害,观察Nogo66-CS疫苗的安全性。 6. Nogo66蛋白眼用疫苗的免疫原性研究。体外培养小胶质细胞,免疫组化法Nogo66-CS疫苗对小胶质细胞CD11b表达的影响;Nogo66-CS疫苗行正常大鼠结膜接种,检测大鼠泪液及血清中IgG抗体滴度,检测大鼠结膜组织抗原特异性IgG型浆细胞增生和淋巴细胞浸润;免疫组化法检测大鼠视网膜CD11b、MHC-Ⅱ、INOS、TNF-α表达,观察Nogo66蛋白眼用疫苗免疫原性。 7.探讨Nogo66蛋白眼用疫苗对大鼠视网膜视神经损伤的保护作用及其机制。给予慢性高眼压模型大鼠和视神经钳夹伤模型大鼠行结膜接种Nogo66蛋白眼用疫苗,0,21,35天各接种1次。49天时荧光金逆行标记视网膜神经节细胞并进行计数,免疫荧光法、免疫组化法检测视网膜GAP43、CD3、BDNF和GDNF表达,免疫印记法检测视网膜BDNF、GDNF表达,探讨Nogo66蛋白眼用疫苗对视网膜视神经损伤免疫保护作用及其机制。主要结果及结论如下: 1.接种不同浓度Nogo66蛋白疫苗后均可诱导大鼠血清IgG抗体滴度增高和T细胞增殖,其中200μg组IgG抗体滴度和T细胞增殖显著高于其他实验组(P0.05)。所有实验大鼠未发现任何类似实验性自身免疫性脑脊髓炎的行为变化,且各器官病理检查未见明显异常。提示200μg/300μl可最佳程度诱导大鼠免疫反应。 2. Nogo66蛋白疫苗组大鼠脾细胞培养上清IL-2、IFN-γ、IL-4水平均增加,明显高于对照组。其中Th1类细胞因子IL-2、IFN-γ增高的幅度显著大于Th2类细胞因子IL-4增高幅度。提示Nogo66疫苗诱导的免疫反应以Th1细胞免疫,即T淋巴细胞介导的免疫效应为主。 3.慢性高眼压大鼠造模后第3天眼压开始升高,此后持续缓慢升高,1周时眼压为(21.5±3.7)mmHg,与对照组存在显著差异(P0.05)。第4周时达最高峰,为(24.8±3.2)mmHg;到12周时眼压未见明显下降,与对照组眼压仍存在显著差异。荧光金逆行染色RGCs发现,高眼压后1m至3m时SD大鼠RGCs视网膜上RGCs密度持续降低、数量持续减少,与相同时间正常对照组比较均差异显著;视神经钳夹伤后1m时SD大鼠RGCs密度降低、数量显著减少,与正常对照呈显著性差异,3m时SD大鼠RGCs数量与1m时比较未见显著差异。慢性高眼压损伤后1、4、8周时荧光显微镜下观察视网膜内未见明显伊文思蓝渗透,视网膜渗透率较对照组无显著差异。视神经钳夹伤后1天视网膜内可见明显的蓝色荧光,且逐渐减弱。视网膜通透性较对照组均增强,存在显著差异。提示本实验建立的慢性高眼压和视神经钳夹伤模型符合疾病特点,RGCs呈特征性数量减少;慢性高眼压尚不能破坏大鼠血视网膜屏障。视神经钳夹伤早期即能引起血视网膜破坏,视网膜血管渗透性增强,血视网膜屏障的这种损伤状态可持续至少1个月。 4.制备了Nogo66-CS眼用疫苗,经检测,其性状、pH值、渗透压、黏度、含量及稳定性均符合滴眼液制剂的药剂学要求,提示以壳聚糖为佐剂的Nogo66蛋白疫苗具有良好的理化性质。 5.眼刺激性试验显示,点药当时和点药后1h、24h、48h、72h、4d、7d,Nogo66-CS疫苗组和生理盐水组大鼠眼刺激性反应评分均3,裂隙灯下检查眼前节无异常,且角膜荧光素钠染色阴性,认为Nogo66-CS眼用疫苗和生理盐水对眼均无刺激性。于第一次点药后第16周行眼球视网膜、心、肝、肾、肺、脑、睾丸病理检查未见明显异常。提示Nogo66-CS疫苗无刺激性,对机体是安全的。 6. Nogo66蛋白眼用疫苗具有免疫原性。体外培养的小胶质细胞,在Nogo66-CS疫苗或Nogo66蛋白刺激后表达大量CD11b,细胞活化;CS刺激后表达少量CD11b,细胞静止;空白组未见CD11b表达。结膜HE染色显示Nogo66-CS组大鼠结膜组织内有大量淋巴细胞浸润和IgG型阳性浆细胞增生;Nogo66组结膜组织可见少量淋巴细胞浸润和IgG型阳性浆细胞;CS组结膜仅偶见淋巴细胞浸润,未见IgG型阳性浆细胞存在。接种疫苗后,Nogo66-CS组泪液IgG抗体滴度从第20天开始持续增高,与对照组有显著差异,血清IgG抗体滴度第34天开始增高,幅度较低;Nogo66组和CS组泪液和血清中IgG抗体滴度无显著变化。Nogo66-CS组视网膜各层小胶质细胞活化,表达CD11b和少量MHC-Ⅱ,未见明显iNOS、TNF-α表达;CS组视网膜仅少量CD11b表达,未见iNOS、TNF-α表达。提示Nogo66-CS疫苗可激活粘膜免疫和系统免疫,并诱导视网膜小胶质细胞活化,具有较强的免疫原性。 7. Nogo66蛋白眼用疫苗可促进视网膜视神经损伤后RGCs的存活和再生。慢性高眼压模型大鼠和视神经钳夹伤模型大鼠Nogo66-CS组RGCs数量显著高于CS组和未接种组;Nogo66-CS组视网膜有大量GAP43,表明视网膜神经元再生活跃。表明Nogo66蛋白眼用疫苗对受损的视网膜视神经具有保护作用。 8、Nogo66蛋白眼用疫苗可诱导视网膜神经营养因子表达增加。Nogo66蛋白眼用疫苗接种后,视网膜CD3阳性淋巴细胞浸润,GDNF和BNDF表达上调。表明Nogo66蛋白眼用疫苗可诱导活化T细胞浸润视网膜,推测T细胞和视网膜被活化的小胶质细胞分泌多种视网膜神经营养因子,对受损的视网膜视神经起到了保护作用。
[Abstract]:Retinal ganglial cells (RGCs) is a major cause of blindness in the eyes of glaucoma and optic nerve injury. The main reason for the difficulty in survival or regeneration after RGCs is due to the existence of myelin protein, and the inhibitory factor Nogo is one of the important members of.Nogo in.Nogo. The normal central nervous system (central nervous system, CNS) has a small amount of expression. After the central nerve is damaged, the release of Nogo factor increases and the expression is enhanced. It not only inhibits the regeneration of the nerve fibers, but also initiates the process of neuronal apoptosis, which causes the neuronal death.Nogo to have a topological structure of 66 amino acid residues in the extracellular matrix, that is, Nogo66, which is Nogo. Active regions, which mediate the inhibitory activity of Nogo's central nerve regeneration, have shown that active or passive immunization repression of myelin or Nogo can induce CNS injured animals to produce autoimmune protection responses, reduce post damage degeneration and promote the regeneration of CNS neurons. The mechanism is not yet clear.
In the previous work, we purified the Nogo66 protein and prepared the Nogo66 protein FOS adjuvant vaccine. It was found that the Nogo66 protein vaccine could induce the high titer specific IgG antibody in the animal body, stimulate the activation and proliferation of the spleen T lymphocyte, and promote the survival and damage of the retinal ganglion cells (RGCs) in the chronic high intraocular pressure rats. After regenerative repair, the further damage of optic nerve fibers was reduced and the nerve function was protected. It was confirmed that the Nogo66 protein vaccine has the immunogenicity and the protection and repair effect after the optic nerve injury.
However, glaucoma and optic nerve injury are diseases that require long-term treatment. The duration of immunization after Nogo66 vaccine inoculation is shorter. It is necessary to strengthen immunity to maintain a certain antibody titer and to activate the number of T cells. It is obviously difficult to ensure that the patient is vaccinated by subcutaneous injection at high frequency for a long time. In addition, Nogo66 is a small molecular protein with a molecular weight of 7.53461kD, and a small dose of subcutaneous inoculation is difficult to activate the body's immune system. To obtain a better immune effect, it is necessary to increase the dose and to induce experimental autoimmune cerebrospinal myelitis (experimental autoimmune encephalomyelitis, EAE). Therefore, we must change the route of inoculation, reduce the dosage of protein in the vaccine, reduce the frequency of vaccination, so that the treatment method of neuroimmune protection can be realized.
The conjunctival vessels of the eyelid are densely distributed and rich in blood supply. There are 2 reticular lymphatic systems in the shallow and deep layers. There are important local lymphoid tissues in the conjunctival folds of the lower eyelids. The study confirms that the conjunctival lymphoid tissues have a variety of antigens to recognize and respond to the mucosal surface. It is an ideal route for vaccination. Since 60s of last century, conjunctival vaccination has been widely used in domestic animals.
Chitosan (CS), which is widely present in nature, has a series of special chemical and biological characteristics, such as non-toxic, good biocompatibility, low immune rejection and so on. And it has the activity of immune stimulation, and can enhance the penetration and absorption of drugs. It is a great potential mucosal immune release material and immune adjuvant.
On the basis of exploring the immune protective mechanism of Nogo66 protein vaccine on retinal optic nerve injury, we improved the adjuvant and inoculation of Nogo66 protein vaccine, and prepared a kind of Nogo66 protein vaccine in the form of eye drops, and studied its physical and chemical properties, immune properties and the mechanism of neuroprotective effect.
The main research contents include the following 7 parts:
1. the effects of different concentrations of Nogo66 protein vaccine on the immune system of rats and the evaluation of the immune status of rats. In normal SD rats, different concentrations of Nogo66 protein vaccine were inoculated. The serum IgG antibody titer and the proliferation rate of spleen T cells were detected at different time points after inoculation. The main organ pathology examination and the evaluation of EAE status were carried out.
The immune effect induced by 2. Nogo66 protein vaccine was induced in rats. After inoculation with the normal SD rats, the titer of serum IgG antibody and the secretion of Th1 cell factor IL-2, IFN- gamma and Th2 cell factor IL-4 in the spleen were detected.
3. the rat model of chronic ocular hypertension and the rat model of optic nerve clamp injury were established. By detecting intraocular pressure, counting retinal ganglion cells with fluorescent gold retrograde staining, and detecting retinal vascular permeability, two models of retinal optic nerve injury were identified.
4. the Nogo66 protein eye vaccine was prepared and the quality control was made. The Nogo66 protein vaccine in the form of eye drops was prepared with chitosan as the adjuvant, and its characters, pH value, osmotic pressure, viscosity value, content and stability were detected.
5. the safety of Nogo66 protein eye vaccine was detected. According to the standard of eye irritation test, the eye irritation score of Nogo66-CS vaccine and the pathological damage of Nogo66-CS vaccine on the main organs such as retina, heart, brain and testis of rats were detected by HE staining, and the safety of Nogo66-CS vaccine was observed.
The immunogenicity of 6. Nogo66 protein eye vaccine. The effect of microglia and immunohistochemistry Nogo66-CS vaccine on the expression of CD11b in microglia in vitro; Nogo66-CS vaccine was inoculated in the conjunctiva of normal rats, and the titer of IgG antibody in the tear and serum of rats was detected, and the antigen specific IgG plasma cell proliferation in conjunctival tissue of rats was detected. Immunohistochemical staining was used to detect the expression of CD11b, MHC- II, INOS and TNF- alpha in rat retina, and to observe the immunogenicity of Nogo66 protein ophthalmic vaccine.
7. to investigate the protective effect and mechanism of Nogo66 protein ophthalmic vaccine on retinal optic nerve injury in rats and to inoculate rats with chronic ocular hypertension model and optic nerve clamp injury model rats with Nogo66 protein eye vaccine. The fluorescent gold retrograde labeling retinal ganglion cells were counted at the time of 1.49 days of 0,21,35 days, and the immunization was counted. The expression of retina GAP43, CD3, BDNF and GDNF were detected by immunofluorescence, and the expression of BDNF and GDNF in retina was detected by immuno imprinting. The protective effect of Nogo66 protein eye vaccine on retinal optic nerve injury and its mechanism were discussed.
The main results and conclusions are as follows:
1. after inoculation with different concentrations of Nogo66 protein, the titer of serum IgG antibody and proliferation of T cells were induced in rats. The titer of IgG antibody and the proliferation of T cells in the 200 g group were significantly higher than those in the other experimental groups (P0.05). No obvious abnormalities were observed. It suggested that 200 mu g/300 l could induce immune response in rats.
The level of IL-2, IFN- gamma and IL-4 increased in the splenocytes culture of 2. Nogo66 protein vaccine group, which was significantly higher than that of the control group. The increase of Th1 cell factor IL-2, IFN- gamma was significantly greater than that of Th2 cell factor IL-4 increase. It suggested that the immune response induced by Nogo66 vaccine was Th1 cell immunity, that is, the immune effect mediated by T lymphocyte Mainly.
3. the intraocular pressure of the chronic high intraocular pressure rats began to rise after third days and then continued to increase slowly. The intraocular pressure was (21.5 + 3.7) mmHg at 1 weeks. There was a significant difference between the control group and the control group (P0.05). The peak was (24.8 + 3.2) mmHg at the fourth week, and the intraocular pressure of the control group was still significantly different from that of the control group at the 12 week. RGCs hair was stained with fluorescent gold retrograde. At the time of high intraocular pressure (1m to 3m), the density of RGCs on the RGCs retina of SD rats continued to decrease and the number continued to decrease, and the difference was significant compared with that of the normal control group. The RGCs density of SD rats was decreased and the number of SD rats decreased significantly after 1m clamp injury, and the number was significantly different from the normal control. The RGCs quantity of SD rats at 3M was not significant compared with that of 1m. There was no obvious Evans blue infiltration in the retina under the fluorescence microscope at 1,4,8 weeks after chronic ocular hypertension injury. There was no significant difference between the retina permeability and the control group. The apparent blue fluorescence was seen in the retina 1 days after the optic nerve clamp injury, and gradually weakened. The chronic high intraocular pressure and the optic nerve clamp injury model established in this experiment conformed to the characteristic of the disease. The characteristic number of RGCs decreased, and the chronic high intraocular pressure could not destroy the retinal barrier of the rat's blood. The retinal blood retinal barrier could be damaged in the early stage of the optic nerve clamp injury, the retinal vascular permeability increased, and the damage state of the blood retinal barrier could be held. Continue for at least 1 months.
4. the Nogo66-CS eye vaccine was prepared. The characteristics, pH value, osmotic pressure, viscosity, content and stability of the vaccine were in accordance with the pharmacology requirements of the eye drops, suggesting that the Nogo66 protein vaccine with chitosan as a adjuvant has good physical and chemical properties.
5. eye irritation tests showed that the eye irritation response scores of 1H, 24h, 48h, 72h, 4D, 7d, Nogo66-CS vaccine group and saline group were all 3, and there were no abnormalities in the anterior segment under the slit lamp, and the cornea fluorescein staining was negative. It was considered that Nogo66-CS eye vaccine and saline were not irritating to the eyes. After the first time, the drug was not irritating to the eye. There was no obvious abnormality in the retina, heart, liver, kidney, lung, brain and testis in sixteenth weeks. It suggested that the Nogo66-CS vaccine was not irritating and safe to the body.
The 6. Nogo66 protein eye vaccine was immunogenic. Microglia cultured in vitro expressed a large number of CD11b and cell activation after the stimulation of Nogo66-CS vaccine or Nogo66 protein. After CS stimulation, a small amount of CD11b was expressed and the cells were stationary. No CD11b expression was found in the blank group. The conjunctival HE staining showed a large number of lymphocyte infiltration in the conjunctival tissue of group Nogo66-CS rats. A small amount of lymphocyte infiltration and IgG type positive plasma cells were found in the conjunctival tissue of group Nogo66, and the conjunctiva of group CS was only infiltrated by lymphocyte and no IgG positive plasma cells existed. After vaccination, the IgG antibody titer of Nogo66-CS group began to increase continuously from twentieth days, which was significantly different from that of the control group, and the serum IgG resistance was significant. The titer of the body began to increase in thirty-fourth days, and the amplitude was lower. The IgG antibody titer in the tears and serum of the Nogo66 and CS groups had no significant changes. The microglia in the retina of the.Nogo66-CS group was activated, expressed CD11b and a small amount of MHC- II, and had no obvious iNOS, TNF- a expression, and a small amount of CD11b expressed in the retina of the CS group, and no iNOS, TNF- alpha expression was not found. Activation of mucosal immunity and systemic immunity, and activation of retinal microglia, have strong immunogenicity.
7. Nogo66 protein eye vaccine can promote the survival and regeneration of RGCs after retinal optic nerve injury. The number of RGCs in rat Nogo66-CS group of chronic ocular hypertension model rats and optic nerve clamp injury model rats is significantly higher than that of group CS and uninoculated group. The retina of Nogo66-CS group has a large number of GAP43, indicating that the regeneration of the optic nerve membrane neurons is active. It indicates Nogo66 protein eye use. The vaccine has protective effects on damaged retinal nerves.
8, Nogo66 protein eye vaccine can induce the expression of retina neurotrophic factor to increase the infiltration of CD3 positive lymphocytes in the retina and up regulation of the expression of GDNF and BNDF after immunization with.Nogo66 protein eye vaccine. It indicates that the Nogo66 protein eye vaccine can induce the activation of T cells to infiltrate the retina, and the secretion of the microglia of the activated T cells and the retina is more secreted. Retinal nerve growth factor has protective effects on damaged retinal nerves.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392

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