發(fā)布時(shí)間：2018-05-19 06:49

  本文選題：Nogo66蛋白疫苗 + 高眼壓��； 參考：《第三軍醫(yī)大學(xué)》2009年博士論文

【摘要】： 視網(wǎng)膜神經(jīng)節(jié)細(xì)胞(retinal ganglial cells, RGCs)損傷后再生困難,這是青光眼、視神經(jīng)損傷等眼部疾病致盲的主要原因。目前認(rèn)為,RGCs受損后存活或再生困難的一個(gè)主要原因是由于髓磷脂蛋白的存在,抑制因子Nogo是其中重要成員之一。Nogo在正常中樞神經(jīng)系統(tǒng)(central nervous system, CNS)有少量表達(dá),中樞神經(jīng)受損后Nogo因子釋放增加、表達(dá)增強(qiáng),不僅抑制神經(jīng)纖維再生,而且可啟動(dòng)神經(jīng)元凋亡過(guò)程,導(dǎo)致神經(jīng)元死亡。Nogo有66個(gè)氨基酸殘基在胞膜外形成的拓?fù)浣Y(jié)構(gòu),即Nogo66,是Nogo的活性區(qū)域,介導(dǎo)了Nogo的中樞神經(jīng)再生抑制活性。目前多項(xiàng)研究表明,用主動(dòng)免疫或被動(dòng)免疫阻遏髓磷脂或Nogo均可誘導(dǎo)CNS損傷動(dòng)物產(chǎn)生自身免疫保護(hù)反應(yīng),減少損傷后變性,促進(jìn)CNS神經(jīng)元再生。其機(jī)制尚不明確。 在前期工作中,我們?cè)吮磉_(dá)純化了Nogo66蛋白,制備了Nogo66蛋白福氏佐劑疫苗,發(fā)現(xiàn)Nogo66蛋白疫苗可誘導(dǎo)動(dòng)物機(jī)體產(chǎn)生較高滴度抗原特異性IgG抗體,刺激脾T淋巴細(xì)胞活化增殖,并可促進(jìn)慢性高眼壓大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞(RGCs)存活、損傷后的再生修復(fù),減少視神經(jīng)纖維進(jìn)一步損害、使神經(jīng)功能得到一定保護(hù)。證實(shí)了Nogo66蛋白疫苗具有免疫原性和視神經(jīng)損傷后的保護(hù)和修復(fù)作用。 然而,青光眼和視神經(jīng)損傷是需要長(zhǎng)期治療的疾病,Nogo66蛋白疫苗接種后產(chǎn)生的免疫效應(yīng)持續(xù)時(shí)間較短,需要不斷加強(qiáng)免疫才能維持一定的抗體滴度和活化T細(xì)胞數(shù)量,長(zhǎng)期高頻率地通過(guò)外周皮下注射接種疫苗的方式顯然很難保證患者的依從性,治療效果可想而知;另外,Nogo66是一個(gè)分子量為7.53461kD的小分子蛋白,小劑量皮下接種很難激活機(jī)體免疫系統(tǒng),要獲得較好的免疫效應(yīng),需要增加劑量,則有致實(shí)驗(yàn)性自身免疫性腦脊髓炎(experimental autoimmune encephalomyelitis, EAE)的風(fēng)險(xiǎn)。因此,必須改變接種途徑,減少疫苗中蛋白的使用劑量,減少接種頻率,使這種神經(jīng)免疫保護(hù)的治療方法得以實(shí)現(xiàn)。 眼瞼結(jié)膜血管分布密集,血液供應(yīng)非常豐富,有淺層和深層2個(gè)網(wǎng)狀淋巴系統(tǒng),在下眼瞼結(jié)膜皺褶中存在重要的局部淋巴組織,研究證實(shí)結(jié)膜的淋巴組織具有攝取粘膜表面多種抗原進(jìn)行識(shí)別,并作出反應(yīng)的功能,是一個(gè)理想的疫苗接種途徑。從上世紀(jì)60年代開(kāi)始,經(jīng)結(jié)膜接種疫苗途徑就已經(jīng)在家養(yǎng)動(dòng)物中廣泛應(yīng)用。 殼聚糖(chitosan, CS)廣泛存在于自然界,具有無(wú)毒、良好的生物相容性、低免疫排斥反應(yīng)等一系列特殊的化學(xué)和生物特性。且其本身具有免疫刺激活性,并可增強(qiáng)藥物滲透和吸收,是一種具有很大潛力的粘膜免疫緩釋材料和免疫佐劑。 本課題在探討Nogo66蛋白疫苗對(duì)視網(wǎng)膜視神經(jīng)損傷的免疫保護(hù)作用機(jī)制的基礎(chǔ)上,改進(jìn)Nogo66蛋白疫苗的佐劑和接種途徑,制備一種滴眼液形式的Nogo66蛋白疫苗,并研究其理化性質(zhì)、免疫性質(zhì)和神經(jīng)保護(hù)作用機(jī)制。 主要研究?jī)?nèi)容包括以下7部分: 1.不同濃度Nogo66蛋白疫苗對(duì)大鼠免疫系統(tǒng)的影響和大鼠免疫狀況評(píng)估。在正常SD大鼠接種不同濃度的Nogo66蛋白疫苗,檢測(cè)接種后不同時(shí)間點(diǎn)血清IgG抗體滴度和脾T細(xì)胞增殖率,主要臟器病理檢查并進(jìn)行EAE狀態(tài)評(píng)價(jià)。 2. Nogo66蛋白疫苗誘導(dǎo)大鼠機(jī)體產(chǎn)生的免疫效應(yīng)。以最佳疫苗接種劑量接種正常SD大鼠后,檢測(cè)大鼠血清IgG抗體滴度和脾分泌Th1類細(xì)胞因子IL-2、IFN-γ和Th2類細(xì)胞因子IL-4的情況。 3.建立慢性高眼壓大鼠模型和視神經(jīng)鉗夾傷大鼠模型,通過(guò)檢測(cè)眼壓、熒光金逆行染色視網(wǎng)膜神經(jīng)節(jié)細(xì)胞計(jì)數(shù),和視網(wǎng)膜血管滲透率檢測(cè)對(duì)兩種視網(wǎng)膜視神經(jīng)損傷模型進(jìn)行鑒定。 4.制備Nogo66蛋白眼用疫苗并進(jìn)行質(zhì)量控制。以殼聚糖為佐劑制備滴眼液形式的Nogo66蛋白疫苗,檢測(cè)其性狀、pH值、滲透壓、黏度值、含量和穩(wěn)定性。 5.檢測(cè)Nogo66蛋白眼用疫苗的安全性。按照眼刺激性試驗(yàn)標(biāo)準(zhǔn),進(jìn)行Nogo66-CS疫苗眼刺激性評(píng)分,HE染色法檢測(cè)Nogo66-CS疫苗對(duì)大鼠視網(wǎng)膜、心、腦、睪丸等主要器官的病理?yè)p害,觀察Nogo66-CS疫苗的安全性。 6. Nogo66蛋白眼用疫苗的免疫原性研究。體外培養(yǎng)小膠質(zhì)細(xì)胞,免疫組化法Nogo66-CS疫苗對(duì)小膠質(zhì)細(xì)胞CD11b表達(dá)的影響;Nogo66-CS疫苗行正常大鼠結(jié)膜接種,檢測(cè)大鼠淚液及血清中IgG抗體滴度,檢測(cè)大鼠結(jié)膜組織抗原特異性IgG型漿細(xì)胞增生和淋巴細(xì)胞浸潤(rùn);免疫組化法檢測(cè)大鼠視網(wǎng)膜CD11b、MHC-Ⅱ、INOS、TNF-α表達(dá),觀察Nogo66蛋白眼用疫苗免疫原性。 7.探討Nogo66蛋白眼用疫苗對(duì)大鼠視網(wǎng)膜視神經(jīng)損傷的保護(hù)作用及其機(jī)制。給予慢性高眼壓模型大鼠和視神經(jīng)鉗夾傷模型大鼠行結(jié)膜接種Nogo66蛋白眼用疫苗,0,21,35天各接種1次。49天時(shí)熒光金逆行標(biāo)記視網(wǎng)膜神經(jīng)節(jié)細(xì)胞并進(jìn)行計(jì)數(shù),免疫熒光法、免疫組化法檢測(cè)視網(wǎng)膜GAP43、CD3、BDNF和GDNF表達(dá),免疫印記法檢測(cè)視網(wǎng)膜BDNF、GDNF表達(dá),探討Nogo66蛋白眼用疫苗對(duì)視網(wǎng)膜視神經(jīng)損傷免疫保護(hù)作用及其機(jī)制。 主要結(jié)果及結(jié)論如下: 1.接種不同濃度Nogo66蛋白疫苗后均可誘導(dǎo)大鼠血清IgG抗體滴度增高和T細(xì)胞增殖,其中200μg組IgG抗體滴度和T細(xì)胞增殖顯著高于其他實(shí)驗(yàn)組(P0.05)。所有實(shí)驗(yàn)大鼠未發(fā)現(xiàn)任何類似實(shí)驗(yàn)性自身免疫性腦脊髓炎的行為變化,且各器官病理檢查未見(jiàn)明顯異常。提示200μg/300μl可最佳程度誘導(dǎo)大鼠免疫反應(yīng)。 2. Nogo66蛋白疫苗組大鼠脾細(xì)胞培養(yǎng)上清IL-2、IFN-γ、IL-4水平均增加,明顯高于對(duì)照組。其中Th1類細(xì)胞因子IL-2、IFN-γ增高的幅度顯著大于Th2類細(xì)胞因子IL-4增高幅度。提示Nogo66疫苗誘導(dǎo)的免疫反應(yīng)以Th1細(xì)胞免疫,即T淋巴細(xì)胞介導(dǎo)的免疫效應(yīng)為主。 3.慢性高眼壓大鼠造模后第3天眼壓開(kāi)始升高,此后持續(xù)緩慢升高,1周時(shí)眼壓為(21.5±3.7)mmHg,與對(duì)照組存在顯著差異(P0.05)。第4周時(shí)達(dá)最高峰,為(24.8±3.2)mmHg;到12周時(shí)眼壓未見(jiàn)明顯下降,與對(duì)照組眼壓仍存在顯著差異。熒光金逆行染色RGCs發(fā)現(xiàn),高眼壓后1m至3m時(shí)SD大鼠RGCs視網(wǎng)膜上RGCs密度持續(xù)降低、數(shù)量持續(xù)減少,與相同時(shí)間正常對(duì)照組比較均差異顯著;視神經(jīng)鉗夾傷后1m時(shí)SD大鼠RGCs密度降低、數(shù)量顯著減少,與正常對(duì)照呈顯著性差異,3m時(shí)SD大鼠RGCs數(shù)量與1m時(shí)比較未見(jiàn)顯著差異。慢性高眼壓損傷后1、4、8周時(shí)熒光顯微鏡下觀察視網(wǎng)膜內(nèi)未見(jiàn)明顯伊文思藍(lán)滲透,視網(wǎng)膜滲透率較對(duì)照組無(wú)顯著差異。視神經(jīng)鉗夾傷后1天視網(wǎng)膜內(nèi)可見(jiàn)明顯的藍(lán)色熒光,且逐漸減弱。視網(wǎng)膜通透性較對(duì)照組均增強(qiáng),存在顯著差異。提示本實(shí)驗(yàn)建立的慢性高眼壓和視神經(jīng)鉗夾傷模型符合疾病特點(diǎn),RGCs呈特征性數(shù)量減少;慢性高眼壓尚不能破壞大鼠血視網(wǎng)膜屏障。視神經(jīng)鉗夾傷早期即能引起血視網(wǎng)膜破壞,視網(wǎng)膜血管滲透性增強(qiáng),血視網(wǎng)膜屏障的這種損傷狀態(tài)可持續(xù)至少1個(gè)月。 4.制備了Nogo66-CS眼用疫苗,經(jīng)檢測(cè),其性狀、pH值、滲透壓、黏度、含量及穩(wěn)定性均符合滴眼液制劑的藥劑學(xué)要求,提示以殼聚糖為佐劑的Nogo66蛋白疫苗具有良好的理化性質(zhì)。 5.眼刺激性試驗(yàn)顯示,點(diǎn)藥當(dāng)時(shí)和點(diǎn)藥后1h、24h、48h、72h、4d、7d,Nogo66-CS疫苗組和生理鹽水組大鼠眼刺激性反應(yīng)評(píng)分均3,裂隙燈下檢查眼前節(jié)無(wú)異常,且角膜熒光素鈉染色陰性,認(rèn)為Nogo66-CS眼用疫苗和生理鹽水對(duì)眼均無(wú)刺激性。于第一次點(diǎn)藥后第16周行眼球視網(wǎng)膜、心、肝、腎、肺、腦、睪丸病理檢查未見(jiàn)明顯異常。提示Nogo66-CS疫苗無(wú)刺激性,對(duì)機(jī)體是安全的。 6. Nogo66蛋白眼用疫苗具有免疫原性。體外培養(yǎng)的小膠質(zhì)細(xì)胞,在Nogo66-CS疫苗或Nogo66蛋白刺激后表達(dá)大量CD11b,細(xì)胞活化;CS刺激后表達(dá)少量CD11b,細(xì)胞靜止;空白組未見(jiàn)CD11b表達(dá)。結(jié)膜HE染色顯示Nogo66-CS組大鼠結(jié)膜組織內(nèi)有大量淋巴細(xì)胞浸潤(rùn)和IgG型陽(yáng)性漿細(xì)胞增生;Nogo66組結(jié)膜組織可見(jiàn)少量淋巴細(xì)胞浸潤(rùn)和IgG型陽(yáng)性漿細(xì)胞;CS組結(jié)膜僅偶見(jiàn)淋巴細(xì)胞浸潤(rùn),未見(jiàn)IgG型陽(yáng)性漿細(xì)胞存在。接種疫苗后,Nogo66-CS組淚液IgG抗體滴度從第20天開(kāi)始持續(xù)增高,與對(duì)照組有顯著差異,血清IgG抗體滴度第34天開(kāi)始增高,幅度較低;Nogo66組和CS組淚液和血清中IgG抗體滴度無(wú)顯著變化。Nogo66-CS組視網(wǎng)膜各層小膠質(zhì)細(xì)胞活化,表達(dá)CD11b和少量MHC-Ⅱ,未見(jiàn)明顯iNOS、TNF-α表達(dá);CS組視網(wǎng)膜僅少量CD11b表達(dá),未見(jiàn)iNOS、TNF-α表達(dá)。提示Nogo66-CS疫苗可激活粘膜免疫和系統(tǒng)免疫,并誘導(dǎo)視網(wǎng)膜小膠質(zhì)細(xì)胞活化,具有較強(qiáng)的免疫原性。 7. Nogo66蛋白眼用疫苗可促進(jìn)視網(wǎng)膜視神經(jīng)損傷后RGCs的存活和再生。慢性高眼壓模型大鼠和視神經(jīng)鉗夾傷模型大鼠Nogo66-CS組RGCs數(shù)量顯著高于CS組和未接種組;Nogo66-CS組視網(wǎng)膜有大量GAP43,表明視網(wǎng)膜神經(jīng)元再生活躍。表明Nogo66蛋白眼用疫苗對(duì)受損的視網(wǎng)膜視神經(jīng)具有保護(hù)作用。 8、Nogo66蛋白眼用疫苗可誘導(dǎo)視網(wǎng)膜神經(jīng)營(yíng)養(yǎng)因子表達(dá)增加。Nogo66蛋白眼用疫苗接種后,視網(wǎng)膜CD3陽(yáng)性淋巴細(xì)胞浸潤(rùn),GDNF和BNDF表達(dá)上調(diào)。表明Nogo66蛋白眼用疫苗可誘導(dǎo)活化T細(xì)胞浸潤(rùn)視網(wǎng)膜,推測(cè)T細(xì)胞和視網(wǎng)膜被活化的小膠質(zhì)細(xì)胞分泌多種視網(wǎng)膜神經(jīng)營(yíng)養(yǎng)因子,對(duì)受損的視網(wǎng)膜視神經(jīng)起到了保護(hù)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陶伊文;;一種克服子孢子疫苗缺點(diǎn)的設(shè)想[J];國(guó)際醫(yī)學(xué)寄生蟲病雜志;1991年03期

2 聞禮永;吳觀陵;;日本血吸蟲候選疫苗研究現(xiàn)狀與展望[J];中國(guó)寄生蟲學(xué)與寄生蟲病雜志;2007年05期

3 張世勇;兒童B群腦膜炎球菌感染的預(yù)防:蛋白疫苗可誘導(dǎo)免疫記憶[J];國(guó)外醫(yī)學(xué).預(yù)防.診斷.治療用生物制品分冊(cè);2002年03期

4 孫玉靜;隋延仿;吳道澄;葛偉;葉菁;張秀敏;;MAGE1納米蛋白疫苗的研制及其抗腫瘤效應(yīng)[J];中華微生物學(xué)和免疫學(xué)雜志;2005年11期

5 史百芬;純化的融合蛋白疫苗在RSV流行季節(jié)保護(hù)囊性纖維變性患兒抵抗下呼吸道疾病的效果[J];國(guó)外醫(yī)學(xué).預(yù)防.診斷.治療用生物制品分冊(cè);1997年01期

6 廖芳;賀超;劉麗江;宋啟發(fā);萬(wàn)沐芬;楊振德;李雍龍;;淋球菌孔蛋白疫苗在動(dòng)物體內(nèi)誘導(dǎo)的免疫應(yīng)答[J];中國(guó)婦幼保健;2005年24期

7 龍軍;楊欣;曹榮月;吳潔;劉景晶;;抗動(dòng)脈粥樣硬化疫苗的免疫學(xué)效應(yīng)研究[J];中國(guó)天然藥物;2007年05期

8 ;治療性乙肝蛋白疫苗有望打破乙肝免疫耐受態(tài)[J];透析與人工器官;2010年02期

9 熊新宇,陳元鼎;丙型肝炎病毒蛋白疫苗的研究進(jìn)展[J];國(guó)外醫(yī)學(xué).病毒學(xué)分冊(cè);2004年03期

10 樊建勇;楊慧蘭;王捷;關(guān)蕾;王穎;成桂明;仕瑤慧;;Hsp70-HSV-2gD融合蛋白疫苗陽(yáng)離子脂質(zhì)體的制備[J];中國(guó)麻風(fēng)皮膚病雜志;2006年11期

相關(guān)會(huì)議論文 前10條

1 陳娟娟;;蛋白疫苗中佐劑應(yīng)用進(jìn)展[A];第六屆全國(guó)免疫學(xué)學(xué)術(shù)大會(huì)論文集[C];2008年

2 鄭黎燕;王國(guó)華;闕海萍;郁毅剛;馬振蓮;李欣;劉少君;;Nogo及其NgR受體在神經(jīng)細(xì)胞及損傷模型中的表達(dá)[A];中國(guó)生理學(xué)會(huì)第21屆全國(guó)代表大會(huì)暨學(xué)術(shù)會(huì)議論文摘要匯編[C];2002年

3 鄂玲玲;周長(zhǎng)滿;馮士生;徐忠濤;楊磊;;Nogo(N-18)在大鼠海馬及大腦皮質(zhì)中分布和發(fā)育的免疫組織化學(xué)研究[A];解剖學(xué)雜志——中國(guó)解剖學(xué)會(huì)2002年年會(huì)文摘匯編[C];2002年

4 姚志彬;汪華僑;鄒俊濤;張革;徐杰;謝瑤;袁群芳;;阿爾茨海默病Aβ_(1-15)疫苗的實(shí)驗(yàn)研究[A];2008年全國(guó)抗衰老與老年癡呆學(xué)術(shù)會(huì)議論文集[C];2008年

5 雷季良;楊磊;趙君朋;劉斯;魏澤峰;周長(zhǎng)滿;于恩華;;正常和視神經(jīng)損傷后Nogo(N-18)在金黃地鼠視網(wǎng)膜分布及變化的免疫組織化學(xué)研究[A];解剖學(xué)雜志——中國(guó)解剖學(xué)會(huì)2002年年會(huì)文摘匯編[C];2002年

6 馮士生;周長(zhǎng)滿;鄂玲玲;;Nogo(N-18)在大鼠脊髓、背根神經(jīng)節(jié)及三叉神經(jīng)節(jié)中的分布的免疫組織化學(xué)研究[A];解剖學(xué)雜志——中國(guó)解剖學(xué)會(huì)2002年年會(huì)文摘匯編[C];2002年

7 何曉文;章亞男;何穎;李凱;姜磊;孫樹漢;;乙肝DNA疫苗聯(lián)合基因工程蛋白疫苗提高免疫效果的研究[A];首屆長(zhǎng)三角科技論壇——長(zhǎng)三角生物醫(yī)藥發(fā)展論壇論文集[C];2004年

8 戴洋;朱蔭昌;王曉婷;唐建霞;趙松;;日本血吸蟲核酸疫苗體內(nèi)電穿孔法的進(jìn)一步研究[A];2011新型疫苗與抗體創(chuàng)制關(guān)鍵技術(shù)及質(zhì)量控制研討會(huì)論文集[C];2011年

9 邢宏義;孫圣剛;梅元武;;Lingo-1：神經(jīng)再生障礙研究中的一種新型分子[A];湖北省神經(jīng)科學(xué)學(xué)會(huì)學(xué)術(shù)年會(huì)論文匯編[C];2007年

10 盛彩虹;劉原君;齊蔓麗;劉全忠;;E型沙眼衣原體主外膜蛋白DNA疫苗和重組蛋白疫苗聯(lián)合免疫誘導(dǎo)出的免疫效應(yīng)[A];中華醫(yī)學(xué)會(huì)第16次全國(guó)皮膚性病學(xué)術(shù)年會(huì)摘要集[C];2010年

相關(guān)重要報(bào)紙文章 前10條

1 本報(bào)記者 董長(zhǎng)青;萬(wàn)克森：研制適合國(guó)人的蛋白疫苗[N];北京日?qǐng)?bào);2011年

2 彭勇;治療性疫苗有望打破乙肝免疫耐受[N];南方日?qǐng)?bào);2005年

3 紅民;艾滋病疫苗距離我們還有多遠(yuǎn)？[N];中國(guó)醫(yī)藥報(bào);2007年

4 康存棟;突破我國(guó)水產(chǎn)疫苗研發(fā)和生產(chǎn)瓶頸[N];中國(guó)漁業(yè)報(bào);2008年

5 喻菲 俞錚;曾毅院士：2010年完成艾滋病疫苗Ⅲ期臨床試驗(yàn)[N];醫(yī)藥經(jīng)濟(jì)報(bào);2006年

6 邵一鳴;國(guó)內(nèi)外艾滋病疫苗的研究進(jìn)展和發(fā)展趨勢(shì)[N];人民政協(xié)報(bào);2008年

7 尹宏毅;功成才能名就？[N];科技日?qǐng)?bào);2003年

8 劉文伊;治療性乙肝疫苗進(jìn)入臨床[N];科技日?qǐng)?bào);2006年

9 峻嶺;人工合成縮氨酸：可促進(jìn)新神經(jīng)生長(zhǎng)[N];醫(yī)藥經(jīng)濟(jì)報(bào);2002年

10 張荔子;中樞神經(jīng)修復(fù)之路有多遠(yuǎn)[N];健康報(bào);2002年

相關(guān)博士學(xué)位論文 前10條

1 程茗;Nogo66蛋白眼用疫苗對(duì)大鼠高眼壓和視神經(jīng)損傷的免疫保護(hù)作用及機(jī)制研究[D];第三軍醫(yī)大學(xué);2009年

2 余思遜;Nogo-A蛋白參與形成大腦皮質(zhì)發(fā)育障礙及其作用機(jī)制研究[D];第三軍醫(yī)大學(xué);2012年

3 米亞靜;探索神經(jīng)元Nogo-A分子的自主性功能[D];上海交通大學(xué);2012年

4 尹小磊;視網(wǎng)膜Nogo氨基末端信號(hào)通路的初步研究[D];第三軍醫(yī)大學(xué);2010年

5 周樹敏;Nogo-B對(duì)Fibulin-5的調(diào)控及其對(duì)細(xì)胞遷移的影響[D];武漢大學(xué);2010年

6 肖飛;Nogo-66受體作為阿爾茨海默病藥物治療新靶標(biāo)的研究[D];暨南大學(xué);2010年

7 宋欣欣;提高HPV融合蛋白疫苗療效策略研究[D];中國(guó)協(xié)和醫(yī)科大學(xué);2010年

8 葉菁;熱休克蛋白70與MAGE-1融合基因疫苗和蛋白疫苗的構(gòu)建及體內(nèi)抑瘤效應(yīng)的研究[D];中國(guó)人民解放軍第四軍醫(yī)大學(xué);2003年

9 李曉楠;手足口病新型疫苗的初步研究[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2010年

10 田建卿;抑胃多肽疫苗的構(gòu)建及主動(dòng)免疫后對(duì)大鼠行為學(xué)和腦功能的影響[D];第二軍醫(yī)大學(xué);2010年

相關(guān)碩士學(xué)位論文 前10條

1 鄧斌;六種抗大鼠Nogo-A分子單克隆抗體的免疫組織化學(xué)染色特性及Nogo-A分子截短體的構(gòu)建[D];第四軍醫(yī)大學(xué);2011年

2 趙莉群;重組金黃色葡萄球菌腸毒素B蛋白疫苗的制備及免疫效果評(píng)價(jià)[D];西北農(nóng)林科技大學(xué);2011年

3 崔珂;CpG ODN對(duì)口蹄疫重組蛋白疫苗增效作用的研究[D];吉林大學(xué);2010年

4 李晟;Nogo基因多態(tài)性與多發(fā)性硬化的相關(guān)性研究[D];昆明醫(yī)學(xué)院;2010年

5 趙明麗;DNA疫苗預(yù)敏蛋白疫苗增強(qiáng)策略對(duì)乙型肝炎病毒表面抗原蛋白免疫應(yīng)答的影響[D];南京醫(yī)科大學(xué);2009年

6 蘇桂云;Nogo-A、nogo受體及其途徑抑制物與新生大鼠HIBD后神經(jīng)再生的相關(guān)性研究[D];山東大學(xué);2010年

7 戴志兵;抗淋病LTB-PorB核酸疫苗與重組蛋白疫苗聯(lián)合免疫的免疫增強(qiáng)效應(yīng)研究[D];南華大學(xué);2010年

8 林麗霞;高原環(huán)境下兔眼球鈍挫傷后視網(wǎng)膜NGF、Nogo表達(dá)的調(diào)控研究[D];蘭州大學(xué);2010年

9 蘇雯靜;乙型肝炎疫苗聯(lián)合B7-H1蛋白疫苗對(duì)HBV轉(zhuǎn)基因小鼠抗HBsAg免疫應(yīng)答的影響[D];第四軍醫(yī)大學(xué);2012年

10 李英輝;異種表皮生長(zhǎng)因子受體胞外段的表達(dá)及其抗腫瘤效果研究[D];西北農(nóng)林科技大學(xué);2008年

，

本文編號(hào)：1909120