本文選題：視網(wǎng)膜中央靜脈阻塞 + YC-1��； 參考：《河北醫(yī)科大學(xué)》2016年博士論文

【摘要】：視網(wǎng)膜中央靜脈阻塞(central retinal vein occlusion,CRVO)是僅次于糖尿病視網(wǎng)膜病變的血管性疾病,患病率在0.7-1.6%之間,常常引起嚴重的視力下降甚至致盲。盡管確切的機制不清,但由于血管通透性增加導(dǎo)致的視網(wǎng)膜廣泛出血及黃斑水腫(cystoids macular edema,CME)是主要的原因之一。目前,已有足夠的證據(jù)表明VEGF在視網(wǎng)膜血管性疾病的發(fā)病過程中發(fā)揮著重要的作用。分子水平上,VEGF通過調(diào)節(jié)血管內(nèi)皮細胞的增殖、移行以及增加血管內(nèi)皮的通透性發(fā)揮病理作用。臨床研究證明玻璃體腔注射抗VEGF藥物,通過抑制VEGF與其受體相結(jié)合,從而抑制脈絡(luò)膜,視網(wǎng)膜新生血管生長及血管滲漏,可以明顯改善CRVO引起的黃斑水腫,不同程度改善患者視力。在包括老年性黃斑變性(AMD),視網(wǎng)膜靜脈阻塞(RVO)及糖尿病視網(wǎng)膜病變(RD)的治療中,抗VEGF藥物的玻璃體腔注射治療已成為一線的治療方案。然而,隨著抗VEGF藥物的廣泛應(yīng)用,仍有諸多問題有待解決:由于藥物半衰期短,需要頻繁行眼內(nèi)注藥治療以穩(wěn)定視力,增加了發(fā)生眼內(nèi)炎的潛在風(fēng)險;部分難治性CRVO患者經(jīng)治療后,視力及黃斑水腫并沒有改善甚至惡化,即所謂“反彈”效應(yīng);長期使用出現(xiàn)了色素上皮細胞及感光細胞層的萎縮現(xiàn)象。另一方面,炎癥因子可能也發(fā)揮了一定的作用,臨床研究證實:多種炎癥因子如:IL1β,IL6,IL8,IL10,IL12,IL13,單核細胞趨化蛋白1(MCP-1),可溶性細胞黏附因子1(Si CAM-1),干擾素等在CRVO患者的玻璃體和房水中明顯升高。通過玻璃體腔注射曲安奈德,有效抑制了VEGF和炎癥因子的升高,減輕CRVO患者黃斑水腫,提高視力。然而,激素的嚴重副作用限制了它的廣泛應(yīng)用,研究表明經(jīng)過曲安奈德玻璃體腔注射后,48-67%的患者出現(xiàn)不同程度的白內(nèi)障,40%有眼內(nèi)壓升高的表現(xiàn),0.1%的眼內(nèi)炎風(fēng)險。因此尋找新的靶向藥物勢在必行。在視網(wǎng)膜血管疾病的病理過程中,缺氧誘導(dǎo)因子1(HIF-1)起到了非常重要的作用,根據(jù)文獻報道,包括VEGF,EPO在內(nèi)的多種血管相關(guān)因子受到HIF-1二聚體的調(diào)控。YC-1作為HIF-1α的抑制劑,通過抑制HIF-1復(fù)合體的產(chǎn)生,在轉(zhuǎn)錄水平提前遏制VEGF等多種血管相關(guān)因子的產(chǎn)生,同時YC-1通過調(diào)控NF-KB通道(目前認為該通道是主要的炎癥因子調(diào)控通道)間接抑制炎癥因子的產(chǎn)生。因此YC-1兼具抗VEGF及抗炎的雙重作用,極具研究價值。獼猴具有與人類類似的黃斑結(jié)構(gòu),通過激光建立CRVO獼猴動物模型后,經(jīng)玻璃體腔注射YC-1,可以觀察眼球組織結(jié)構(gòu),特別是黃斑結(jié)構(gòu)的變化,以及玻璃體和視網(wǎng)膜中分子水平的變化,探討YC-1玻璃體腔注射治療CRVO疾病的可行性。第一部分YC-1藥物在正常獼猴玻璃體腔中的藥物動力學(xué)研究目的:在成年獼猴玻璃體腔中注射YC-1藥物,觀察其藥物動力學(xué)變化,為進一步應(yīng)用提供初步研究數(shù)據(jù)。方法:選取正常獼猴3只,共6只眼睛,應(yīng)用YC-1;化學(xué)名稱為3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole(中文名稱:3-(5-羥甲基-2-呋喃基)-1-苯甲基吲唑)(intel number.81560 5mg Cayman Chemical);溶解在DMSO(二甲基氬砜,濃度為0.01%)藥物中,稀釋成200μM濃度后,抽取90μl注入獼猴玻璃體腔中,并分別在注射后0.25小時,0.5小時,1小時,2小時,3小時,4小時,5小時,6小時,8小時,10小時,抽取玻璃體標(biāo)本100μl,經(jīng)樣品預(yù)處理后,應(yīng)用高效液相色譜技術(shù)+質(zhì)譜聯(lián)用法檢測各時間點玻璃體中YC-1的濃度變化。并應(yīng)用DAS 3.0藥物動力學(xué)軟件及PKSolver 2.0軟件,計算YC-1的藥物消除半衰期等藥物動力學(xué)指標(biāo)。結(jié)果:結(jié)果表明,獼猴眼玻璃體液的內(nèi)源性物質(zhì)對YC-1及內(nèi)標(biāo)的測定沒有干擾,保留時間分別為1.25和1.80 min,表明該方法專屬性良好。相關(guān)系數(shù)r均大于0.995,表明線性良好。藥物動力學(xué)研究結(jié)果顯示在給藥后0.25小時,藥物濃度為1.217μM,0.5小時為0.824μM,1小時為0.497μM,2小時為0.368μM,3小時為0.326μM,4小時為0.265μM,5小時為0.196μM 6小時為0.145μM,8小時為0.083μM。經(jīng)軟件分析YC-1藥物半衰期為2.79小時。結(jié)論:通過液質(zhì)聯(lián)用方法測量玻璃體內(nèi)YC-1藥物動力學(xué)的變化,專屬性和精密度良好,可以應(yīng)用于YC-1在獼猴玻璃體腔中的藥物動力學(xué)研究,結(jié)果符合預(yù)期的實驗設(shè)計。本研究通過觀察不同時間組YC-1在玻璃體內(nèi)的質(zhì)量濃度隨時間變化的規(guī)律,發(fā)現(xiàn)玻璃體內(nèi)YC-1質(zhì)量濃度隨時間的延長而迅速下降。YC-1在玻璃體內(nèi)的消除半衰期t1/2為2.79小時。第二部分YC-1玻璃體腔注射對正常獼猴眼部組織的毒性研究目的:對YC-1玻璃體腔注射后其對眼部組織毒性問題進行研究,以期對該藥在眼部應(yīng)用的安全性進行評價。方法:共3只實驗用成年獼猴共6只眼睛,其中每只猴子右眼作為實驗組,行YC-1 90μl(濃度為200μM)玻璃體腔注射,左眼作為對照組,行二甲基氬砜90μl(DMSO,濃度為0.01%)玻璃體腔注射。觀察時間點為術(shù)前和術(shù)后1天,1周,2周,1月。測量各實驗眼眼壓、裂隙燈觀察結(jié)膜反應(yīng)、前房炎癥反應(yīng)以及角膜透明性,眼底照相機觀察眼底病變情況。相干光斷層掃描(optic coherence tomography,OCT)觀察黃斑區(qū)結(jié)構(gòu),眼底熒光血管造影檢查血管病變情況。進行暗適應(yīng)及明適應(yīng)ERG檢查。并在術(shù)后1月對摘除的眼球行光鏡組織學(xué)檢查。結(jié)果:YC-1玻璃體腔注射后各檢查時間點,所有眼球角膜透明,無前房反應(yīng),也未見玻璃體積血、視網(wǎng)膜脫離等并發(fā)癥。眼壓,黃斑中心厚度無明顯差異(P0.05)。眼底血管造影未見血管異常改變。ERG檢查各組各時間點,實驗組眼視桿細胞b波和視錐細胞a波和b波振幅與對照組眼相比差異無統(tǒng)計學(xué)意義(P0.05)。各時間點視網(wǎng)膜組織學(xué)檢查未見異常改變。結(jié)論:應(yīng)用YC-1(90μl濃度為200μM)注射于正常獼猴眼玻璃體內(nèi),無明顯炎癥反應(yīng),經(jīng)過1月的隨訪觀察,對視網(wǎng)膜結(jié)構(gòu)和功能無顯著性影響。第三部分獼猴視網(wǎng)膜中央靜脈阻塞的模型的建立及視網(wǎng)膜功能及結(jié)構(gòu)和玻璃體相關(guān)因子的測定目的:探討多波長激光誘導(dǎo)獼猴視網(wǎng)膜中央靜脈阻塞的可行性,觀察視網(wǎng)膜結(jié)構(gòu)和功能變化,為臨床進一步認識該疾病的轉(zhuǎn)歸提供參考。方法:選擇6只健康成年獼猴,首先行裂隙燈,眼底檢查,OCT檢查,眼底熒光血管造影,ERG檢查及眼壓檢查,并確認無眼部疾患后,應(yīng)用多波長激光的黃光封閉6只獼猴的右眼視盤周圍所有主干靜脈,制成CRVO模型,隨后即刻行眼底熒光血管造影證實,并于術(shù)后6小時,1天,1周,2周,1月行相同檢查。同時在造模前和造模后各時間點行玻璃體腔抽取玻璃體液200μl。分別行CBA及ELASA檢測玻璃體細胞因子表達變化,包括:IL6,IL8,MCP-1及VEGF和HIF-1α。1個月后摘除眼球(每只獼猴摘除1眼,共3只左眼,3只右眼),進行視網(wǎng)膜的HE染色及VEGF,HIF-1α,Caspase3的免疫組化觀察研究。結(jié)果:6只獼猴眼睛經(jīng)激光制作的CRVO模型經(jīng)FFA檢查均成功,結(jié)果顯示眼壓在術(shù)后1天及1周有輕度升高,但無顯著性差異(P0.05),虹膜面可見新生血管,黃斑水腫在1天,1周,2周有顯著性升高(P0.05),并于1月恢復(fù)到術(shù)前水平(P0.05)。眼底可見廣泛火焰出血,ERG顯示視桿細胞b波和視錐細胞a波和b波顯著下降(P0.05),眼底造影提示視網(wǎng)膜缺血滲漏明顯,并可見無灌注區(qū)及新生血管,符合人視網(wǎng)膜中央靜脈阻塞的基本變化。通過CBA及ELASA檢測玻璃體炎癥因子IL-6,IL-8及MCP-1表達水平在術(shù)后不同時間點出現(xiàn)了顯著升高(IL-6和IL-8從術(shù)后1天,MCP-1從術(shù)后2周)(P0.05)。VEGF與HIF-1α表達也一致性升高,免疫組化研究發(fā)現(xiàn),HIF-1α和VEGF在視網(wǎng)膜中表達明顯升高,并伴有凋亡蛋白Caspase3的表達升高,光密度值均顯示三種蛋白表達顯著性升高(P0.05)。結(jié)論:激光制作的CRVO獼猴動物模型在術(shù)后出現(xiàn)了典型的黃斑水腫,視網(wǎng)膜出血及無灌注區(qū)等體征。視網(wǎng)膜電圖顯示視網(wǎng)膜功能不同程度受到破壞。測量玻璃體和視網(wǎng)膜中VEGF,HIF-1α表達顯著升高,同時,玻璃體中炎癥因子IL-6,IL-8,MCP-1表達上調(diào)。以上結(jié)果表明血管生長因子及炎癥因子均參與了實驗性CRVO的發(fā)病過程,激光所致獼猴CRVO可以模擬人類CRVO的發(fā)病過程,并為進一步藥物干預(yù)治療提供理想的動物模型。第四部分YC-1玻璃體腔注射治療獼猴實驗性視網(wǎng)膜中央靜脈阻塞的研究目的:探討YC-1玻璃體腔注射后對CRVO獼猴動物模型眼的視網(wǎng)膜結(jié)構(gòu),功能及視網(wǎng)膜和玻璃體內(nèi)各種細胞因子的影響。方法:選用健康成年獼猴6只,每只獼猴右眼為YC-1實驗組,行玻璃體腔注射YC-1,左眼為DMSO對照組,行DMSO玻璃體腔注射。行激光制作CRVO動物模型后,在術(shù)后1周,分別給予實驗組和對照YC-1和DMSO玻璃體腔注射,分別在注射前和注射后1天,1周,2周、1月使用裂隙燈顯微鏡觀察角膜、房水、晶狀體、前節(jié)炎癥反應(yīng)情況;使用眼底照相,OCT及熒光眼底血管造影評估視網(wǎng)膜結(jié)構(gòu)變化情況,并行眼壓檢查。同時采取玻璃體樣本200μl,應(yīng)用CBA及ELASA檢測各時間點玻璃體樣本中VEGF、HIF-1α、IL-6、IL-8、MCP-1等細胞因子的表達。并行ERG視網(wǎng)膜電圖檢查評估視網(wǎng)膜功能情況。隨后在1個月時摘除眼球行HE染色及視網(wǎng)膜免疫組化研究。通過免疫組化方法檢測視網(wǎng)膜中VEGF、HIF-1、Caspase3的表達。結(jié)果:YC-1實驗組與DMSO對照組,經(jīng)注射藥物后,兩組均可見虹膜面新生血管,視網(wǎng)膜出血,無灌注區(qū)及新生血管。各時間點,兩組間眼壓未見顯著性差異(P0.05)。OCT檢查可見黃斑區(qū)明顯水腫,給予YC-1和DMSO注射后,發(fā)現(xiàn)在YC-1實驗組,黃斑區(qū)水腫消退顯著。在注射后1天,1周和2周均較DMSO對照組有顯著性差異。ERG檢查發(fā)現(xiàn),給予YC-1玻璃體腔注射后1周視網(wǎng)膜電圖的rod b波和cone a和b波均較DMSO組有顯著改善。應(yīng)用YC-1玻璃體腔注射后,IL-6、IL-8均得到明顯的抑制,而MCP-1盡管有輕度降低,但與DMSO對照組比較沒有顯著性差異。此外,在應(yīng)用YC-1后,玻璃體中HIF-1α和VEGF的表達顯著降低,HE染色發(fā)現(xiàn)YC-1和DMSO二組的視網(wǎng)膜內(nèi)核層和外核層細胞均較正常視網(wǎng)膜顯著減少。免疫組化研究發(fā)現(xiàn)在YC-1治療組,視網(wǎng)膜中HIF-1α,VEGF及Caspase3三種抗體的表達均顯著受到抑制,應(yīng)用圖像分析系統(tǒng)測量了三種抗體在視網(wǎng)膜表達的光密度值,證實了三種蛋白表達均較DMSO組顯著降低。結(jié)論:在造模后一周時,YC-1玻璃體腔注射后,可以顯著改善黃斑水腫的程度,加快水腫吸收,改善ERG中視桿細胞b波和視錐細胞的a波和b波的波幅。同時抑制IL-6、IL-8、VEGF、HIF-1α在玻璃體內(nèi)的表達,輕度抑制MCP-1的表達。在視網(wǎng)膜中降低VEGF和HIF-1α的表達,減少細胞凋亡,對視網(wǎng)膜細胞有保護作用�；谏鲜鲅芯拷Y(jié)果可以得出以下結(jié)論:1應(yīng)用YC-1玻璃體腔注射對獼猴眼睛無明顯毒性反應(yīng),對視網(wǎng)膜功能及結(jié)構(gòu)無明顯影響。2多波長激光制作獼猴CRVO動物模型,可實現(xiàn)模擬人眼CRVO的表現(xiàn),其視網(wǎng)膜體征和功能均有一定相似性,可作為理想的實驗?zāi)Ｐ?進行相關(guān)的藥物研究。3應(yīng)用YC-1玻璃體腔注射治療激光誘導(dǎo)的獼猴CRVO動物模型,結(jié)果表明YC-1可以減輕黃斑水腫程度,改善視網(wǎng)膜功能,迅速抑制CRVO引起的玻璃體和視網(wǎng)膜中各種炎癥因子及VEGF和HIF-1α的升高,降低視網(wǎng)膜細胞的凋亡程度,具有潛在的臨床使用價值。
[Abstract]:Central retinal vein occlusion (CRVO) is a vascular disease second only to diabetic retinopathy. The prevalence rate is between 0.7-1.6% and often causes severe visual loss and even blindness. Although the exact mechanism is not clear, there is extensive retinal hemorrhage and macular edema caused by increased vascular permeability (Cys Toids macular edema, CME) is one of the main reasons. At present, there is sufficient evidence to show that VEGF plays an important role in the pathogenesis of retinal vascular diseases. At the molecular level, VEGF plays a pathological role by regulating the proliferation and migration of vascular endothelial cells and increasing the permeability of the intravascular skin. Anti VEGF drugs are injected into the cavity of the body. By inhibiting the combination of VEGF and its receptor, thus inhibiting the choroid membrane, the growth of retinal neovascularization and vascular leakage, it can obviously improve the macular edema caused by CRVO and improve the visual acuity of patients in varying degrees. It includes senile macular degeneration (AMD), retinal vein occlusion (RVO) and diabetic retinopathy (RD). In the treatment of the treatment, the vitreous intravitreal injection of anti VEGF drugs has become a first-line treatment. However, with the widespread use of anti VEGF drugs, there are still many problems to be solved: due to the short half-life of the drug, frequent intraocular injection is needed to stabilize the vision and increase the potential risk of endophthalmitis; some refractory CRVO patients After treatment, visual acuity and macular edema did not improve or even deteriorate, namely the so-called "rebound" effect; long term use of the atrophy of pigment epithelial cells and photoreceptor cells. On the other hand, the inflammatory factors may also play a role. Clinical studies have confirmed that a variety of inflammatory factors such as IL1 beta, IL6, IL8, IL10, IL12, IL13, mononuclear Cell chemoattractant protein 1 (MCP-1), soluble cell adhesion factor 1 (Si CAM-1), interferon in vitreous and aqueous humor of CRVO patients were significantly increased. The injection of Cu Ann Ned through the vitreous cavity effectively inhibited the increase of VEGF and inflammatory factors, alleviated macular edema and improved visual acuity in patients with CRVO. However, the severe side effects of the hormone restricted it. Widely used, studies have shown that after the injection of Cu Ann Ned's vitreous cavity, patients with 48-67% have different degrees of cataract, 40% with elevated intraocular pressure and 0.1% of the risk of endophthalmitis. Therefore, it is imperative to find new target drugs. In the pathological process of retinal vascular disease, hypoxia inducible factor 1 (HIF-1) is very important. As reported in the literature, a variety of vascular related factors, including VEGF, EPO, are regulated by the HIF-1 two polymer as an inhibitor of HIF-1 alpha. By inhibiting the production of the HIF-1 complex, the production of a variety of vascular related factors, such as VEGF, is inhibited in advance at the transcriptional level, and at the same time YC-1 is regulated by the NF-KB channel (currently believed to be the channel). The main inflammatory factor regulation channel indirectly inhibits the production of inflammatory factors. Therefore, YC-1 has both anti VEGF and anti-inflammatory effects and has great research value. Rhesus monkey has a macular structure similar to that of human. After establishing a CRVO macaque animal model by laser, the structure of eyeball tissue, especially macular knot, is observed by intravitreal injection of YC-1. The changes in the structure and the changes in the molecular level in the vitreous and retina, the feasibility of YC-1 intravitreal injection for the treatment of CRVO disease is discussed. Part 1 the pharmacokinetics of YC-1 in the normal rhesus monkey's vitreous cavity is studied in order to observe the pharmacokinetics of YC-1 in the adult rhesus monkey's glass cavity. Methods: a total of 3 normal macaques and 6 eyes were selected to use YC-1. The chemical name was 3- (5-hydroxymethyl-2-furyl) -1-benzyl indazole (Chinese Name: 3- (5- hydroxymethyl -2- furanyl) -1- benzyl indazole) (Intel number.81560 5mg), and dissolved in the drug (two methyl argon sulfoxide, 0.01%), After diluted to 200 M concentration, 90 Mu l was injected into the glass cavity of macaque, and 0.25 hours, 0.5 hours, 1 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 6 hours, 6 hours, 8 hours, 10 hours, respectively, were extracted from the vitreous body of the rhesus macaque, respectively. After sample pretreatment, high performance liquid chromatography and mass spectrometry were used to detect YC in the vitreous body of each time point. The concentration changes of -1. And using the DAS 3 pharmacokinetic software and the PKSolver 2 software to calculate the pharmacokinetic indexes of the drug elimination half life of YC-1. Results: the results showed that the endogenous substances of the macaque eye glass body fluid were not interfered with the determination of YC-1 and the internal standard, and the retention time was 1.25 and 1.80 min respectively, indicating that the method was exclusive and good. The correlation coefficient r was more than 0.995, indicating a good linearity. The pharmacokinetic study showed that the drug concentration was 1.217 mu M, 0.5 hours was 0.824 mu M, 1 hours 0.497 mu M, 2 hours 0.368 u M, 3 hours 0.326 M, 4 hour 0.265 M, 2 hour M hour M.. The half-life of YC-1 was 2.79 hours. Conclusion: the changes in the pharmacokinetics of YC-1 in the vitreous body were measured by the method of liquid mass spectrometry, and the specificity and precision were good. It could be applied to the pharmacokinetics study of YC-1 in the macaque glass cavity. The results accorded with the expected experimental design. This study was conducted by observing different time groups of YC-1 in the glass body. It was found that the mass concentration of YC-1 in the vitreous body rapidly decreased with time and reduced the half-life of.YC-1 in the glass body for 2.79 hours. The toxicity of the second part of the YC-1 glass cavity injection on the eye tissues of normal rhesus monkeys: the toxicity to the ocular tissue after the injection of YC-1 vitreous cavity. The problem was studied in order to evaluate the safety of the drug in the eye application. Methods: a total of 6 eyes were used in 3 adult rhesus monkeys, of which the right eye of each monkey was taken as the experimental group. The YC-1 90 Mu L (concentration 200 M) was injected into the glass cavity, the left eye was used as the control group, and the two methyl argon sulfoxide 90 L (DMSO, 0.01%) was injected into the glass body cavity. The observation time was 1 days before and 1 days, 1 weeks, 2 weeks, January. The intraocular pressure of the experimental eyes was measured, the conjunctiva reaction was observed in the slit lamp, the inflammation of the anterior chamber and the corneal transparency, the ocular fundus camera observed the lesion of the fundus. The optic coherence tomography (OCT) was used to observe the macular region, and the fundus fluorescein angiography was used to examine the blood vessels. Pathological changes. Dark adaptation and adaptive ERG examination. And the ocular microscopy examination of the exucleation of the eyeball in January. Results: every examination time after the injection of YC-1 glass body cavity, all cornea of the eyeball was transparent, no anterior chamber reaction, no vitreous blood, retinal detachment and other complications. There was no significant difference in intraocular pressure and macular center thickness (P0.0 5). No abnormal changes of blood vessels in the ocular fundus angiography were found in each time point of.ERG examination. There was no significant difference in the B and b wave amplitudes of the eye rod cells in the experimental group compared with the control group (P0.05). There was no abnormal change in the retinal histological examination at all time points. Conclusion: the application of YC-1 (the concentration of 90 Mu l to 200 mu M) was injected into the normal Actinidia. There is no obvious inflammatory response in the monkey eye glass. After the follow-up observation in January, there is no significant effect on the structure and function of the retina. The establishment of the model of central retinal vein occlusion in third macaques and the determination of retinal function and structure and the related factors of vitreous body: To explore the central vein of rhesus macaque induced by multi wavelength laser The feasibility of blocking, observing the changes of retinal structure and function, provides reference for further understanding of the prognosis of the disease. Methods: 6 healthy adult macaques were selected, first of which were slit lamp, fundus examination, OCT examination, fundus fluorescein angiography, ERG examination and intraocular pressure examination, and the application of multi wavelength laser yellow light after no ocular disease was confirmed. All the main veins around the right eye disc around 6 rhesus monkeys were closed, and the CRVO model was made. Then the fundus fluorescein angiography was performed immediately, and the same examination was performed at 6 hours, 1 days, 1 weeks and 2 weeks after the operation. At the same time, the vitreous body fluid was extracted by the vitreous body fluid 200 mu before and after the modeling, and the vitreous cells were detected by CBA and ELASA respectively. Factor expression changes, including: IL6, IL8, MCP-1 and VEGF and HIF-1 alpha.1 months after the removal of the eyeball (1 eyes from each rhesus monkey, 3 left eyes, 3 right eyes), HE staining of the retina and immunohistochemical observation of VEGF, HIF-1 alpha and Caspase3. Results: 6 monkeys' eyes were successfully made by laser for FFA, and the results showed intraocular pressure. There was a slight increase in the 1 and 1 weeks after operation, but there was no significant difference (P0.05). The neovascularization was seen on the iris surface. The macular edema had a significant rise in 1 days, 1 weeks and 2 weeks (P0.05), and was restored to the preoperative level (P0.05) in January. Extensive flame hemorrhage was found in the fundus, and ERG showed a significant decrease in the A and B waves (P0.05) of the B and conical cells of the optic cells (P0.05). The findings suggest that the retinal ischemic leakage is obvious, and the non perfusion area and neovascularization can be seen in accordance with the basic changes in the central retinal vein occlusion. The levels of vitreous inflammatory factors IL-6, IL-8 and MCP-1 are significantly increased by CBA and ELASA (IL-6 and IL-8 from 1 days after operation, MCP-1 from 2 weeks after operation) (P0.05).VE (P0.05).VE The expression of GF and HIF-1 alpha was also elevated. The immunohistochemical study found that the expression of HIF-1 alpha and VEGF increased significantly in the retina, accompanied by the increase of the expression of apoptotic protein Caspase3, and the value of light density showed a significant increase in the expression of three proteins (P0.05). Conclusion: the laser made CRVO macaque animal model appeared typical Huang Banshui after the operation. Swelling, retinal hemorrhage and instillation areas. Electroretinogram showed that retinal function was damaged in varying degrees. The expression of VEGF, HIF-1 a in the vitreous and retina was significantly increased, and the expression of inflammatory factors IL-6, IL-8, and MCP-1 in the vitreous body was up-regulated. The above results showed that both vascular growth factor and inflammatory factor were involved in experimental CR. The pathogenesis of VO, laser induced macaque CRVO can simulate the pathogenesis of human CRVO, and provide an ideal animal model for further drug intervention. Fourth part YC-1 vitreous intravitre injection for experimental retinal vein occlusion in rhesus monkeys: To explore the model eye of CRVO rhesus monkeys after YC-1 vitreous injection. Methods: the effects of retinal structure, function and various cytokines in the retina and vitreous body. Methods: 6 healthy adult macaques were selected, the right eye of each macaque was YC-1 experimental group, YC-1 was injected into the vitreous cavity, the left eye was DMSO control group, and DMSO glass cavity was injected. After the laser production of CRVO animal model, the experimental group was given 1 weeks after the operation, and the experimental group and the pair were given respectively. YC-1 and DMSO vitreous intravitre were injected before and 1 days, 1 weeks and 2 weeks after injection. In January, corneal, aqueous humor, lens, and anterior inflammatory reaction were observed by slit light microscope, retinal structure changes were evaluated by fundus photography, OCT and fluorescein angiography, and intraocular pressure was checked in parallel with 200 l of vitreous samples. CBA and ELASA were used to detect the expression of VEGF, HIF-1 alpha, IL-6, IL-8, MCP-1 in the vitreous samples of each time point. The retinal function was evaluated by parallel ERG electroretinogram. Then the eyeball was extirpated with HE staining and retina immunohistochemical study at 1 months. VEGF, HIF-1, Caspase3 in retina were detected by immunization. Results: YC-1 experimental group and DMSO control group, after the injection of drugs, two groups of iris neovascularization, retinal hemorrhage, no perfusion area and neovascularization. At all time points, there was no significant difference in intraocular pressure between the two groups (P0.05).OCT examination showed obvious edema in the macular area. After the injection of YC-1 and DMSO, it was found in the YC-1 experimental group, macula 1 days after injection, 1 and 2 weeks after injection, there was significant difference between the.ERG and the DMSO control group. The rod b wave and cone a and b wave of the electroretinogram after the injection of YC-1 glass body cavity were significantly better than those in the DMSO group. But there was no significant difference compared with the DMSO control group. In addition, after the application of YC-1, the expression of HIF-1 alpha and VEGF in vitreous decreased significantly, HE staining.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R774.1

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相關(guān)期刊論文 前2條

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本文編號：1937608