CN113057142A - 一种视网膜内和/或视网膜下纤维化动物模型的构建方法 - Google Patents
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Abstract
本发明公开了一种视网膜内和/或视网膜下纤维化动物模型的构建方法,它是向非人灵长类动物的眼部给予重组腺病毒AAV‑VEGF得到动物模型。本发明选择非人灵长类恒河猴,通过眼局部给予AAV‑VEGF得到的动物模型可以同时模拟nAMD的长期CNV和SRF,并且在短时间内就能得到能够长期模拟nAMD的晚期表型SRF。
Description
技术领域
本发明具体涉及一种视网膜内和/或视网膜下纤维化动物模型的构建方法。
背景技术
年龄相关性黄斑变性(age-related macular degeneration,AMD)是导致发达地区50岁以上人群不可逆性盲的首要病因。中国由于人口老龄化日趋严重,AMD也已成为重要的致盲性眼病。由于发病机制不清,一直是眼科临床工作的重点及难点。AMD根据临床病理特征分为干性(萎缩性)和湿性(nAMD)两类,而SRF(视网膜下纤维化,又称为黄斑盘状变性)被认为是湿性AMD的晚期阶段,是造成不可逆性视力受损的主要原因及nAMD抗VEGF(抗血管内皮生长因子)治疗的重要瓶颈。因此,迫切需要研究SRF发病机制,以期阻止nAMD进展以指导有效治疗。
目前已有运用大小鼠,兔,猪和猴等物种建立了能够模拟AMD部分重要的病理特征,但是仍未有任何动物模型可以重现人nAMD的所有表型。Ambati等于2003年报道了兼具类似干性AMD及湿性AMD(CNV)病理改变的基因敲除动物模型,即Ccl-2-/-和Ccr-2-/-小鼠模型,然而作为一种免疫缺陷动物,此类小鼠的存活率以及成模率不甚理想。也有多项研究利用激光诱导CNV啮齿动物模型来研究AMD晚期SRF的发病机制,但是首先,激光诱导的CNV持续时间较短(不超过1个月)并能自行恢复,不足与进行长期的药效学观察尤其是进行SRF的观察及研究;其次,不是所有激光诱导的CNV病灶都能够出现SRF;再者,解剖结构和人类相比,啮齿动物由于寿命短及眼球解剖差异大(眼球小,无黄斑区)等缺点,也不能真实反应AMD病人CNV和SRF特点。
由于nAMD病因复杂、病程长及临床病变多样,目前还没有任何动物模型可以重现人nAMD所有表型,尤其是早期和晚期nAMD病变都能够模拟的体内动物模型,并且纤维化的动物模型耗时均较长,这些都给建立该疾病动物模型带来了挑战。
发明内容
为解决上述问题,本发明提供了一种视网膜内和/或视网膜下纤维化动物模型的构建方法,它是向非人灵长类动物的眼部给予重组腺病毒AAV-VEGF得到动物模型。
进一步地,它是向非人灵长类动物眼部巩膜位置给予重组腺病毒AAV-VEGF得到动物模型。
进一步地,所述非人灵长类动物为新大陆猴或旧大陆猴,优选旧大陆猴。
更进一步地,所述旧大陆猴为猕猴,所述猕猴为恒河猴、熊猴、短尾猴、台湾岩猴或平顶猴,优选恒河猴。
进一步地,所述重组腺病毒AAV-VEGF的剂量为1.6*107~1.6*109μg/眼。
本发明还提供了一种视网膜内和/或视网膜下纤维化动物模型,它是眼部被给予重组腺病毒AAV-VEGF的非人灵长类动物。
进一步地,它是眼部巩膜下的脉络膜上腔位置被给予重组腺病毒AAV-VEGF的非人灵长类动物。
进一步地,所述非人灵长类动物是旧大陆猴;所述旧大陆猴为猕猴。
进一步地,所述猕猴为恒河猴、熊猴、短尾猴、台湾岩猴或平顶猴,优选恒河猴。
本发明还提供了一种前述方法构建的动物模型在筛选治疗视网膜内和/或视网膜下纤维化的药物中的应用。
本发明还提供了一种前述方法构建的动物模型在研究视网膜内和/或视网膜下纤维化病理机制中的应用。
本发明最后提供了一种重组腺病毒AAV-VEGF在构建视网膜内和/或视网膜下纤维化动物模型中的应用。
本发明重组腺病毒AAV-VEGF,是指携带VEGF基因的重组腺病毒。VEGF,是血管内皮生长因子(vascular endothelial growth factor,VEGF)。
本发明一种视网膜内和/或视网膜下纤维化动物模型的构建方法,选择非人灵长类恒河猴,通过巩膜外路,向其眼部给予AAV-VEGF,得到的动物模型可以同时模拟nAMD的长期CNV和SRF,并且在短时间内就能得到能够长期模拟nAMD的晚期表型SRF,进一步地,由于是通过巩膜外路给药,不会对眼球和视网膜造成损伤,操作简单,易于工业化推广应用。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1给予重组腺病毒AAV-VEGF 2月后眼底炫彩照相(Multicolor)及荧光造影检查
图2给予重组腺病毒AAV-VEGF 18月后眼底炫彩照相(Multicolor)及荧光造影检查
图3给予重组腺病毒AAV-VEGF 2月后眼底OCT检查
图4给予重组腺病毒AAV-VEGF 18月后眼底OCT检查
图5给予重组腺病毒AAV-VEGF 2月后组织病理学检测HE染色
图6给予重组腺病毒AAV-VEGF 2月后组织病理学检测Masson染色
具体实施方式
实施例1、本发明模型的构建
取恒河猴,在其巩膜下的脉络膜上腔位置给予1.6*108vg/眼重组腺病毒AAV-VEGF,给药后抗感染护理,即得。
实施例2、本发明模型的构建
取恒河猴,在其巩膜下的脉络膜上腔位置给予1.6*109vg/眼重组腺病毒AAV-VEGF,给药后抗感染护理,即得。
以下通过试验例来说明本发明的有益效果。
试验例1
1实验对象
1)对象:4-6岁年龄,体重均值范围5.0~10.0kg,普通级成年恒河猴(动物由四川大学华西医院提供,雌雄不限);
饲养条件:双层不锈钢猴笼(L×W×H:800mm×900mm×2080mm),2笼/架,1只/笼;饲料(猴维持饲料,京科澳协力饲料有限公司提供)、饮用水,自由摄取;
饲养环境条件:室温16~26℃(日温差≤4℃),相对湿度40~70%,人工照明,12/12小时昼夜明暗交替。
2)试验分组设计:4眼/组(每组2只,每只注射2眼)
恒河猴随机平均分为AAV空载对照组2只、溶媒对照组2只、低剂量模型组2只,高剂量模型组2只,每只给药2眼相应剂量试剂,具体如下表1:
表1试验设计
注:眼局部巩膜外路即在避免眼球损伤的情况下,用注射器轻扎巩膜,给予巩膜下方的脉络膜上腔重组腺病毒AAV-VEGF。
2、主要试剂与耗材
1)重组腺相关病毒(AAV-VEGF)(结构示意图rAAV-CMV-VEGFA-hGH pA,货号:2-1499-K190307),由武汉枢密脑科学技术有限公司提供
2)试验所需主要试剂、仪器、器械:
主要试剂列表:
表2试剂
主要仪器
表3仪器
试验所用主要计算机软件或数据系统
表4计算机系统
3、造模设计
动物麻醉后,根据表1分组,动物眼通过巩膜外路眼局部给予AAV+VEGF或空载AAV对照或溶媒对照,给药后给与抗感染护理(即:注射眼给予左氧氟沙星眼膏眼内涂,并每天电筒光照或手持裂隙灯观察眼情况,确保无异常眼分泌物及眼部感染)。造模后每周做1次眼底检查(包括眼底照相、荧光素血管造影FFA和OCT),连续4周,以出现晚期荧光素渗漏作为造模成功与否判断依据。以后每月一次检查连续半年,之后每2月一次检查。建模当天定义为试验第1天。
4、眼科检查
检测时间:第1月:1次/周;1次/月,连续5月;之后1次/2月,连续18月;
检查动物:各组动物;
检查内容:眼底炫彩照相、荧光造影检查FFA、光学相干断层扫描(OCT)
5、眼球组织病理学检查
解剖时间:建模后3个月;
每组各摘除1只眼球,沿角巩膜缘穿刺一小口,置于改良的Davidson′s固定液中4度过夜固定用于石蜡切片制备。病理染色:将各组切片进行苏木精-伊红染色及Masson三色染色并行显微镜检查和分析。
6、结果
给药2月后眼底炫彩照相(Multicolor)结果及荧光造影检查FFA结果见图1,由图1可见AAV-VEGF高低剂量组均有墨绿色的视网膜下隆起(蓝色箭头),提示有血管和纤维组织增生;荧光造影(FFA)显示ND对照组和AAV-ND对照组无明显渗漏改变,AAV-VEGF高和低剂量组血管迂曲,新生血管形成,造影晚期高剂量组的荧光渗漏面积较大。
给药18月后眼底炫彩照相(Multicolor)及荧光造影检查FFA结果见图2,从图2可见AAV-VEGF高低剂量组的墨绿色视网膜下隆起区域的面积较之前均有扩大,高剂量组最明显(蓝色箭头),黄色箭头提示纤维组织增生累及周围视网膜区域;荧光造影(FFA)示ND对照组和AAV-ND对照组仍无明显渗漏改变,AAV-VEGF高低剂量组血管严重迂曲,新生血管簇较之前明显增多,造影晚期高剂量组的荧光渗漏面积明显增大(蓝色箭头示)。
给药2月后眼底OCT检查结果见图3,从图3可见ND对照组和AAV-ND对照组的注射点有视网膜凹陷区,AAV-VEGF高低剂量组均出现视网膜隆起,并有视网膜下高反射物质(右蓝色箭头),同时有视网膜下渗漏脱离区域(左蓝色箭头)。
给药18月后眼底OCT检查结果见图4,从图4可见ND对照组和AAV-ND对照组的注射点视网膜凹陷,较前无明显变化,而AAV-VEGF高低剂量组的视网膜下高反射物质增多(蓝色箭头),视网膜水肿明显,尤其高剂量组显示视网膜内层劈裂(黄色箭头),可能为视网膜新生血管渗漏导致。
给药2月后组织病理学检测HE染色结果见图5,从图5可见显示与对照组(ND、AAV-ND)比较,AAV-VEGF高低剂量出现视网膜水肿,层次紊乱,视网膜下和视网膜内血管增生(黄色箭头示),尤其高剂量组出现视网膜内层劈裂(红色星号示),外核层结构消失。
给药2月后组织病理学检测Masson染色结果见图6,从图6可见显示与对照组(ND、AAV-ND)比较,AAV-VEGF高低剂量组除了视网膜下和视网膜内血管增殖外(红色箭头示),还出现纤维组织明显增生(黄色箭头示蓝染的胶原纤维),即视网膜下纤维化和视网膜内纤维化(红色星号示)形成。
本发明通过巩膜外路眼局部给予AAV-VEGF的方法,2-3月后能够诱导恒河猴眼出现CNV,并有明显的视网膜下纤维化形成:FFA示视网膜新生血管形成,眼底炫彩照相和OCT提示有新生血管和纤维组织增生,经病理检测即HE和Masson染色证实CNV与视网膜下和视网膜内的纤维化并存。随访观察18月,眼底影像学显示病变持续存在,并且视网膜下CNV和视网膜内新生血管在不断增多的同时,视网膜下和视网膜内纤维化也在增生,范围扩大。因此说明通过巩膜外路眼局部给予AAV-VEGF可在短期内得到nAMD早期(CNV)和晚期(SRF)病变表型的体内动物疾病模型,并且恒河猴可最大程度模拟人的疾病表型,可重复性良好。
综上,本发明通过巩膜外路眼局部给予AAV-VEGF,可在短期内(2-3月)就出现除CNV之外的视网膜下纤维化(SRF)病变,均符合早期和晚期nAMD的表型特征,并且病变可长期(大于18月)存在,因此本发明建立的长期CNV和SRF动物疾病模型能为AMD疾病尤其是晚期视网膜下纤维化的药效学和病理机制研究提供一个强有力的工具,应用前景广泛。
Claims (10)
1.一种视网膜内和/或视网膜下纤维化动物模型的构建方法,其特征在于:
它是向非人灵长类动物的眼部给予重组腺病毒AAV-VEGF得到动物模型。
2.根据权利要求1所述的构建方法,其特征在于:它是向非人灵长类动物眼部巩膜下的脉络膜上腔位置给予重组腺病毒AAV-VEGF得到动物模型;
所述非人灵长类动物为新大陆猴或旧大陆猴,优选旧大陆猴。
3.根据权利要求2所述的构建方法,其特征在于:所述旧大陆猴为猕猴,所述猕猴为恒河猴、熊猴、短尾猴、台湾岩猴或平顶猴,优选恒河猴。
4.根据权利要求1所述的构建方法,其特征在于:所述重组腺病毒AAV-VEGF的剂量为1.6*107~1.6*109μg/眼。
5.一种视网膜内和/或视网膜下纤维化动物模型,其特征在于:它是眼部被给予重组腺病毒AAV-VEGF的非人灵长类动物。
6.根据权利要求5所述的动物模型,其特征在于:它是眼部巩膜下的脉络膜上腔被给予重组腺病毒AAV-VEGF的非人灵长类动物;所述非人灵长类动物是旧大陆猴;所述旧大陆猴为猕猴。
7.根据权利要求6所述的动物模型,其特征在于:所述猕猴为恒河猴、熊猴、短尾猴、台湾岩猴或平顶猴,优选恒河猴。
8.权利要求1~4任意一项所述方法构建的动物模型在筛选治疗视网膜内和/或视网膜下纤维化的药物中的应用。
9.权利要求1~4任意一项所述方法构建的动物模型在研究视网膜内和/或视网膜下纤维化病理机制中的应用。
10.重组腺病毒AAV-VEGF在构建视网膜内和/或视网膜下纤维化动物模型中的应用。
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