CN111990339A - 高糖饮食诱导的早期干性年龄相关性黄斑变性色素兔模型 - Google Patents
高糖饮食诱导的早期干性年龄相关性黄斑变性色素兔模型 Download PDFInfo
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Abstract
本发明公开了一种高糖饮食诱导的早期干性年龄相关性黄斑变性色素兔模型的构建方法,属于眼科疾病动物模型领域。本发明的方法包括:在青紫蓝兔维持饲料中添加重量比40%的蔗糖,持续饲喂青紫蓝兔24周。本发明的方法仅需饮食控制,即可使动物产生年龄相关性黄斑变性的症状,简单便捷,易于产业化应用。
Description
技术领域
本发明属于眼科疾病动物模型领域。
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根据发病阶段分为早期AMD和晚期AMD,前者以早期的玻璃膜疣、视网膜色素上皮层(RPE)和光感受器细胞改变,即干性(萎缩性)AMD为特征,也会进展为脉络膜新生血管(CNV),即湿性(渗出性)AMD;晚期AMD以累及黄斑中心的地图样萎缩为特点。临床上约90%的患者为干性AMD。虽然目前对于湿性AMD的诊断和治疗研究(如抗VEGF治疗)有了很大的进展,但是对于早期干性AMD和晚期地图样萎缩,迄今仍无有效的治疗和预防措施。此外,由于医学伦理的限制,多数对AMD在视网膜脉络膜结构、功能及生化方面的研究不能直接以人作为研究对象,临床标本来源困难,干性AMD成为AMD研究的瓶颈,因此需要选择合适的动物疾病模型来指导其研究,通过模拟人早期干性AMD的结构、功能及代谢特征来探索其发病机制和开发新型治疗以期阻止或者延缓干性AMD进程。
目前的早期干性AMD动物模型主要有基因敲除模型、药物诱导模型、光损伤视网膜模型等。
转基因/基因编辑模型主要针对于AMD相关基因进行基因敲除(例如:单核细胞趋化因子2、载脂蛋白E),但其周期长,成模率低,对人员设备要求高,不适合推广应用。
药物诱导模型是通过眼注射碘酸钠、聚乙二醇等药物,对人员技术要求高,且容易导致视网膜发生不可逆的急性损坏,不适合AMD模型研究。
光损伤模型是用蓝光照射动物,光线入眼后可诱发视紫红质、脂褐素等感光分子产生活性氧,导致感光细胞外节盘膜的脂质过氧化,诱导感光细胞凋亡,对视网膜造成损伤。该方法需要精准可控的蓝光发生装置,对设备有较高要求。
上述主流方法存在周期长、对人员设备要求高或对动物伤害过重等问题,不利于产业化应用。
在动物日常饮食中额外添加脂肪和糖(即“高脂高糖饮食”),曾被认为是一种简单可行的早期干性AMD造模方法。发明人前期用8-13岁雄性老年恒河猴作为对象,发现喂养18-24月后,均未见脉络膜及视网膜色素上皮的显著改变,即无法成功造模。CN107787912A公开了一种干性AMD造模方法,是将蔗糖、猪油、胆固醇、胆酸盐按质量比5~15:5~15:0.5~1.5:0.1~1.0的比例进行混合,制备成半流质膳食,通过鼻饲管每天灌注一次(该配方必须人工灌注,否则猴子在摄入一定量饲料后就不再摄入),每天灌注9.2~17.2g/kg体重,持续15个月。其周期长,人工灌注操作繁琐,不适用于产业化应用。
发明内容
本发明要解决的问题是:提供一种简单有效的早期干性AMD动物模型的构建方法。
本发明提供了如下技术方案:
本发明首先提供了一种早期干性黄斑变性色素兔模型的构建方法,它包括:
每日使色素兔摄入36~67.5g蔗糖,持续16~30周。
进一步的,所述构建方法包括:
每日使色素兔摄入40~60g蔗糖,持续16~30周。
进一步的,所述蔗糖是按重量比36%~45%添加到兔维持饲料中,制成高糖饲料,对色素兔进行饲喂,每日饲喂高糖饲料100~150g;
优选地,所述蔗糖按重量比40%添加到兔维持饲料中。
进一步的,所述兔维持饲料含有:
20~27%(w/w)蛋白质、8~14%(w/w)脂肪和60~70%(w/w)碳水化合物;
优选地,所述兔维持饲料含有:
23.5%(w/w)蛋白质、11%(w/w)脂肪和65.5%(w/w)碳水化合物。
进一步的,所述色素兔为青紫蓝兔;和/或,所述模型是早期干性年龄相关性黄斑变性。
进一步的,所述饲喂的具体方式为:对每只兔上午饲喂40-60g,下午饲喂60-90g。
本发明还提供了前述方法得到的模型在药物筛选中的用途,所述药物为治疗早期干性年龄相关性黄斑变性的药物。
本发明还提供了一种用于构建早期干性年龄相关性黄斑变性色素兔模型的兔饲料,
所述兔饲料是在兔维持饲料中添加重量比36~40%的蔗糖制备而成;
所述兔维持饲料含有:
20~27%(w/w)蛋白质、8~14%(w/w)脂肪和60~70%(w/w)碳水化合物。
进一步的,所述兔饲料含有重量比40%的蔗糖。
进一步的,述兔维持饲料含有:
23.5%(w/w)蛋白质、11%(w/w)脂肪和65.5%(w/w)碳水化合物。
术语“色素兔”指,含有色素眼的兔品种(即非白化兔)。
本发明的有益效果:
本发明以色素兔青紫蓝兔作为对象,饲喂高糖饮食,仅需24周,即可成功使青紫蓝兔表现出早期干性AMD症状。该方法克服了基因敲除模型、药物诱导模型、光损伤视网膜模型等方法的周期长、对人员设备要求高或对动物伤害过重等问题,且无需人工灌注干预进食,十分快捷便利。
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:血糖水平;左,高糖组(HSD)和对照组之间血糖水平比较;右,高糖组(HSD)和对照组之间IVGTT的AUC值比较。
图2:血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)和高密度脂蛋白(HDL)水平比较。
图3:眼底炫彩照相(Multicolor)、光学相干断层扫描(OCT)和自发荧光检测图;标尺=500μm。
图4:荧光素血管造影(FFA)检测图;标尺=500μm。
图5:HE染色、甲苯胺蓝染色结果;标尺=100μm
图6:免疫荧光染色和血管覆盖面积统计;标尺=100μm
图7:视网膜电图(ERG)检测结果。
具体实施方式
实施例1模型构建
1.方法
1.1建模
将16周龄体重3.5±0.5kg的成年雄性青紫蓝兔分成两组,各10只,对照组(ND)饲喂兔维持饲料(北京科澳协力饲料有限公司),高糖组(HSD)则饲喂高糖饲料,每天饲喂的量为每天饲喂的量为上午40-60g,下午60-90g。
所述维持饲料的营养成分:23.5%(w/w)蛋白质,11%(w/w)脂肪以及65.5%(w/w)碳水化合物;能量:2.7kcal/g。
所述高糖饲料是在维持饲料的基础上,按重量比加入40%的蔗糖。
1.2检测
1)血液检查
检测时间:建模前及建模后8、12、16、24周;
检测指标:血糖(GLU)、总胆固醇(CHOL)、甘油三酯(TG)、游离脂肪酸(NFFA)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)
2)葡萄糖耐量试验(OGTT)
测定时间:建模后24周;
测定方法:采样检测前禁食至少12小时,不禁水。每只兔经腹腔注射1g/kg剂量葡萄糖(给药体积:2mL/kg,给药浓度:500mg/mL),注射后0、0.5、1.5、2小时耳缘静脉穿刺取血10微升,罗氏血糖仪(卓越型)虹吸芯片吸取检测。
3)眼科检查
检测时间:建模前及建模后24周;
检测项目:眼底炫彩照相(multicolor),荧光素血管造影(FFA),眼底自发荧光,光学相干断层扫描(SD-OCT)。
4)眼球组织病理学检查
检测时间:第24周。
摘除各组兔眼球,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(HE染色)、甲苯胺蓝染色及免疫荧光染色(针对Brn3和IB4蛋白),并行显微镜检查和计数分析。
5)检测时间:第24周。
将动物分别于暗适应30min后用国际临床视觉电生理会标准参数刺激,常规程序记录暗适应ERG(包括Scotopic 0.01ERG、Scotopic 3.0ERG、Scotopic3.0OPS),之后将动物进行明适应10min后记录明适应ERG(Photopic 3.0flicker)。
2.结果
2.1血液检查结果
高糖组血糖值在第2月开始观察到明显高于对照组,其第6月IVGTT的AUC值也明显高于对照组(图1)。
总体上,高糖组血清总胆固醇、甘油三酯、低密度脂蛋白从喂养高糖饲料60天后,有显著升高,高密度脂蛋白无显著改变(图2)。
上述结果表明,本发明的饲喂方法可提高青紫蓝兔的血脂血糖。
2.2眼科检查结果
眼底炫彩照相显示,高糖组视乳头周围神经纤维束明显减少(图3,Multicolor);光学相干断层扫描(OCT)显示,RPE(视网膜色素上皮)层不连续,RPE层下部有指状凸起(图3,OCT栏箭头所指);自发荧光显示,RPE下有强荧光,为脂样物质沉积(图3,自发荧光栏箭头所指),可见产生玻璃膜疣(Drusen)样沉积物。
荧光素血管造影(FFA)结果显示,高糖组后视乳头早期周边血管荧光增强,晚期血管末端有新生血管样强荧光渗漏(图4箭头所示)。
上述结果中,RPE指状凸起和RPE下脂样物质沉积与干性AMD的RPE改变症状相吻合,RPE形态不规则,不连续与早期干性AMD的RPE受损一致;神经纤维束减少可能与老龄化诱导的AMD和视网膜神经纤维损失有关;此外,乳头周边血管渗漏可能与糖代谢脂代谢异常引起视网膜内外屏障破坏的血管增生反应有关,也对湿性AMD特征的模拟有一定参考意义。
2.3眼球组织病理学检查
HE染色显示高糖组RPE不连续,其下有脂样物质沉淀(红色箭头),光感受器外节空泡样变(黄色箭头);甲苯胺蓝染色显示高糖组RPE下有脂样物质沉淀(红色箭头),并且光感受器外节出现畸形和变性(图5)。
免疫荧光染色显示,高糖组RPE不连续,Rhodopsin阳性的视杆细胞外节形态不规则,间隙增宽内有空泡,IB4染色的视乳头周边血管簇面积变大。
该部分病理结果表明高糖组RPE细胞和光感受器(视杆细胞)病变均与早期干性AMD的RPE和光感受器细胞受损一致。
2.4ERG
ERG结果显示,高糖组的暗适应b波振幅较对照组明显降低,暗适应、明适应b波潜伏期也较对照延长;闪光VEP振幅比对照组明显降低。
该结果表明:高糖组视功能尤其是RPE和视网膜外层功能明显下降,符合早期干性AMD症状。
本发明的模型还可选择其他含有色素眼的兔品种(即非白化兔)来替代青紫蓝兔。
综上,本发明以青紫蓝兔作为对象,饲喂高糖饮食,仅需24周,即可成功使青紫蓝兔表现出早期干性AMD症状。该方法克服了基因敲除模型、药物诱导模型、光损伤视网膜模型等方法的周期长、对人员设备要求高或对动物伤害过重等问题,且无需人工灌注干预进食,十分快捷便利。
Claims (10)
1.一种早期干性黄斑变性色素兔模型的构建方法,其特征在于,它包括:
每日使色素兔摄入36~67.5g蔗糖,持续16~30周。
2.如权利要求1所述的构建方法,其特征在于:它包括:
每日使色素兔摄入40~60g蔗糖,持续16~30周。
3.如权利要求1所述的构建方法,其特征在于:所述蔗糖是按重量比36%~45%添加到兔维持饲料中,制成高糖饲料,对色素兔进行饲喂,每日饲喂高糖饲料100~150g;
优选地,所述蔗糖按重量比40%添加到兔维持饲料中;
进一步优选地,饲喂时间为24周。
4.如权利要求3所述的方法,其特征在于:
所述兔维持饲料含有:
20~27%(w/w)蛋白质、8~14%(w/w)脂肪和60~70%(w/w)碳水化合物;
优选地,所述兔维持饲料含有:
23.5%(w/w)蛋白质、11%(w/w)脂肪和65.5%(w/w)碳水化合物。
5.如权利要求1所述的方法,其特征在于:所述色素兔为青紫蓝兔;和/或,所述模型是早期干性年龄相关性黄斑变性。
6.如权利要求1~5任一所述的方法,其特征在于:所述饲喂的具体方式为:对每只兔上午饲喂40-60g,下午饲喂60-90g。
7.权利要求1~6任一所述方法得到的模型在药物筛选中的用途,所述药物为治疗早期干性年龄相关性黄斑变性的药物。
8.一种用于构建早期干性年龄相关性黄斑变性色素兔模型的兔饲料,其特征在于:
所述兔饲料是在兔维持饲料中添加重量比36~40%的蔗糖制备而成;
所述兔维持饲料含有:
20~27%(w/w)蛋白质、8~14%(w/w)脂肪和60~70%(w/w)碳水化合物。
9.如权利要求8所述的兔饲料,其特征在于:所述兔饲料含有重量比40%的蔗糖。
10.如权利要求8所述的兔饲料,其特征在于:述兔维持饲料含有:
23.5%(w/w)蛋白质、11%(w/w)脂肪和65.5%(w/w)碳水化合物。
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