CN109430156A - 胰岛素抵抗动物模型的建立方法 - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
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- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/158—Fatty acids; Fats; Products containing oils or fats
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/168—Steroids
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/30—Oligoelements
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/50—Feeding-stuffs specially adapted for particular animals for rodents
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
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- Food Science & Technology (AREA)
- Environmental Sciences (AREA)
- Birds (AREA)
- Inorganic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Fodder In General (AREA)
Abstract
本发明公开了胰岛素抵抗动物模型的建立方法,使用高硒高糖高脂饲料和低浓度果糖饮用水诱导遗传背景高度相似的近交系C57BL/6小鼠,造成其糖脂代谢紊乱,进而诱导胰岛素抵抗的发生,建立胰岛素抵抗动物模型。通过对饲料的独特配比,高度模拟由于饮食结构发生改变而导致的胰岛素抵抗的发病过程,所诱导的动物模型与对照组比,模型组动物口服糖耐量、胰岛素耐量、动物胰岛素抵抗指数及胰岛素敏感指数与正常对照组动物有显著差异,表明模型组动物已经发生明显的胰岛素抵抗。有益效果为:本发明采用独特饲料,两种方式饲喂,排除了化学试剂产生的不良反应,具有造模方法简单、成模率高、成本低以及重现性好的优势。
Description
技术领域
本发明涉及动物模型构建技术领域,尤其是涉及胰岛素抵抗动物模型的建立方法。
背景技术
动物模型的构建是医学研究中的一项很重要的技术。构建与人类发病相似的疾病模型,可以克服人类本身发生疾病潜伏期长、影响因素多、发生发展缓慢、发病机制复杂等因素的制约,更好地反应疾病发生、发展的规律,并有利于研究不便于在人类本身进行研究的疾病。常用的动物模型主要包括自发性动物模型、转基因动物模型和实验性动物模型。自发性动物模型、转基因动物模型应用价值较高,但其造模成本高、技术饲养条件要求高等特点限制了这些动物模型的普遍应用。实验性动物模型操作简单、成本低、成模率较高,目前,这类动物模型被广泛使用。
近年来,糖尿病患病率快速、大幅上升,成为全球范围内的一个重大公共卫生问题。2型糖尿病患者占糖尿病总人数的90%以上,胰岛素抵抗是2型糖尿病的基本病理生理机制。胰岛素抵抗是指肝、肌肉、脂肪等胰岛素作用的靶组织,不能响应胰岛素导致胰岛素的功能减弱甚至缺失的病理状态。除2型糖尿病之外,胰岛素抵抗还与高血压、动脉粥样硬化、代谢综合征的发生发展密切相关。因此,对胰岛素抵抗的发病机制及防治研究显得尤为重要。目前,胰岛素抵抗的发病机制还不明确。
现有技术如授权公告号为CN 103211834 B的中国发明专利,公开了一种造模方法简单、成本低、周期短的高硒诱导胰岛素抵抗动物模型的应用及构建方法,利用SD大鼠,每天灌胃100μg/kg-200μg/kg的硒,6周后得到胰岛素抵抗动物模型。对糖耐量和胰岛素耐量的统计分析、胰岛素信号传导和糖异生关键酶的测定表明:利用硒酸钠构建的胰岛素抵抗大鼠模型是可行的,可作为高硒诱导胰岛素抵抗以及2型糖尿病的发病机制和药物作用机理的研究模型。但是该高硒诱导胰岛素抵抗动物模型是由单一高硒因素诱导所致,造出的动物模型稳定性不强,所用单一高硒因素也易导致结果差别过大,致使造模的致病程度存在一定差异。
发明内容
本发明的目的在于提供一种利用C57BL/6小鼠建立经高糖高脂高硒饲料和果糖溶液诱导的,造模方法简单、成模率高、重现性好的胰岛素抵抗动物模型的建立方法。
本发明针对上述技术中提到的问题,采取的技术方案为:
胰岛素抵抗动物模型的建立方法,包括选取鼠种、分组与造模、采血与检测,通过同时饲喂高糖高脂高硒饲料与饮用果糖溶液造成糖脂代谢紊乱,进而诱导雄性小鼠产生胰岛素抵抗,得到胰岛素抵抗动物模型,具体步骤为:
选取鼠种:SPF级雄性C57BL/6小鼠,4-6周龄,体重22±2g,饲养于本部屏障环境内,温度22-25℃,湿度45-55%,12小时昼夜交替,适应性喂养1周;C57BL/6小鼠为近交系小鼠,遗传背景高度相似,在一定程度上保证了模型的稳定性;
分组与造模:适应性喂养1周后,在分组上模型组与对照组均采取正态分布分组方法,即以小鼠体重为依据,将小鼠划分为高、中、低三个体重区间,将每个区间随机分配到模型组和对照组中,模型组和对照组,每组15只,模型组喂食高糖高脂高硒饲料、饮用果糖溶液,对照组喂食标准饲料、饮用蒸馏水,在饲养方法上采取单只单笼饲养措施,饲料定量定投,早晚各一次,维持25g/笼,保证较小的组内差异,2-3天换垫料,给笼具消毒,垫料为刨花木屑,里面含有小木块,要求高压灭菌,造模10周,每周检测体重和随机血糖值;
采血与检测步骤为:连续饲喂10周,第10周结束后,按2g/kg体重灌服40%D-葡萄糖溶液进行口服糖耐量试验;禁食8小时后眼球取血留血清,检测空腹血清胰岛素水平,五项血脂指标,分别为胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白-胆固醇(LDL)、高密度脂蛋白-胆固醇(HDL)及游离脂肪酸(FFA);计算胰岛素抵抗指数及胰岛素敏感指数,得到胰岛素抵抗模型。
作为优选,分组与造模步骤中,饲喂的高脂高糖高硒饲料包含硒酸钠、山梨酸钾。
进一步优选,分组与造模步骤中,饲喂的高脂高糖高硒饲料由以下组分按重量百分比构成,基础饲料40-60%,蔗糖15-25%,猪油15-25%,胆固醇8-15%,硒酸钠0.5-2.5%,猪胆盐0.05-0.15%,山梨酸钾0.05-0.15%;所述的果糖溶液浓度为10-30%;猪油提供脂肪,加速生物体脂肪堆积,导致体重、血液中甘油三酯和总胆固醇浓度的增加,同时导致糖耐量受损,血糖异常,诱发胰岛素抵抗;高浓度蔗糖导致血液中血糖升高,刺激胰腺长期分泌大量胰岛素,最终致使胰腺受损、碳水化合物和脂肪代谢紊乱,诱发胰岛素抵抗;果糖摄入过多时,会刺激脂质生成,导致甘油三酯堆积,并介导氧化应激反应,影响胰岛素与其受体结合、影响葡萄糖转运并干预游离脂肪酸代谢,进而引起胰岛素敏感性下降、肝胰岛素抵抗以及肥胖;硒酸钠保证高硒摄入,使蛋白激酶B磷酸化水平降低,磷酸烯醇式丙酮酸羧基酶和葡萄糖6磷酸脱氢酶水平升高,造成血糖水平升高,糖脂代谢紊乱,进而诱导胰岛素抵抗。
更进一步优选,分组与造模步骤中,饲喂的高糖高脂高硒饲料由以下组分按重量百分比构成,基础饲料50%,蔗糖18%,猪油18%,胆固醇12%,硒酸钠1.5%,猪胆盐0.1%,山梨酸钾0.1%;所述的果糖溶液浓度为20%。
与现有技术相比,本发明的优点在于:本发明使用高糖高脂高硒饲料和低浓度果糖饮用水诱导C57BL/6小鼠的糖脂代谢紊乱,进而诱导胰岛素抵抗的发生,建立胰岛素抵抗动物模型。应用本方法建立的胰岛素抵抗动物模型高度模拟由于饮食结构发生改变而导致的胰岛素抵抗的发病过程,具有与人类高糖高脂高硒胰岛素抵抗相似的特征表现,结果可信度高,并且排除了化学试剂产生的不良反应,避免了单纯食物饲喂时模型不稳定,成模率低的问题,对于探究胰岛素抵抗和2型糖尿病发病机理及其应对方法有着重要的意义。同时C57BL/6小鼠为近交系小鼠,遗传背景高度相似,在一定程度上保证了模型的稳定性。
进一步的,通过对饲料的独特配比,模拟人类饮食的条件;其中以猪油和蔗糖摄入实现对小鼠的血糖值升高的影响;以猪油的摄入影响脂肪堆积和胰岛素分泌;以蔗糖摄入影响糖原合成;以硒酸钠摄入影响胰岛素分泌;通过饮水中添加的果糖来增加甘油三脂的浓度和升高血压,三者的相互配合,实现对模型的构建。
本发明中胰岛素抵抗小鼠模型的评价指标包括体重变化指标,口服糖耐量检测,胰岛素耐量检测,血脂四项和游离脂肪酸检测,空腹血清胰岛素水平检测以及胰岛素抵抗指数及胰岛素敏感指数的计算等,所诱导的动物模型与对照组比,模型组动物口服糖耐量、胰岛素耐量、动物胰岛素抵抗指数及胰岛素敏感指数与正常对照组动物有显著差异,表明模型组动物已经发生明显的胰岛素抵抗。
本发明具有造模方法简单、成模率高、造模周期短、成本低以及重现性好的优势。
附图说明
图1为本发明实施例1糖耐量测试中血糖水平的检测结果图。
具体实施方式
下面通过实施例对本发明方案作进一步说明:
实施例1:
胰岛素抵抗动物模型的建立方法,包括选取鼠种、分组与造模、采血与检测,通过同时饲喂高糖高脂高硒饲料与饮用果糖溶液造成糖脂代谢紊乱,进而诱导雄性小鼠产生胰岛素抵抗,得到胰岛素抵抗动物模型。本发明中高糖高脂高硒饲料由常规实验动物饲料添加猪油、蔗糖、胆固醇、硒酸钠、山梨酸钾和猪胆盐配制而成;果糖溶液采用分析纯加蒸馏水配制。具体构建过程及检测结果如下:
1.实验试剂和仪器
小鼠胰岛素酶联免疫分析试剂盒,北京科盈美科技有限公司,批号:17072401。胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白-胆固醇(LDL)、高密度脂蛋白-胆固醇(HDL)试剂盒由长春汇力生物技术有限公司提供,生产批号分别是:20170219、20170223、20170224、20170232。游离脂肪酸(FFA)测试盒,南京建成生物工程研究所,生产批号:20170723。GLM-77博仕珑血糖分析仪,博仕珑血糖试纸片,厚美德生物科技股份有限公司(台北)。一次性无菌采血针,北京瑞成医疗器械有限公司。全自动生化分析仪器等常见实验室设备。
2.实验步骤
选取鼠种:SPF级雄性C57BL/6小鼠,4-6周龄,体重22±2g,饲养于本部屏障环境内,温度22-25℃,湿度45-55%,12小时昼夜交替,适应性喂养1周;
分组与造模:适应性喂养1周后,在分组上模型组与对照组均采取正态分布分组方法,即以小鼠体重为依据,将小鼠划分为高、中、低三个体重区间,将每个区间随机分配到模型组和对照组中,模型组和对照组,每组15只,模型组喂食高糖高脂高硒饲料、饮用果糖溶液,对照组喂食标准饲料、饮用蒸馏水,在饲养方法上采取单只单笼饲养措施,饲料定量定投,早晚各一次,维持25g/笼,保证较小的组内差异,2-3天换垫料,给笼具消毒,垫料为刨花木屑,里面含有小木块,要求高压灭菌,造模10周,每周检测体重和随机血糖值;
模型组小鼠的高糖高脂高硒饲料配方:按重量份数比,基础饲料40-60%,蔗糖15-25%,猪油15-25%,胆固醇8-15%,硒酸钠0.5-2.5%,猪胆盐0.05-0.15%,山梨酸钾0.05-0.15%;所述的果糖溶液浓度为10-30%;在取值范围中,就可以进行配比。本实施例中优选的采用,基础饲料50%,蔗糖18%,猪油18%,胆固醇12%,硒酸钠1.5%,猪胆盐0.1%,山梨酸钾0.1%;所述的果糖溶液浓度为20%;
先将猪油放置锅中熔化,然后加入蔗糖直至与猪油混匀,然后将称好的胆固醇和猪胆盐倒入混和的猪油和蔗糖中,然后将普通饲料倒入锅中,炒拌均匀,冷却至40-50摄氏度,加入硒酸钠和山梨酸钾,炒拌均匀,放置瓷盘中晾干,即得模型组小鼠的高糖高脂高硒饲料;模型组小鼠的果糖溶液浓度20%,即将20g果糖溶解在100ml蒸馏水中;
采血与检测步骤为:连续饲喂10周,第10周结束后,按2g/kg体重灌服40%D-葡萄糖溶液进行口服糖耐量试验;禁食8小时后眼球取血留血清,检测空腹血清胰岛素水平,五项血脂指标,分别为胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白-胆固醇(LDL)、高密度脂蛋白-胆固醇(HDL)及游离脂肪酸(FFA);计算胰岛素抵抗指数及胰岛素敏感指数,得到胰岛素抵抗模型;
血糖值:采用博士珑公司提供的血糖仪和试纸。第10周结束后,进行OGTT试验。实验前动物禁食12h,然后按2g/kg体重比灌服40%D-葡萄糖溶液进行口服糖耐量试验,测定灌胃前空腹血糖值(FBG)及灌胃后30min、60min和120min血糖值(BG30min、BG60min和BG120min)。据公式:AUC=(FBG+BG30min)×1/4+(BG30min+BG60min)×1/4+(BG60min+BG120min)×1/2,计算血糖曲线下面积(area under curve,AUC);
空腹血清胰岛素水平及五项血脂:禁食8小时后眼球取血留血清,参照上述各种试剂盒的说明书,检测总胆固醇、甘油三酯、低密度脂蛋白、高密度脂蛋白和游离脂肪酸,空腹血清胰岛素水平(FINS);并计算胰岛素抵抗指数(HOMA-IR);其中,HOMA-IR=(FBG×FINS)/22.5。
3.结果分析
1.如图1所示,与正常组相比,模型组体重、随机血糖、空腹血清胰岛素、TC、TG、HDL、LDL明显升高,并且糖耐量受损,均具有统计学差异。具体的结果如下表1、表2和表3所示,其中,表1为本发明实例中所述的两组实验小鼠的体重、随机血糖、空腹血糖、空腹血清胰岛素水平、HOMA-IR的差别比较;表2为本发明实例中所述的两组实验小鼠的糖耐量的变化值;表3为本发明实例中所述的两组实验小鼠的TC、TG、HDL、LDL、FFA指标(mmol/L)。
2.与正常组相比,造模小鼠胰岛素抵抗指数(HOMA-IR)升高。
表1两组实验小鼠的体重、随机血糖、空腹血糖、空腹血清胰岛素水平、HOMA-IR
组别 | 正常组 | 模型组 |
体重(g) | 31.64 | 41.02 |
随机血糖(mmol/L) | 7.86 | 12.82 |
空腹血糖(mmol/L) | 6.36 | 10.46 |
空腹血清胰岛素(μU/mL) | 22.78 | 43.96 |
HOMA-IR | 6.44 | 20.44 |
空腹血清胰岛素水平(FINS);并计算胰岛素抵抗指数(HOMA-IR);其中,HOMA-IR=(FBG×FINS)/22.5。
表2两组实验小鼠的糖耐量
表3两组实验小鼠的TC、TG、HDL、LDL、FFA指标(mmol/L)
组别 | TC | LDL | HDL | TG | FFA |
正常组 | 1.65 | 1.56 | 3.92 | 1.93 | 1.95 |
模型组 | 2.41 | 3.41 | 1.52 | 3.04 | 2.22 |
本发明的操作步骤中的常规操作为本领域技术人员所熟知,在此不进行赘述。
以上所述的实施例对本发明的技术方案进行了详细说明,应理解的是以上所述仅为本发明的具体实施例,并不用于限制本发明,凡在本发明的原则范围内所做的任何修改、补充或类似方式替代等,均应包含在本发明的保护范围之内。
Claims (7)
1.胰岛素抵抗动物模型的建立方法,包括选取鼠种、分组与造模、采血与检测,其特征在于:同时饲喂高糖高脂高硒饲料与饮用果糖溶液诱导雄性小鼠产生胰岛素抵抗,得到胰岛素抵抗动物模型。
2.根据权利要求1所述的胰岛素抵抗动物模型的建立方法,其特征在于:所述高糖高脂高硒饲料包含硒酸钠、山梨酸钾。
3.根据权利要求1所述的胰岛素抵抗动物模型的建立方法,其特征在于:所述高糖高脂高硒饲料由以下组分按重量百分比构成,基础饲料40-60%,蔗糖15-25%,猪油15-25%,胆固醇8-15%,硒酸钠0.5-2.5%,猪胆盐0.05-0.15%,山梨酸钾0.05-0.15%。
4.根据权利要求1所述的胰岛素抵抗动物模型的建立方法,其特征在于:所述的果糖溶液浓度为10-30%。
5.根据权利要求1所述的胰岛素抵抗动物模型的建立方法,其特征在于:所述选取鼠种步骤为:SPF级雄性C57BL/6小鼠,4-6周龄,体重22±2g,饲养于本部屏障环境内,温度22-25℃,湿度45-55%,12小时昼夜交替,适应性喂养1周。
6.根据权利要求1所述的胰岛素抵抗动物模型的建立方法,其特征在于:所述分组与造模步骤为:适应性喂养1周后,在分组上模型组与对照组均采取正态分布分组方法,即以小鼠体重为依据,将小鼠划分为高、中、低三个体重区间,将每个区间随机分配到模型组和对照组中,模型组和对照组,每组15只,模型组喂食高糖高脂高硒饲料、饮用果糖溶液,对照组喂食标准饲料、饮用蒸馏水,在饲养方法上采取单只单笼饲养措施,饲料定量定投,早晚各一次,维持25g/笼,保证较小的组内差异,2-3天换垫料,给笼具消毒,垫料为刨花木屑,里面含有小木块,要求高压灭菌,造模10周,每周检测体重和随机血糖值。
7.根据权利要求1所述的胰岛素抵抗动物模型的建立方法,其特征在于:所述采血与检测步骤为:连续饲喂10周,第10周结束后,按2g/kg体重灌服40%D-葡萄糖溶液进行口服糖耐量试验;禁食8小时后眼球取血留血清,检测血脂四项和游离脂肪酸,空腹血清胰岛素水平;计算胰岛素抵抗指数及胰岛素敏感指数,得到胰岛素抵抗模型。
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