WO2014202023A1 - 一种恒河猴代谢综合征模型的制备方法 - Google Patents

一种恒河猴代谢综合征模型的制备方法 Download PDF

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Publication number
WO2014202023A1
WO2014202023A1 PCT/CN2014/080413 CN2014080413W WO2014202023A1 WO 2014202023 A1 WO2014202023 A1 WO 2014202023A1 CN 2014080413 W CN2014080413 W CN 2014080413W WO 2014202023 A1 WO2014202023 A1 WO 2014202023A1
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metabolic syndrome
feed
model
cholesterol
animal
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PCT/CN2014/080413
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English (en)
French (fr)
Inventor
陈又南
程惊秋
陆燕蓉
刘敬平
李新丽
杨光
张�杰
廖光能
李红霞
王莉
钟治晖
李兰
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四川大学华西医院
四川康城生物科技有限公司
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Publication of WO2014202023A1 publication Critical patent/WO2014202023A1/zh

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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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/02—Breeding vertebrates
    • 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
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/25—Animals on a special diet
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/106—Primate
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes

Definitions

  • the invention relates to a preparation method of a rhesus monkey metabolic syndrome model.
  • Metabolic Syndrome is a group of clinical syndromes involving lipid metabolism and glucose metabolism, involving obesity, insulin resistance, dyslipidemia and hypertension. It causes severe cardiovascular and cerebrovascular diseases by causing atherosclerosis. Its complications are caused by genetic heterogeneity and environmental factors. There are still many problems to be studied in the pathogenesis of MetS. A large number of animal experiments and clinical studies are looking for effective methods for intervention in the treatment of MetS. Establishing an ideal MetS animal model remains an urgent issue for MetS basic and preclinical experiments.
  • the present invention provides a method for preparing a rhesus monkey metabolic syndrome model.
  • the method for preparing a rhesus monkey metabolic syndrome model of the present invention is to feed a rhesus monkey with a high-fat and high-cholesterol feed, which is fed twice a day, and the feeding amount is 0.3-0.413 ⁇ 4/time*, and the feeding time is not For less than 6 months, the feed contains the following raw materials by weight: 78 parts of standard monkey feed, 15 parts of animal fat, 5 parts of sugar, 2 parts of cholesterol.
  • the animal fat is lard; the sugar is sucrose.
  • Standard monkey feed refers to the monkey compound feed specified in the National Standard of China's Republic of China GB14924.8-2001.
  • Animal fats and oils refer to oils derived from animals, such as butter, sheep oil, and lard.
  • the invention establishes a feed for a model of rhesus monkey metabolic syndrome, which comprises the following raw materials with a weight ratio: 78 standard monkey feeds, 15 animal fats, 5 sugars, and 2 cholesterol.
  • the animal fat is lard; the sugar is sucrose.
  • the present invention also provides a rhesus monkey metabolic syndrome model prepared by the aforementioned method and its use in screening for a medicament for treating metabolic syndrome.
  • the invention screens a method for treating a drug for a metabolic syndrome model, which comprises the following steps: a. Establishing a rhesus monkey metabolic syndrome model according to the foregoing method;
  • the modeling method of the invention can induce the disorder of sugar and lipid metabolism of rhesus monkey, and the clinical manifestation of metabolic syndrome, and the administration method is simple and reproducible, and is an effective method for modeling the rhesus MetS model.
  • Figure 1 Changes in sebum thickness, abdominal circumference, body weight and body fat in the experimental group and the control group.
  • Control group VS experimental group ***: p ⁇ o.001; *: p ⁇ 0.05; A: subcutaneous fat thickness; B: abdominal circumference; C: body weight; D: percentage of whole body fat
  • FIG. 3 Tissue HE staining of rhesus liver and skeletal muscle.
  • A Liver HE staining in the control group;
  • B Liver HE staining in the experimental group (arrow indicates inflammatory cell infiltration);
  • C Liver HE staining in the experimental group (arrow indicates hepatic cell mild edema);
  • D Control group skeletal muscle HE staining;
  • E Experimental group skeletal muscle HE staining;
  • Figure 4 Rhesus monkey liver function and renal function test
  • Figure 9 Changes in serum high-density lipoprotein cholesterol parameters at different times; control group VS experiment Group: ***: ⁇ .001; **: p ⁇ 0.01; *: p ⁇ 0.05;#: experimental group 6mVS 12m
  • Figure 10 Changes in serum ApoA parameters at different times; Control group vs experimental group: ***: pO .001; ** : p ⁇ 0.01; * :p ⁇ 0.05;#: experimental group 6m VS 12m
  • Figure 12 Changes in serum free fatty acid parameters at different times; control group VS experimental group: ***: pO.001; **: p ⁇ 0.01; *: p ⁇ 0.05; #: experimental group 6m VS 12m
  • Figure 13 Changes in serum lipoprotein lipase levels. Standard curve of lipoprotein lipase; (B) serum lipoprotein lipase concentration in the experimental group and the control group as a function of time; control group VS experimental group: ***: p ⁇ 0.001; **: p ⁇ 0.01; * :p ⁇ 0.05.
  • Control group Monkey standard monkey feed, one of the commercially available monkey pellet feeds (Institute of Animal Science, Sichuan Academy of Medical Sciences, the formula is the same as the monkey compound feed specified in the National Standard of Chinese People's Republic of China GB14924.8-2001).
  • High fat and high cholesterol feed 2% (w/w) Edible cholesterol (Anhui Tianqi Chemical Technology Co., Ltd.), 15% (w/w) refined lard (commercially available), 5% (w/w) sucrose (commercially available), 78% (w/w) monkey standard monkey feed.
  • Blood glucose detector Luo Kang full vitality blood glucose detector, Luo Kang full vitality blood glucose test strips Other tests are completed in the 4th Division of the University of West China Hospital Laboratory.
  • the experimental group was fed a rouge high cholesterol diet, and the control group was fed with regular monkey feed, fed twice a day, and the feeding amount was 0.3-0.413 ⁇ 4/time.
  • HbACl Glycated hemoglobin
  • IVGTT Intravenous glucose tolerance test
  • Double-energy X-ray absorptive test (DEXA) was used to measure body fat distribution, B-ultrasound examination of abdominal organs and large vessels, liver wear guided by B-ultrasound and skeletal muscle biopsy at 18 months of feeding. Pathological examination.
  • the pancreatic function of the animals was tested by the intravenous glucose tolerance test (IVGTT) to understand the range of blood glucose fluctuations and to rule out the possibility of spontaneous diabetes in animals.
  • IVGTT intravenous glucose tolerance test
  • ketamine 50 mg/ml was intramuscularly injected at 15 mg/Kg ;
  • the upper and lower limbs of the rhesus monkey are fixed on the operating table, and the saphenous vein of the lower leg of the lower leg is exposed to the saphenous vein, and the iodine fluoride is disinfected;
  • Rhesus monkeys were fasted for more than 16 hours, routine anesthesia, weighing, and collecting 4 ml of lower extremity venous blood into a separate gel-promoting tube.
  • the venous cannula was placed from the median vein of the elbow and the two veins were used to construct two venous channels for infusion of insulin and glucose.
  • the rate is continuously infused, and 20% glucose solution is simultaneously input using the infusion pump.
  • the venous blood glucose level was measured every 5 minutes, and the infusion rate of 200 mL/L glucose solution was adjusted according to the blood glucose level, so that the blood glucose level of the subject was maintained at about 4.5 mmol/L.
  • Venous blood was measured every 30 min to determine serum insulin concentration.
  • the insulin sensitivity index SIClamp M / ⁇ I / fat-free mass (Kg) is calculated, M is the glucose infusion rate at steady state, and ⁇ ⁇ is the difference between the insulin level at steady state and the basal insulin level before the experiment. Value ( ⁇ / ⁇ 1).
  • the body weight of the experimental group was significantly lower than that of the control group (PO.001), and the abdominal circumference was higher than the control group ( P ⁇ 0.05), at 18 months, the sebum thickness of the experimental group was significantly higher than that of the control group (P ⁇ 0.05).
  • the experimental results showed that the experimental group showed clinical manifestations of metabolic syndrome such as increased sebum thickness and elevated abdominal circumference.
  • HE staining showed that the hepatocyte arrangement in the experimental group was mildly disordered, local inflammatory cells invaded, some cells showed mild edema, and no obvious abnormalities in skeletal muscle (Fig. 3). Longitudinal routine serum biochemical monitoring results showed that there was no abnormal change in liver function and renal function of rhesus monkeys in the experimental group during the experiment (Fig. 4).
  • the experimental results showed that the experimental group showed clinical manifestations of metabolic syndrome such as lipid droplet accumulation.
  • the serum cholesterol level of rhesus monkeys in the experimental group was always significantly higher than that of the control group, reaching a peak at 6 months, 3.5 times that of the control group. (8.98 ⁇ 3.30mmol/L vs 2.85 ⁇ 0.37mmol/L, experimental group vs control group, p ⁇ 0.01), then stabilized and maintained at 2.5 ⁇ 3 times of the control group.
  • the serum triglyceride (TG) level of the experimental group showed an upward trend, which was significantly higher than that of the control group at 6 months (0.82 ⁇ 0.28mmol/L vs 0.50 ⁇ 0.07mmol/L, experimental group vs control group).
  • p ⁇ 0.01 peaked at 12 months (1.21 ⁇ 0.75mmol/L vs 0.54 ⁇ 0.10mmol/L, experimental group vs control group, p ⁇ 0.01), and significantly higher than the control group at 18 months.
  • LDL-C is the major lipoprotein carrying TC
  • serum LDL-C level in the experimental group was significantly higher than that in the control group (p ⁇ 0.01).
  • ApoB was the main carrier of LDL. Compared with the control group, the serum ApoB level of the experimental group was also significantly increased (p ⁇ 0.01), and it showed an increasing trend with the prolongation of the rouge high cholesterol diet.
  • ApoA was the main carrier for transporting HDL-C.
  • the change of serum ApoA was similar to that of TG, which was significantly higher than that of the control group (PO.05), which was significantly higher than that of June (P ⁇ 0.05). ⁇ 0.05).
  • TAA total bile acid
  • Serum free fatty acid (FFA) as a catabolic product of TG, as shown in Figure 12, experiment The FFA of the group increased linearly, and was nearly 10 times higher than that of the control group at 6 months (1010.78 ⁇ 289.83 umol/L vs 119.17 ⁇ 35.74 umol/L, experimental group vs control group, p ⁇ 0.01), and Keep at a high level.
  • the diet did not cause changes in fasting blood glucose levels in rhesus monkeys during the first 6 months (4.96 ⁇ 0.69 mmol / L vs 4.88 ⁇ 0.33 mmol / L, experimental group vs control group), 6 months later, the experiment The fasting blood glucose level of rhesus monkeys gradually increased, reaching 6.33 ⁇ in 12 months.
  • the serum insulin level of the experimental group was significantly higher than that of the control group at 6 months (10.32 ⁇ 5.00 mmol / L vs 5.47 ⁇ 2.38 mmol / L, HFHCD vs control, p ⁇ 0.05), and then remained in one The state of hyperinsulinemia.
  • glycated hemoglobin HbACl was also significantly higher in the experimental group than in the control group (3.22 ⁇ 0.36 % vs 2.28 ⁇ 0.39 %, HFHCD vs control, p ⁇ 0.01).
  • IVGTT intravenous glucose tolerance test
  • Serum insulin was maintained at a level of 145 ⁇ 42.59 uIU/L (Fig. 15B).
  • the glucose clearance rate and the area under the insulin curve in the experimental group were significantly lower than those in the control group (p ⁇ 0.001), indicating that the experimental group had impaired glucose tolerance in the rhesus monkey; the peripheral tissue glucose metabolism rate in the experimental group
  • the insulin sensitivity index was lower than that of the control group, and the insulin resistance index was higher than that of the control group, indicating that the experimental group developed insulin resistance.
  • the experimental results showed that the glucose metabolism in the control group was normal, and the glucose metabolism disorder occurred in the experimental group.
  • the method of the invention is modeled by feeding high-fat and high-cholesterol diet, and when fed for 6 months, rhesus monkeys have disorder of glucose metabolism and lipid metabolism, and various clinical manifestations of metabolic syndrome are obvious, indicating that the rhesus monkey metabolism is successfully established. Syndrome model; continued feeding with the high-fat and high-cholesterol feed of the present invention, and various indexes were measured at the 12th month and the 18th month, respectively, and the results showed that various clinical symptoms of the metabolic syndrome continued to exist stably, indicating that the present invention The mode method is stable and effective.
  • Example 2 Screening for the treatment of metabolic syndrome using the model of the present invention
  • the modeling method of the present invention induces the disorder of sugar and lipid metabolism of rhesus monkeys through long-term high-fat and high-cholesterol feed, and the clinical manifestation of metabolic syndrome is an effective method for establishing the MetS model of rhesus monkey.
  • the modeling method of the invention feeds the long-term high-fat and high-cholesterol feed, induces the disorder of sugar and lipid metabolism of the rhesus monkey, and shows the clinical manifestation of the metabolic syndrome, and establishes the rhesus MetS model, which can be used for screening the therapeutic drugs for metabolic syndrome. Suitable for industrial applications.

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Abstract

一种恒河猴代谢综合征模型的制备方法，它是向恒河猴饲喂高脂高胆固醇饲料，每天饲喂2次，饲喂量为0.3-0.4kg/次*只，饲喂时间不少于6个月，所述饲料包含如下重量配比的原料：标准猴饲料78份、动物油脂15份、糖5份、胆固醇2份。通过采用长期喂养高脂高胆固醇饲料的方式，成功地诱导恒河猴糖、脂代谢紊乱，出现代谢综合征的临床表现，是建立恒河猴MetS模型的有效方法。

Description

一种恒河猴代谢综合征模型的制备方法 技术领域
本发明涉及恒河猴代谢综合征模型的制备方法。
背景技术
代谢综合征 （Metabolic Syndrome, MetS) 是一组存在脂代谢和糖代谢紊 乱， 涉及肥胖、 胰岛素抵抗、 血脂紊乱及高血压的临床综合征， 通过引发动 脉粥样硬化导致严重的心脑血管疾病及其并发症， 由遗传异质性和环境因素 所导致。 MetS的发病机制仍然还有很多尚待研究的问题，大量动物实验和临 床研究都在寻找干预治疗 MetS的有效方法。建立理想的 MetS动物模型仍然 是 MetS基础研究和临床前实验急需解决的问题。
目前， 虽然已有近百种不同种属不同种类的 MetS动物模型， 但是动物 实验的结果转化到临床试验和实际临床治疗中还非常艰难， 主要问题在于目 前的动物模型， 如， 现在普遍应用的大小鼠模型与人代谢综合征的病理特征 存在极大的差异。很多研究证明， 大小鼠的脂代谢， 脂肪组织的生物学特性， 脂肪因子的分泌等都与人有明显的差异， 并且， 小鼠不能模拟人在炎性反应 时的基因表达变化 (PNAS,2013,Jan)， 对人类炎性疾病的研究必须依赖更高端 的大动物模型。 建立高端的非人灵长类 MetS模型， 对于其基础和临床研究， 特别是转化医学研究都具有重要价值。
从大规模种群中筛选自然发生 MetS 的非人灵长类依然是获得这类动物 模型的主要方法， 但是这种方法需要非常费时费力， 筛选成功的比例不足 10%， 很难满足目前研究的需要， 急需寻找一种快速稳定的建模方法。
发明内容
为了解决上述问题， 本发明提供了一种恒河猴代谢综合征模型的制备方 法。
本发明恒河猴代谢综合征模型的制备方法， 它是向恒河猴饲喂高脂高胆 固醇饲料， 每天饲喂 2次， 饲喂量为 0.3-0.41¾/次*只， 饲喂时间不少于 6个 月， 所述饲料包含如下重量配比的原料： 标准猴饲料 78份、 动物油脂 15 份、 糖 5份、 胆固醇 2份。
优选地， 所述动物油脂是猪油； 所述糖是蔗糖。
标准猴饲料， 是指《中华人名共和国国家标准 GB14924.8-2001》规定的 猴配合饲料。
动物油脂， 是指来源于动物的油脂， 如， 牛油、 羊油、 猪油。
本发明建立恒河猴代谢综合征模型用饲料，它包含如下重量配比的原料： 标准猴饲料 78份、 动物油脂 15份、 糖 5份、 胆固醇 2份。
优选地， 所述动物油脂是猪油； 所述糖是蔗糖。
本发明还提供了前述方法制备的恒河猴代谢综合征模型及其在筛选治疗 代谢综合征药物中的用途。
本发明筛选治疗代谢综合征模型的药物的方法， 它包括如下歩骤： a、 按照前述方法， 建立恒河猴代谢综合征模型；
b、 将候选药物施用于动物模型；
c、 用动物模型评价潜在的治疗谢综合征疾病的药物。
本发明造模方法可以诱导恒河猴糖、 脂代谢紊乱， 出现代谢综合征的临 床表现， 给药方法简单， 可重复性强， 是一种有效的恒河猴 MetS模型造模 方法。
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歩 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 实验组和对照组的皮脂厚度、腹围、体重及体脂的变化。对照组 VS 实验组： *** :p<o.001; * :p<0.05;A: 皮下脂肪厚度； B: 腹围; C: 体重； D: 全身脂肪百分比
图 2 恒河猴肝脏和骨骼肌的组织油红 0染色
图 3 恒河猴肝脏和骨骼肌的组织 HE染色。 A. 对照组肝脏 HE染色； B. 实验组肝脏 HE染色（箭头示炎性细胞浸润）； C.实验组肝脏 HE染色（箭 头示肝细胞轻度水肿）; D.对照组骨骼肌 HE染色; E. 实验组骨骼肌 HE染色; 图 4 恒河猴肝功能以及肾功能检测
图 5 不同时期血清胆固醇参数的变化； 对照组 VS 实验组： *** : pO.001; ** :p<0.01; * :p<0.05;#: 实验组 6m VS 12m
图 6 不同时期血清甘油三酯参数的变化； 对照组 VS 实验组： *** : pO.001; ** :p<0.01; * :p<0.05;#: 实验组 6m VS 12m
图 7 不同时期血清低密度脂蛋白胆固醇参数的变化； 对照组 VS 实验 组： ***： pO.001; ** :p<0.01; * :p<0.05;#: 实验组 6mVS 12m
图 8 不同时期血清 ApoB参数的变化； 对照组 VS 实验组： *** : pO.001; ** :p<0.01; * :p<0.05;#: 实验组 6mVS 12m
图 9 不同时期血清高密度脂蛋白胆固醇参数的变化； 对照组 VS 实验 组： *** : ρθ.001; ** :p<0.01; * :p<0.05;#: 实验组 6mVS 12m 图 10 不同时期血清 ApoA参数的变化； 对照组 VS 实验组： *** : pO.001; ** :p<0.01; * :p<0.05;#: 实验组 6m VS 12m
图 11 不同时期血清总胆汁酸参数的变化； 对照组 VS 实验组： ***： pO.001; ** :p<0.01; * :p<0.05;#: 实验组 6m VS 12m
图 12 不同时期血清游离脂肪酸参数的变化；对照组 VS 实验组： ***： pO.001; ** :p<0.01; * :p<0.05;#: 实验组 6m VS 12m
图 13 血清脂蛋白脂酶水平的变化。 脂蛋白脂酶的标准曲线; (B)实验组 和对照组血清脂蛋白脂酶浓度随时间变化;对照组 VS 实验组：*** :p<0.001; ** :p<0.01; * :p<0.05。
图 14 葡萄糖代谢稳态的变化
图 15 胰岛细胞的功能检测结果
具体实施方式
实施例 1 本发明恒河猴代谢综合征模型的制备
1实验材料和仪器
6月龄恒河猴 8只（雌雄各半）， 无 B疱疹病毒、 猴逆转录病毒、 猴白血 病病毒和免疫缺陷病毒感染， 购自成都平安动物繁育基地。
饲料配方：
对照组： 猴标准猴饲料一一市售的猴颗粒饲料 （四川省医学科学院动物 研究所， 配方与《中华人名共和国国家标准 GB14924.8-2001》规定的猴配合 饲料相同）。
实验组： 高脂高胆固醇饲料： 2% (w/w) 食用胆固醇 （安徽天启化工 科技有限公司）， 15% (w/w) 精炼猪油 （市售）， 5% (w/w) 蔗糖 （市售）， 78% (w/w) 猴标准猴饲料。
血糖检测仪： 罗康全活力型血糖检测仪， 罗康全活力型血糖试纸 其他检测均在四 ) 11大学华西医院检验科完成。
2实验方法
实验组给予髙脂高胆固醇饮食喂养， 对照组给予常规猴饲料喂养， 每天 饲喂 2次， 饲喂量为 0.3-0.41¾/次 ·只。
(1)定期采集血样进行常规血生化、 血常规、 血清胰岛素水平、 血清脂蛋 白和脂蛋白脂酶 （LPL) 检测。 每月一次， 采血前进行体重、 腹围和皮脂厚 度测量。 连续监测 12个月后， 改为两个月一次。
(2)每隔三个月测定糖化血红蛋白 （HbACl)。
(3)分别在喂养 12和 18个月时， 进行血清和脂质代谢产物的分析。
(4)喂养 18个月后进行静脉葡萄糖耐量试验 （IVGTT) 和高胰岛素 -正常 血糖钳夹试验。
(5)喂养 18个月时进行双能 X线吸收测试（DEXA)测量体脂分布、腹腔 脏器及大血管的 B超检査、 B超引导下的肝穿及骨骼肌的活检， 进行组织病 理学检査。
2.1 恒河猴静脉葡萄糖耐受实验
通过静脉葡萄糖耐受实验（The intravenous glucose tolerance test, IVGTT ) 测试动物胰腺功能， 了解血糖波动范围，排除动物患有自发性糖尿病的可能。
动物禁食 10-12h， 不禁水；
根据动物购买时体重， 按 15mg/Kg肌肉注射氯氨酮 （50 mg/ml ) ;
待动物麻醉后， 准确称量体重， 并记录；
将恒河猴上下肢固定于手术台上， 下肢小腿后侧备皮暴露大隐静脉， 碘 氟消毒；
20G留滞针穿剌大隐静脉， 5ml注射器抽血 6ml (分别做生化、 血常规、 血糖和空腹胰岛素， 并标记为 0分钟数值）；
立即用 5ml注射器静脉推注 50%葡萄糖高渗溶液 0.5g/Kg (在 30sec内推 完），并开始记时，再注射生理盐水 5ml, 向留滞针内推肝素（250 U/ml ) lml; 分别于记时后的第 1， 3， 5， 10， 30min时采血 1.5ml， 放入红头采血管， 测定胰岛素水平； 同时分别于第 0， 1， 3， 5， 10， 30， 60， 120 min时测定 血糖；
检测结束， 将动物放回饲养笼中， 观察动物状况至苏醒。
2.2 高胰岛素 -正常血糖钳夹试验
恒河猴空腹 16h以上， 常规麻醉， 称重、 采集下肢静脉血 4ml注入分离 胶促凝管。
取平卧位， 逆心方向行一侧手臂静脉穿剌， 置入留置导管， 以生理盐水 维持通道,以备取血测定血糖。 不采血时缓慢静滴生理盐水， 采血前临时关闭 输液器。 通过电热垫加热以维持手的温度在 50〜60°C。
从另一侧肘正中静脉置入静脉插管利用三通管组成 2条静脉通道以备 输注胰岛素和葡萄糖。 钳夹开始 10 min内以 4 mU / ( kg · min)速率输注人 胰岛素溶液（优泌林， 40U/mL;>， 使血液胰岛素浓度迅速升高， 随后 l lOmin 内以 2 mU/ ( kg · πιιη:)速率持续输注， 并同时用输液泵输入 20% 葡萄糖液。
在此期间每 5 min测一次静脉血糖值， 根据血糖值调整 200 mL/L萄糖 液输注率， 使受试者血糖值维持在 4.5mmol/L左右。每 30 min采静脉血测定 血清胰岛素浓度。
当血糖水平变化小于 10% 时， 视为达到稳态， 稳态持续约 60 min后结 束试验。 检测胰岛素、 C肽的血标本离心分离血清后置 - 20°C冰箱保存， 统 根据 Steel's 公式计算钳夹试验过程中外周组织葡萄糖代谢率 (glucose disposal rate, M), 计算公式： M=钳夹试验最后 40 min每分钟每公斤体重的 葡萄糖输注量 +(G80— G120) X 0.0625， 其中 G80和 G120分别为钳夹试验 80min和 120 min时的血糖浓度 (mg I dl)。 根据 M值计算胰岛素敏感指数 SIClamp = M/ Δ I / fat-free mass (Kg), M是稳态时的葡萄糖输注率， Δ Ι 是稳 态时胰岛素水平与实验前的基础胰岛素水平的差值（μΐυ/πι1)。
3实验结果
3.1 恒河猴基础生理参数及饮食对体重、 体脂的影响
分组实验前对 16只 6个月的幼年恒河猴体重、腹围、皮脂厚度、血液生 化及血常规进行了测量， 以此作为生理基线， 具体如表 1所示：
表 1 恒河猴基础生化特征 （η=16 )
Figure imgf000007_0001
由表 1可以看出， 生化及血液学检査显示， 6个月的幼年恒河猴均在正 常生理范围内， 发育生长良好。 如图 1A~C所示， 分组后给予不同的饮食， 经过一定的时间， 髙脂高胆 固醇喂养的恒河猴皮脂厚度和腹围高于正常饲料喂养的恒河猴 （图 1 AB )。 这种趋势随着时间的延长逐渐明显， 而体重却低于对照组 （图 1C); 喂养 6 个月时，实验组体重明显低于对照组（PO.001 ),腹围高于对照组（P<0.05 ) ,18 个月时， 实验组的皮脂厚度显著高于对照组 （P<0.05 )。
实验结果说明， 实验组出现皮脂厚度增加、 腹围升高等代谢综合征的临 床表现。
3.2 组织活检
持续喂养 18个月后，与对照组比，实验组肝细胞和骨骼肌肌细胞间出现 明显的脂滴堆积 (图 2)。
HE染色显示， 实验组肝细胞排列结构轻度紊乱， 局部出现炎性细胞侵 润， 部分细胞出现轻度水肿， 骨骼肌没见明显异常（图 3 )。 纵向常规血清生 化监测结果显示， 实验期间实验组恒河猴的肝功和肾功一直未出现异常变化 (图 4 )。
实验结果说明， 实验组出现脂滴堆积等代谢综合征的临床表现。
3.3血脂变化
如图 5所示， 实验组恒河猴血清胆固醇水平一直显著高于对照组， 6个 月时达高峰,是对照组的 3.5倍。（8.98±3.30mmol/L vs 2.85 ±0.37mmol/L， 实 验组 vs对照组， p<0.01 )， 随后趋于平稳， 维持在对照组 2.5~3倍的水平。
如图 6所示， 实验组血清甘油三酯 （TG) 水平呈上升趋势， 6个月时显 著高于同期对照组（0.82±0.28mmol/L vs 0.50±0.07mmol/L, 实验组 vs对照 组 p<0.01 )， 12个月时达到高峰（1.21 ±0.75mmol/L vs 0.54 ±0.10mmol/L, 实 验组 vs对照组， p<0.01 )， 18个月时也显著高于对照组。
如图 7所示， LDL-C是携带 TC的主要脂蛋白，同期的实验组血清 LDL-C 水平显著高于对照组 （p<0.01 )。
如图 8所示， ApoB作为 LDL的主要载体， 与对照组相比， 实验组的血 清 ApoB水平也显著增加 （p<0.01 )， 随着髙脂高胆固醇饮食的延长， 呈现出 递增趋势。
如图 9所示， 实验组血清 HDL-C水平显著高于对照组。
如图 10所示， ApoA作为运输 HDL-C的主要载体， 血清 ApoA的变化 与 TG相似， 一直明显高于对照组 （PO.05 ) ,与 6月相比， 12月时显著升高 (P<0.05 )。
血清中胆固醇主要的一个代谢途径是参与胆汁酸的合成， 如图 11所示， 实验组的血清总胆汁酸 （TBA) 水平在 12个月内高于对照组。
血清游离脂肪酸 （FFA) 作为 TG的分解代谢产物， 如图 12所示， 实验 组的 FFA—直进行性增加， 6个月时比同期的对照组高了近 10倍 （1010.78 ±289.83umol/Lvs 119.17 ±35.74 umol/L, 实验组 vs对照组， p<0.01)， 并且 一直保持在一个较高的水平。
如图 13所示， ELISA法测定血清脂蛋白脂酶（LPL)结果显示， 标准曲 线相关性好， 结果可信， 与普通饮食比较， 髙脂高胆固醇饮食导致恒河猴血 清 LPL明显升高（p<0.01)， 6个月时， 实验组血清 LPL水平是对照组的近 4 倍。随着时间延长， LPL维持在高于同期对照组的水平上。（8.83±1.82ng/mL vs 4.43 + 1.53 ng/mL vs 2.81 +0.42ng/mL, 6m vs 12m vs 18m, p<0.01)。
实验结果说明， 实验期间， 对照组血清脂质没有出现明显变化， 一直保 持在生理水平， 而实验组恒河猴的脂代谢紊乱， 表现出明显的饮食依赖性的 混合型髙脂血症 （高 TC+高 TG+高脂蛋白）。
3.4葡萄糖代谢稳态的影响
如图 14A所示，最初的 6个月内饮食并未引起恒河猴空腹血糖水平变化 (4.96±0.69mmol/Lvs4.88±0.33mmol/L, 实验组 vs对照组）， 6个月以后 实验组恒河猴的空腹血糖水平出现逐渐升高， 12个月时达到 6.33士
1.14mmol/mL， 显著高于对照组 4.55±0.99mmol/mL (p<0.01)， 并一直持续 到 18个月。
如图 14B所示， 血清胰岛素水平实验组在 6个月时即显著高于对照组， ( 10.32 ±5.00mmol/Lvs 5.47 ±2.38mmol/L,HFHCDvs control, p<0.05)， 随 后一直保持在一个高胰岛素血症的状态。
如图 14C所示， 随着实验组空腹血糖的增加， 糖化血红蛋白 （HbACl) 也显著高于对照组 （3.22±0.36 % vs 2.28±0.39 %, HFHCD vs control, p<0.01
胰岛细胞的功能检测结果：
静脉葡萄糖耐量实验 （IVGTT) 结果显示， 给予高糖剌激以后， 对照组 血糖明显升高， lmin就升到 14.15±1.17mmol/L， 3min达到高峰 16.53士 0.90mmol/L, 然后逐渐下降， 60min血糖降到 6.29±0.69mmol/L ； 实验组 lmin血糖升高到 12.74±3.58mmol/L， 3min血糖降至 10.25±2.14mmol/L， 随后一直保持这个水平， 血糖未见明显下降， 60min血糖为 10.15士
3.18mmol/L图 15A：)。 实验结果说明， 实验组恒河猴的胰岛 β细胞的分泌功 能受损。
高胰岛素 -正常血糖钳夹实验发现， 实验组血糖浓度维持在
4.3~4.6mmol/L,CV<5%。 血清胰岛素保持在 145 ±42.59uIU/L水平（图 15B)。
根据 IVGTT和钳夹实验的数据进行相关代谢变量的计算， 结果见表 2。 表 2 实验组和对照组葡萄糖代谢变量的均值
Variables Control (n =8) HFHCD (n =8) 葡萄糖清除率 Keiu(%) 1.73±0.37 0.70±0.33*** 胰岛素曲线下面积 AUC-insulin(^U/ 387.24±122.39 105.50±66.04***
L* min _1) 外周组织葡萄糖代谢率 M value(mg. 16.35±2.06 13.78±1.21 kg · min 、 ) 胰岛素敏感指数 SIdamp (mg - kg 1 - 0.12±0.05 0.07±0.03 min - 1 /μΐυ/ L)
0.85±0.55
胰岛素抵抗指数 HOMA-IR 1.55±1.12
Note: HFHCD vs. Control： *** P <0.001.HOMA-IR= ( FPGxFPI ) 172.5
由表 2可以看出， 实验组的葡萄糖清除率和胰岛素曲线下面积显著低于 对照组 （p<0.001 )， 说明实验组恒河猴已出现糖耐量受损； 实验组的外周组 织葡萄糖代谢率和胰岛素敏感指数低于对照组，胰岛素抵抗指数高于对照组， 说明实验组出现了胰岛素抵抗。
实验结果说明， 对照组的糖代谢正常， 实验组出现了糖代谢紊乱。 本发明方法通过饲喂高脂高胆固醇饲料造模， 饲喂 6个月时， 恒河猴出 现糖代谢和脂代谢紊乱， 代谢综合征的各种临床表现明显， 说明成功建立了 恒河猴代谢综合征模型；用本发明高脂高胆固醇饲料继续饲养，在第 12个月 和第 18个月时分别测定各种指标，结果表明代谢综合征的各种临床症状持续 稳定存在， 说明本发明造模方法稳定有效。 实施例 2 用本发明模型筛选治疗代谢综合征的药物
a、 按照实施例 1方法建立的恒河猴代谢综合征模型；
b、 将候选药物施用于动物模型；
c、观察候选药物对代谢综合征的各种指标的影响情况，评价潜在的治疗 代谢综合征疾病的药物。 综上，本发明造模方法通过长期高脂高胆固醇饲料喂养，诱导恒河猴糖、 脂代谢紊乱， 出现代谢综合征的临床表现， 是建立恒河猴 MetS模型的有效 方法。 工业应用性
本发明造模方法通过长期高脂高胆固醇饲料喂养， 诱导恒河猴糖、 脂代 谢紊乱， 出现代谢综合征的临床表现， 建立了恒河猴 MetS模型， 可用于代 谢综合征治疗药物的筛选， 适于工业应用。

Claims

权 利 要 求 书
1、一种恒河猴代谢综合征模型的制备方法， 其特征在于： 它是向恒河猴 饲喂高脂高胆固醇饲料， 每天饲喂 2次， 饲喂量为 0.3-0.41¾/次*只， 饲喂时 间不少于 6个月， 所述饲料包含如下重量配比的原料： 标准猴饲料 78份、 动物油脂 15份、 糖 5份、 胆固醇 2份。
2、根据权利要求 1所述的制备方法，其特征在于：所述动物油脂是猪油； 所述糖是蔗糖。
3、一种建立恒河猴代谢综合征模型的饲料， 其特征在于： 它包含如下重 量配比的原料： 标准猴饲料 78份、 动物油脂 15份、糖 5份、 胆固醇 2份。
4、 根据权利要求 3所述的饲料， 其特征在于： 所述动物油脂是猪油； 所 述糖是蔗糖。
5、 权利要求 1或 2所述方法制备的恒河猴代谢综合征模型。
6、权利要求 5所述恒河猴代谢综合征模型在筛选治疗代谢综合征药物中 的用途。
7、一种筛选治疗代谢综合征模型药物的方法， 其特征在于： 它包括如下 歩骤：
a、 按照权利要求 1或 2所述方法， 建立恒河猴代谢综合征模型； b、 将候选药物施用于动物模型；
c、 用动物模型评价潜在的治疗谢综合征疾病的药物。
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