CN105918673A - 一种高热量饲料喂饲建立巴马小型猪高脂血症模型的制作方法 - Google Patents
一种高热量饲料喂饲建立巴马小型猪高脂血症模型的制作方法 Download PDFInfo
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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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Fodder In General (AREA)
Abstract
本发明属于实验动物学和基础医学领域,特别涉及一种高热量饲料及其喂饲建立巴马小型猪高脂血症模型的制作方法。高热量饲料配方具体方案如下:各成分占重量百分比为牛油10%、花生油6%、胆盐0.5%、胆固醇3%,安赛蜜2.5%,基础饲料78%。用上述高热量饲料喂饲建立巴马小型猪高脂血症模型的制作方法,包括以下步骤:(1)选取健康广西巴马小型猪,月龄6‑8个月,体重(29.88±2.27)kg,雄性,肠道菌群正常;(2)高热量饲料喂饲,共48周,每日对巴马小型猪进行体重测量,日进食量为所测体重的3%,自由饮水;(3)对巴马小型猪的体征指标进行检测。采用高热量饲料喂饲建立的动物模型重复性和稳定性好,可适用于高脂血症的基础研究。
Description
技术领域
本发明属于实验动物学和基础医学领域,特别涉及一种高热量饲料,以及用其喂饲建立巴马小型猪高脂血症模型的制作方法。
背景技术
高脂血症也称血脂异常,是指由于脂质转运或代谢异常,使血浆中的总胆固醇(TC)、总甘油三脂(TG)、低密度脂蛋白(LDL-C)、极低密度脂蛋白(VLDL-C)等致动脉粥样硬化脂质中的一种或多种水平高于正常,高密度脂蛋白(HDL-C)水平低于正常的一类代谢疾病。高脂血症是动脉粥样硬化的重要成因,也是导致心脑血管疾病的主要病理基础,其防治已在全球受到广泛关注,成为目前研究热点。随着人民生活水平的提高,高脂血症的发病率逐年攀升,严重危害着人类健康。
西医高脂血症动物经典模型的制备方法通常是以高脂饲料饲喂动物(GranadaJF, Kaluza GL, Wilensky RL, Biedermann BC, Schwartz RS, Falk E. Porcinemodels of coronary atherosclerosis and vulnerable plaque for imaging andinterventional research. EuroIntervention. 2009. 5(1): 140-8),研究表明,已涉及动物种类很多,如鹌鹑、大鼠、家兔、犬和猪等;李欣志,刘建勋,任建勋,于震,李磊,史跃,马彦雷. 痰瘀互结证冠心病小型猪模型的建立[J]. 中国中西医结合杂志,2009,03:228-232.研究表明,猪相比于其它动物有以下优势:在血管形态、血小板凝聚系统、脂质代谢和消化系统等方面与人类有许多共同之处,可自发形成动脉粥样硬化,且斑块分布和病变过程近似于人类。然而,以往的研究中由于饲料配方的成分与比例各不相同,动物模型重复性和稳定性较差, 造模时间较长。
发明内容
针对现有技术的不足,本发明提供一种采用高热量饲料喂饲制作高脂血症动物模型。该动物模型重复性和稳定性好,可适用于高脂血症的基础研究。
为了实现上述目的,本发明基于现有的小型猪动高脂血症模型,利用高热量饲料喂饲法,具体技术方案如下:所述高热量饲料各成分按重量的百分比为牛油10%、花生油6%、胆盐0.5%、胆固醇3%,安赛蜜2.5%,基础饲料78%。基础饲料其具体配方如下:玉米80.20%、豆饼18.00%、鱼粉0.30%、氢钙0.40%、石粉0.40%、盐0.28%、预混剂0.42%。
所述的预混剂中的各成分百分比:赖氨酸0.20%、多种维生素0.05%、微量元素0.10%、胆碱0.05%、植酸酶0.02%;预混剂中多种维生素为:维生素 A,维生素 D-3,维生素E,维生素 K-3,泛酸,烟酰胺,维生素 B-1,维生素 B-2,维生素 B-6,维生素 B-12,生物素;微量元素为:五水硫酸铜,一水硫酸亚铁,一水硫酸锌,一水硫酸锰,1%碘酸钙,1%亚硒酸钠。
为了实现上述目的,本发明还提供了一种用上述高热量饲料喂饲建立巴马小型猪高脂血症模型的制作方法,包括以下步骤:(1)选取健康广西巴马小型猪,月龄6-8个月,体重(29.88±2.27)kg,雄性,肠道菌群正常;(2)采用前述高热量饲料喂饲,共48周,每日对巴马小型猪进行体重测量,日进食量为所测体重的3%,自由饮水;(3)定期对巴马小型猪的血症指标进行检测。
本发明对于现有技术有以下有益效果。
(1)在大量关于建立高脂血症动物模型的方法中,动物饲料均用到猪油以助高脂血症的形成,但因小型猪食用猪油在伦理上不符合中华人民共和国卫生部动物实验管理条例(No.55,2001),因此改用牛油。国内外文章中饲料配方大多仅使用一种油(除了常用的动物类的猪油、牛油,国外一些文章有报道使用了植物类的花生油、椰子油、饱和脂肪酸等),且油类占饲料总比例从5%-25%不等。我们在使用动物类牛油(占总饲料10%)的基础上又配以植物类花生油(占总饲料6%)。动物油(除鱼油)中含有大量的饱和脂肪酸,而植物油中则含有大量不饱和脂肪酸。动脉粥样硬化斑块的主要组成物质是不饱和脂肪酸,而非饱和脂肪酸。但饱和脂肪酸会提高血清中的HDL-C水平,从而使胆固醇沉积在动脉管壁的几率增加,因此动物油与植物油联合应用更有利于造模成功。
(2)猪胆囊不仅浓缩胆汁的能力低,而且产生胆汁的量少,因此本技术为了促进小型猪对胆固醇的吸收,在饲料中加入胆盐。同时胆盐味苦,加入后使得小猪进食量减少,因此加入安赛蜜改善适口性。长期饲喂饲该高脂饲料造成小猪血脂异常,从而复制出高脂血症特有病变。
(3)以25kg的小猪为例,体重为25kg的小猪维持体重日参考喂食量为0.62kg,维持所需热量为1410.98 kcal。我们实验小猪的日喂食量为体重的3%,即25kg小猪的日喂食量为0.75kg,基础饲料和高脂饲料所能产生的热量分别为2316.53kcal和3050.81kcal,均远高于维持热量1410.98kcal,属于高热量高脂喂饲与现代人的高脂高热量饮食的生活习惯十分相似。正常小猪的日喂食量应为体重的2%- 4%,过低不能满足小猪维持代谢所需能量,过高又给饲养和实验的操作增加了难度。因此我们将日喂食量定在小猪体重的3%,这样既不给饲养和实验操作增加负担又能满足维持小猪正常代谢所需能量。
(4)与现有技术相比,本发明所构建的动物模型与高脂血症临床病变症状、体征相似,并经过重复建模,证明其重复性和稳定性良好,适用于高脂血症疾病机理机制的探讨,可为进一步开展相关的新药研发提供实验依据。
附图说明
图1-1为本发明24、28、35、48周时正常组和模型组小型猪TG水平变化折线图。
图1-2为本发明24、28、35、48周时正常组和模型组小型猪TC水平变化折线图。
图1-3为本发明24、28、35、48周时正常组和模型组小型猪LDL-C水平变化折线图。
图1-4为本发明24、28、35、48周时正常组和模型组小型猪HDL-C水平变化折线图。
图1-5为本发明24、28、35、48周时正常组和模型组小型猪oxLDL-C水平变化折线图。
图2-1为本发明24、28、35、48周时正常组和模型组小型猪hs-CRP水平变化折线图。
图2-2为本发明24、28、35、48周时正常组和模型组小型猪IL-6水平变化折线图。
图2-3为本发明24、28、35、48周时正常组和模型组小型猪MCP-1水平变化折线图。
图2-4为本发明24、28、35、48周时正常组和模型组小型猪VCAM-1水平变化折线图。
图2-5为本发明24、28、35、48周时正常组和模型组小型猪sP-selectin水平变化折线图。
图3-1为本发明正常组24周时40×光镜下的动脉血管。
图3-2为本发明正常组24周时40×光镜下的动脉血管。
图3-3为本发明正常组48周时40×光镜下的动脉血管。
图3-4为本发明正常组48周时40×光镜下的动脉血管。
图3-5为本发明模型组24周时40×光镜下的动脉血管。
图3-6为本发明模型组24周时40×光镜下的动脉血管。
图3-7为本发明模型组48周时40×光镜下动脉血管。
图3-8为本发明模型组48周时40×光镜下的动脉血管。
其中: 3、血管内弹力膜 4、泡沫细胞 5、纤维组织。
具体实施方式
下面以实施例对本发明做进一步详细说明,这些实施例仅用以说明本发明,并不限制本发明的范围,凡基于本发明上述内容实现的技术均属于本发明的范围。
实施例1。
1.实验材料:健康广西巴马小型猪55只,月龄6-8个月,体重(29.88±2.27)kg,雄性,肠道菌群正常,由泰州泰和生物科技有限公司提供。随机分为正常组5只(基础饲料喂饲)和模型组50只(高脂饲料喂饲)。
2.实验方法。
高热量饲料喂饲:高热量饲料(配方中各成分的重量百分比为:牛油10%、花生油6%、胆盐0.5%、胆固醇3%,安赛蜜2.5%,基础饲料78%)喂饲模型组巴马小型猪,共48周。每日对巴马小型猪进行体重测量,正常组和模型组日进食量均为所测体重的 3%,自由饮水。
其中基础饲料的原料为玉米80.20%、豆饼18.00%、鱼粉0.30%、氢钙0.40%、石粉0.40%、盐0.28%、预混剂0.42%。
预混剂中的各成分占高脂饲料重量百分比:赖氨酸0.20%、多种维生素0.05%、微量元素0.10%、胆碱0.05%、植酸酶0.02%,共计0.42%。
预混剂中多种维生素包括:12500IU维生素A,5500IU维生素 D-3,维生素 E 60g,维生素 K-3 10g,泛酸 10g,烟酰胺 60g,维生素 B-1 10g,维生素 B-2 30g,维生素 B-610g,维生素 B-12 0.1g,生物素 0.6g;微量元素包括:五水硫酸铜 200g,一水硫酸亚铁260g,一水硫酸锌 260g,一水硫酸锰 80g,1%碘酸钙 5g,1%亚硒酸钠 3g。饲料载体:膨润土,统糠。
3.指标检测方法。
3.1 理化检测。
全部小型猪分别于24、28、36、48周由耳缘静脉取血(取血前禁食 16h),常规制备血清。胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)应用全自动生化分析仪进行检测;氧化修饰低密度脂蛋白(ox-LDL)、超敏C反应蛋白(hs-CRP)、白细胞介素-6(IL-6)、血管细胞粘附分子-1(VCAM-1)、单核细胞趋化蛋白-1(MCP-1)、P选择素(sP-selectin)采用ELISA法测定,具体操作严格按照试剂盒说明书进行。
3.1.1血脂指标。
第24周时,模型组TC、TG、HDL-C、LDL-C、ox-LDL水平明显高于正常组,统计学差异显著(p<0.01)。随着造模时间延长,模型组TC、TG、LDL-C、ox-LDL逐渐升高,在28、36、48周时明显高于同期正常组,统计学差异显著(p<0.01)。随着造模时间延长,模型组HDL-C水平呈下降趋势但仍高于正常组,在28、36周时两组比较差异有统计学意义(p<0.01,p<0.05),48周时两组比较差异无统计学意义(p>0.05),两组各时间点脂质指标TC、TG、HDL-C、LDL-C水平比较参见表1。图1为两组各时间点TG、TC、LDL-C、HDL-C、ox-LDL水平(其中第35周小猪各项指标检测结果与第36周相似)。
表1 两组各时间点脂质指标TC、TG、HDL-C、LDL-C、ox-LDL水平比较。
项目 | 时间(周) | 正常组 | 模型组 |
TC(mmol/L) | 24 | 3.88±1.89 | 14.89±5.56** |
28 | 4.00±1.69 | 15.30±4.64** | |
36 | 4.31±1.63 | 16.01±1.86** | |
48 | 4.63±1.60 | 16.42±4.11** | |
TG(mmol/L) | 24 | 0.35±0.09 | 1.00±0.36** |
28 | 0.39±0.06 | 1.01±0.42** | |
36 | 0.39±0.12 | 1.08±0.33** | |
48 | 0.46±0.14 | 1.28±0.10** | |
HDL-C(mmol/L) | 24 | 0.77±0.17 | 1.97±0.61** |
28 | 0.77±0.18 | 1.56±0.31** | |
36 | 0.75±0.24 | 1.47±0.43* | |
48 | 0.78±0.09 | 1.34±0.52* | |
LDL-C(mmol/L) | 24 | 1.09±0.25 | 8.00±3.17** |
28 | 1.12±0.45 | 8.69±2.43** | |
36 | 1.58±0.33 | 9.45±2.62** | |
48 | 1.73±0.44 | 10.08±0.43** | |
ox-LDL(ug/L) | 24 | 8.04±0.25 | 8.84±0.86** |
28 | 8.20±0.44 | 10.24±1.74* | |
36 | 8.50±0.64 | 10.42±1.38** | |
48 | 8.68±0.49 | 12.38±2.72** |
注:*P<0.05,**P<0.01,与正常对照组同期比较。
3.1.2炎症因子。
第24周,模型组hs-CRP、IL-6、VCAM-1、MCP-1、sP-selectin水平明显高于同期正常组,差异有统计学意义(p<0.01或p<0.05)。随着造模时间延长,模型组hs-CRP、IL-6、VCAM-1、MCP-1、sP-selectin水平逐渐升高,两组28、36、48周进行比较,模型组明显高于同期正常组,差异有统计学意义(p<0.01或p<0.05)。两组各时间点炎症指标hs-CRP、IL-6、VCAM-1、MCP-1、sP-selectin水平比较见表2,两组各时间点炎症指标hs-CRP、IL-6、MCP-1、VCAM-1、sP-selectin水平见图2(其中第35周小猪各项指标检测结果与第36周相似)。
表2 两组各时间点炎症指标hs-CRP、IL-6、VCAM-1、MCP-1、sP-selectin水平比较。
项目 | 时间(周) | 正常组 | 模型组 |
hs-CRP(ug/L) | 24 | 6.42±0.51 | 7.09±0.89* |
28 | 6.55±0.27 | 7.75±1.16* | |
36 | 6.93±0.58 | 8.14±0.67* | |
48 | 6.80±0.57 | 9.92±1.99** | |
IL-6(ng/L) | 24 | 9.85±0.89 | 11.91±1.16** |
28 | 9.54±038 | 11.56±1.34** | |
36 | 8.66±0.53 | 11.51±1.55** | |
48 | 8.80±0.77 | 12.89±3.49** | |
MCP-1(ng/L) | 24 | 11.38±0.74 | 12.68±0.74* |
28 | 11.47±1.07 | 13.39±1.46* | |
36 | 11.10±0.85 | 13.43±1.46** | |
48 | 9.24±0.81 | 14.21±4.48** | |
VCAM-1(ug/L) | 24 | 10.87±0.74 | 12.74±0.98** |
28 | 10.70±0.61 | 13.52±1.99** | |
36 | 10.02±1.10 | 13.89±1.87** | |
48 | 9.01±0.47 | 14.87±4.18** | |
sP-selectin(ng/L) | 24 | 9.32±0.71 | 10.39±0.65* |
28 | 9.65±1.14 | 11.25±0.60* | |
36 | 9.26±0.62 | 11.75±0.71** | |
48 | 7.57±0.51 | 11.59±2.27** |
注:*P<0.05,**P<0.01,与正常对照组同期比较。
3.2 冠状动脉HE染色。
在24周时,于麻醉状态下肝门静脉放血处死正常组小型猪1只,模型组小型猪2只,48周时所有小型猪全部处死,快速分离冠状动脉前降支,4%多聚甲醛固定后,常规石蜡包埋、切片、苏木精和伊红(HE)染色,进行病理组织学分析,主要观测管腔狭窄程度、内膜损伤情况等。
3.2.1 HE染色结果。
正常组24、48周结果可见冠状动脉管腔规则,管壁厚薄均匀,无形态结构异常,冠状动脉内皮细胞完整,组织层次清楚,肌层纵横排列有序,内弹力膜明显,具体见图3-1、图3-2、图3-3、图3-4。模型组24周可见冠状动脉内膜局部不规则增厚,动脉管腔略有狭窄,增厚的内膜中存在泡沫细胞,具体见图3-5、图3-6;48周可见冠状动脉内膜不规则增厚,粥样斑块形成,动脉管腔狭窄,增厚的内膜中存在泡沫细胞及增生的纤维组织,动脉中膜受压变薄,由于脂质在染色过程中被有机溶剂洗掉,泡沫细胞常常显示为空泡状或空白区。具体见图3-7、图3-8。
综上所述,本实验通过高热量喂饲法在短时间内成功建立巴马小型猪高脂血症硬化模型,模型组中的50只小猪血症指标全部如表1和表2的数据结果,具体数值变动在误差范围内,由此可见该方法建立的动物模型重复性较好;并根据模型组24周和48周的冠状动脉HE染色结果可以说明该动物模型的建立方法保证了模型的稳定性与可靠性,在评价方法上也实现了在体实时可视化与离体病理、理化检查相结合,使模型评价方法更加完整,为相关的基础研究提供了可靠模型,有利于进一步开展高脂血症的相关研究。
Claims (6)
1.一种用于建立高血脂动物模型的高热量饲料,其特征在于,包括牛油10%、花生油60%、蛋盐0.5%、胆固醇3%,安赛蜜2.5%,基础饲料78%。
2.如权利要求1所述的高热量饲料,其特征在于,所述的基础饲料配方如下:玉米80.20、豆饼18.00、鱼粉0.30、氢钙0.40、石粉0.40、盐0.28、预混剂0.42。
3.如权利要求2所述的高热量饲料,其特征在于,所述的预混剂中的各成分百分比:赖氨酸0.20%、多种维生素0.05%、微量元素0.10%、胆碱0.05%、植酸酶0.02%。
4.如权利要求1所述的高热量饲料,其特征在于,所述的多种维生素包括:维生素 A,维生素 D-3,维生素 E,维生素 K-3,泛酸,烟酰胺,维生素 B-1,维生素 B-2,维生素 B-6,维生素 B-12,生物素。
5.如权利要求1所述的高热量饲料,其特征在于,所述的微量元素包括:五水硫酸铜,一水硫酸亚铁,一水硫酸锌,一水硫酸锰,1%碘酸钙,1%亚硒酸钠。
6.如权利要求1所述的高热量饲料,其特征在于,包括以下步骤:(1)选取健康广西巴马小型猪,月龄6-8个月,体重(29.88±2.27)kg,雄性,肠道菌群正常;(2)高热量饲料喂饲,共48周,每日对巴马小型猪进行体重测量,日进食量为所测体重的3%,自由饮水;(3)定期对巴马小型猪的血症指标进行检测。
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CN109924168A (zh) * | 2019-04-01 | 2019-06-25 | 凯斯艾生物科技(苏州)有限公司 | 一种儿童及青少年代谢综合征的动物模型及构建方法 |
CN109924168B (zh) * | 2019-04-01 | 2021-06-01 | 凯斯艾生物科技(苏州)有限公司 | 一种儿童及青少年代谢综合征的动物模型及构建方法 |
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