CN114375905A - 一种肝纤维化动物模型的构建方法 - Google Patents
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- A—HUMAN NECESSITIES
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- 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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- A23K50/50—Feeding-stuffs specially adapted for particular animals for rodents
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- Biodiversity & Conservation Biology (AREA)
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Abstract
本发明提供了一种肝纤维化动物模型的制备方法,以含有500IU/kg维生素A的饲料喂养小鼠4周,造成动物体内维生素A缺乏(血清视黄醇浓度为0.7‑1.0μmol/L)的基础上,给予5%低浓度CCL4,10ml/kg剂量,腹腔注射每周2次,造模8‑12周,可获得中重度的肝纤维化模型,模型成功率为100%,且血生化指标ALT/AST及肝纤维化组织病理结构稳定,停药8周不发生自发逆转。该制备方法的肝纤维化模型避免了肝脏自发修复对肝纤维化程度评估的影响,可应用于肝纤维化发生发展的分子机制研究,寻找肝纤维化治疗药物等研究领域。
Description
技术领域
本发明涉及实验动物学的应用领域,具体涉及一种肝纤维化动物模型的构建方法。
背景技术
肝纤维化是与慢性肝脏损伤相关的肝脏瘢痕修复反应,是多种慢性肝病进展至肝硬化的中间过程,乙型和丙型肝炎、酒精性肝炎、非酒精性脂肪性肝炎、血吸虫病、自身免疫性肝病、药物性肝损伤等均可导致肝纤维化,其机制是持续性的肝实质细胞的损伤和(或)炎症,导致富含Ⅰ型和Ⅲ型胶原蛋白的细胞外基质(ECM)合成与降解失衡,大量沉积的ECM可破坏正常的肝脏结构。
肝纤维化的动物模型对于深入研究肝纤维化发生发展的分子生物学机制,筛选防治肝纤维化药物等具有重要的意义。四氯化碳(CCL4)是一种肝毒性药物,可导致肝细胞损伤、变性、坏死,通过灌胃、皮下注射或腹腔注射CCL4的方式是肝纤维化模型制备的常用方法,诱导的肝纤维化模型在组织形态学,病理生理学等方面与人的肝纤维化相似,适合肝硬化发生发展过程的动态研究。CCL4造模的时间由给药途径和剂量决定,一般为8周-4个月不等,如:40-60%CCL4, 3ml/kg皮下注射,12周形成肝纤维化;造模10周,0.5ml/kg CCL4仅在汇管区形成纤维化,而1ml/kg CCL4可形成早期肝硬化。CCL4肝毒性强,1ml/kg剂量 CCL4灌胃方式死亡率在10%左右,而腹腔注射死亡率在80%以上。CCL4腹腔注射的浓度和频率会影响其作用部位和毒性,CCL4(40%CCL4,2ml/kg)联合饮用酒精,造模成功率高,死亡率仍有7.9%-26.9%。CCL4造模停药后,损伤的肝脏组织有自然恢复的趋势,不适合用于周期长的实验研究,在肝纤维化药物治疗的研究中,肝脏自发修复不能准确判断药物的实际效果。因此,成功率高、死亡率低、重复性好且稳定的肝纤维化动物模型非常重要。
肝脏参与维甲酸代谢,也是维甲酸作用的一个重要靶器官。维甲酸在肝脏合成,并可以与维甲酸受体相互作用,从而控制大量肝代谢相关基因的表达。肝星状细胞(HSC)活化是肝纤维化形成的中心环节,HSC是体内维生素A储存最主要的细胞,以维生素A酯的形式储存于脂滴中,HSC活化可导致维甲酸存储丢失及维甲酸信号的失调。脂多糖(LPS)处理可激活肝星状细胞,显著降低 HSC的脂质含量,包括脂滴数目和脂质染色面积,LPS导致HSC细胞内维甲酸水平降低、维甲酸受体RARs及相关靶基因表达也降低。维生素A调节脂质积累和基因转录,肝纤维化发生发展过程中,储存维生素A的肝星状细胞转分化为缺乏维生素A的肌纤维母细胞。用维生素A处理静止期的HSCs,可部分维持脂滴的积聚和静止的HSC标记物,并通过激活JAK2/STAT5信号通路和上调甾醇调节元件结合蛋白-1,完全抑制肌纤维母细胞标记物(α-SMA、热休克蛋白47 和Ⅰ型胶原Col1a1)的表达,提示维生素A参与维持HSCs在生理条件下的静止状态。专利CN 102526012 B也公开了视黄酸及其衍生物在制备治疗肝纤维化药物中的应用。维生素A与肝纤维化密切相关,但我们的前期研究显示维生素A 缺乏的动物模型中,肝脏大体样本及组织学未见纤维化表现。
发明内容
为了解决的现有技术中肝纤维化动物模型造模时间较长,死亡率高,模型不稳定等缺陷,本发明提供了一种肝纤维化动物模型的制备方法。
本发明的目的是通过以下措施实现的:
本发明提供了一种肝纤维化动物模型的制备方法,包括以下步骤:
步骤1,选择模型动物,采用维生素A缺乏饲料喂养4周以上;
步骤2,腹腔注射CCL4溶液,10ml/kg剂量,每周2次,持续8-12周,持续维生素A缺乏饲料喂养;
步骤3,检测血生化指标ALT/AST,肝组织H.E及Masson染色。
优选的,CCL4浓度为5%,将5ml CCL4溶于95ml辛癸酸甘油酯,紫外消毒30分钟获得。
优选的,上述模型动物包括但不限于小鼠、大鼠、兔、狗、猴。
优选的,上述维生素A缺乏饲料的组分包括:动物蛋白20%,脂肪5%,葡萄糖61.3%,纤维3%,无机盐组合物6.8%,维生素组合物0.22%,水3.68%,以重量百分数计算。其中维生素组合物各组分在1kg饲料中的含量为:维生素A 500IU、维生素B1 8mg、维生素B2 10mg、维生素B6 6mg、维生素B12 0.02mg、维生素D 800IU、维生素E 64IU、维生素K3mg、叶酸4mg、生物素0.1mg、烟酸45mg、泛酸17mg、胆碱1.25g。
有益效果:
本发明提供了一种肝纤维化动物模型的制备方法,在给予维生素A缺乏饲料造成动物体内维生素A缺乏的基础上,给予5%低浓度CCL4,10ml/kg注射剂量造模,药物分散度好,动物死亡率低,为了避免含有维生素A的溶剂对动物体内维生素A的影响,采用不含维生素A的辛癸酸甘油酯配制CCL4溶液,保证动物血清内维生素A保持在稳定的较低水平,造模时间短,成功率高、死亡率低、重复性好、模型稳定性好,肝纤维化程度为中重度,纤维化形成后8周不发生自发逆转,可用于较长时间的实验研究,避免肝组织结构自发修复对肝纤维化程度评估的影响,适合各实验室推广,可应用于肝纤维化发生发展的分子机制研究,寻找肝纤维化治疗药物等研究领域。
附图说明
图1:CCL4造模8周后各组肝脏组织大体标本、H.E染色及Masson染色(从左至右依次为:VAN未造模组、VAD未造模组1、VAD未造模组2、VAN造模组、VAD造模组1、VAD造模组2)
图2:CCL4停药4周后各组肝脏组织大体标本、H.E染色及Masson染色(从左至右依次为:VAN未造模组、VAD未造模组1、VAD未造模组2、VAN造模组、VAD造模组1、VAD造模组2)
图3:CCL4停药8周后各组肝脏组织大体标本、H.E染色及Masson染色(从左至右依次为:VAN未造模组、VAD未造模组1、VAD未造模组2、VAN造模组、VAD造模组1、VAD造模组2)
具体实施方式
1、不同维生素A缺乏饲料配制
基础饲料的配方参见实验动物配合饲料的国家标准GB 14924.3-2001及专利 CN101911923 B,其中1kg基础饲料中:动物蛋白20%,脂肪5%,葡萄糖61.3%,纤维3%,无机盐组合物6.8%,维生素组合物0.22%,水3.68%,以重量百分数计算。其中维生素组合物各组分在1kg饲料中的含量为:维生素A 500IU、维生素B1 8mg、维生素B2 10mg、维生素B66mg、维生素B12 0.02mg、维生素D 800IU、维生素E 64IU、维生素K 3mg、叶酸4mg、生物素0.1mg、烟酸45mg、泛酸17mg、胆碱1.25g。
具体配方如下表:
维生素A正常饲料:含维生素A 7000IU/kg,在10kg基础饲料中加入14粒维生素A软胶囊中的油剂,每一粒软胶囊含5000IU维生素A;
维生素A缺乏饲料1:含维生素A 500IU/kg,在10kg基础饲料中加入1粒维生素A软胶囊中的油剂,每一粒软胶囊含5000IU维生素A;
维生素A缺乏饲料2:含维生素A 200IU/kg,在10kg基础饲料中加入0.4 粒维生素A软胶囊中的油剂,每一粒软胶囊含5000IU维生素A;
2、不同饲料喂养的血清维生素A水平检测
选取Balb/c小鼠126只,雄性,6-8周龄,体重22g±3.0g,分为三组:维生素A正常(VAN)组、维生素A缺乏(VAD)组1、维生素A缺乏(VAD)组 2,每组42只,分别给予以上维生素A正常饲料、维生素A缺乏饲料1、维生素A缺乏饲料2喂养4周。
VAN组神态自然、皮毛光泽度好,活动自如,眼睛有神;
VAD组1毛发粗糙、色泽偏黄、活动频繁,眼神呆滞;
VAD组2毛发粗糙、并有少许脱落、色泽偏黄、活动频繁、眼神呆滞、易激怒。
各组取6只小鼠,取血检测血清视黄醇水平(μmol/L),结果见表1:
表1.不同VA饲料喂养4周后血清视黄醇水平
b:与VAN组比较,p<0.05
c:与VAD组1比较,p<0.05
3、CCL4诱导的肝纤维化模型
将5ml CCL4溶于95ml辛癸酸甘油酯(H860153,上海麦克林生化科技有限公司),紫外照射30分钟消毒备用。各组随机选取18只小鼠,按10ml/kg剂量,腹腔注射CCL4,每周2次,共8周。造模过程中,各组分别给予维生素A正常饲料、维生素A缺乏饲料1、维生素A缺乏饲料2喂养。VAN造模组及VAD 造模组1无死亡,VAD造模组2有4只小鼠死亡,造模8周后,检测肝脏指数及血生化指标ALT、AST,肝组织纤维化评分依据METAVIR评价系统(F0=无纤维化;F1=汇管区纤维性扩大,但无纤维间隔形成;F2=汇管区纤维性扩大,少数纤维间隔形成;F3=多数纤维间隔形成,无硬化结节;F4=肝硬化)。
VAD组血清视黄醇水平均显著低于VAN组,且VAD组2低于VAD组1,有统计学差异(表2,p<0.05.)。未造模组,三组的肝脏指数无差异,血清ALT 和AST浓度均正常,大体肝脏组织未见差异,VAN组的肝脏组织结构正常,肝小叶完整;VAD组可见较多空泡,VAD组2空泡数量更多,肝小叶完整,未见纤维化。造模组,肝脏指数及ALT/AST增高,大体肝脏标本可见白色颗粒状突起,病理切片显示肝索结构紊乱、核固缩或消失,有较多岛屿状假小叶结构形成,汇管区有较多炎性细胞,Masson染色门静脉周围有大量蓝色纤维化,纤维间隔形成,小叶结构部分保留。与VAN组相比,VAD组的血清ALT和AST增高明显(表2,p<0.05.),VAD组1的蓝染纤维化更多,有纤维间隔伴形成假小叶形成;VAD组2肝脏坏死严重,假小叶纤维间隔处有大量炎性细胞浸润(图1)。
表2 CCL4造模8周后各组血清视黄醇、ALT/AST及肝纤维化程度
a:与未造模组比较,p<0.05
b:与VAN组比较,p<0.05
c:与VAD组1比较,p<0.05
4、停止CCL4给药后,肝纤维化模型的稳定性评估
造模成功,停止CCL4给药,各组继续给予相应饲料喂养,4周内VAN组及VAD组1无死亡,VAD组2有2只小鼠死亡,第4周末进行检测。未造模组,各指标无变化。造模组,肝脏指数无变化,VAN造模组的血清ALT、AST较4 周前有恢复,大体标本可见少量颗粒状,纤维化减少,仍可见纤维间隔。VAD 造模组的血清ALT、AST水平,肝脏大体及病理无明显变化,VA缺乏造模组1 仍可见肝索结构紊乱和假小叶,VA缺乏造模组2腹腔黏连严重,仍可见假小叶及大量炎性细胞浸润(表3,p<0.05;图2)。
表3 CCL4停药4周后各组血清视黄醇、ALT/AST及肝纤维化程度
a:与未造模组比较,p<0.05
b:与VAN组比较,p<0.05
c:与VA缺乏1组比较,p<0.05
各组继续给予相应饲料喂养,4周末-8周末,各组均无小鼠死亡,第8周末进行检测。未造模组,各指标无变化。造模组,肝脏指数均有轻微下降,与前一时间点相比无统计学差异,VAN造模组的血清ALT、AST基本恢复,大体标本肝脏几乎无颗粒状,肝索结构恢复正常,仅在汇管区有纤维化,几乎无纤维间隔。 VAD造模组的血清ALT、AST及肝脏病理较前一时间点无明显变化,VAD造模组1仍可见肝索结构紊乱和假小叶,VAD造模组2仍可见假小叶,炎性细胞浸润较前减少(表4,p<0.05;图3)。
表4 CCL4停药8周后各组血清视黄醇、ALT/AST及肝纤维化程度
a:与未造模组比较,p<0.05
b:与VAN组比较,p<0.05
c:与VA缺乏1组比较,p<0.05
以上结果说明:以含有200IU/kg维生素A的饲料喂养小鼠4周,CCL4干预8周,获得中重度的肝纤维化模型,模型成功率为66.67%,肝脏损伤较重,病理结构稳定,停药8周无自发逆转。以含有500IU/kg维生素A的饲料喂养小鼠4周,动物血清视黄醇浓度为0.7-1.0μmol/L,CCL4干预8周,获得中重度的肝纤维化模型,模型成功率为100%,无死亡,且血生化指标ALT/AST及肝纤维化病理结构稳定,停药8周无自发逆转。
此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其他实施方式。
Claims (4)
1.一种肝纤维化动物模型的制备方法,其特征在于,包括以下步骤:
步骤1,选择模型动物,采用维生素A缺乏饲料喂养4周以上;
步骤2,腹腔注射CCL4溶液,10ml/kg剂量,每周2次,持续8-12周,持续维生素A缺乏饲料喂养;
步骤3,检测血生化指标ALT/AST,肝组织H.E及Masson染色。
2.如权利要求1所述的制备方法,其特征在于:CCL4浓度为5%,将5ml CCL4溶于95ml辛癸酸甘油酯,紫外消毒30分钟获得。
3.如权利要求1所述的制备方法,其特征在于:所述模型动物包括但不限于小鼠、大鼠、兔、狗、猴。
4.如权利要求1-3所述的制备方法,其特征在于:所述维生素A缺乏饲料的组分包括:蛋白20%,脂肪5%,葡萄糖61.3%,纤维3%,无机盐组合物6.8%,维生素组合物0.22%,水3.68%,以重量百分数计算。维生素组合物各组分在1kg饲料中的含量为:维生素A 500IU、维生素B1 8mg、维生素B2 10mg、维生素B6 6mg、维生素B12 0.02mg、维生素D 800IU、维生素E 64IU、维生素K 3mg、叶酸4mg、生物素0.1mg、烟酸45mg、泛酸17mg、胆碱1.25g。
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