CN112961882A - 一种肝脏纤维化疾病模型的构建方法和应用 - Google Patents
一种肝脏纤维化疾病模型的构建方法和应用 Download PDFInfo
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Abstract
本发明提供了一种肝脏纤维化疾病模型的构建方法和应用。本发明方法通过敲除目标动物肝脏实质细胞中的基因组中的NF‑YA基因序列,使目标动物表现出典型的肝脏纤维化疾病特征。该目标动物既可作为肝脏纤维化疾病模型,用于肝脏纤维化疾病的研究,可以帮助阐明肝脏纤维化疾病的发病过程及机制,并为该疾病的治疗或预防提供新的靶标。
Description
技术领域
本发明属于生物技术领域,具体涉及一种肝脏纤维化疾病模型的构建方法和应用。
背景技术
肝纤维化是大多数慢性肝病的共有的主要特征。临床上常见于病毒性肝炎、酒精肝、脂肪肝、自身免疫性疾病、自身代谢综合征等。其主要特征表现为细胞外基质(Extracellular Matrix)增多,尤其以胶原纤维为主等过度增生。当纤维组织过度增生形成交联的锁链状,破坏了原有的肝小叶结构后,可进一步发展成为肝硬化。当形成肝硬化后其过程便不可逆转,肝脏功能丧失,最终多数患者发展成为原发性肝癌。而肝纤维化作为一种修复机制在发展成肝硬化之前是可以逆转的。因此,找出纤维化发生的刺激因子、以及抗纤维化治疗一直是肝病研究和防治的重要方向。
为了更好地研究和治疗肝脏纤维化,以及筛选出可有效治疗肝脏纤维化的药物,本领域需要能够模拟出肝脏纤维化特征的动物模型。
目前,对肝脏纤维化疾病模型的构建主要包括以下几类:
化学损伤肝纤维化模型的建立目前最广泛应用的是利用四氯化碳(CCl4)建立肝纤维化模型,利用四氯化碳直接溶解肝细胞膜,经干细胞细胞色素p450依赖性混合功能氧化酶的代谢,生成活性的三氯甲基自由基和氯甲基自由基,启动脂质过氧化作用,导致肝细胞损伤。但是,单独使用CCl4诱导肝纤维化模型虽然简单易行,但死亡率较高(NabeshimaY,Tazuma S,Kanno K,et al.Anti-fibrogenic function of angiotensin II type 2receptor in CCl4-induced liver fibrosis.[J].Biochem Biophys Res Commun,2006,346(3):658-664)。此外,还有利用二甲基亚硝胺(DMNA)、D-氨基半乳糖(DGA)、二乙基亚硝胺(DEN)、硫代乙酰胺(TAA)等物质诱导肝纤维化的报道,但由于DMNA、DEN是致癌药物,TAA毒性大,DGA价格昂贵、造模时间长等缺点,均很难推广应用。
酒精性肝纤维化动物模型则是利用了肝脏是酒精代谢、降解的主要场所的特点,过度饮酒使自由基产生、脂肪积蓄、脂质过氧化对肝细胞造成损伤诱导肝纤维化。但实际造模过程中由于动物厌酒,难以控制摄入量等问题,也难以推广应用。
营养性肝纤维化动物模型主要通过膳食不平衡或缺乏特种营养素引起干细胞脂肪变性,进而形成肝纤维化,但耗时长、饲料配制复杂且花费大。
复合损伤肝纤维化模型建立目前主要是上述方法的结合,通过采用高脂低蛋白食物、饮用酒精,以及皮下注射CCl4的方式,诱导但肝纤维化,能够获得较高的成型率,但是死亡率仍能达到20%左右。
可见,目前构造肝纤维化疾病模型的方法均存在诸多缺陷,需要较长的建模时间,而且建模成型率、死亡率的不可控因素较强。因此,进一步研究建模成型率高、耗时短、死亡率低的肝纤维化疾病模型的构建方法,将为深入全面地研究肝纤维化发病机制,为临床筛选防治肝纤维化药物提供研究基础。
发明内容
本发明的目的在于提供一种肝脏纤维化疾病模型的构建方法。本发明的发明人首次发现,在目标动物的肝脏实质细胞中敲除NF-YA基因即沉默NF-YA基因的表达,可以使得目标动物表现出肝脏纤维化疾病相关的特征例如肝脏表面有明显的纹理分膈、纤维增生、肝叶边缘锐化、肝小叶界板破坏明显、形成假小叶。因此,肝脏实质细胞中的NF-YA基因被敲除的动物可以作为肝脏纤维化疾病模型,用于肝脏纤维化疾病研究等领域中,为该疾病的研究例如发病过程、机制以及相关药物的筛选提供一种新的模型。
本发明提供了一种肝脏纤维化动物模型的构建方法,包括如下步骤:通过基因工程技术敲除目标动物肝脏实质细胞中的基因组中的NF-YA基因序列。
基因片段:是指基因的一部分序列,比如外显子。
进一步地,上述NF-YA基因序列是NF-YA基因的外显子序列,优选地,所述NF-YA基因的外显子序列是指NF-YA基因上第3、第4、第5、第6、第7和第8外显子中的任意一个或多个外显子序列。
进一步地,上述基因工程技术为基因编辑技术、基因敲除技术、RNA干扰技术中的任意一种技术或多种技术的组合,优选为基因敲除技术。
更进一步地,上述的构建方法包括如下步骤:
(1)通过基因敲除技术敲除目标动物肝脏实质细胞中的基因组中的NF-YA基因第3、第4、第5、第6、第7或第8外显子中的任意一个或多个外显子序列,得到NF-YA基因条件性纯合子动物NF-YALoxp/Loxp;
(2)将步骤(1)得到的NF-YA基因条件性纯合子动物与转Alb-Cre基因动物交配得到后代,将后代中同时携带Alb-Cre基因和NF-YA条件性敲除基因的动物再与步骤(1)得到的NF-YA基因条件性纯合子动物交配,即可得到肝脏实质细胞NF-YA基因条件性敲除的纯合子动物,作为肝脏纤维化动物模型。
更进一步地,上述目标动物选自小鼠、大鼠、狗、猪、猴、猿中的任意一种。
本发明还提供了一种肝脏纤维化动物模型,其特征在于,它是NF-YA基因在肝脏实质细胞中不表达或表达受到抑制的动物模型,优选地,它是肝脏实质细胞中的基因组中的NF-YA基因敲除的动物模型。进一步地,它是通过上述的构建方法构建得到的。
本发明还提供了NF-YA基因沉默或缺失的动物模型作为肝脏纤维化动物模型的用途。
本发明还提供了上述肝脏纤维化动物模型在肝脏纤维化疾病研究中的应用,所述研究是以非疾病的诊断或治疗为目的。
进一步地,本发明提供了上述肝脏纤维化动物模型在筛选用于预防或治疗肝脏纤维化疾病药物中的应用。
实验结果表明,在肝脏实质细胞条件性敲除NF-YA基因,可以使目标动物表现出肝脏纤维化疾病。肝脏实质细胞条件性敲除NF-YA基因的动物,可以作为肝脏纤维化疾病模型。本发明方法通过小鼠自然交配,获得肝脏纤维化疾病模型,相比于现有的构建肝纤维化模型的方法而言,不需要额外的化学试剂或人工干预,具有成型率高、模型稳定、死亡率低、无毒无害环境友好等优势。通过本发明的构建方法得到的动物模型其具有典型肝脏纤维化疾病特征,其具有非常广阔的应用前景,例如将其用于研究肝脏纤维化的发病过程、发病机制,为深入了解研究肝脏纤维化提供基础。又或者将其用于筛选用于预防或治疗肝脏纤维化药物、用于评价药物的疗效或预后等。
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:NF-YA基因敲除小鼠的策略及鉴定;A:NF-YA基因敲除的路线。B:NF-YA基因敲除小鼠的基因型鉴定结果。C:免疫印记实验分析NF-YA敲除小鼠肝脏中NF-YA的含量。
图2:NF-YA基因敲除小鼠体重、摄食量检测;A:小鼠体重随周龄变化情况;B:小鼠每天平均摄食量。
图3:NF-YA基因敲除小鼠肝脏解剖图。
图4:NF-YA基因敲除小鼠肝脏病理学检测;A:HE染色。B:Masson染色。C:Siriusred染色。
图5:NF-YA基因敲除小鼠肝脏纤维化相关指标检测;A:免疫印记实验分析星状细胞活化标志蛋白α-SMA和胶原酶I(Collagen-1)。B:免疫组化染色检测α-SMA蛋白。C:Realtime qPCR定量分析肝纤维化相关基因的表达。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
NF-YA基因:即Nuclear transcription factor Y subunit alpha,Gene ID:18044。
NF-YA基因的第3-8个外显子序列:从NF-YA基因第3外显子至第8外显子。
Alb-Cre基因:由Albumin基因启动子驱动的Cre重组酶基因表达,Albumin(Alb):Gene ID:11657;Cre:Gene ID:2777477。
实施例1、本发明肝纤维化模型的构建
以小鼠为目标动物,进行如下操作:
1、用Cre-loxP敲除技术一次性同步敲除小鼠NF-YA基因的第3-8个外显子序列得到NF-YALoxp/Loxp条件性纯合子小鼠。将转Alb-Cre基因小鼠和NF-YALoxp/Loxp条件性纯合子小鼠交配,后代有一半的动物同时携带Alb-Cre和NF-YA条件性敲除基因。此动物再与NF-YALoxp/Loxp条件性纯合子小鼠交配,可得到肝脏实质细胞NF-YA基因条件性敲除纯合子小鼠(NF-YALoxp/Loxp/Alb-Cre)(图1A),即作为本发明肝纤维化模型。
上述实施例的敲除策略是采用Cre-loxP敲除技术敲除NF-YA基因。但在其他的实施例中,实现基因敲除的技术手段有很多,例如CRISPR/Cas9技术、人工核酸酶介导的锌指核酸酶技术(zinc-finger nucleases,ZFN)、转录激活因子样效应物核酸酶技术(transceription activator-like effector nucleases,TALEN)等。因此,在其他的实施例中采用CRISPR/Cas9技术或其他的技术手段敲除NF-YA基因,也是属于本发明的保护范围。
上述实施例选用小鼠作为目标动物,但无论选用何种动物,只要是具有NF-YA基因的动物,均可作为本发明所述构建方法中的目标动物,在其肝脏实质细胞中敲除NF-YA基因,使其表现出肝脏纤维化特征,作为肝脏纤维化疾病模型,用于肝脏纤维化疾病研究领域中,均属于本发明的保护范围。
以下通过实验例证明本发明方法的有益效果。
实验例1、肝脏NF-YA基因条件性敲除小鼠的基因鉴定
1、实验方法
(1)小鼠基因组DNA提取:剪取本发明实施例1得到的肝脏NF-YA基因条件性敲除小鼠的尾尖,提取基因组DNA。
(2)PCR扩增:PCR反应体系包括2μL 2×Master Mix;2μL基因组DNA;引物1(NF-YAF1或F2或Alb F)和引物2(NF-YA R或Alb R)各1μL;6μL ddH2O。PCR反应程序:94℃预变性2min后,94℃变性3min,55℃退火30s,72℃延伸30s,25-40个循环后,在72℃延伸20min,最后于4℃保存。引物序列:NF-YA F1,5’-GTAAGTCAGGCTCCAGGG-3’;NF-YA F2,5'AGGCAAGGCAGATTTAGGAAGGTC-3';NF-YA R,5'-GGGTTGTCAGGATGTTCGCAG-3';Alb F,5’-ATTTGCCTGCATTACCGGT-3’;Alb R,5’-ATCAACGTTTTCTTTTCGG-3’。
(3)凝胶电泳:取10μl PCR产物于加样孔中,140V恒压、1.5%的琼脂糖凝胶电泳15min后,用凝胶成像系统成像。
2、实验结果
条件性纯合子NF-YALoxp/Loxp的条带大小为250bp,Alb-Cre大小为290bp。从图1B的本发明实施例1的NF-YA条件性敲除鉴定结果可以看出,本发明方法构建的NF-YA条件性敲除小鼠同时携带Alb-cre和NF-YALoxp/Loxp条件性纯合子小鼠,即成功得到了肝脏NF-YA基因条件性敲除小鼠(NF-YAloxp/loxp/Alb-Cre)。
以上结果说明本发明方法可快速、易操作地成功建立肝脏NF-YA基因条件性敲除的动物模型。
实验例2、免疫印记实验(Western blot)检测肝脏实质细胞NF-YA基因条件性敲除小鼠肝脏中NF-YA蛋白表达
1、实验方法
(1)制备肝脏组织蛋白质样品。分离实施例2肝脏NF-YA基因条件性敲除纯合子小鼠(NF-YALLoxp/Loxp/Alb-Cre)的肝脏组织,同时分离NF-YAwt/wt野生型基因小鼠、NF-YALoxp/Loxp条件性纯合子小鼠、NF-YALoxp/wt/Alb-Cre的肝脏组织作为对照,在肝脏组织中加入蛋白裂解液RIPA,经超声破碎、冰上裂解20min后,于4℃、12000g离心10min。吸取上清,采用BCA法测定上清液中蛋白的浓度。
(2)聚丙烯酰胺凝胶电泳(SDS-PAGE)分离蛋白。取20μL蛋白质样品,80V恒压电泳15min左右,把电压调整到150~200V,直到溴酚蓝跑出分离胶为止。
(3)转膜。待SDS-PAGE结束后,切掉浓缩胶,分离胶放到转膜缓冲液中。裁剪适当大小的硝酸纤维素膜,按顺序铺上滤纸、胶、硝酸纤维素膜及滤纸,并赶去气泡,转膜槽放入冰水浴中,在100V恒压条件下转膜1.5~2h。
(4)抗体孵育。转膜完毕后,用5%的脱脂牛奶封闭2h。封闭完成后,加入一定量的按一定比例(按照抗体使用说明书)稀释于封闭液的一抗,4℃孵育过夜。用1×TBST缓冲液洗膜3次(每次10min),根据一抗来源,选择合适二抗,用1×TBST稀释辣根过氧化氢酶(HRP)标记的二抗,缓慢摇晃孵育40-60min。
(5)显影。二抗孵育结束后,用1×TBST洗膜3次(每次10min),将PVDF膜放置在玻璃板上,滴加适量的ECL显影液(Thermo公司)显影拍照。
2、实验结果
如图1C所示,结果表明本发明方法构建的模型小鼠肝脏的NF-YA水平显著降低,说明本发明方法的确制备得到了肝脏NF-YA基因条件性敲除的动物模型。
实验例3、本发明方法建模小鼠对生长发育的影响
1、小鼠体重监测
选取9周龄的正常(NF-YALoxp/Loxp)雄性小鼠6只作为对照组,同时选取实施例2的NF-YA基因条件性敲除纯合子(NF-YALoxp/Loxp/Alb-Cre)雄性小鼠6只,分别记录初始体重。在后续的连续9周,每周称重并记录。
结果见图2A。与对照小鼠相比,在检测周期内(9-18周),NF-YA肝脏敲除小鼠与野生型小鼠的体重没有显著差异。因此敲除小鼠的生长状况没有发生改变。
2、小鼠摄食量监测
不同基因型的小鼠单笼饲养,每组5-6只小鼠,喂食前,饲料要称重并记录,第二天称量剩余的饲料,记录消耗饲料量。同样的方法持续记录一周,然后计算小鼠一周平均每天的摄食量。
结果见图2B。与对照小鼠相比,NF-YA肝脏敲除小鼠与对照小鼠的体重没有显著差异,说明本建模方法不会引起小鼠的不良反应,动物存活率高。
实验例4、本发明方法建模小鼠对肝脏形态的影响
1、小鼠肝脏解剖学观察
选取10周龄、体重在20.00g~23.00g左右的实施例2得到的肝脏NF-YA基因条件性敲除纯合子小鼠(NF-YALoxp/Loxp/Alb-Cre)的雄性小鼠。解剖后取小鼠的肝脏,进行观察。
结果见图3。与正常小鼠(NF-YALoxp/Loxp)肝脏相比,NF-YA肝脏敲除小鼠(NF-YALoxp /Loxp/Alb-Cre)的肝脏表面有肉眼可见的损伤痕迹,其肝脏出现明显纹理分膈、纤维增生、肝叶边缘锐化,疑似出现假小叶。
2、小鼠肝脏组织病理学检测
制作肝脏组织的固定和石蜡切片,并对其进行HE染色,Sirius red染色、Masson染色。
结果见图4A图4B和图4C。
肝脏组织HE染色发现:正常小鼠(NF-YALoxp/Loxp)肝组织中,肝小叶是类圆形体,内部为中央静脉,其不远处为门静脉、及小关组成的汇管区,肝细胞放射性排列形成肝索,肝细胞核为多边形,包浆丰富。本发明建模的NF-YA肝脏敲除小鼠(NF-YALoxp/Loxp/Alb-cre)的肝小叶失去正常形态,肝小叶的界板破坏明显,形成假小叶,在中央静脉处聚集;肝细胞核存在点状坏死,肝细胞数量明显增加。通过Masson和Sirius red染色,发现本发明建模的NF-YA肝脏敲除小鼠相较于正常小鼠,可以明显看到胶原纤维沿界板破坏处向肝小叶沿伸。
上述结果说明本发明方法建模小鼠表现出典型的肝脏纤维化疾病相关特征,本发明方法可高效、稳定地构建肝脏纤维化动物模型。
实验例5、本发明方法建模小鼠的肝纤维化相关指标验证
检测实施例2的肝脏NF-YA基因条件性敲除纯合子小鼠(NF-YALoxp/Loxp/Alb-cre)肝纤维化相关指标,与正常小鼠(NF-YALoxp/Loxp)对照。
1.免疫印记实验分析星状细胞活化标志蛋白α-SMA和胶原酶I(Collagen-1),方法同实施例1.3。
2.免疫组化染色检测小鼠肝脏组织α-SMA蛋白分布情况。
肝脏组织石蜡切片经脱蜡和复水后,进行抗原修复和封闭。然后加入α-SMA抗体于4℃过夜孵育。并用DAB染色法进行细胞核染色,经中性树脂或荧光封片剂封片后,用Confocal观察。
3.Real time定量PCR(qPCR)定量分析肝纤维化相关基因的表达
(1)提取小鼠肝脏总RNA,取3μl RNA样品进行琼脂糖凝胶电泳检测完整性。取2μlRNA样品,在nanodrop 2000上测定分析RNA纯度和浓度。
(2)RNA逆转录成cDNA
(3)实时荧光定量PCR(qPCR)检测基因表达
结果见图5A图5B和图5C。
相较于正常小鼠,NF-YA肝脏敲除小鼠肝脏中纤维化标志蛋白α-SMA和胶原酶I(Collagen-1)表达显著升高(图5A)。同时,在纤维沿肝索方向沿伸处α-SMA显著表达(图5B),星状细胞异常活化,提示肝纤维化发生。qPCR检测发现Ⅲ型胶原蛋白Collagen-3表达升高,基质金属蛋白(MMP)是胞外基质(ECM)降解的关键基因,NF-YA肝脏敲除小鼠肝脏组织MMP2和MMP9的基因表达显著降低,但是抑制基质金属蛋白(TIMP)TIMP-1和TIMP-2则显著升高(图5C)。
以上结果说明:本发明方法构建的肝脏纤维化动物模型稳定,成型率高。
综上,本发明实施例以小鼠为例,通过Cre-LoxP敲除技术在其肝脏实质细胞中特异性敲除NF-YA基因,使小鼠肝脏表面有明显的纹理分膈、纤维增生、肝叶边缘锐化、肝小叶界板破坏明显、形成假小叶。这些特征都是肝脏纤维化疾病的典型特征。由此充分说明,在肝脏实质细胞条件性敲除NF-YA基因,可以使目标动物表现出肝脏纤维化疾病。肝脏实质细胞条件性敲除NF-YA基因的动物,可以作为肝脏纤维化疾病模型。该疾病模型可以用于视网膜新生血管性疾病研究等领域中,为该疾病的研究例如发病过程、机制以及相关药物的筛选提供一种新的模型。
Claims (10)
1.一种肝脏纤维化动物模型的构建方法,其特征在于,包括如下步骤:通过基因工程技术,敲除动物肝脏实质细胞基因组中的NF-YA基因或者NF-YA基因的基因片段。
2.如权利要求1所述的构建方法,其特征在于,所述NF-YA基因的基因片段是NF-YA基因的外显子序列,优选地,所述NF-YA基因的外显子序列是指NF-YA基因上第3、第4、第5、第6、第7和第8外显子中的任意一个或多个外显子序列。
3.如权利要求1或2所述的构建方法,其特征在于,所述基因工程技术为基因编辑技术、基因敲除技术、RNA干扰技术中的任意一种技术或多种技术的组合,优选为基因敲除技术。
4.如权利要求1~3任一项所述的构建方法,其特征在于,包括如下步骤:
(1)通过基因敲除技术敲除目标动物肝脏实质细胞中的基因组中的NF-YA基因第3、第4、第5、第6、第7或第8外显子中的任意一个或多个外显子序列,得到NF-YA基因条件性纯合子动物NF-YALoxp/Loxp;
(2)将步骤(1)得到的NF-YA基因条件性纯合子动物与转Alb-Cre基因动物交配得到后代,将后代中同时携带Alb-Cre基因和NF-YA条件性敲除基因的动物再与步骤(1)得到的NF-YA基因条件性纯合子动物交配,即可得到肝脏实质细胞NF-YA基因条件性敲除的纯合子动物,作为肝脏纤维化动物模型。
5.根据权利要求1~4任一项所述的构建方法,其特征在于,所述目标动物选自小鼠、大鼠、狗、猪、猴、猿中的任意一种。
6.一种肝脏纤维化动物模型,其特征在于,它是NF-YA基因在肝脏实质细胞中不表达或表达受到抑制的动物模型,优选地,它是肝脏实质细胞中的基因组中的NF-YA基因敲除的动物模型。
7.如权利要求6所述的动物模型,其特征在于,它是通过权利要求1~5任一项所述的构建方法构建得到的。
8.动物肝脏实质细胞基因组中的NF-YA基因或者NF-YA基因的基因片段沉默或缺失的动物模型作为肝脏纤维化动物模型的用途。
9.权利要求6或7所述的肝脏纤维化动物模型在肝脏纤维化疾病研究中的应用,所述研究是以非疾病的诊断或治疗为目的。
10.权利要求6或7所述的肝脏纤维化动物模型在筛选用于预防或治疗肝脏纤维化疾病药物中的应用。
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