CN113424800B - 一种免疫检查点抑制剂相关心肌炎小鼠模型及构建方法 - Google Patents
一种免疫检查点抑制剂相关心肌炎小鼠模型及构建方法 Download PDFInfo
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Abstract
本发明涉及一种免疫检查点抑制剂相关心肌炎小鼠模型及构建方法,属于医学动物模型技术领域。本发明通过给予小鼠皮下注射小鼠心肌肌钙蛋白I肽段的完全弗氏佐剂和给予小鼠腹腔注射PD‑1抑制剂构建了一种免疫检查点抑制剂相关心肌炎小鼠模型;本发明通过确定合理的剂量和给药间隔,有效地提高了建模的成功率,且模型构建方法简单易行,易于推广应用。通过本发明构建的一种免疫检查点抑制剂相关心肌炎小鼠模型,为临床免疫检查点抑制剂相关心肌炎发病机制的研究,相关心肌炎的治疗药物的筛选提供了可实验的对象。
Description
技术领域
本发明涉及一种免疫检查点抑制剂相关心肌炎小鼠模型及构建方法,属于医学动物模型技术领域。
背景技术
免疫检查点抑制剂(immune checkpoint inhibitors,ICIs)是近年来肿瘤免疫疗法的一种新的治疗选择,其通过激活免疫系统将表达肿瘤抗原的肿瘤细胞杀死,达到抗肿瘤的目的。然而,由于免疫检查点在自身免疫耐受中也发挥着重要的作用,因此ICIs激活免疫系统会引起广泛的自身免疫反应,称为免疫相关不良反应(immune-related adverseeffects,irAEs)。与肿瘤抗原相关的免疫检查点包括细胞毒性T淋巴细胞相关抗原4(cytotoxic T lymphocyte associated antigen-4,CTLA-4)、免疫检查点程序性死亡分子1(programmed death-1,PD-1)及程序性死亡分子配体1(programmed cell death-ligand1, PD-L1)。其中,ICIs引起的心脏毒性虽然十分罕见,但却是最致命的irAEs之一。现有的临床研究数据提示,由ICIs引起的免疫性心肌炎发病率从0.09%到 1.14%不等,提示免疫性心肌炎的发生可能已经比预计的更为普遍。另外,ICIs 引起的免疫性心肌炎其临床表现为非特异性的,其严重程度也从无症状的心肌损伤标志物的升高、疲劳、全身不适的亚临床症状到胸痛、呼吸困难、多器官衰竭、心源性休克和心脏骤停不等,目前亟需进一步研究其发病机制为临床防治提供有效的靶点。
由于临床免疫检查点抑制剂相关心肌炎的发病率低,致死性高,目前已有的小鼠心肌炎模型包括自身免疫性心肌炎模型及PD-1抑制剂诱导的心肌损伤模型,但现有的模型引起的心肌炎成模率低、病变范围小,均不能很好的模拟临床免疫检查点抑制剂相关心肌炎的临床表现,且在发病机制上也不尽相同。然而,随着ICIs在肿瘤治疗中的兴起,临床免疫检查点抑制剂相关心肌炎的发生率也逐渐升高,亟需一种能模拟其发病情况的小鼠模型,为其机制的研究及药物的筛选评价建立基础。
发明内容
本发明的目的是为解决如何获得一种免疫检查点抑制剂相关心肌炎小鼠模型的技术问题。
为达到解决上述问题的目的,本发明所采取的技术方案是提供一种免疫检查点抑制剂相关心肌炎小鼠模型的构建方法,包括以下步骤:
步骤1:选取雄性BALB/c小鼠,6周龄,体重20-25g,分别于第0天和第7 天给予小鼠皮下注射0.1ml含有0.25mg小鼠心肌肌钙蛋白I肽段的完全弗氏佐剂;
步骤2:自第7天起,每2天给予小鼠腹腔注射PD-1抑制剂,剂量为5mg/kg/ 次;
步骤3:共注射PD-1抑制剂5次,获得免疫检查点抑制剂相关心肌炎小鼠模型。
优选地,上述步骤1中小鼠心肌肌钙蛋白I(troponin I,TnI)肽段的氨基酸序列为SEQ ID NO:1;
SEQ ID NO:1序列为:
HARVDKVDEERYDVEAKVTKNITEIADLTQKIYDLRGKFKRPTLRRVRIS。
本发明提供一种免疫检查点抑制剂相关心肌炎小鼠模型,采用如上述的一种免疫检查点抑制剂相关心肌炎小鼠模型的构建方法构建。
本发明提供一种免疫检查点抑制剂相关心肌炎小鼠模型在针对免疫检查点抑制剂相关心肌炎的发病机制的研究中的应用。
本发明提供一种免疫检查点抑制剂相关心肌炎小鼠模型在针对免疫检查点抑制剂相关心肌炎的治疗药物的筛选中的应用。
本发明提供一种免疫检查点抑制剂相关心肌炎小鼠模型在针对免疫检查点抑制剂相关心肌炎的药物疗效评价中的应用。
相比现有技术,本发明具有如下有益效果:
本发明通过多次实验,确定了合理的剂量和给药间隔,从而有效地提高了建模的成功率,且模型构建方法简单易行,技术手段相对容易,易于推广应用。本建模方法所用的试剂无显著毒性,不会引起额外的药物不良反应。此外,本小鼠模型也避免了构建嵌合体小鼠模型所引起的重型免疫缺陷。相较于既往模型,本发明具有更显著的心肌炎表现及心肌损伤标志物的异常。
本发明构建的一种免疫检查点抑制剂相关心肌炎小鼠模型,为今后临床上免疫检查点抑制剂相关心肌炎发病机制的研究,相关心肌炎的治疗药物的筛选提供了可实验的对象。借助本发明能够为免疫检查点抑制剂相关心肌炎的防治研究提供更多的理论和实验依据。
附图说明
图1为各试验组的小鼠急性心肌炎炎症期心脏组织HE染色结果。
图2为各试验组小鼠肌酸激酶水平变化图。
图中横坐标为各试验组;纵坐标为肌酸激酶(CK)含量。
图3为各试验组小鼠肌酸激酶同工酶水平变化图。
图中横坐标为各试验组;纵坐标为肌酸激酶同工酶(CK-MB)含量。
图4为各试验组小鼠于造模第56天彩色多普勒超声实时影像图。
图5为各试验组小鼠于造模第56天扩张型心肌病期心脏功能变化图。
图中横坐标为各试验组;纵坐标为左室射血分数。
具体实施方式
为使本发明更明显易懂,兹以优选实施例,并配合附图作详细说明如下:
如图1-5所示,本发明提供一种免疫检查点抑制剂相关心肌炎小鼠模型的构建方法,包括以下步骤:
步骤1:选取雄性BALB/c小鼠,6周龄,体重20-25g,分别于第0天和第7 天给予小鼠皮下注射0.1ml含有0.25mg小鼠心肌肌钙蛋白I肽段的完全弗氏佐剂;
步骤2:自第7天起,每2天给予小鼠腹腔注射PD-1抑制剂,剂量为5mg/kg/ 次;
步骤3:共注射PD-1抑制剂5次,获得免疫检查点抑制剂相关心肌炎小鼠模型。
上述步骤1中小鼠心肌肌钙蛋白I(troponin I,TnI)肽段的氨基酸序列为SEQ IDNO:1。即TnI肽段序列为:
HARVDKVDEERYDVEAKVTKNITEIADLTQKIYDLRGKFKRPTLRRVRIS。
本发明提供一种免疫检查点抑制剂相关心肌炎小鼠模型,采用如上述的一种免疫检查点抑制剂相关心肌炎小鼠模型的构建方法构建。
本发明提供一种免疫检查点抑制剂相关心肌炎小鼠模型在针对免疫检查点抑制剂相关心肌炎的发病机制的研究中的应用。
本发明提供一种免疫检查点抑制剂相关心肌炎小鼠模型在针对免疫检查点抑制剂相关心肌炎的治疗药物的筛选中的应用。
本发明提供一种免疫检查点抑制剂相关心肌炎小鼠模型在针对免疫检查点抑制剂相关心肌炎的药物疗效评价中的应用。
本发明提供一种免疫检查点抑制剂相关心肌炎小鼠模型的构建方法,包括以下步骤:选取雄性BALB/c小鼠,6周龄,体重20-25g,分别于第0天和第7天给小鼠皮下注射0.1ml含有0.25mg小鼠心肌肌钙蛋白I(troponin I,TnI)肽段的完全弗氏佐剂。
TnI肽段序列为HARVDKVDEERYDVEAKVTKNITEIADLTQKIYDLRGKFKRPTLRRVRIS,由生工生物工程(上海)股份有限公司合成。
完全氟式试剂购自美国Sigma公司。
自第7天起,每2天给予腹腔注射PD-1抑制剂(InVivoMab anti-mouse PD-1) 剂量为5mg/kg/次,共5次,得到免疫检查点抑制剂相关心肌炎小鼠模型。
既往文献报道,TnI是引起免疫检查点抑制相关心肌炎的主要自身免疫反应抗原,因此,通过构建小鼠TnI肽段,诱导小鼠自身免疫性心肌炎。本发明提供的模型在此基础上增加了TnI的剂量,并通过腹腔注射PD-1抑制剂的方式模拟临床肿瘤的免疫治疗,通过抑制免疫检查点的作用,激活自身免疫反应,使得小鼠体内生物学变化更接近于临床病人的真实情况。
实施例
1.实验方法:
1)选取30只雄性6周龄BALB/c小鼠共分为3组,每组10只:分为对照组 (control)、自身免疫性心肌炎组(TnI组),ICI相关心肌炎组(TnI+anti-PD-1 组);除对照组外,分别于第0天和第7天给小鼠皮下注射0.1ml含有0.25mg 小鼠心肌肌钙蛋白I(troponin I,TnI)肽段的完全弗氏佐剂。ICI相关心肌炎组自第7天起,每2天给予腹腔注射PD-1抑制剂(InVivoMab anti-mouse PD-1) 剂量为5mg/kg/次,共5次。各组小鼠分别于第21天和第56天检测急性心肌炎炎症期和扩张型心肌病期心脏功能及分子水平变化。
2)采用小鼠心脏固定后石蜡包埋切片,HE染色评估心脏炎性表现;
3)采用Vevo2100超高分辨率小动物彩色多普勒超声实时影像系统检测小鼠左心室收缩功能,主要指标:左室射血分数(Left Ventricular Ejection Fractions,LVEF);
4)采用ELISA法检测外周血血浆中心肌酶谱水平,包括肌酸激酶(Creatinekinase,CK)、肌酸激酶同工酶(creatine kinase-MB,CK-MB);
5)统计分析:所有数据均表示为平均值±标准差(SD)。多组间分析采用单因素方差分析(ANOVA),使用Graphpad Prism 8.0软件进行Turkey法检验统计分析,*p<0.05被设定为具有统计学差异。
实验结果:
1)各组小鼠急性心肌炎炎症期心脏组织HE染色结果如图1所示,control 组小鼠心肌细胞排列整齐,心肌组织致密;TnI组可见心外膜下少量炎症细胞浸润;心肌细胞形态结构尚完整;TnI+anti-PD-1组可见心外膜下大量炎症细胞浸润,心肌细胞边界不清,心肌细胞坏死,间隙水肿形成。显微镜放大倍数为400 倍。
2)各组小鼠心肌酶谱水平变化如图2、3所示,TnI+anti-PD-1组诱导后引起小鼠血浆CK、CK-MB水平显著性升高,且较TnI组升高更为显著。
3)各组小鼠于造模第56天扩张型心肌病期心脏功能变化如图4、5所示。 TnI+anti-PD-1组小鼠相较于control组,左室EF显著下降,且较TnI组下降更为显著。
试验结论:
建模成功,获得免疫检查点抑制剂相关心肌炎小鼠模型。
以上所述,仅为本发明的较佳实施例,并非对本发明任何形式上和实质上的限制,应当指出,对于本技术领域的普通技术人员,在不脱离本发明的前提下,还将可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员,在不脱离本发明的精神和范围的情况下,当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化,均为本发明的等效实施例;同时,凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变,均仍属于本发明的技术方案的范围内。
序列表
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Claims (5)
1.一种免疫检查点抑制剂相关心肌炎小鼠模型的构建方法,其特征在于:包括以下步骤:
步骤1:选取雄性BALB/c小鼠,6周龄,体重20-25g,分别于第0天和第7天给予小鼠皮下注射0.1ml含有0.25mg小鼠心肌肌钙蛋白I肽段的完全弗氏佐剂;小鼠心肌肌钙蛋白I肽段的氨基酸序列为SEQ ID NO:1;
步骤2:自第7天起,每2天给予小鼠腹腔注射PD-1抑制剂,剂量为5mg/kg/次;
步骤3:共注射PD-1抑制剂5次,获得免疫检查点抑制剂相关心肌炎小鼠模型。
2.一种免疫检查点抑制剂相关心肌炎小鼠模型,其特征在于:采用如权利要求1所述的一种免疫检查点抑制剂相关心肌炎小鼠模型的构建方法构建。
3.如权利要求2所述的一种免疫检查点抑制剂相关心肌炎小鼠模型在针对免疫检查点抑制剂相关心肌炎的发病机制的研究中的应用。
4.如权利要求2所述的一种免疫检查点抑制剂相关心肌炎小鼠模型在针对免疫检查点抑制剂相关心肌炎的治疗药物的筛选中的应用。
5.如权利要求2所述的一种免疫检查点抑制剂相关心肌炎小鼠模型在针对免疫检查点抑制剂相关心肌炎的药物疗效评价中的应用。
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