CN118020714A - 一种移植相关血栓性微血管病小鼠模型的构建方法和应用 - Google Patents
一种移植相关血栓性微血管病小鼠模型的构建方法和应用 Download PDFInfo
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Abstract
本发明公开了一种移植相关血栓性微血管病小鼠模型的构建方法和应用,所述构建方法为:先构建小鼠异基因造血干细胞移植模型,移植后第14天腹腔注射小剂量LPS+H2O2诱导,即得。本发明通过多种实验手段验证了小剂量LPS与H2O2联合诱导TA‑TMA模型的可行性,填补了TA‑TMA小鼠模型研究领域的空白,并为深入研究TA‑TMA的发病机理和治疗策略提供了实验工具和平台。本发明有助于推动TA‑TMA相关药物研究的进展,为临床上治疗TA‑TMA提供新的思路和方法,具有重要的实际应用价值。
Description
技术领域
本发明属于动物模型构建技术领域,具体涉及一种移植相关血栓性微血管病小鼠模型的构建方法和应用。
背景技术
移植相关血栓性微血管病(TA-TMA)是一类造血干细胞移植(HSCT)后的严重的血栓并发症。其病因尚不清楚,目前认为TA-TMA的发病可能与补体系统激活,形成膜攻击复合物引起内皮受损有关。TA-TMA与血栓性血小板减少性紫癜(TTP)、非典型溶血尿毒综合征(aHUS)有着类似的临床特征,主要表现为微血管性溶血性贫血、外周血血小板减少、乳酸脱氢酶升高、血肌酐升高、外周血破碎红细胞增多、微循环纤维蛋白沉积、微血管血栓形成,最终导致多器官功能衰竭。回顾既往文献,TA-TMA的发病率为0.5%~76%,一项大型综述发现死亡率为75%,大多数患者在TA-TMA诊断后3个月内死亡。它影响肾脏、胃肠道、中枢神经系统、心脏、肺和浆膜表面等。TA-TMA发生的危险因素包括钙调神经磷酸酶抑制剂、移植物抗宿主病(GVHD)、感染、静脉血栓栓塞疾病、ABO血型不相容、人类白细胞抗原不匹配等。
TA-TMA的治疗基于对潜在病理生理学的认识,分为预防措施和支持治疗。预防措施,如避免内皮毒素,避免感染,优化移植预处理方案,可最大限度地减少内皮损伤。积极的支持治疗包括尽量减少输血、控制高血压和任何潜在感染,支持治疗是TA-TMA治疗的核心。其他方法包括停用钙调神经磷酸酶抑制剂、治疗性血浆交换(TPE)、利妥昔单抗、去纤肽和依库珠单抗,疗效和生存效益各不相同。
目前治疗TA-TMA的药物仅依靠探索性的临床病例应用,而TA-TMA的小鼠模型方面仍是空缺。
发明内容
针对现有技术的不足,本发明提供一种移植相关血栓性微血管病小鼠模型的构建方法和应用,通过应用小剂量的炎症加氧化应激构建TA-TMA小鼠模型,填补了TA-TMA小鼠模型的空白,为探究治疗TA-TMA的药物研究提供了可靠的技术支持。
本发明是通过以下技术方案实现的:
一种移植相关血栓性微血管病小鼠模型的构建方法,包括以下步骤:
步骤1)构建小鼠异基因造血干细胞移植模型:选择不同品种的小鼠分别作为受体和供体;移植前第7天开始每天对受体小鼠给予庆大霉素+左氧氟沙星的酸化水喂养,含有抗生素的酸化水持续喂养至移植后第7天,之后更改为普通酸化水继续喂养;移植当天接受6.5Gy致死性辐照,辐照后4~6h内回输供体小鼠来源的骨髓细胞,完成造血干细胞移植;
步骤2)构建移植相关血栓性微血管病小鼠模型:移植后第14天,小鼠血象基本重建,此时先对小鼠腹腔注射3% H2O2,30min后,再腹腔注射脂多糖LPS,即得。
优选地,所述步骤1)中,作为受体的小鼠为6~8周野生型Balb/C小鼠,作为供体的小鼠为6~8周野生型C57BL/6小鼠。
优选地,步骤1)所述庆大霉素的剂量为40万U/L,所述左氧氟沙星的浓度为50mg/L。
优选地,步骤1)所述骨髓细胞为去除红细胞,有核细胞数为5×106个的骨髓细胞。
优选地,步骤2)所述3% H2O2的注射量为5mL/kg;所述脂多糖LPS的注射量为2.5mg/kg。
优选地,步骤2)所述脂多糖LPS来源于大肠杆菌0111:B4。
上述构建方法构建得到的小鼠模型在制备治疗移植相关血栓性微血管病的药物中的应用。
上述构建方法构建得到的小鼠模型在筛选治疗移植相关血栓性微血管病的药物中的应用。
上述构建方法构建得到的小鼠模型在与移植相关血栓性微血管病有关的药物药效、副作用研究中的应用。
上述构建方法构建得到的小鼠模型在移植相关血栓性微血管病发生、发展的病因及病理机制的研究中的应用。
本发明的有益效果如下:
本发明通过综合运用小鼠生存情况观察、血小板计数分析、生化指标检测、外周血涂片查找破碎红细胞以及肾脏切片HE染色等多种实验手段,全面验证了小剂量LPS与H2O2联合诱导TA-TMA模型的可行性,填补了TA-TMA小鼠模型研究领域的空白,并为深入研究TA-TMA的发病机理和治疗策略提供了实验工具和平台。通过本发明的方法构建的TA-TMA小鼠模型,研究人员能够更加准确地模拟人类TA-TMA疾病的病理过程,从而有助于揭示其发病机制和病理生理变化。同时,该模型还可用于评估不同药物对TA-TMA的治疗效果,为药物筛选和优化提供可靠依据。此外,本发明所建立的TA-TMA小鼠模型具有操作简便、重复性好、稳定性高等优点,适用于大规模的药物筛选和深入研究。因此,本发明有助于推动TA-TMA相关药物研究的进展,为临床上治疗TA-TMA提供新的思路和方法,具有重要的实际应用价值。
附图说明
图1为构建TA-TMA小鼠模型及对照组的流程图;
图2为小鼠生存预后分析;
图3为小鼠腹腔注射药物4h后外周血血小板计数;
图4为小鼠腹腔注射药物4h后外周血生化指标:a为小鼠血清肌酐统计图;b为小鼠血清乳酸脱氢酶统计图;
图5为小鼠外周血涂片刘氏染色100×镜观察图:红色箭头所指为破碎红细胞和异性红细胞;
图6为小鼠组织脏器HE染色观察图:a为肾小球100×镜观察图,红色箭头所指为纤维样物质沉积,红色三角所指为血小板血栓;b为肺部20×镜观察图,红色箭头所指为炎细胞浸润;c为肺部100×镜观察图,红色箭头所指为炎细胞浸润,红色三角所指为血小板聚集。
具体实施方式
下面结合附图与具体实施例对本发明做进一步详细说明。
以下实施例中涉及的实验鼠来源如下:
野生型Balb/C小鼠和野生型C57BL/6小鼠均购自北京维通利华实验动物技术有限公司。
实施例1
TA-TMA是一种因微血管内血栓形成而导致多脏器损伤的综合征,临床上以微血管溶血性贫血(伴红细胞碎片)、外周血小板减少为主要表现,并且常伴有急性肾功能损伤和中枢神经系统异常,是移植后的严重并发症,与移植病人的预后密切相关。辛辛那提儿童医院的Jodele教授对TA-TMA患者的RNA-seq数据进行差异分析显示,TA-TMA患者存在炎症、氧化应激的改变。TA-TMA患者的中位发病时间为造血干细胞移植后第40天,造血已基本重建。因此我们在小鼠异基因造血干细胞移植模型的基础上,待其造血基本重建时,给予小剂量的炎症与氧化应激,检测TA-TMA的相关指标,判断小鼠是否发病。具体方法如下:
1、TA-TMA小鼠模型的构建
(1)构建小鼠异基因造血干细胞移植模型
选择6~8周SPF级Balb/C野生型雌性小鼠作为受体,6~8周SPF级C57BL/6野生型雌性小鼠作为供体。移植前第7天开始每天对受体小鼠给予庆大霉素(40万U/L)+左氧氟沙星(50mg/L)的酸化水喂养,含有抗生素的酸化水持续喂养至移植后第7天,之后更改为普通酸化水继续喂养。移植当天接受6.5Gy致死性(清髓性)辐照,辐照后4~6h内回输供体小鼠来源的骨髓细胞(去除红细胞,有核细胞数5×106)。
(2)移植后造血重建评估
移植后第14天,取小鼠外周血测外周血三分类,经检测,小鼠白细胞、血红蛋白、血小板基本重建,可以作为TA-TMA小鼠模型的基线数值。
(3)构建TA-TMA小鼠模型
如图1所示,移植后第14天,血象基本重建,进行实验分组,分别为诱导TA-TMA组(LPS+H2O2)、H2O2组、LPS组、对照组(PBS),具体如下:
①诱导TA-TMA组:腹腔注射3% H2O2 5mL/kg,30min后,腹腔注射小剂量脂多糖LPS(大肠杆菌0111:B4来源,下同)2.5mg/kg;
②H2O2组:腹腔注射3% H2O2 5mL/kg;
③LPS组:腹腔注射小剂量脂多糖LPS2.5mg/kg;
④对照组:腹腔注射PBS。
(4)给药后观察小鼠生存,另外小鼠腹腔给药4h后小鼠眼眶静脉采血,通过血小板计数、生化指标、外周血涂片找破碎红细胞、肾脏切片HE染色多个指标验证小鼠TA-TMA模型。
2、TA-TMA小鼠模型的验证分析
(1)小鼠生存状态观察
如图2所示,PBS对照组与H2O2组小鼠没有死亡,小鼠均存活到最终观察时间即腹腔注射后14天;小剂量LPS+H2O2诱导TA-TMA模型组小鼠最早在用药后7h内出现死亡,且所有小鼠均在24h内死亡,中位生存时间只有14h;小剂量LPS组小鼠从用药后2天开始出现死亡,在观察期14天内未达到中位生存。因此,与PBS对照组小鼠相比,小剂量LPS+H2O2诱导TA-TMA模型组小鼠生存期明显缩短(****P<0.0001)。
(2)小鼠外周血血小板计数
因H2O2组小鼠和PBS组均没有死亡,此后实验改为3组,分别为TA-TMA组(LPS+H2O2)、LPS组、对照组(PBS)。小鼠腹腔注射给药4h后,取小鼠眼眶内眦静脉血,测血常规记录小鼠血小板计数。
如图3所示,与腹腔注射PBS对照组相比,小剂量LPS+H2O2诱导TA-TMA模型组小鼠血小板计数明显下降(162±76vs.456±91×109/L,***P<0.001)。由此说明,小剂量LPS+H2O2组小鼠出现血小板减少。
(3)小鼠外周血生化指标
TA-TMA模型的微循环血栓同血栓性血小板减少性紫癜(TTP)五联症的临床表现类似,不仅包含发热、血小板计数,还有微血管病性溶血性贫血、肾功能损害、神经精神异常。其中肾功能损害,包含外周血生化指标中与肾脏相关的指标异常,以及肾脏病理活检发现微循环血栓。TA-TMA的各个版本的诊断标准中均纳入了乳酸脱氢酶升高。因此我们检测了小鼠外周血血清中肌酐(CREA)、乳酸脱氢酶(LDH)两个指标,如图4所示。
血清肌酐水平如图4中a所示,与腹腔注射PBS对照组相比,小剂量LPS+H2O2诱导TA-TMA模型组小鼠肌酐显著升高(50.39±5.39vs.31.29±0.92μmol/L,**P<0.01);单药LPS并不能升高血清肌酐水平(32.73±5.06vs.31.29±0.92μmol/L,P>0.05)。
血清乳酸脱氢酶水平如图4中b所示,与腹腔注射PBS对照组相比,小剂量LPS+H2O2诱导TA-TMA模型组小鼠乳酸脱氢酶显著升高(5554±1727vs.626±91U/L,**P<0.01);单药LPS对血清乳酸脱氢酶水平的改变无统计学差异(1171±653vs.626±91U/L,P>0.05)。
综合以上实验结果提示,小剂量LPS+H2O2诱导TA-TMA模型组小鼠血清肌酐和乳酸脱氢酶均升高,符合TA-TMA的生化指标。
(4)小鼠外周血涂片刘氏染色
随后我们将小鼠进行外周血涂片,给予刘氏染色后,显微镜下在单层细胞处寻找破碎红细胞。
如图5所示,红细胞是外周血涂片中最多的细胞,呈橘红色,中央淡染,符合双凹圆盘状的细胞形态。在小剂量LPS+H2O2诱导TA-TMA模型组外周血涂片染色可见破碎的红细胞及异形红细胞(红色箭头所指),其余2组破碎红细胞及异形红细胞均<1%。该实验进一步证明了以小剂量LPS+H2O2诱导TA-TMA模型的可行性。
(5)小鼠组织脏器HE染色
随后将小鼠颈椎脱臼处死,取小鼠肾脏及肺组织,用多聚甲醛固定后,进行石蜡包埋,切片后HE染色,显微镜下观察肾小球和肺组织,如图6所示。
肾小球大多数分布于肾皮质和靠近髓质部分,肾小球由肾小囊和囊内的毛细血管球组成。肾小球于100×镜(油镜)下观察,如图6中a所示,在小剂量LPS+H2O2诱导TA-TMA模型的小鼠肾脏中可见血小板微血栓(红色三角所指),局部有纤维样物质沉积(红色箭头所指);其余2组未见血小板血栓。
肺组织于20×镜下观察,如图6中b所示,与腹腔注射PBS组小鼠相比,小剂量LPS+H2O2诱导TA-TMA模型组小鼠肺组织结构明显受损,肺泡结构紊乱,大量炎细胞浸润(红色箭头所指)。
肺组织于100×镜下观察,如图6中c所示,与腹腔注射PBS组小鼠相比,小剂量LPS+H2O2诱导TA-TMA模型组小鼠肺部可见血小板聚集的微血栓(红色三角所指)及炎细胞浸润(红色箭头所指)。
以上组织切片染色表明,小剂量LPS+H2O2诱导TA-TMA模型证据完整,病理可见微血栓。
上述实验数据从小鼠生存、血小板计数、生化指标、外周血涂片找破碎红细胞、肾脏切片HE染色多个方面验证了小剂量LPS+H2O2诱导TA-TMA模型的可行性。该模型有助于揭示TA-TMA的发病机制和病理生理变化,还可用于评估不同药物对TA-TMA的治疗效果,为药物筛选和优化提供可靠依据,并为临床上治疗TA-TMA提供新的思路和方法。
以上所描述的实施例仅是本发明的一部分实施例,而不是全部的实施例。本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围,而是仅仅表示本发明的选定实施例。本发明的保护范围以权利要求书要求的范围为准,基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
Claims (10)
1.一种移植相关血栓性微血管病小鼠模型的构建方法,其特征在于,包括以下步骤:
步骤1)构建小鼠异基因造血干细胞移植模型:选择不同品种的小鼠分别作为受体和供体;移植前第7天开始每天对受体小鼠给予庆大霉素+左氧氟沙星的酸化水喂养,含有抗生素的酸化水持续喂养至移植后第7天,之后更改为普通酸化水继续喂养;移植当天接受6.5Gy致死性辐照,辐照后4~6h内回输供体小鼠来源的骨髓细胞,完成造血干细胞移植;
步骤2)构建移植相关血栓性微血管病小鼠模型:移植后第14天,小鼠血象基本重建,此时先对小鼠腹腔注射3%H2O2,30min后,再腹腔注射脂多糖LPS,即得。
2.根据权利要求1所述的一种移植相关血栓性微血管病小鼠模型的构建方法,其特征在于,所述步骤1)中,作为受体的小鼠为6~8周野生型Balb/C小鼠,作为供体的小鼠为6~8周野生型C57BL/6小鼠。
3.根据权利要求1所述的一种移植相关血栓性微血管病小鼠模型的构建方法,其特征在于,步骤1)所述庆大霉素的剂量为40万U/L,所述左氧氟沙星的浓度为50mg/L。
4.根据权利要求1所述的一种移植相关血栓性微血管病小鼠模型的构建方法,其特征在于,步骤1)所述骨髓细胞为去除红细胞,有核细胞数为5×106个的骨髓细胞。
5.根据权利要求1所述的一种移植相关血栓性微血管病小鼠模型的构建方法,其特征在于,步骤2)所述3%H2O2的注射量为5mL/kg;所述脂多糖LPS的注射量为2.5mg/kg。
6.根据权利要求1所述的一种移植相关血栓性微血管病小鼠模型的构建方法,其特征在于,步骤2)所述脂多糖LPS来源于大肠杆菌0111:B4。
7.如权利要求1-6任一项所述的一种移植相关血栓性微血管病小鼠模型的构建方法构建得到的小鼠模型在制备治疗移植相关血栓性微血管病的药物中的应用。
8.如权利要求1-6任一项所述的一种移植相关血栓性微血管病小鼠模型的构建方法构建得到的小鼠模型在筛选治疗移植相关血栓性微血管病的药物中的应用。
9.如权利要求1-6任一项所述的一种移植相关血栓性微血管病小鼠模型的构建方法构建得到的小鼠模型在与移植相关血栓性微血管病有关的药物药效、副作用研究中的应用。
10.如权利要求1-6任一项所述的一种移植相关血栓性微血管病小鼠模型的构建方法构建得到的小鼠模型在移植相关血栓性微血管病发生、发展的病因及病理机制的研究中的应用。
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