CN112889713B - 斑马鱼肺炎克雷伯菌感染模型、其构建方法及评价方法 - Google Patents
斑马鱼肺炎克雷伯菌感染模型、其构建方法及评价方法 Download PDFInfo
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Abstract
本案涉及一种斑马鱼肺炎克雷伯菌感染模型、其构建方法及评价方法,构建方法包括挑选发育正常的斑马鱼通过浸入细菌液的手段获得斑马鱼炎克雷伯菌感染模型,评价方法包括采用苏丹黑B和中性红染色观察炎症细胞的变化情况。本发明采用细菌浸入实验代替传统斑马鱼模型中的显微注射细菌液的感染方法,细菌浸入实验中斑马鱼的感染率高达90%;独创性的设计了对斑马鱼进行染色的方法,染色后可以显微镜下直观的观察到嗜中性粒细胞和巨噬细胞的变化,方便直观的对肺炎毒力做出评估;建立了完整的评价方法,可以充分评估肺炎克雷伯菌毒力和致病性。
Description
技术领域
本发明属于生物技术模型构建领域,具体涉及一种斑马鱼肺炎克雷伯菌感染模型、其构建方法及评价方法。
背景技术
肺炎克雷伯菌是一种革兰氏阴性菌,可侵害任何动物的肠道和呼吸道等器官,从而引起肺部感染、肝脓肿、败血症、脑膜炎、坏死性筋膜炎,严重时甚至导致死亡。肺炎的毒性有很多原因,主要是因为其表面胶囊、脂质多糖、皮利和铁运输系统。这些细菌的一些表型是不同抗原特性的基础,也是细菌对人体机理有害的原因。编码这些外部毒性表型的基因被称为毒力基因。
目前,有许多生物模型可以评估肺炎克雷伯菌的毒性,包括小鼠、大鼠、猪、盘基网柄菌和蜡螟,前三个价格昂贵,不适合高通量实验,后两种是无脊椎动物,实验数据参考量不是很大。斑马鱼是一种小型热带观赏鱼。斑马鱼具有产卵能力大、体外发育、胚胎透明等优点,已成为生物医学研究中继老鼠之后的第三大模型生物,由此,斑马鱼可以作为宿主来评估研究肺炎克雷伯菌的毒力和病理学机制。
发明内容
针对现有技术中的不足之处,本发明利用细菌浸入的手段获得了斑马鱼肺炎克雷伯菌感染模型并通过苏丹黑B和中性红染色来评价其炎症细胞感染效果。
为实现上述目的,本发明提供如下技术方案:
一种斑马鱼肺炎克雷伯菌感染模型的构建方法,包括:挑选发育正常的斑马鱼通过浸入细菌液的手段获得斑马鱼肺炎克雷伯菌感染模型。
进一步地,所述斑马鱼为受精3天后的斑马鱼幼鱼。
进一步地,所述细菌液的制备方法如下:将肺炎克雷伯菌细菌培养物生长到指数相OD600=0.4±0.6,离心、洗涤,随后分散于灭菌的E3培养液中,得到细菌液。
进一步地,所述浸入细菌液的手段具体为将斑马鱼浸入细菌液中,28.5℃下恒温孵育72h。
本发明提供一种如上所述的构建方法构建出的斑马鱼肺炎克雷伯菌感染模型。
本发明进一步提供一种使用如上所述的斑马鱼肺炎克雷伯菌感染模型对肺炎炎症细胞的评价方法,其特征在于,至少包括以下步骤:
1)对斑马鱼肺炎克雷伯菌感染模型进行巨噬细胞和中性粒细胞染色;
2)于显微镜下观察中性粒细胞和巨噬细胞的变化,以此获得对肺炎克雷伯菌感染斑马鱼模型后炎症细胞的评价结果;
其中,步骤1)中的巨噬细胞染色方法为:将斑马鱼肺炎克雷伯菌感染模型浸于4%甲醛溶液中,用PBST缓冲液冲洗,随后用苏丹黑B染色,观察巨噬细胞;
中性粒细胞染色方法为:将斑马鱼肺炎克雷伯菌感染模型浸于4%甲醛溶液中,用PBST缓冲液冲洗,之后用70%乙醇洗涤,置于2.5mg/L的中性红染料溶液中培养,观察中性粒细胞。
本发明的有益效果是:与现有技术相比,本发明对斑马鱼幼鱼的感染途径做出了改进,大大提高了感染效率,省时省力;独创性的设计了对斑马鱼的染色方法,可以直观的观察炎症细胞的变化情况;
一、通过受精3天后的斑马鱼幼鱼细菌浸入实验代替传统斑马鱼模型中的显微注射细菌液的感染方法,细菌浸入实验中斑马鱼的感染率高达90%,虽然不能像显微注射途径一样感染效率非常高,但是在对斑马鱼模型的群体研究中,未感染的10%左右的个体对实验结果的影响很小,可以忽略,却大大提高了感染效率,省时省力,能够高效,快速,高通量进行临床分离的克雷伯菌进行毒力评估。
二、在对感染后的斑马鱼进行炎症分析时,利用苏丹黑B和中性红对斑马鱼进行染色的方法,可以在显微镜下直观的观察到巨噬细胞和中性粒细胞的变化,方便直观的对肺炎克雷伯菌的毒力做出评估。
三、在对细菌浸入实验后的斑马鱼的分析过程中,本发明建立了完整的评价方法,通过对孵育后的斑马鱼的存活率、心率、鱼鳔的大小和宽度、炎症细胞数量的统计分析和转录组测序中一种或多种方法分析了肺炎克雷伯菌对斑马鱼的影响,可以充分评估其毒性和致病性,为了解临床肺炎克雷伯菌的病理机理提供参考,用于充分评估肺炎克雷伯菌的毒性和对器官造成病理损伤。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为三组肺炎克雷伯菌感染组和空白对照组中斑马鱼存活率随时间变化趋势图。
图2为三组肺炎克雷伯菌感染组和空白对照组中斑马鱼心率随时间变化趋势图。
图3为三组肺炎克雷伯菌感染组和空白对照组中斑马鱼48h后的鱼鳔变化:(a)为斑马鱼图;(b)为斑马鱼鱼鳔的尺寸随时间变化趋势图。
图4为三组肺炎克雷伯菌感染组和空白对照组中斑马鱼染色后巨噬细胞数量统计图。
图5为三组肺炎克雷伯菌感染组和空白对照组中斑马鱼染色后中性粒细胞的数量统计图。
具体实施方式
下面将结合附图对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做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。
此外,下面所描述的本发明不同实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互结合。
实施例1:肺炎克雷伯菌的获取
从两名患者中分离出三株肺炎克雷伯菌KP1053、KP1196和KP1195。
实施例2:斑马鱼肺炎克雷伯菌感染模型的构建
1)斑马鱼的繁殖:将斑马鱼按14小时光照/10小时黑暗的条件喂养3天,随后将性成熟的雌雄斑马鱼在黑暗条件下交配,10h后光照条件下产卵,将鱼卵放置于含有甲基蓝培养液的培养皿中,并在28℃下按照14小时光照/10小时黑暗的条件培养3天;
2)肺炎克雷伯菌的准备:将实施例1中获取的三种肺炎克雷伯菌细菌培养物生长到指数相OD600=0.4±0.6,离心、洗涤,随后分散于灭菌的E3培养液中,得到三种细菌液;
3)斑马鱼肺炎克雷伯菌感染模型的构建:挑选1)中发育正常的斑马鱼用E3培养基洗涤,随后按10条/每孔的密度放入多孔板中,设置空白对照组和细菌感染组,向所述空白对照组中加入E3培养基,向所述细菌感染组中加入2)中所述细菌液(5~8×108CFU/mL),28.5℃下恒温孵育72h,得到三组肺炎克雷伯菌感染组(KP1053组、KP1196组和KP1195组)以及空白对照组(control)。
实施例3:斑马鱼感染肺炎克雷伯菌的毒力验证
观察实施例2中三组肺炎克雷伯菌感染组以及空白对照组的斑马鱼的存活情况,其存活率如图1所示。与空白对照组相比,KP1195的斑马鱼在72h后存活率明显下降,KP1053和KP1196的斑马鱼则在24h后就明显下降,且KP1196的存活率下降幅度最大。在感染24h后以及48h后,对斑马鱼的心率进行统计分析,结果如图2所示,所有受感染的斑马鱼的心率在这两个时间点都有明显降低。观察图3(a),与空白对照组相比,感染48h后,KP1195组的斑马鱼鱼鳔发育明显受到了抑制,而KP1053组和KP1196组的斑马鱼几乎没有影响;记录鱼鳔的体积如图3(b)所示,鱼鳔的体积均大幅下降,这些数据表明,肺炎克雷伯菌三种菌株对斑马鱼有明显的发育毒性。
实施例4:斑马鱼感染肺炎克雷伯菌的细胞影响
从三组肺炎克雷伯菌感染组以及空白对照组中取10条斑马鱼,然后用4%的甲醛固定斑马鱼,用PBST缓冲液冲洗,并用苏丹黑B染色,观察巨噬细胞。
从三组肺炎克雷伯菌感染组以及空白对照组中取10条斑马鱼,然后用4%的甲醛固定斑马鱼,随后用70%乙醇洗涤,之后分别培养在含有2.5mg/L的中性红染料溶液中,在显微镜下观察中性粒细胞的数目变化。
染色后的巨噬细胞和中性粒细胞的数量统计分别如图4和图5所示,与对照组相比,三组肺炎克雷伯菌感染组的巨噬细胞和中性粒细胞数量显著增加,但KP1053和KP1196组的斑马鱼中性粒细胞数量明显高于KP1195组。这些数据表明,三种肺炎克雷伯菌可以显著增加斑马鱼中的炎症细胞数量,并且KP1053和KP1196的毒力明显高于KP1195。
实施例5:斑马鱼感染肺炎克雷伯菌的病理学机制
三组肺炎克雷伯菌感染组以及空白对照组分别进行RNA-se样,在受精后60和120小时下取样;100条幼鱼作为转录组库。总共有12个转录库进行RNA-seq。与对照组相比,KP1053组有1132个分表达基因,KP1196组有1486个分表达基因,KP1195组有884个分表达基因。
利用GO和KEGG的统计分析,研究发现,在GO分析中,差异表达基因富集于肺发育、肺上皮发育、慢性炎症反应、慢性炎症反应调节、刺激反应、巨分子代谢过程、免疫系统过程、对细菌的反应、细胞外基质、肝形态生成等生物过程。KEGG信号通路分析表明,不同表达的基因可丰富到噬菌体、细胞因子受体相互作用、心肌收缩、PPAR信号通路、自噬、TGF-beta信号通路、内质神经通理中的蛋白质处理、疱疹单纯病毒感染、神经活性配结受体相互作用等细胞通路。
由上述实施例1-5可见,本发明除了死亡率和炎症细胞的变化、心率、鱼鳔的变化等指标外,还丰富了克雷伯菌肺炎的毒性检测,也为肺炎克雷伯菌引起的病理提供了参考。本发明提供的模型构建方法简单,成本低廉,准确性高,具有较高的稳定性和可靠性,可实现高通量筛选。
尽管本发明的实施方案已公开如上,但其并不仅仅限于说明书和实施方式中所列运用,它完全可以被适用于各种适合本发明的领域,对于熟悉本领域的人员而言,可容易地实现另外的修改,因此在不背离权利要求及等同范围所限定的一般概念下,本发明并不限于特定的细节和这里示出与描述的图例。
Claims (3)
1.一种斑马鱼肺炎克雷伯菌感染模型的构建方法,其特征在于,包括:挑选发育正常的斑马鱼通过浸入细菌液的手段获得斑马鱼肺炎克雷伯菌感染模型;所述斑马鱼为受精3天后的斑马鱼幼鱼;所述细菌液的制备方法如下:将肺炎克雷伯菌细菌培养物生长到指数相OD600=0.4±0.6,离心、洗涤,随后分散于灭菌的E3培养液中,得到细菌液;所述浸入细菌液的手段具体为将斑马鱼浸入细菌液中,28.5℃恒温孵育72h。
2.一种如权利要求1所述的构建方法构建出的斑马鱼肺炎克雷伯菌感染模型。
3.一种使用权利要求2所述的斑马鱼肺炎克雷伯菌感染模型对炎症细胞的评价方法,其特征在于,至少包括以下步骤:
1)对斑马鱼肺炎克雷伯菌感染模型进行巨噬细胞和中性粒细胞染色;
2)于显微镜下观察中性粒细胞和巨噬细胞的变化,以此获得对肺炎克雷伯菌感染斑马鱼模型后炎症细胞的评价结果;
其中,所述巨噬细胞染色方法为:将斑马鱼肺炎克雷伯菌感染模型浸于4%甲醛溶液中,用PBST缓冲液冲洗,随后用苏丹黑B染色,观察巨噬细胞;
所述中性粒细胞染色方法为:将斑马鱼肺炎克雷伯菌感染模型浸于4%甲醛溶液中,用PBST缓冲液冲洗,之后用70%乙醇洗涤,置于2.5mg/L的中性红染料溶液中培养,观察中性粒细胞。
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