CN114009401A - 一种建立乙型脑炎病毒感染诱导周围神经损伤小鼠模型方法 - Google Patents
一种建立乙型脑炎病毒感染诱导周围神经损伤小鼠模型方法 Download PDFInfo
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Abstract
一种建立乙型脑炎病毒感染诱导周围神经损伤小鼠模型方法,步骤为:1.4‑6周龄的雄性小鼠,将其随机分为12组,每组5只,两组为对照组,其余10组为实验组;2.人源基因Ⅰ型乙型脑炎病毒NX1889株由原浓度稀释为102~106pfu五个不同的病毒滴度;3.给予实验组前五组小鼠皮下注射病毒;4.注射前进行初始体重的称量;5.观察小鼠的一般情况,以判断其肢体运动功能;6.分别在注射后第4,8,12,16,20天时选取各组部分小鼠进行麻醉后行双侧坐骨神经肌电图检查;7.待肌电图操作完毕取一侧坐骨神经行透射电镜检查;8.取另一侧坐骨神经行免疫荧光检测,判定坐骨神经髓鞘及轴突的损伤。本发明为乙脑周围神经损害的机制研究建立了良好的基础。
Description
技术领域
本发明属于疾病防控技术应用领域,尤其是涉及一种建立乙型脑炎病毒感染诱导周围神经损伤小鼠模型方法。
背景技术
乙型脑炎病毒是一种单股正链的RNA病毒,属于黄病毒科黄病毒属,感染人类后可引起急性脑炎综合征,发病率及死亡率均较高,即使存活下来的患者大多也伴发严重的精神神经系统的后遗症,是威胁亚洲地区人类公共安全的一个重大问题。乙型脑炎病毒有很强的神经嗜性和神经侵袭性,自1935年首次被分离以来,在亚洲地区引起过多次的爆发流行。随着该病毒从最早的以基因Ⅲ型为主要致病基因型到近年来逐渐被基因Ⅰ型所取代,其发病的模式也出现了变化。2018年宁夏北部地区出现了流行性乙型脑炎的爆发流行,这期间有很多患者伴发了吉兰-巴雷综合征的表现,经研究确定乙脑病毒感染人类后可导致周围神经损伤,并且发病人群从过去以儿童为主,转变为以成年人,尤其是中老年人为主。然而对于该病毒如何导致周围神经损伤的机制尚不清楚。该疾病尚没有行之有效的治疗方法。本发明提供了一种乙脑病毒诱导的周围神经损伤的动物模型,提供了可靠的检测方式及病毒感染的最适浓度,为进一步研究乙脑病毒诱导周围神经损伤的相关机制及寻找预防及治疗的靶点提供了可靠的研究基础。
现有的对于乙脑病毒所造成的损伤的研究仅针对于中枢神经系统,有相应运用于中枢神经系统损害的动物模型,然后对于乙脑病毒诱导的周围神经损害还没有相应的动物模型用于研究。
发明内容
本发明的目的是针对现有技术存在的问题提供一种建立乙型脑炎病毒感染诱导周围神经损伤小鼠模型方法。
本发明的技术方案为:一种建立乙型脑炎病毒感染诱导周围神经损伤小鼠模型方法,所述方法包括如下步骤:
1.4-6周龄的C57BL/6雄性小鼠作为实验动物,将其随机分为12组,每组5 只(n=5),设置其中两组为对照组,其余10组为实验组;
2.人源基因Ⅰ型乙型脑炎病毒NX1889株由原浓度稀释为102~106pfu五个不同的病毒滴度;
3.给予实验组前五组小鼠左侧腹股沟区皮下注射病毒,每个病毒滴度注射一组,共五组为皮下注射组,实验组后五组小鼠予以腹腔注射病毒,每个病毒滴度注射一组,共五组为腹腔注射组,注射剂量均为50μl;两组对照组分别予以皮下及腹腔注射两种方式注射同等剂量的PBS或生理盐水;
4.病毒注射前进行初始体重的称量,之后对各组小鼠进行体重的称量并记录;
5.观察小鼠的一般情况,包括毛发、精神状态、活动情况、有无肢体活动障碍等;对所有小鼠进行病毒瘫痪量表的评分及吊线实验,以判断其肢体运动功能;
6.分别在注射病毒后第4,8,12,16,20天时根据以上测评的结果选取各组小鼠中部分小鼠进行麻醉后行双侧坐骨神经肌电图检查,检测的内容主要为坐骨神经波幅、潜伏期及传导速度;
7.待肌电图操作完毕后取一侧坐骨神经行透射电镜检查,主要检测坐骨神经轴突及髓鞘等超微结构的改变;
8.取另一侧坐骨神经行免疫荧光检测,主要检测JEV-E蛋白的表达明确病毒的侵染,以及髓鞘碱性蛋白MBP(髓鞘特异性蛋白),神经丝重链NF-H(轴突特异性蛋白)的表达情况,从而判定坐骨神经髓鞘及轴突的损伤。
本发明显著效果是:本发明针对现有技术存在的缺陷,提供了一种乙脑病毒感染诱导周围神经损伤的小鼠模型。该模型构建是基于C57BL/6雄性小鼠给于不同滴度的病毒后,通过动物行为学、神经电生理学及组织病理学等手段来检测小鼠周围神经病变情况。该动物模型的构建为乙脑周围神经损害的机制研究建立了良好的基础。
附图说明
图1为本发明模型小鼠坐骨神经免疫荧光示意图;
图2为本发明模型小鼠坐骨神经透射电镜示意图;
图3为本发明建立模型方法技术路线图。
具体实施方式
下面将结合实施例对本发明的技术方案具体实施方式进行清楚、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。
一种建立乙型脑炎病毒感染诱导周围神经损伤小鼠模型方法,所述方法包括如下步骤:
1.4-6周龄的C57BL/6雄性小鼠作为实验动物,将其随机分为12组,每组5只 (n=5),设置其中两组为对照组,其余10组为实验组;
2.人源基因Ⅰ型乙型脑炎病毒NX1889株由原浓度稀释为102~106pfu五个不同的病毒滴度;
3.给予实验组前五组小鼠左侧腹股沟区皮下注射病毒,每个病毒滴度注射一组,共五组为皮下注射组,实验组后五组小鼠予以腹腔注射病毒,每个病毒滴度注射一组,共五组为腹腔注射组,注射剂量均为50μl;两组对照组分别予以皮下及腹腔注射两种方式注射同等剂量的PBS或生理盐水;
4.病毒注射前进行初始体重的称量,之后每日对各组小鼠进行体重的称量并记录;
5.观察小鼠的一般情况,包括毛发、精神状态、活动情况、有无肢体活动障碍等;对所有小鼠进行病毒瘫痪量表的评分及吊线实验,以判断其肢体运动功能;
6.分别在注射病毒后第4,8,12,16,20天时根据以上测评的结果选取各组中部分小鼠进行麻醉后行双侧坐骨神经肌电图检查,检测的内容主要为坐骨神经波幅、潜伏期及传导速度;
7.待肌电图操作完毕后取一侧坐骨神经行透射电镜检查,主要检测坐骨神经轴突及髓鞘等超微结构的改变;
8.取另一侧坐骨神经行免疫荧光检测,主要检测JEV-E蛋白的表达明确病毒的侵染,以及髓鞘碱性蛋白MBP(髓鞘特异性蛋白),神经丝重链NF-H(轴突特异性蛋白)的表达情况,从而判定坐骨神经髓鞘及轴突的损伤。
如图3所示,建立模型方法的技术路线说明如下:
说明1:实验组小鼠出现体重下降或体重增加不明显,并出现毛发松散,粗糙,无光泽,活动减少,精神萎靡,行走缓慢,尾巴低平甚至拖曳,弓背状,单侧或双后肢不同程度的跛行,拖曳,走路摇晃,关节僵硬,甚至不能支撑身体。
说明2:实验组小鼠病毒瘫痪量表评分升高,吊线实验时间下降。
说明3:坐骨神经波幅及潜伏期反应轴突功能,传导速度反应髓鞘功能。实验组小鼠出现坐骨神经波幅降低,传导速度减慢。
说明4:JEV-E蛋白在实验组小鼠中的免疫荧光强度增高,MBP及NF-H蛋白在实验组小鼠中免疫荧光强度降低,代表坐骨神经有病毒侵染,且髓鞘及轴突均有损伤。
说明5:坐骨神经透射电镜观察到对照组坐骨神经的轴突及髓鞘的结构基本正常。实验组中可见有不同程度的神经组织水肿,髓鞘板层松散,脱髓鞘改变,施万细胞轻度水肿,部分线粒体轻度水肿,仅有部分伴有轴突变性,萎缩。病毒瘫痪量表:
表1病毒瘫痪量表
Table
1
Virus Paralysis Scale
备注:1)足底踏步:踩的时候爪子平放在地上,它不会向一侧卷曲或打滑,脚趾/脚也不会在任何点卷曲或拖曳;
吊线实验:使用一高50cm无顶的透明盒子,底部覆盖棉布,以防小鼠摔伤。顶部覆盖45×30cm的铁丝网架。铁丝直径约2mm,每个网格为1×1cm大小。铁丝网架刚好覆盖盒子顶部,将小鼠放置于网架上方,让其在网架上自由活动,随后将网架翻转180°放置于盒子顶部,以观察小鼠在铁丝网架上停留的时间,最长时间为180秒,达到180秒被视为正常,并被放回笼中,期间如有掉落,记录掉落的时间,再行两次实验,每两次实验间隔2分钟,记录三次的平均时间。肌电图操作步骤:将小鼠腹腔注射10%水合氯醛1μl/g,待麻醉满意后将小鼠俯卧位固定于手术台。用剪刀剪开其双股侧皮毛,暴露术区,用分离针及镊子仔细分离双侧坐骨神经,分离全长的坐骨神经,局部予以生理盐水保持湿润。将肌电图仪的地线钳夹于小鼠的身体,记录电极插入一侧腓肠肌肌腹,刺激电极分别放置在坐骨神经近端(脊柱侧)及远端(坐骨神经切迹),自0.1mA给于电流刺激,并逐渐增加电流量,以达到最大波幅,在肌电图仪上分别记录两侧坐骨神经近端及远端的波幅及潜伏期,测量坐骨神经近远端之间的距离,计算出传导速度。
以上所揭露的仅为本发明较佳实施例而已,当然不能以此来限定本发明之权利范围,本领域普通技术人员可以理解实现上述实施例的全部或部分流程,并依本发明权利要求所作的等同变化,仍属于发明所涵盖的范围。
Claims (1)
1.一种建立乙型脑炎病毒感染诱导周围神经损伤小鼠模型方法,其特征在于,所述方法包括如下步骤:
(1)4-6周龄的C57BL/6雄性小鼠作为实验动物,将其随机分为12组,每组5只(n=5),设置其中两组为对照组,其余10组为实验组;
(2)人源基因Ⅰ型乙型脑炎病毒NX1889株由原浓度稀释为102~106pfu五个不同的病毒滴度;
(3)给予实验组前五组小鼠左侧腹股沟区皮下注射病毒,每个病毒滴度注射一组,共五组为皮下注射组,实验组后五组小鼠予以腹腔注射病毒,每个病毒滴度注射一组,共五组为腹腔注射组,注射剂量均为50μl;两组对照组分别予以皮下及腹腔注射两种方式注射同等剂量的PBS或生理盐水;
(4)病毒注射前进行初始体重的称量,之后每日对各组小鼠进行体重的称量并记录;
(5)观察小鼠的一般情况,包括毛发、精神状态、活动情况、有无肢体活动障碍等;对所有小鼠进行病毒瘫痪量表的评分及吊线实验,以判断其肢体运动功能;
(6)分别在注射病毒后第4,8,12,16,20天时根据以上测评的结果选取各组中部分小鼠进行麻醉后行双侧坐骨神经肌电图检查,检测的内容主要为坐骨神经波幅、潜伏期及传导速度;
(7)待肌电图操作完毕后取一侧坐骨神经行透射电镜检查,主要检测坐骨神经轴突及髓鞘等超微结构的改变;
(8)取另一侧坐骨神经行免疫荧光检测,主要检测JEV-E蛋白的表达明确病毒的侵染,以及髓鞘碱性蛋白MBP(髓鞘特异性蛋白),神经丝重链NF-H(轴突特异性蛋白)的表达情况,从而判定坐骨神经髓鞘及轴突的损伤。
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