CN110841059A - 老年性痴呆小鼠模型的制备方法 - Google Patents
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Abstract
本发明公开了一种老年性痴呆小鼠模型的制备方法；本发明注射AD P‑tau后10个月的C57BL/6小鼠，可以检测出小鼠大脑的不同区域有tau病理性的改变，可成为靶标是tau的AD模型，亦可用于对AD疾病的分子机制的研究。通过该动物模型的建立，研究AD发病的分子机制，tau蛋白的病理发展过程以及传播特征；也可以使用该动物模型来研究某些因素，例如创伤性脑损伤、过表达或沉默磷酸酯酶来研究对tau病理发生发展的影响；更有意义的是，可以通过该模型的建立，有助于筛选出可以抑制或逆转tau病理发生发展及传播的药物。
Description
技术领域
本发明属于动物模型技术领域，特别涉及一种老年性痴呆小鼠模型的制备方法。
背景技术
AD是多因素相关的疾病，老化是其最主要的病因。AD拥有病理特征：细胞外β-淀粉样蛋白（β-amyloid protein, Aβ）聚集形成的老年斑（senile plaque, SP）的沉积和细胞内大量的tau蛋白聚集形成神经原纤维缠结（neurofibrillary tangles, NFTs）和广泛神经元退变。一直以来在AD研究领域都是以Aβ为中心，以Aβ为靶标作为药物开发的重点。遗憾的是AD一直是新药研发的重灾区，该领域临床试验失败率高达99.6%，过去20年来在该领域的研究几乎全军覆没，所以寻找新的靶点非常必要。
越来越多的研究提示，人脑中tau病理传播是导致AD病程进展的关键原因之一，抑制 tau 病理可能对延缓或阻止AD病理传播起到积极作用(Iqbal et al., 2018)。因此，研究tau病理发生、发展的分子机制、开发以tau为靶标的AD药物，是当今AD领域的研究热点。现有技术从 AD 患者脑中提取的异常过度磷酸化 tau或体外表达重组 tau 蛋白经肝素诱导形成的聚集物，注射到小鼠脑海马区，诱发小鼠tau 蛋白发生类AD样tau病理, 并向相关脑区传播，可作为AD的疾病模型小鼠，用于AD相关致病机制研究，药物开发和筛选等(Clavaguera et al., 2009 Clavaguera, F., Bolmont, T., Crowther, R.A.,Abramowski, D., Frank, S., Probst, A., Fraser, G., Stalder, A.K., Beibel, M.,Staufenbiel, M., Jucker, M., Goedert, M., Tolnay, M., 2009. Transmission andspreading of tauopathy in transgenic mouse brain. Nat Cell Biol 11, 909-913.Hu, W., Zhang, X., Tung, Y.C., Xie, S., Liu, F., Iqbal, K., 2016.Hyperphosphorylation determines both the spread and the morphology of taupathology. Alzheimers Dement 12, 1066-1077。NFTs的聚集是AD另一病理特征，其数量和患者的痴呆程度呈明显的正相关，被认为是 AD 患者神经元纤维退化（neurofibrillarydegeneration)的病理基础。
研究的AD动物模型大致有以下几种: （1）损伤性动物模型, 包括前脑胆碱能系统损伤模型，冈田酸( OA )损害模型，铝损害模型，D-半乳糖损害模型，脑缺血痴呆动物模型，β淀粉样肽(Aβ)损害模型，秋水仙碱损伤模型等；（2）自然衰老认知障碍动物模型；（3）快速老化小鼠( SAM ) 模型；（4）复合型A D模型；（5）转基因动物模型。而目前通常使用转基因小鼠，但是从转基因小鼠的遗传背景、操作难易度及经济角度考虑，用于对AD分子机制的研究或治疗效果的判断有一定的限制。
C57BL/6小鼠也被称为C57 black 6，当然有的人也把它叫做B6，1921年被培育出来，属于近交品系。该品系的最主要的两个特点就是品系稳定和易于繁殖，是经济型动物，亦可以用于临床疾病模型，另外C57BL/6小鼠是第一个完成基因组测序的小鼠品系，这也为它增加了不少的知名度。以C57BL/6小鼠为研究对象，使其获得AD所特有的tau病理特征，成为研究AD的发病机制和靶标为tau的药物筛选经济型动物模型。
发明内容
为了解决现有技术中的问题，本发明提供一种老年性痴呆小鼠模型的制备方法，该方法以tau病理改变为主的AD动物模型，为针对以tau为靶标的AD治疗以及探究AD发病的分子机制提供经济有效的动物模型。
为实现上述目的，本发明采用的技术方案为：
一种老年性痴呆小鼠模型的制备方法，包括以下步骤：
步骤1、AD患者脑内提取的病理性tau，即AD p-tau的分离：
10 %的AD脑匀浆液，在4℃下，27000 xg离心30 min，取上清，在4℃下， 235000 xg离心45 min，取沉淀（Oligo-tau）用生理盐水洗三次，用生理盐水重悬，-80℃保存，得到的tau为AD O-tau；AD O-tau经盐析、尿素处理，透析、离子交换层析，收集磷酸化的fractions，透析，-80℃保存，得到的tau为AD P-tau。
步骤2、海马注射AD P-tau：四月龄的C57BL/6小鼠，腹腔注射2.5%的 2-Avertin麻醉，根据小鼠脑图谱，使用脑立体定位仪、微量注射泵和 31-gauge Hamilton 微量注射器，海马注射AD P-tau，注射的AD P-tau中蛋白浓度11mg/ml，tau的量600ng/2.5ul；
步骤3、小鼠4月龄时脑注射后，采用常规的饲养方式自由摄取水和食物，室温20℃，12小时光照/黑暗循环环境。
步骤4、组织固定与切片：取小鼠，先经腹腔注射2.5%的 2-Avertin 麻醉剂，麻醉后，开胸暴露心脏，自心尖部将灌注针头经左心室插至升主动脉，灌注生理盐水后，改灌注含有4%多聚甲醛的0.1 MPBS（pH7.2），待动物全身抽搐后，调慢灌注速度至50滴/min，持续时间30 min后，取出大脑放入含有4%多聚甲醛的0.1 MPBS（pH7.2）中后固定4 h，再先后转入含20%和30%蔗糖溶液中，待组织块下沉后，行冠状位冰冻连续切片，片厚40 μm，漂于0.01M PBS中。
步骤5、免疫组织化学，
501、挑取海马结构清晰的漂片，置于0.01 M PBS缓冲液（pH7.2）中漂洗3遍后，放入含有10% triton-100液体中20min，0.01 M PBS缓冲液（pH7.2）中漂洗3遍，放入含有10%山羊血清的封闭液中，在室温下振摇2 h；
502、将封闭好的切片转入到1:1000稀释的对磷酸化tau（tau病理）的特异性抗体mouse-AT8 中，室温下轻轻振摇1 h后放入4℃冰箱中过夜；
503、次日晨吸去一抗，用PBS清洗3遍后，加入Alexa-555荧光标记的抗小鼠二抗，室温下轻轻振摇2 h，加入1:5000 Hochest孵育15 min；
504、0.01M PBS（pH 7.2）避光清洗3次，每次10min；
505、荧光封片液封片后激光共聚焦显微镜下观察。
所述步骤2中，注射的坐标如下：前囟向后2.5 mm，向左/右旁开2.0 mm，自硬膜向腹侧进针1.67 mm，注射体积2.5μl，注射速度1.25μl/min，注射后，留针3 min 以防液体外溢。
与现有技术相比，本发明具有以下有益效果：
本发明注射AD P-tau后10个月的C57BL/6小鼠，可以检测出小鼠大脑的不同区域有tau病理性的改变，可成为靶标是tau的AD模型，亦可用于对AD疾病的分子机制的研究。通过该动物模型的建立，研究AD发病的分子机制，tau蛋白的病理发展过程以及传播特征；也可以使用该动物模型来研究某些因素，例如创伤性脑损伤、过表达或沉默磷酸酯酶来研究对tau病理发生发展的影响；更有意义的是，可以通过该模型的建立，有助于筛选出可以抑制或逆转tau病理发生发展及传播的药物。
附图说明
图1是本发明中C57BL/6小鼠海马注射AD P-tau的示意图；
图2是本发明中注射AD P-tau不同时间（10w，10m）后tau的病理改变的图；
图3是本发明中注射AD P-tau10个月后在C57BL/6小鼠脑的不同区域出现了tau病理改变的图；
图4是本发明中C57BL/6小鼠tau病理改变的表现图。
具体实施方式
下面结合实施例对本发明作更进一步的说明。
实施例1
本实施例1，以tau病理模型的应用举例，包括以下步骤：
（1）筛选可抑制tau病理的基因。
构建靶基因腺相关病毒载体，注射入4月龄C57BL/6小鼠海马区，使靶基因高表达。2周后，同一位置注射AD P-tau，诱导tau病理产生。14月龄时，取鼠脑进行免疫荧光，比较注射靶基因病毒和对照病毒的鼠脑tau病理强度（tau病理特异性抗体mouse-AT8阳性细胞的数目），判断靶基因是否影响tau病理产生，从而筛选出可有效抑制tau病理的基因。
（2）筛选可抑制tau病理的药物：
4月龄C57BL/6小鼠海马区注射AD P-tau，诱导tau病理产生。注射后第二天，通过腹腔注射或灌胃给小鼠施加相应浓度的药物，持续10个月。取鼠脑进行免疫荧光，比较给药组和对照组之间tau病理强度（tau病理特异性抗体mouse-AT8阳性细胞的数目），判断药物是否影响tau病理传播，从而筛选出可有效抑制tau病理的药物。
如图1所示，C57BL/6小鼠海马注射AD P-tau：
如图2所示，注射AD P-tau不同时间（10w，10m）后tau的病理改变，具体地讲，C57BL/6小鼠在注射AD P-tau10w时没有出现tau的病理改变，10个月之后，注射AD P-tau的小鼠明显出现了tau的病理改变，而注射生理盐水的小鼠无论是10w还是10m均没有出现tau病理改变。
如图3所示，注射AD P-tau10个月后在C57BL/6小鼠脑的不同区域出现了tau病理改变，具体地讲，在海马CA1注射AD P-tau的小鼠10m后在海马以及皮层的其它区域均出现了不同程度的tau病理改变。
如图4所示，C57BL/6小鼠tau病理改变的表现，C57BL/6小鼠tau的病理改变与其病变程度有关，可以表现为不同的tau病理特征。可以是在胞浆中散在的点在聚集（A-D），随着病程进展，可以发展为絮状的聚集（E-H），甚者可以看到典型的神经纤维的缠结（I-L）。
以上所述仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (2)
1.一种老年性痴呆小鼠模型的制备方法，其特征在于，包括以下步骤：
步骤1、AD患者脑内提取的病理性tau，即AD p-tau的分离：
10 %的AD脑匀浆液，在4℃下，27000 xg离心30 min，取上清，在4℃下， 235000 xg离心45 min，取沉淀Oligo-tau用生理盐水洗三次，用生理盐水重悬，-80℃保存，得到的tau为ADO-tau；AD O-tau经盐析、尿素处理，透析、离子交换层析，收集磷酸化的fractions，透析，-80℃保存，得到的tau为AD P-tau；
步骤2、海马注射AD P-tau：四月龄的C57BL/6小鼠，腹腔注射2.5%的 2-Avertin 麻醉，根据小鼠脑图谱，使用脑立体定位仪、微量注射泵和 31-gauge Hamilton 微量注射器，海马注射AD P-tau，注射的AD P-tau中蛋白浓度11mg/ml，tau的量600ng/2.5ul；
步骤3、小鼠4月龄时脑注射后，采用常规的饲养方式自由摄取水和食物，室温20℃，12小时光照/黑暗循环环境；
步骤4、组织固定与切片：取小鼠，先经腹腔注射2.5%的 2-Avertin 麻醉剂，麻醉后，开胸暴露心脏，自心尖部将灌注针头经左心室插至升主动脉，灌注生理盐水后，改灌注含有4%多聚甲醛的0.1 MPBS，待动物全身抽搐后，调慢灌注速度至50滴/min，持续时间30 min后，取出大脑放入含有4%多聚甲醛的0.1 MPBS中后固定4 h，再先后转入含20%和30%蔗糖溶液中，待组织块下沉后，行冠状位冰冻连续切片，片厚40 μm，漂于0.01 M PBS中；
步骤5、免疫组织化学，
501、挑取海马结构清晰的漂片，置于0.01 M PBS缓冲液中漂洗3遍后，放入含有10%triton-100液体中20min，0.01 M PBS缓冲液中漂洗3遍，放入含有10%山羊血清的封闭液中，在室温下振摇2 h；
502、将封闭好的切片转入到1:1000稀释的对磷酸化tau的特异性抗体mouse-AT8 中，室温下轻轻振摇1 h后放入4℃冰箱中过夜；
503、次日晨吸去一抗，用PBS清洗3遍后，加入Alexa-555荧光标记的抗小鼠二抗，室温下轻轻振摇2 h，加入1:5000 Hochest孵育15 min；
504、0.01M PBS（pH 7.2）避光清洗3次，每次10min；
505、荧光封片液封片后激光共聚焦显微镜下观察。
2.根据权利要求1所述的老年性痴呆小鼠模型的制备方法，其特征在于，所述步骤2中，注射的坐标如下：前囟向后2.5 mm，向左/右旁开2.0 mm，自硬膜向腹侧进针1.67 mm，注射体积2.5μl，注射速度1.25μl/min，注射后，留针3 min 以防液体外溢。
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