CN116965376B - 一种子痫前期小鼠模型的构建方法与应用 - Google Patents
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Abstract
本发明公开了一种子痫前期小鼠模型的构建方法与应用，涉及动物模型构建技术领域。本发明提供一种子痫前期小鼠模型的构建方法，包括以下步骤：(1)将雌鼠与雄鼠合笼，第二天观察到阴道栓确定该雌鼠怀孕并定为怀孕0.5天；(2)于孕中期开始对步骤(1)孕鼠进行C2神经酰胺给药至妊娠结束；(3)通过监测孕鼠血压确定是否成功构建子痫前期小鼠模型。本发明的子痫前期小鼠模型可用于子痫前期病因、发生发展机制和开发相关治疗药物的研究；本发明的子痫前期小鼠模型的构建方法操作简单，构建成功率高，构建周期短。
Description
技术领域
本发明涉及动物模型构建技术领域，具体涉及一种子痫前期小鼠模型的构建方法与应用。
背景技术
子痫前期是一种累及多系统的妊娠期进行性疾病，多发生于妊娠20周后。临床症状为孕妇新发高血压，严重时伴随多器官功能损伤，肾功能严重损害，子宫胎盘血流灌注不足，胎儿生长受限，是目前导致孕产妇及围生儿死亡的主要原因之一。子痫前期发生机制尚不明确，但胎盘功能异常和胎盘滋养细胞侵袭障碍被认为是与发病相关的主要特征。
动物模型是研究子痫前期发病机制，以及探索防治措施对于母儿短、长期效应的重要工具。子痫前期动物模型可以模拟临床一部分病症，实现母体高血压、蛋白尿、其他脏器病变和胎儿生长受限表征。目前小鼠和大鼠是构建子痫前期疾病模型的首选实验动物。
现有的子痫前期的动物模型是由手术、药物诱导、转基因和基因敲除构建。RUPP手术制作的子痫前期模型为利用手术狭窄大鼠腹主动脉和卵巢动脉，减少子宫供血造成胎盘氧化应激，然而RUPP手术难度高，对孕鼠创伤大容易造成流产和死亡。药物诱导的模型为使用不同药物诱导动物产生模拟子痫前期的症状，如：目前研究中广泛使用的一氧化氮合成酶抑制剂L-NAME诱导构建的小鼠子痫前期模型，可诱导孕鼠高血压和胎鼠生长受限，然而其机理为抑制血管扩张素一氧化氮的合成，通过诱导血管收缩引起高血压症状，不能完全模拟子痫前期的发生。转基因和基因敲除构建的子痫前期动物模型，为敲除或过表达被认为调控子痫前期发病的单个基因，检测基因缺陷动物是否出现子痫前期样症状，然而单个基因仅能改变其调控的下游信号通路，不能体现子痫前期多病因的特点，且基因缺陷动物模型构建周期长、造价高。因此，发展一种简易操作且模拟子痫前期发展进程的动物模型十分必要。
发明内容
本发明的目的在于克服现有技术的不足，提供一种子痫前期小鼠模型的构建方法与应用。
为实现上述目的，本发明采取的技术方案为：一种子痫前期小鼠模型的构建方法，包括以下步骤：
（1）将雌鼠与雄鼠合笼，第二天观察到阴道栓确定该雌鼠怀孕并定为怀孕0.5天；
（2）于孕中期开始对步骤（1）孕鼠进行C2神经酰胺给药至妊娠结束；
（3）通过监测孕鼠血压确定是否成功构建子痫前期小鼠模型。
C2神经酰胺（C2 Ceramide，C2-Cer），又名N-乙酰基神经鞘氨醇、N-乙酰鞘氨醇，是一种短链神经酰胺，在生物体内由乙酰转移酶将乙酰基团从血小板活化因子（PAF）转移至鞘氨醇合成。C2-Cer可激活蛋白质磷酸酶1（PP1）、蛋白质磷酸酶2（PP2A）和神经酰胺激活的蛋白磷酸酶（CAPP），对多种肿瘤细胞有促凋亡、抑制侵袭迁移，以及抑制线粒体呼吸的作用。在子痫前期病人的血和胎盘中，数种长链神经酰胺均水平升高，如C16-Cer、C18-Cer、C24-Cer。本申请发明人经过大量的实验发现，与正常孕妇样本相比，C2-Cer在子痫前期病人粪便代谢组中水平升高，且通过靶向质谱验证了C2-Cer也在子痫前期病人胎盘中含量升高，表明C2-Cer与子痫前期发生发展相关。因此，本发明向孕鼠施加C2-Cer，发现孕鼠出现高血压、胎儿生长受限、胎盘坏死增多的子痫前期特征，表明C2-Cer可以用于构建子痫前期小鼠模型。
作为本发明所述子痫前期小鼠模型的构建方法的优选实施方式，所述C2神经酰胺的给药方式包括灌胃。
作为本发明所述子痫前期小鼠模型的构建方法的优选实施方式，所述C2神经酰胺的给药剂量为100 μg /kg/day。
作为本发明所述子痫前期小鼠模型的构建方法的优选实施方式，所述步骤（2）孕中期为雌鼠怀孕第7.5天。
作为本发明所述子痫前期小鼠模型的构建方法的优选实施方式，所述步骤（1）中的雌鼠与雄鼠周龄为10-12周。
作为本发明所述子痫前期小鼠模型的构建方法的优选实施方式，所述步骤（1）中的雌鼠与雄鼠的比例为雌鼠：雄鼠=2：1。
本发明还提供所述的构建方法构建得到的子痫前期小鼠模型。
本发明还提供所述子痫前期小鼠模型在筛选或制备治疗子痫前期的药物中的应用。
本发明还提供C2神经酰胺在构建子痫前期小鼠模型中的应用。
作为本发明所述应用的优选实施方式，所述C2神经酰胺的浓度为20μg/mL。
本发明的有益效果：本发明提供一种子痫前期小鼠模型的构建方法与应用，本发明通过对孕中期的小鼠施加C2神经酰胺构建子痫前期小鼠模型，建立得到的子痫前期小鼠模型呈现高血压、胎儿生长受限、胎盘坏死增多的子痫前期特点，本发明的子痫前期小鼠模型可用于子痫前期病因、发生发展机制和开发相关治疗药物的研究；本发明的子痫前期小鼠模型的构建方法操作简单，构建成功率高，构建周期短。
附图说明
图1为子痫前期患者与正常孕妇的粪便代谢组分析结果，其中A为偏最小二乘法判别分析模型图（Partial Least Squares Discrimination Analysis, PLS-DA）；B为两组样品的差异代谢物火山图，图中红色点为显著上调代谢物，绿色点为显著下调代谢物。PE：子痫前期。
图2为靶向质谱检测结果，其中A为靶向质谱检测正常孕妇与子痫前期孕妇胎盘样品中C2-Cer的含量，代表性离子流图；C9：对照组样品9，Qing1：轻度子痫前期组样品1；B为15例正常孕妇胎盘与21例子痫前期孕妇胎盘中C2-Cer浓度。PE：子痫前期。
图3为利用C2-Cer构建子痫前期小鼠的构建策略图：于孕鼠怀孕的第7.5天（D7.5），通过灌胃方法给药100μg /kg/day C2-Cer直至D17.5，D18.5终止妊娠。
图4为C2-Cer诱导的子痫前期小鼠和对照组小鼠不同孕期的收缩压测定结果，*：p<0.05。
图5为孕D18.5胎鼠的身长、体重，胎盘重量测定结果。
图6为小鼠D18.5胎盘石蜡切片的H&E染色结果，C2-Cer组孕鼠的胎盘出现坏死区域面积增多（黑色箭头所示）。
具体实施方式
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
实施例1
本实施例通过非靶向质谱和靶向质谱检测C2-Cer在子痫前期患者样本中的含量，具体方法如下：（1）对30例子痫前期（PE）孕妇与30例正常孕妇的粪便样品进行非靶向质谱（LC-MS/MS）分析。入组样品均排除慢性高血压史、代谢疾病史、吸烟史、饮酒史及其他妊娠合并症，确保采集样品前病人短期内均未接受药物治疗。（2）通过靶向质谱，分析21例子痫前期（PE）孕妇胎盘和15例正常孕妇胎盘中C2-Cer含量。
结果如图1-2所示。由图1可知，子痫前期（PE）孕妇粪便中代谢物C2-Cer的水平升高。由图2可知，子痫前期（PE）孕妇胎盘中C2-Cer水平较正常孕妇升高1.82倍。由上述可知，C2-Cer与子痫前期发生发展相关。
实施例2
本实施例提供一种子痫前期动物模型的构建方法，如图3所示，具体包括以下步骤：
（1）选取10-12周龄，体重20-26g的C57BL/6小鼠，雌鼠与雄鼠按照2:1比例合笼，第二天观察到阴道栓确定该雌鼠怀孕并定为怀孕0.5天；
（2）将确认怀孕的孕鼠分为Saline组，和C2-Cer组，每组6只；对Saline组孕鼠灌胃生理盐水，对C2-Cer组孕鼠灌胃100μg /kg/day C2-Cer；灌胃自D7.5开始至D17.5结束，每天一次。
（3）对孕鼠进行如下检测：
血压检测：从孕前一天开始，每隔一天监测孕鼠血压；
胎鼠特征检测：于孕D18.5取胎鼠和胎盘，记录其重量和胎仔身长；
胎盘病理形态检测：对孕D18.5的胎盘石蜡切片进行H&E染色。
结果如图4-6所示。由图4可知，Saline组在孕期血压维持在100-105mmHg间相对稳定的范围；C2-Cer组血压自D9.5起开始升高，维持在120-140mmHg，明显高于Saline对照组，表明C2-Cer诱导孕鼠出现了高血压的子痫前期特征。由图5可知，C2-Cer组胎仔身长与体重明显偏小，胎盘重量偏大，胎仔重量与胎盘重量的比值显著降低，表明C2-Cer诱导的模型鼠出现胎儿生长受限的子痫前期特征。由图6可知，C2-Cer组胎盘迷路区出现大量坏死与血栓区域（黑色箭头指示），表明C2-Cer诱导的模型鼠出现胎盘发育异常的特征。由上述可知，本实施例成功构建了子痫前期动物模型。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (9)
1.一种子痫前期小鼠模型的构建方法，其特征在于，包括以下步骤：
（1）将雌鼠与雄鼠合笼，第二天观察到阴道栓确定该雌鼠怀孕并定为怀孕0.5天；
（2）于孕中期开始对步骤（1）孕鼠进行C2神经酰胺给药至妊娠结束；
（3）通过监测孕鼠血压确定是否成功构建子痫前期小鼠模型。
2.根据权利要求1所述的构建方法，其特征在，所述C2神经酰胺的给药方式包括灌胃。
3. 根据权利要求2所述的构建方法，其特征在，所述C2神经酰胺的给药剂量为100 μg/kg/day。
4.根据权利要求1所述的构建方法，其特征在，所述步骤（2）孕中期为雌鼠怀孕第7.5天。
5.根据权利要求1所述的构建方法，其特征在，所述步骤（1）中的雌鼠与雄鼠周龄为10-12周。
6.根据权利要求1所述的构建方法，其特征在，所述步骤（1）中的雌鼠与雄鼠的比例为雌鼠：雄鼠=2：1。
7.权利要求1-6任一项所述子痫前期小鼠模型的构建方法在筛选或制备治疗子痫前期的药物中的应用。
8. C2神经酰胺在构建子痫前期小鼠模型中的应用。
9.根据权利要求8所述的应用，其特征在于，所述C2神经酰胺的浓度为20μg/mL。
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