CN112400799A - 评估hdac5作为孕期感染子代大鼠模型质量早期外周标志物的试验方法 - Google Patents
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Abstract
本发明提供一种评估HDAC5作为孕期感染子代大鼠模型质量早期外周标志物的试验方法,通过在孕9天尾静脉注射Poly I:C激活孕鼠免疫反应制备孕期感染子代大鼠模型,综合行为学与HDAC5表达分析,发现模型大鼠HDAC5异常表达现象早于其异常行为出现,且外周血HDAC5表达的变化与中枢反应一致,故HDAC5可作为评估孕期感染子代大鼠模型质量控制的早期外周标记分子。本发明构思巧妙,方案科学,对人类流行病学研究以及药物的研发具有重要意义。
Description
技术领域
本发明涉及孕期感染子代大鼠模型质量早期外周标志物的研究技术领域,具体涉及评估HDAC5作为孕期感染子代大鼠模型质量早期外周标志物的试验方法。
背景技术
流行病学研究显示母孕期感染是许多精神疾病的环境危险因素,母孕期感染的子代大鼠在成年期表现异常行为,很好的模拟许多精神疾病的临床表现,是病因学研究、药物研发的很好模型,但缺乏早期模型质量的评价标准。
动物水平的研究证实广谱HDACs抑制剂具有改善认知以及缓解神经退行性疾病的病理状态的作用,显示HDACs功能紊乱在精神疾病中发挥作用,提示其可能作为疾病的潜在生物标记分子。
发明内容
本发明的目的是针对现有技术的不足,提供一种评估HDAC5作为孕期感染子代大鼠模型质量早期外周标志物的试验方法,具体方案如下:
一种评估HDAC5作为孕期感染子代大鼠模型质量早期外周标志物的试验方法,包括以下步骤:
步骤1:孕鼠准备:选择SPF级10-14周龄的SD种鼠,新环境适应2周后开始育种,每3-5只雄鼠与1只雌鼠至于一个分段笼里,前三天只允许嗅觉接触无身体接触,3天后放一起交配,次日早上通过阴道栓检查雌鼠是否成功受孕,成功受孕者入组;
步骤2:孕期感染:确定怀孕后第 9 天尾静脉注射10mg/kg 的聚肌胞苷酸(Poly I:C)或等体积生理盐水(对照组),模型组和对照组各随机选取3只,在注射3小时后处死,检测孕鼠外周血血清 IL-1β、IL-6、TNF-α的蛋白水平,通过免疫激活状态确定注射效果,其余孕鼠继续饲养至产下子鼠;
步骤3:行为学检测:将12-15只仔鼠于40天(青春期)、56天(成年期)采用小动物行为采集和分析系统进行Y迷宫、前脉冲抑制实验,分别分析大鼠的学习记忆状态、感觉门控作用,比较两组动物随年龄增长在行为上的差异;
步骤4:对孕期感染子代大鼠模型进行转录组测序及荧光定量PCR分析;
步骤5:对孕期感染子代大鼠模型外周血单核细胞中HDAC5水平进行验证。
基于上述,步骤2中ELISA检测孕鼠血浆IL-6 、IL-1β和TNF-α的表达水平,当结果显示模型组孕鼠血浆炎症因子水平相比于对照组显著升高,则提示孕期免疫激活。
基于上述,步骤3中行为学检测时间控制在8:00-18:00之间,房间内控制适宜的温度、湿度及照明。
基于上述,步骤4中将孕期感染子代大鼠模型青春子代前额叶皮质进行转录组测序分析HDAC5转录水平,并将测序结果通过荧光定量PCR进一步验证。
基于上述,收集孕期感染大鼠模型外周血样,分离单核淋巴细胞,提取RNA,定量分析HDCA5表达水平。
本发明相对现有技术具有突出的实质性特点和显著的进步,具体地说,本发明具有以下优点:
本发明前期通过在孕9天尾静脉注射Poly I:C激活孕鼠免疫反应制备孕期感染子代大鼠模型,在青春期及成年期进行行为学检测,结合青春期转录组测序,定量PCR分析等方法,发现模型大鼠HDAC5异常表达现象早于其行为异常,且外周血与中枢反应一致,故HDAC5可作为评估孕期感染子代大鼠模型质量的早期外周标志物。
附图说明
图1是本发明中检测孕鼠血浆中IL-6 、IL-1β和TNF-α中表达水平的实验对比图。
图2是本发明中孕期感染子代大鼠模型行为学检测的实验对比图。
图3是本发明中孕期感染子代大鼠模型的转录组测序及荧光定量PCR检测分析对比图。
图4是本发明中荧光定量PCR检测孕期感染子代大鼠模型HDCA5的mRNA水平对比图。
具体实施方式
下面通过具体实施方式,对本发明的技术方案做进一步的详细描述。
实施例
本发明提供评估HDAC5作为孕期感染子代大鼠模型质量早期外周标志物的试验方法,包括以下步骤:
步骤1:孕鼠准备:选择SPF级10-14周龄的SD种鼠,新环境适应2周后开始育种,每3-5只雄鼠与1只雌鼠至于一个分段笼里,前三天只允许嗅觉接触无身体接触,3天后放一起交配,次日早上通过阴道栓检查雌鼠是否成功受孕,成功受孕者入组;
步骤2:孕期感染:确定怀孕后第 9 天尾静脉注射10mg/kg 的聚肌胞苷酸(Poly I:C)或等体积生理盐水,模型组和对照组各随机选取3只,在注射3小时后处死,检测孕鼠外周血血清 IL-1β、IL-6、TNF-α的蛋白水平,通过免疫激活状态确定注射效果,其余孕鼠继续饲养至产下子鼠;
步骤3:行为学检测:将12-15只仔鼠于40天(青春期)、56天(成年期)采用小动物行为采集和分析系统进行Y迷宫、前脉冲抑制实验,分别分析大鼠的学习记忆状态、感觉门控作用,比较两组动物随年龄增长在行为上的差异;
步骤4:对孕期感染子代大鼠模型进行转录组测序及荧光定量PCR分析;
步骤5:对孕期感染子代大鼠模型外周血单核细胞中HDAC5水平进行验证。
如图1所示,上述步骤2中通过ELISA检测孕鼠血浆IL-6 、IL-1β和TNF-α的表达水平,当结果显示模型组孕鼠血浆炎症因子水平相比于对照组显著升高,则提示孕期免疫激活。
需要注意的是,步骤3中行为学检测时间控制在8:00-18:00之间,房间内控制适宜的温度、湿度及照明以确保检测效果。
图2为孕期感染子代大鼠模型青春期和成年期行为学检测分析对比图。图2(A和B)为开放旷场实验,大鼠进入中心区域的时间与其焦虑情绪负相关,结果显示,青春期大鼠无明显焦虑,而在成年期焦虑水平增加。Y迷宫实验(C和D),大鼠进入新异臂的次数、时间与空间记忆能力正相关,结果显示模型组大鼠在青春期无明显差异,而在成年期,进入新异臂的次数显著降低,提示成年期模型组大鼠空间记忆能力受损。图2(E和F)为前脉冲抑制效率(PPI),依据此可反应大鼠的感觉门控作用,结果显示,青春期和成年期模型组大鼠PPI水平低于对照组,同时差异在成年期更加显著,提示模型组大鼠感觉门控功能随时间增加受损。
步骤4中将孕期感染子代大鼠模型青春子代前额叶皮质进行转录组测序分析HDAC5转录水平,并将测序结果通过荧光定量PCR进一步验证,具体结果如下:
将孕期感染子代大鼠模型青春子代前额叶皮质进行转录组测序分析,结果显示一共有461个差异表达基因图3(A),红色代表上调基因,蓝色代表下调基因,颜色深浅代表差异程度,与对照组相比上调表达262个,下调表达199个。该测序结果通过荧光定量PCR进一步验证,通过青春期图3(B)可以看出,HDAC5转录水平(mRNA水平)在模型组大鼠显著上调,这一发现迄今未见公开报导,同时该上调现象还出现在海马区域,并持续到成年期图3(C)。结合行为学结果,可以得出在疾病早期,异常行为还未充分显现阶段,HDAC5的变化就已经出现的启示,提示HDAC5可以作为疾病早期诊断的分子标志物。
鉴于脑组织样本的特殊性,取材较难,以及实验的可延续性,故收集孕期感染大鼠模型外周血样,分离单核淋巴细胞,提取RNA,定量分析HDCA5转录水平。
如图4所示,结果显示,外周反应与中枢一致,青春期和成年期,孕期感染子代大鼠模型外周血单核细胞HDAC5表达水平升高,提示外周HDAC5反应与中枢一致,故外周血单核细胞HDAC5表达水平可作为评价早期模型质量的标志物。
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其限制;尽管参照较佳实施例对本发明进行了详细的说明,所属领域的普通技术人员应当理解:依然可以对本发明的具体实施方式进行修改或者对部分技术特征进行等同替换;而不脱离本发明技术方案的精神,其均应涵盖在本发明请求保护的技术方案范围当中。
Claims (5)
1.评估HDAC5作为孕期感染子代大鼠模型质量早期外周标志物的试验方法,其特征在于,包括以下步骤:
步骤1:孕鼠准备:选择SPF级10-14周龄的SD种鼠,新环境适应2周后开始育种,每3-5只雄鼠与1只雌鼠至于一个分段笼里,前三天只允许嗅觉接触无身体接触,3天后放一起交配,次日早上通过阴道栓检查雌鼠是否成功受孕,成功受孕者入组;
步骤2:孕期感染:确定怀孕后第 9 天尾静脉注射10mg/kg 的聚肌胞苷酸(Poly I:C)或等体积生理盐水(对照组),模型组和对照组各随机选取3只,在注射3小时后处死,检测孕鼠外周血血清 IL-1β、IL-6、TNF-α的蛋白水平,通过免疫激活状态确定注射效果,其余孕鼠继续饲养至产下子鼠;
步骤3:行为学检测:将12-15只仔鼠于40天(青春期)、56天(成年期)采用小动物行为采集和分析系统进行Y迷宫、前脉冲抑制实验,分别分析大鼠的学习记忆状态、感觉门控作用,比较两组动物随年龄增长在行为上的差异;
步骤4:对孕期感染子代大鼠模型进行脑组织转录组测序及荧光定量PCR分析;
步骤5:对孕期感染子代大鼠模型外周血单核细胞中HDAC5水平进行验证。
2.根据权利要求1所述的评估HDAC5作为孕期感染子代大鼠模型质量早期外周标志物的试验方法,其特征在于:步骤2中ELISA检测孕鼠血浆IL-6 、IL-1β和TNF-α的表达水平,当结果显示模型组孕鼠血浆炎症因子水平相比于对照组显著升高,则提示孕期免疫激活。
3.根据权利要求1所述的评估HDAC5作为孕期感染子代大鼠模型质量早期外周标志物的试验方法,其特征在于:步骤3中行为学检测时间控制在8:00-18:00之间,房间内控制适宜的温度、湿度及照明。
4.根据权利要求1所述的评估HDAC5作为孕期感染子代大鼠模型质量早期外周标志物的试验方法,其特征在于:步骤4中将孕期感染子代大鼠模型青春子代前额叶皮质进行转录组测序分析HDAC5转录水平,并将测序结果通过荧光定量PCR进一步验证。
5.根据权利要求1所述的评估HDAC5作为孕期感染子代大鼠模型质量早期外周标志物的试验方法,其特征在于:收集孕期感染大鼠模型外周血样,分离单核淋巴细胞,提取RNA,定量分析HDCA5表达水平。
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