CN109022486A - 一种癫痫动物模型的构建方法 - Google Patents
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Abstract
本发明涉及一种癫痫动物模型的构建方法，属于动物模型构建技术领域。该方法将FGF9fl/fl小鼠与GABA能神经元Cre小鼠交配，获得FGF9fl/+VGAT‑Cre杂合敲除小鼠，之后再与FGF9fl/fl纯合小鼠交配，获得FGF9纯合敲除小鼠(FGF9fl/ flVGAT‑Cre)，即癫痫动物模型。该癫痫动物模型更贴近临床癫痫症状及机制，为临床癫痫病因学的研究，以及进一步的诊断和治疗提供了良好的研究工具。
Description
技术领域
本发明涉及一种动物模型构建方法，更具体地，涉及一种癫痫动物模型的构建方法，属于动物模型构建技术领域。
背景技术
癫痫(epliepsy)是一组由于脑部神经元异常过度放电所引起的突然、短暂、反复发作的中枢神经系统(Central nervous system，CNS)功能失常的慢性疾病和综合征，是最常见的严重的神经系统疾病之一。据世界卫生组织估计，全球大约有7千多万癫痫患者，约占了世界人口的1％。我国癫痫的患病率在4％到7％之间，约有600万左右的活动性癫痫患者，同时每年有40万左右新发癫痫患者。癫痫可严重降低患者生活质量、加重患者家庭及社会经济负担，死亡危险性为一般人群的2-3倍，已经成为重要的社会问题。但是，癫痫的发病机制极其复杂，迄今为止尚未完全阐明。因此，研究癫痫发病机制以及开发新的治疗药物迫在眉睫。
建立良好的动物模型是研究疾病的基础。目前，癫痫动物模型主要分为点燃模型和基因敲除模型。点燃模型(kindling model)是通过重复给予一个阈下电或化学刺激引起部分或全面性癫痫发作的模型。电刺激过程十分复杂，且所埋置电极的一端暴露体外，长期保持在确切位置十分困难，因此成功率并不高。化学药物也能够引出点燃现象，比如利多卡因、戊四氮、印方以毒素和红藻氨酸等。化学药物点燃需要反复给与刺激，劳动强度太大，整个过程耗时耗力太长。此外，如果需要诱导出自发癫痫则需要过度点燃，需要的刺激量更大，小鼠死亡率较高，也缺乏一个可靠的标准。目前基因敲除和转基因技术已经在癫痫模型研究取得很大进展，成功制作了多个癫痫模型，例如cystatin B基因敲除模型、钾通道基因Kv1.1敲除模型、UBE3a基因敲除模型等，这些癫痫遗传学动物模型为遗传性癫痫的分子病理研究，以及为人类癫痫筛选候选基因提供了捷径。
FGF9是一种成纤维细胞生长因子，具有神经胶原激活因子活性，参与了神经发育、细胞凋亡、胚胎发育和癌症进展等多项生理和病理过程。但是到现在为止，国内外未见FGF9与癫痫相关的报道。我们工作显示在GABA能神经元中敲除FGF9能够诱导小鼠癫痫。因此，研究FGF9条件敲除小鼠的癫痫机制，对阐明癫痫的作用机制以及寻找新的靶点提供依据具有非常重要的研究意义。
发明内容
本发明所要解决的技术问题是克服现有技术存在的缺陷提供一种癫痫动物模型的构建方法。
本发明的目的是通过以下技术方案予以实现的。
γ-氨基丁酸(gamma-Aminobutyric acid,GABA)是中枢神经系统内最具代表性的抑制性神经递质，在中枢神经系统广泛分布，主要作用是平衡谷氨酸递质的兴奋性活性。FGF9是一种分泌性的成纤维细胞生长因子，参与了神经发育、细胞凋亡、胚胎发育和癌症进展等多项生理和病理过程，对脑神经元有维持生存和促进生长及修复受损的作用。小鼠FGF9基因在14号染色体正链上，全长54.2kb，发明人通过研究发现GABA能神经元中条件性敲除FGF9基因(FGF9fl/flVGAT-cre)小鼠出现后肢阵挛，全身强直-阵挛性发作等一系列癫痫症状，部分小鼠死于癫痫持续状态。脑电图(EEG)显示FGF9fl/flVGAT-cre小鼠出现显著的癫痫波。
于是，本发明在上述研究的基础上，提供GABA能神经元中条件性敲除FGF9基因的方法以及在构建癫痫动物模型中的应用。
首先，所述GABA能神经元中条件性敲除FGF9基因的方法，即利用GABA能神经元特异性Cre小鼠与FGF9基因外显子1两侧携带LoxP的Flox allele小鼠(FGF9fl/fl)杂交，制备FGF9fl/flVGAT-cre小鼠(CKOVGAT)，从而实现在GABA能神经元中条件性敲除FGF9基因。
该方法主要利用Cre/loxP重组酶系统。Cre重组酶是一种位点特异性重组酶，能介导两个LoxP位点之间的特异性重组，使LoxP位点间的基因序列被删除或重组。LoxP序列具有两个13bp反向重复序列和中间间隔的8bp序列共同组成，8bp的间隔序列同时也确定了LoxP的方向。如果两个LoxP位点位于一条DNA链上，且方向相同，Cre重组酶能有效切除两个LoxP位点间的序列，从而实现基因敲除。
本发明所述FGF9fl/fl小鼠制备步骤如下：
1)构建FGF9条件性敲除的打靶载体
分析FGF9基因的结构，exon1(ENSMUSE00000337574)可被条件性敲除，敲除后FGF9基因不表达，在5’端和3’端loxP位点分别插入在启动子区和intron2的非保守区；
2)ES细胞电转和正确重组的ES细胞克隆筛选
打靶载体线性化后(序列边缘限制性内切酶酶切打靶载体)，进行2次电转ES细胞，共挑选出600个克隆，经PCR和Southern blot筛选，共筛选到10个两端均正确重组的ES细胞克隆；
3)ES细胞显微注射囊胚及子宫移植
挑选5只小鼠，共注射3批ES细胞克隆，共出生小鼠51只，其中克隆4-A6(2#)产生第一只阳性F1小鼠，基因型鉴定为阳性杂合小鼠，将杂合子小鼠与Flp-deleter小鼠交配，得到带有Flp转基因的floxed(fl)杂合子小鼠，即FGF9fl/+小鼠，FGF9fl/+小鼠互相交配得到FGF9fl/fl纯合小鼠。
其次，本发明提供GABA能神经元中条件性敲除FGF9基因在癫痫动物模型构建中的应用，即一种癫痫动物模型的构建方法。
一种癫痫动物模型的构建方法，包括如下步骤：
将FGF9fl/fl小鼠与GABA能神经元Cre小鼠交配，获得FGF9fl/+VGAT-Cre杂合敲除小鼠，之后再与FGF9fl/fl纯合小鼠交配，获得FGF9纯合敲除小鼠(FGF9fl/flVGAT-Cre)，即癫痫动物模型。
以同窝FGF9fl/fl小鼠为对照小鼠，维持小鼠C57BL/6背景。C57BL/6背景常被认作"标准"的近交系，是肿瘤学、生理学、免疫学、遗传学研究中常用的品系。通过PCR对小鼠进行基因型鉴定，检测FGF9小鼠的LoxP位点，westblot验证脑组织中FGF9蛋白表达降低。
本发明首次在GABA能神经元中条件性敲除FGF9基因，并通过GABA能神经元中条件性敲除FGF9基因构建癫痫动物模型，目前在国内外未见相关报道；该方法构建小鼠模型具有以下优点：
(1)构建得到的FGF9fl/flVGAT-Cre癫痫动物模型稳定，癫痫发作重复率高达100％；(2)该癫痫动物模型不仅癫痫症状比较典型(如小鼠出现包括点头、抽搐、后肢阵挛、全身强直-全身强直阵挛发作而跌倒、癫痫持续状态等癫痫症状)，而且脑电图也显示典型的癫痫波(重复高振幅持续10s以上的棘波)；(3)构建得到的癫痫动物模型小鼠生存周期长，除早期部分死于癫痫持续状态，其余小鼠生存可达24个月；(4)构建得到的癫痫动物模型主要由于GABA/Glu递质失衡造成，与临床上认为神经递质失衡是癫痫发作的主要原因一致，利用本发明所述癫痫动物模型可以进一步完善癫痫发病机制以及寻找筛选抗癫痫药物。总之，该GABA能神经元中条件性敲除FGF9基因癫痫动物模型更贴近临床癫痫症状及机制，为临床癫痫病因学的研究，以及进一步的诊断和治疗提供了良好的研究工具。
附图说明
图1为利用Cre/loxP重组酶系统建立FGF9条件敲除动物模型的示意图。
图2为FGF9fl/flVGAT-Cre小鼠出现典型的癫痫症状和癫痫波。
图3为正常小鼠和FGF9fl/flVGAT-Cre小鼠海马C3区GABA含量变化。
图4为FGF9fl/flVGAT-Cre小鼠给予加巴喷丁治疗后显著降低癫痫发作频率。
图5为FGF9fl/flVGAT-Cre小鼠癫痫的主要信号通路是cAMP信号通路。
图6为给与cAMP信号通路抑制剂能够降低FGF9fl/flVGAT-Cre小鼠癫痫发作频率。
具体实施方式
下面结合附图对本发明的较佳实施例进行详细阐述，以使本发明的优点和特征能更易于被本领域技术人员理解，从而对本发明的保护范围做出更为清楚明确的界定。
实施例1GABA能神经元中条件性敲除FGF9基因的方法以及癫痫动物模型的构建
首先，FGF9fl/fl小鼠制备步骤如下：
1)构建FGF9条件性敲除的打靶载体
分析FGF9基因的结构，exon1(ENSMUSE00000337574)可被条件性敲除，敲除后FGF9基因不表达，在5’端和3’端loxP位点分别插入在启动子区和intron2的非保守区；
2)ES细胞电转和正确重组的ES细胞克隆筛选
打靶载体线进限制性酶切线性化后，进行2次电转ES细胞，共挑选出600个克隆，经PCR和Southern blot筛选，共筛选到10个两端均正确重组的ES细胞克隆；
3)ES细胞显微注射囊胚及子宫移植
挑选5只小鼠，共注射3批ES细胞克隆，共出生小鼠51只，其中克隆4-A6(2#)产生第一只阳性F1小鼠，基因型鉴定为阳性杂合小鼠，将杂合子小鼠与Flp-deleter小鼠交配，得到带有Flp转基因的floxed(fl)杂合子小鼠，即FGF9fl/+小鼠。FGF9fl/+小鼠互相交配得到FGF9fl/fl纯合小鼠。
其次，FGF9纯合敲除小鼠(FGF9fl/flVGAT-Cre)制备方法，即本发明所述癫痫动物模型的构建方法，步骤如下：
1)FGF9fl/+VGAT-Cre杂合敲除小鼠
将获得的FGF9fl/fl小鼠，与GABA能神经元Cre小鼠(VGAT-Cre，购于Jackson StockNo:017535)交配，获得FGF9fl/+VGAT-Cre杂合敲除小鼠；
2)FGF9纯合敲除小鼠(FGF9fl/flVGAT-Cre)
将步骤1)所得FGF9fl/+VGAT-Cre杂合敲除小鼠与FGF9fl/fl纯合小鼠交配，获得FGF9纯合敲除小鼠(FGF9fl/flVGAT-Cre)，即本发明所述癫痫动物模型。
本发明所述方法将获得的FGF9fl/fl小鼠，与GABA能神经元Cre小鼠杂交，从而将LoxP位点中外显子1敲除，获得FGF9基因敲除小鼠，见图1。
以同窝FGF9fl/fl小鼠为对照小鼠，维持小鼠C57BL/6背景(C57BL/6背景常被认作"标准"的近交系，是肿瘤学、生理学、免疫学、遗传学研究中常用的品系)，通过PCR对小鼠进行基因型鉴定，检测FGF9小鼠LoxP位点，westblot验证脑组织中FGF9蛋白表达降低。
FGF9fl/flVGAT-Cre(CKOVGAT)小鼠从出生后16天起出现典型的癫痫症状，包括点头、抽搐、后肢阵挛、全身强直-全身强直阵挛发作而跌倒等。脑电图显示敲除小鼠出现持续大于10秒的重复高振幅棘波(典型癫痫波)，见图2。
与对照FGF9fl/fl小鼠相比，FGF9fl/flVGAT-Cre小鼠海马C3区GABA荧光明显减少，说明影响了GABA的合成，见图3。
视频统计FGF9fl/flVGAT-Cre小鼠48小时癫痫发作次数，之后每12小时腹腔注射0.1ml加巴喷丁(100mg/kg)。比较给药后48小时的癫痫发作次数，明显降低，见图4。
我们对FGF9fl/flVGAT-Cre小鼠和对照FGF9fl/fl小鼠进行RNA测序，比较差异基因参与的信号通路，KEGG分析显示差异基因主要参与了神经活性配体-受体相互作用，钙信号通路，cAMP信号通路和GABA能神经突触信号途径，见图5。
统计FGF9fl/flVGAT-Cre小鼠48小时内癫痫发作次数，之后给与SQ22536和布比卡因-HCl(分别为AC和cAMP抑制剂，剂量均为10mg/kg)。FGF9fl/flVGAT-Cre小鼠给药后48小时的癫痫发作次数明显降低，见图6。
观察统计FGF9fl/flVGAT-Cre小鼠癫痫发作频次、持续时间、发作等级以及死亡率，进行脑电图(EEG)分析，以棘波、棘慢波及爆发性高波幅节律性活动为痫样放电。
统计了30只FGF9fl/flVGAT-Cre小鼠，全部癫痫发作，对3只癫痫小鼠进行脑电图检测，全部出现典型的癫痫波。FGF9fl/flVGAT-Cre敲除小鼠出生两周后出现癫痫症状，3周-4周平均每天发作9-26次，部分小鼠死于癫痫持续状态(如表1)。此外，与GABA能神经元功能密切相关的神经元(比如可以分化成GABA能神经元的前体细胞Nestin-Cre和Olig1-Cre以及生化关系及其密切的VGLUT1-Cre)敲除FGF9也会出现癫痫发作，但癫痫的严重程度及发作频率差异较大。
表1.不同组织特异性敲除FGF9小鼠的癫痫发作情况和癫痫发作特征。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。
Claims (4)
1.GABA能神经元中条件性敲除FGF9基因的方法，其利用GABA能神经元特异性Cre小鼠与FGF9基因外显子1两侧携带LoxP的Flox allele小鼠(FGF9fl/fl)杂交，得到FGF9fl/flVGAT-cre小鼠，实现在GABA能神经元中条件性敲除FGF9基因。
2.根据权利要求1所述的GABA能神经元中条件性敲除FGF9基因的方法，其特征在于，所述FGF9fl/fl小鼠制备步骤如下：
1)构建FGF9条件性敲除的打靶载体
分析FGF9基因的结构，exon1(ENSMUSE00000337574)可被条件性敲除，敲除后FGF9基因不表达，在5’端和3’端loxP位点分别插入在启动子区和intron2的非保守区；
2)ES细胞电转和正确重组的ES细胞克隆筛选
打靶载体被序列边缘限制性内切酶酶切从而线性化，进行2次电转ES细胞，挑选出600个克隆，经PCR和Southern blot筛选，筛选到10个两端均正确重组的ES细胞克隆；
3)ES细胞显微注射囊胚及子宫移植
挑选5只小鼠，共注射3批ES细胞克隆，共出生小鼠51只，其中克隆4-A6(2#)产生第一只阳性F1小鼠，基因型鉴定为阳性杂合小鼠，将杂合子小鼠与Flp-deleter小鼠交配，得到带有Flp转基因的floxed(fl)杂合子小鼠，即FGF9fl/+小鼠，FGF9fl/+小鼠互相交配得到FGF9fl /fl纯合小鼠。
3.根据权利要求1所述的GABA能神经元中条件性敲除FGF9基因在癫痫动物模型构建中的应用。
4.一种癫痫动物模型的构建方法，其特征在于，包括步骤如下：
1)FGF9fl/+VGAT-Cre杂合敲除小鼠
将FGF9fl/fl小鼠，与GABA能神经元Cre小鼠交配，获得FGF9fl/+VGAT-Cre杂合敲除小鼠；
2)FGF9fl/flVGAT-Cre小鼠
将步骤1)所得FGF9fl/+VGAT-Cre杂合敲除小鼠与FGF9fl/fl纯合小鼠交配，获得FGF9纯合敲除小鼠(FGF9fl/flVGAT-Cre)，即癫痫动物模型。
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| CN111700034A (zh) * | 2020-05-22 | 2020-09-25 | 中国人民解放军空军军医大学 | 一种基于中枢神经系统髓鞘功能改变的精神分裂症动物模型的构建方法和应用 |
| CN112941102A (zh) * | 2021-01-26 | 2021-06-11 | 南方医科大学 | 一种子痫前期小鼠动物模型的构建方法及其应用 |
| CN114946762A (zh) * | 2022-04-18 | 2022-08-30 | 安徽大学 | 一种气道纤毛上皮细胞缺失的动物模型的构建及应用 |
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| CN110951781A (zh) * | 2019-12-18 | 2020-04-03 | 武汉大学 | 一种癫痫动物模型的构建方法及应用 |
| CN110951781B (zh) * | 2019-12-18 | 2021-06-04 | 武汉大学 | 一种癫痫动物模型的构建方法及应用 |
| CN111700034A (zh) * | 2020-05-22 | 2020-09-25 | 中国人民解放军空军军医大学 | 一种基于中枢神经系统髓鞘功能改变的精神分裂症动物模型的构建方法和应用 |
| CN112941102A (zh) * | 2021-01-26 | 2021-06-11 | 南方医科大学 | 一种子痫前期小鼠动物模型的构建方法及其应用 |
| CN114946762A (zh) * | 2022-04-18 | 2022-08-30 | 安徽大学 | 一种气道纤毛上皮细胞缺失的动物模型的构建及应用 |
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