CN116411069B - 检测生物标志物的产品在制备诊断神经管畸形产品的用途 - Google Patents
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Abstract
本发明涉及医药技术领域，具体涉及一种检测生物标志物的产品在制备诊断神经管畸形的产品的用途。本发明提供的一种检测生物标志物的产品在制备辅助诊断或诊断神经管畸形的产品中的用途，所述生物标志物包括DNA的N6‑甲基腺嘌呤（6mA）。经本发明研究发现，DNA 6mA在胚胎发育早期母体低叶酸的状态下诱导的神经管畸形样本中异常降低，说明DNA 6mA的水平和神经管畸形显著相关，进而DNA 6mA可以作为神经管畸形早期诊断或辅助诊断的生物标志物。
Description
技术领域
本发明涉及医药技术领域，具体涉及一种检测生物标志物的产品在制备诊断神经管畸形的产品的用途。
背景技术
神经管畸形（NTDs）是指胚胎期间神经管发育不完全或发育异常所导致的一类畸形。常见的神经管畸形包括脊柱裂、无脑儿、脑膨出等。现阶段神经管畸形的诊断主要包括产前超声检查、羊水穿刺和胎儿磁共振成像（MRI）等，但这些常规产前检查存在胎儿发育早期检出率低的弊端。人类胎儿，从胚胎发育第16天起，脊索诱导与之相邻的外胚层细胞形成神经板，在此基础上经历不断增殖、迁移、分化和细胞形态变化等过程，神经板两侧边缘向上隆起增厚形成神经嵴，中央凹陷形成神经沟，最后，神经嵴逐渐融合并沿其中轴形成中空的神经管。在胚胎发育第27天左右，前后神经孔先后闭合而实现神经管的闭合。小鼠胚胎中，神经管闭合的窗口期在胚胎发育的第7.5-10.5天。以上说明神经管闭合异常在胚胎发育较早期就已经形成。在胚胎发育早期发生神经管闭合异常，常规的超声等诊断方法无法在早期诊断，找到一种在胚胎发育早期可以辅助诊断神经管畸形的标志物，对于神经管畸形的早期辅助诊断，减少家庭和社会的负担具有重要意义。
发明内容
因此，本发明要解决的技术问题在于提供一种检测生物标志物的产品在制备辅助诊断或诊断神经管畸形的产品中的用途。
为此，本发明提供了如下的技术方案：
一种检测生物标志物的产品在制备辅助诊断或诊断神经管畸形的产品中的用途，所述生物标志物包括DNA的N6-甲基腺嘌呤。
可选的，所述生物标志物包括DNA 6mA去甲基化酶ALKBH1的基因或其转录产物或其表达产物。
可选的，所述神经管畸形为低叶酸导致的。
可选的，所述神经管畸形包括神经管闭合异常导致的神经管畸形。
可选的，检测生物标志物的产品为检测DNA的N6-甲基腺嘌呤修饰水平或DNA 6mA去甲基化酶ALKBH1的基因、转录产物或表达产物的水平的产品。
可选的，所述检测生物标志物的产品包括试剂盒、试纸、试剂、引物、生物芯片或探针。
可选的，所述制备辅助诊断或诊断神经管畸形的产品包括试剂盒、试纸、试剂、引物、生物芯片或探针。
可选的，待测样本为外周血或血清。
本发明技术方案，具有如下优点
1.本发明提供的一种检测生物标志物的产品在制备辅助诊断或诊断神经管畸形的产品中的用途，所述生物标志物包括DNA的N6-甲基腺嘌呤（6mA）。经本发明研究发现，DNA6mA在胚胎发育早期母体低叶酸的状态下诱导的神经管畸形样本中异常降低，说明DNA 6mA的水平和神经管畸形显著相关，进而DNA 6mA可以作为神经管畸形早期诊断或辅助诊断的生物标志物。
2.本发明提供的一种检测生物标志物的产品在制备辅助诊断或诊断神经管畸形的产品中的用途，所述生物标志物包括DNA 6mA去甲基化酶ALKBH1；经本发明研究发现，DNA6mA在胚胎发育早期母体低叶酸的状态下诱导的神经管畸形样本中异常降低的同时，伴随着DNA 6mA的去甲基化酶ALKBH1的显著上调，说明去甲基化酶ALKBH1的水平和神经管畸形显著相关，进而DNA 6mA去甲基化酶ALKBH1可以作为神经管畸形早期诊断或辅助诊断的生物标志物。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1 是本发明实施例中正常胚胎（CON）以及低叶酸导致神经管畸形的小鼠胚胎（NTD）；
图2是本发明实施例中各组小鼠胚胎脑组织样本中DNA 6mA水平的斑点杂交（Dotblot）（图中a图）及MeDIP检测结果（图中b图和c图）；
图3是本发明实施例中各组脑组样本DNA 6mA MeDIP测序差异甲基化区（DMRs）对应基因的GO通路富集结果；
图4是本发明实施例中各组脑组织样本的蛋白质免疫印记结果；
图5是本发明实施例中低叶酸胚胎干细胞与正常叶酸胚胎干细胞DNA 6mA 差异甲基化区（DMRs）对应的神经发育相关基因；
图6是本发明实施例中低叶酸胚胎干细胞与正常叶酸胚胎干细胞DNA 6mA 差异甲基化区（DMRs）对应基因GO通路富集，主要在鼓泡、局部开链的DNA的结合以及神经发育相关通路。
具体实施方式
提供下述实施例是为了更好地进一步理解本发明，并不局限于所述最佳实施方式，不对本发明的内容和保护范围构成限制，任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的产品，均落在本发明的保护范围之内。
实施例中未注明具体实验步骤或条件者，按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。
实施例
1.细胞和动物模型
1.1、低叶酸Sv/129细胞模型的建立：小鼠胚胎干细胞(SV129)复苏后，以 含4mg/L叶酸浓度的培养基（4mg/L叶酸、8.2wt%DMEM（Sigma，D2429）、15 wt % FBS、1 wt % NEAA、1wt % L-谷氨酰胺、10 wt % NaHCO3、0.7%葡萄糖、0.001 wt %β-巯基乙醇，余量为蒸馏水）培养6代（细胞密度为1×106个细胞/mL），作为正常叶酸组; 以含 0mg/L 叶酸浓度培养基（8.2wt% DMEM（Sigma，D2429）、15wt% FBS、1 wt % NEAA、1wt% L-谷氨酰胺、10wt% NaHCO3、0.7%葡萄糖、0.001 wt %β-巯基乙醇，余量为蒸馏水）培养 6 代（细胞密度为1×106个细胞/mL），作为低叶酸组。
1.2、低叶酸喂养联合甲氨蝶呤诱导的NTDs小鼠模型建立：
小鼠饲养温度22～26℃，湿度40～60％，采用12h：12h 昼夜间断照明，饲养室条件保持稳定，以确保试验结果的可靠性，小鼠自由进食饮水。
实验组（低叶酸致神经管畸形组）：选择6-8周的SPF级C57/6J雌性小鼠，喂养低叶酸饲料（低叶酸饮食配方包括酪蛋白 23重量份，胱氨酸 0.3重量份，玉米淀粉 38重量份，麦芽糖糊精 13重量份，蔗糖10重量份，纤维素5重量份，豆油8重量份，酒石酸氢胆碱0.3重量份，AIN93多矿 3份，AIN93多维 1份，该部分已公开在[1]裴培,崔小岱,张霆,王珊.低叶酸联合甲氨蝶呤诱导神经管畸形小鼠模型的建立[J].中华神经外科杂志,2019(02):193-196.)4周，按2：1（雌：雄）比例选择性成熟雄性SPF级C57/6J小鼠于头天晚上8：00合笼，交配过夜，次日晨8时取出。检孕栓并称重，将发现阴栓孕鼠分笼喂养，并将检栓当天12时定为孕0.5天（E0.5d），于孕7.5天给孕鼠称重后，小鼠腹腔注射甲氨蝶呤一次，甲氨蝶呤应用剂量为1.5mg/kg（小鼠体重）。待孕10.5天时，颈椎脱臼处死孕鼠，75%乙醇擦拭腹部，沿腹部中线剪开皮肤，打开腹腔，分离出子宫，置于预冷的PBS平皿中，将子宫清洗2次，体视显微镜下，依次剥离子宫壁、蜕膜级卵黄囊，分离出胚胎，观察对照组级实验组小鼠胚胎情况及神经管发育情况，拍照并记录。
对照组：选择6-8周的SPF级C57/6J雌性小鼠，喂养正常繁育饲料4周（购自北京维通利华实验动物技术有限公司），按2：1比例选择性成熟雄性SPF级C57/6J小鼠于头天晚上8：00合笼，交配过夜，次日晨8时取出。检孕栓并称重，将发现阴栓孕鼠分笼喂养，并将检栓当天12时定为孕0.5天（E0.5d），于孕7.5天给孕鼠称重后，小鼠腹腔注射等体积的生理盐水。待孕10.5天时，颈椎脱臼处死孕鼠，同实验组操作进行解剖取胎鼠。
2.实验方法和结果
2.1、 DNA 6mA水平检测和差异基因GO通路富集
将1.2中的实验组和对照组小鼠，在孕10.5天（小鼠神经管闭合窗口期）处死后，取出小鼠胚胎进行拍照如图1所示，说明实验组构建神经管畸形小鼠模型成功。取出的小鼠胚胎的脑组织样本（每组10只混合）应用斑点杂交（Dot blot）及MeDIP（委托上海云序生物科技有限公司）检测脑组织DNA 6mA水平，以及利用启动子区（TSS-2000 ~ TSS+2000）的差异甲基化区（DMRs）对应的基因进行GO功能分析，以注释并推测这些甲基化区可能的作用。斑点杂交具体方法为：将待测DNA样品浓度统一稀释为200 ng/μL，将样品95℃加热3分钟后立即冰上冷却，将2μL的 DNA样品按顺序滴在硝酸纤维素（NC）膜上，254nm紫外线灯下照射30min-1h，TBST缓冲液清洗膜5min，室温摇动封闭1小时，一抗（m6A antibody,货号202003，购自SYSY）4℃孵育过夜，洗膜后二抗室温孵育1小时，显影仪下拍照，白光采集亚甲蓝染色后的图像确定负载对照均一。GO分析方法：将在两组间差异甲基化区（DMRs）对应的表达基因向GO数据库(http://www.geneontology.org/)的各词条（term）映射，并计算每个词条（term）的基因数，从而得到具有某个GO功能的基因列表及基因数目统计；然后应用超几何检验，找出与整个基因组背景相比，在差异表达基因中显著富集的GO条目。
检测结果如图2所示，斑点杂交结果（图2中a图）显示相比于正常组，DNA 6mA水平在母体低叶酸导致的神经管畸形小鼠神经管闭合窗口期E10.5天的脑组织中异常降低，且DNA 6mA MeDIP测序（图2中b图和c图）证实DNA 6mA整体修饰水平在E10.5神经管畸形胎鼠脑组织中下调，进而DNA 6mA可以作为神经管畸形早期诊断或辅助诊断的生物标志物。
如图3所示，DNA 6mA在两组间差异甲基化区（DMRs）对应基因的 GO 分析显示，差异表达基因主要富集在DNA结合以及神经发育相关通路，提示这些差异甲基化区的作用是参与DNA结合以及神经发育。
2.2、DNA 6mA去甲基化酶ALKBH1水平检测
ALKBH1是DNA 6mA的去甲基化酶。将人类神经管畸形样本人脑组织（7个，由首都儿科研究所提供）以及正常的人脑组织（7个，由首都儿科研究所提供）、小鼠神经管畸形样本鼠脑组织（10个混合样本，1.2中实验组小鼠）、鼠脊组织（10个混合样本，1.2中实验组小鼠）以及叶酸缺乏胚胎干细胞系(SV129)、正常叶酸胚胎干细胞系(SV129)进行去甲基化酶ALKBH1的蛋白质免疫印迹检测，方法为蛋白提取物在SDS-PAGE聚丙烯酰胺凝胶上通过电泳分离，然后通过湿转法(220 mA)转到PVDF膜上，封闭1 h，孵育一抗(Anti-ALKBH1，ab195376，1:2000)并4 ℃过夜，TBST洗膜后二抗(ab205718，Abcam ，1:8000)室温孵育1 h,洗膜后进行条带显影和图像采集。
检测结果如图4所示，可以看到在人类神经管畸形样本、小鼠神经管畸形样本以及叶酸缺乏胚胎干细胞系中发现DNA 6mA的去甲基化酶ALKBH1均上调, 说明去甲基化酶ALKBH1可以诊断低叶酸导致的神经管畸形。
2.3、DNA 6mA及ALKBH1对低叶酸致神经管畸形具有潜在诊断意义
既往结构生物学分析研究表明，ALKBH1更倾向于将鼓泡、局部开链的DNA(bubbledor bulged DNA)作为底物，而不是单链(ss-)或双链(ds-) DNA。鼓泡、局部开链的 DNA（bubbled DNA）是一种DNA结构，它是由于DNA在某些条件下局部开链后形成的一种结构，具体来说，ALKBH1可以通过催化反应将甲基基团从鼓泡、局部开链的 DNA中去除。叶酸缺乏时，DNA会发生双链不稳定，在本发明中，将1.1中的低叶酸和正常叶酸组的细胞系进行DNA6mA测序（委托上海云序生物科技有限公司），结果发现，DNA 6mA在两组间差异甲基化区（DMRs）对应基因大多为神经发育相关基因（图5所示）且 GO 富集在鼓泡、局部开链的 DNA结合以及神经发育相关通路，提示这些差异甲基化区的作用是参与鼓泡、局部开链的 DNA结合以及神经发育（图6所示），证明叶酸缺乏与鼓泡、局部开链的 DNA相关，进一步证明了ALKBH1和DNA 6mA在低叶酸导致的神经管畸形中潜在的诊断作用。
综上，本发明研究发现DNA 6mA在胚胎发育早期母体低叶酸的状态下异常降低，且伴随着DNA 6mA的去甲基化酶ALKBH1的上调，说明DNA 6mA及其去甲基化酶ALKBH1可以作为早期诊断低叶酸致神经管畸形的一种生物标记物。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (3)
1.一种检测生物标志物的产品在制备辅助诊断或诊断低叶酸导致的神经管畸形的产品中的用途，其特征在于，所述生物标志物为在样本中水平上调的去甲基化酶ALKBH1，低叶酸是导致神经管畸形的唯一原因。
2.根据权利要求1所述的用途，其特征在于，所述检测生物标志物的产品包括试剂盒、试纸、试剂、引物、生物芯片或探针。
3.根据权利要求1或2所述的用途，其特征在于，所述样本为外周血或血清。
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