CN111378744B - miR-148a-3p作为热性惊厥诊治标志物的应用 - Google Patents
miR-148a-3p作为热性惊厥诊治标志物的应用 Download PDFInfo
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Abstract
本发明涉及miR‑148a‑3p作为热性惊厥诊治标志物的应用。本发明首次发现了分子标志物miR‑148a‑3p与热性惊厥诊断和治疗相关，通过检测受试者miR‑148a‑3p的变化，可用于热性惊厥的诊断；通过以miR‑148a‑3p为靶点可以制备治疗热性惊厥的药物。
Description
技术领域
本发明属于生物医学领域，涉及miR-148a-3p作为热性惊厥诊治标志物的应用。
背景技术
热性惊厥(Febrile seizures,FS)是儿童期的常见疾病之一，也是门诊就诊的常见原因。全球有2％-5％的儿童至少经历过一次热性惊厥。FS在各地区的发病率有所不同，据研究表明，在欧洲及美国儿童中的患病率为2％-5％；日本为6-9％，西太平洋马丽亚群岛则为11.3％。我国各地区发病率报道不一，最低1％，最高达13％。对于绝大多数患儿来说预后良好，但仍有约2％-10％的热性惊厥患儿最终可进一步发展为癫痫。热性惊厥持续状态和反复发作可导致一过性或长期的损害，影响神经系统的发育，进而造成不同程度的脑损伤。对于热性惊厥患儿来说，惊厥时间越长，则损伤脑细胞的几率就越大。
从发病机制方面考虑，热性惊厥可能与遗传、年龄、发热、感染、免疫等因素有关。热性惊厥好发于6个月-5岁，高峰年龄为1-2岁，大部分患儿6岁后终止发作，复发率高，首次发作后约1/3的儿童有再发风险，伴有高危因素的复发率更高，可达75％-80％。分析这种年龄相关性可能与小儿脑发育不完善、神经细胞结构简单、皮层分化不全、神经元的树突、轴突发育不完善因年龄而异等有关。研究发现热性惊厥有明显的遗传倾向，患儿中父母或同代有惊厥性疾病史的占26％-53.8％。双亲均有热性惊厥病史者，其子女发病率为55.6％；双亲单方阳性病史者，子女患病率为21.7％，发病率明显高于普通人群。常染色体显性伴不完全外显遗传、多基因遗传、多因素遗传是目前所认可的热性惊厥的遗传模式。感染因素是热性惊厥发生不容忽略的重要原因，发热、低血糖症、低血钙症、胃肠道感染、呼吸道感染、头部损伤、中毒及药物副作用等可能为导致热性惊厥发生的间接原因。
近年来，对于分子标志物与热性惊厥易感性的研究较为热门，本申请的目的在于筛选一种可用于热性惊厥诊断的分子标志物，为日后的临床工作提供指导。
发明内容
为了克服现有技术不足，本发明的目的在于提供一种诊治热性惊厥的分子标志物。
为了实验上述目的，本发明采用了如下技术方案：
本发明提供了miR-148a-3p的应用，用于制备诊断热性惊厥的产品。
进一步，所述产品通过测定样本中miR-148a-3p的表达水平来诊断热性惊厥。
进一步，miR-148a-3p在热性惊厥患者中表达高。
进一步，所述产品包括通过测序技术、核酸杂交技术、核酸扩增技术检测miR-148a-3p表达的变化。
进一步，所述核酸扩增技术选自聚合酶链式反应、逆转录聚合酶链式反应、转录介导的扩增、连接酶链式反应、链置换扩增和基于核酸序列的扩增。
本发明提供了一种诊断热性惊厥的产品，所述产品能够通过检测样本中miR-148a-3p表达来诊断热性惊厥。
进一步，所述产品包括芯片、试剂盒或制剂。
其中，所述芯片包括基因芯片；所述基因芯片包括固相载体以及固定在固相载体的寡核苷酸探针，所述寡核苷酸探针包括用于检测miR-148a-3p水平的针对miR-148a-3p的寡核苷酸探针；所述试剂盒包括基因检测试剂盒；所述基因检测试剂盒包括用于检测miR-148a-3p水平的试剂。
本发明提供了miR-148a-3p在制备治疗热性惊厥的药物中的应用。
进一步，所述药物包括抑制miR-148a-3p表达的物质。
所述物质包括蛋白、寡核苷酸、小分子化合物。
在本发明的具体实施方案中，所述物质是miR-148a-3p的inhibitor。
miRNA inhibitor是化学修饰的专门针对细胞中特异的靶miRNA的抑制剂。miRNAinhibitors，特异的靶向和敲除单个的miRNA分子，可以削弱内源miRNA的基因沉默效应，提高蛋白表达量，进行功能缺失性(loss-of-function)研究，可以用来筛选miRNA靶位点，筛选调控某一基因表达的miRNA，筛选影响细胞发育过程的miRNAmiRNA inhibitor是化学修饰的RNA单链，能够与成熟miRNA序列竞争性结合，其易于获得，操作简便，实验周期短，可以很好地应用于miRNA功能分析研究，如细胞增殖、细胞凋亡、细胞分化、细胞迁移、干细胞生长等方面功能研究。miRNA inhibitor只需用转染试剂包裹即可转染进入细胞，无需构建载体的繁琐操作，无需病毒防护方面的担忧，用转染对照即可观察其转染效率。
本发明所述药物还包括药物学上可接受的载体。通过常规制药工艺将有效成分-抑制所述生物标志物表达的物质与药物学上可接受的载体制备成本发明的药物。
本发明的优点和有益效果：
本发明首次发现了分子标志物miR-148a-3p与热性惊厥诊断和治疗相关，通过检测受试者miR-148a-3p的变化，可用于热性惊厥诊断；以miR-148a-3p为靶点可以开发治疗热性惊厥的药物。
附图说明
图1显示利用QPCR检测海马组织中miR-148a-3p的表达情况的统计图；
图2显示利用免疫荧光检测海马神经元细胞中miR-148a-3p定位的荧光图，其中，A：miR-148a-3p；B：DAPI；C：Merge；
图3显示利用TUNEL染色检测海马神经元细胞凋亡情况的结果图，其中，A：TUNEL染色图；B：凋亡率统计图；
图4显示利用流失细胞仪检测miR-148a-3p表达对海马神经元细胞凋亡影响的流式图，其中，A：对照；B：miR-148a-3p mimics；C：miR-148a-3p mimics NC；D：miR-148a-3pinhibitor；E：miR-148a-3p inhibitor NC；
图5显示miR-148a-3p表达对海马神经元细胞凋亡影响的统计图。
具体的实施方式
本发明经过广泛而深入的研究，通过大量筛选，首次发现了在热性惊厥患者中miR-148a-3p呈现特异性高表达。
miR-148a-3p
本发明的miR-148a-3p可以是天然的或是人工合成的，或者使用可以表达miR-148a-3p的DNA片段的载体转染细胞获得。所述载体包括病毒载体、真核载体。
病毒载体可以是任何适当的载体，包括但不限于逆转录病毒载体、腺病毒载体、腺病毒相关病毒载体、疱疹病毒(例如单纯疱疹病毒、痘苗病毒及EB病毒)载体、甲病毒载体。
真核表达载体可以是任何适当的表达载体，包括但不限于pCMV-Myc表达载体、pcDNA3.0表达载体、pcDNA3.1表达载体、pEGFP表达载体、pEF Bos表达载体、pTet表达载体、pTRE表达载体、或者在公知表达载体的基础上经改造的载体，比如pBin438、pCAMBIA1301等。
可以表达miR-148a-3p的DNA片段可以通过如下方式获取：从miRNA数据库中(http://microrna.sanger.ac.uk/sequences/)寻找miR-148a-3p在基因组上的位置及具体序列信息，根据基因组序列确定miR-148a-3p初始miRNA的位置，在miR-148a-3p初始miRNA位置的上下游500-800bp区间内设计特异性引物，扩增引物中间的序列即可获得表达miR-148a-3p的DNA片段。
试剂盒
本发明中的试剂盒可用于检测miR-148a-3p的表达，优选的，所述的试剂盒包括检测有效量的检测miR-148a-3p的试剂，选自下组的一种或多种物质：容器、使用说明书、阳性对照物、阴性对照物、缓冲剂、助剂或溶剂。例如用于混悬或固定细胞的溶液，可检测的标签或标记，使核酸易于杂交的溶液，用于裂解细胞的溶液，或用于核酸纯化的溶液。
本发明的试剂盒中还可附有试剂盒的使用说明书，其中记载了如何采用试剂盒进行检测，和如何利用检测结果对疾病预后进行判断。
采用本发明的试剂盒，可通过选自下组的各种方法(包括但不限于)检测miR-148a-3p：实时定量反转录PCR、生物芯片检测法、DNA印迹法、或RNA印迹法或原位杂交法。本领域普通技术人员可根据实际条件和需要对检测方式进行调整和改变。
芯片
制备本发明的芯片的所述固相载体包括基因芯片领域的各种常用材料，例如但不限于尼龙膜，经活性基团(如醛基、氨基等)修饰的玻片或硅片、未修饰的玻片、塑料片等。
所述的miRNA芯片的制备可采用本领域已知的生物芯片的常规制造方法，例如，如果固相载体采用的是修饰玻片或硅片，探针的5’端含有氨基修饰的聚dT串，可将寡核苷酸探针配制成溶液，然后采用点样仪将其点在修饰玻片或硅片上，排列成预定的序列或阵列，然后通过放置过夜来固定，就可得到本发明的miRNA芯片。如果核酸不含氨基修饰，则其制备方法也可参照：王申五主编的《基因诊断技术-非放射性操作手册》；J.L.erisi，V.R.Iyer，P.O.BROWN.Exploring the metabolic and genetic control of geneexpression on a genomic scale.Science，1997；278：680和马立人，蒋中华主编.生物芯片.北京：化学工业出版社，2000，1-130。
检测技术(方法)
本发明的基因使用本领域普通技术人员已知的多种检测技术进行检测，这些技术包括但不限于：核酸测序、核酸杂交、核酸扩增技术。
核酸测序技术的示例性非限制性实例包括但不限于链终止子(Sanger)测序和染料终止子测序。本领域的普通技术人员将认识到，由于RNA在细胞中不太稳定并且在实验中更易受到核酸酶攻击，因此在测序前通常将RNA逆转录成DNA。
核酸测序技术的另一示例性非限制性实例包括下一代测序(深度测序/高通量测序)，高通量测序技术是一种基于单分子簇的边合成边测序技术，基于专有的可逆终止化学反应原理。测序时将DNA的随机片段附着到光学透明的玻璃表面，这些DNA片段经过延伸和桥式扩增后，在玻璃表面形成数以亿计的簇，每个簇是具有数千份相同模板的单分子簇，然后利用带荧光基团的四种特殊脱氧核糖核苷酸，通过可逆性的边合成边测序技术对待测的模板DNA进行测序。
核酸杂交技术的示例性非限制性实例包括但不限于原位杂交(ISH)、微阵列和Southern或Northern印迹。原位杂交(ISH)是一种使用标记的互补DNA或RNA链作为探针以定位组织一部分或切片(原位)或者如果组织足够小则为整个组织(全组织包埋ISH)中的特异性DNA或RNA序列的杂交。DNA ISH可用于确定染色体的结构。RNA ISH用于测量和定位组织切片或全组织包埋内的mRNA和其他转录本(例如，ncRNA)。通常对样本细胞和组织进行处理以原位固定靶转录本，并增加探针的进入。探针在高温下与靶序列杂交，然后将多余的探针洗掉。分别使用放射自显影、荧光显微术或免疫组织化学，对组织中用放射、荧光或抗原标记的碱基标记的探针进行定位和定量。ISH也可使用两种或更多种通过放射性或其他非放射性标记物标记的探针，以同时检测两种或更多种转录本。
本发明可在检测前或与检测同时地对核酸(例如，ncRNA)进行扩增。核酸扩增技术的示例性非限制性实例包括但不限于：聚合酶链式反应(PCR)、逆转录聚合酶链式反应(RT-PCR)、转录介导的扩增(TMA)、连接酶链式反应(LCR)、链置换扩增(SDA)和基于核酸序列的扩增(NASBA)。本领域的普通技术人员将认识到，某些扩增技术(例如，PCR)需要在扩增前将RNA逆转录成DNA(例如，RT-PCR)，而其他扩增技术则直接扩增RNA(例如，TMA和NASBA)。
通常称为PCR的聚合酶链式反应使用变性、引物对与相反链的退火以及引物延伸的多个循环，以指数方式增加靶核酸序列的拷贝数；TMA的转录介导的扩增(在基本上恒定的温度、离子强度和pH的条件下自身催化地合成靶核酸序列的多个拷贝，其中靶序列的多个RNA拷贝自身催化地生成另外的拷贝；LCR的连接酶链式反应使用与靶核酸的相邻区域杂交的两组互补DNA寡核苷酸；其他扩增方法包括例如：通常称为NASBA的基于核酸序列的扩增；使用RNA复制酶(通常称为Qβ复制酶)扩增探针分子本身的扩增；基于转录的扩增方法；以及自我维持的序列扩增。
本发明中术语“样本”包括任何细胞、组织或体液取样，其中包括但不限于，组织或细胞样本的来源可以来自新鲜、冷冻和/或保存的器官或组织样本的固体组织，或者活组织检查或者抽吸物，血液或任何血液成分；体液如脑脊髓液、羊水、腹腔液或间质液。组织样本可以是初级或体外培养的细胞或细胞株。可选地，组织或细胞样本从疾病组织/器官获取。组织样本可能包含所述组织自然混合的化合物，诸如防腐剂、抗凝血剂、缓冲剂、固定剂、营养剂、抗生素、或类似化合物。
药物
短语“药学上可接受的载体”是本领域认可的并且包括例如参与从身体的一个器官或部分携带或运输任何主题组合物至身体的另一个器官或部分的药学上可接受的材料、组合物或赋形剂，如液体或固体填充剂、稀释剂、溶剂或包囊材料。每种载体必须在与主题组合物的其他成分相容的意义上是“可接受的”并且对患者无害。在某些实施方案中，药学上可接受的载体是无热原的。可以用作药学上可接受的载体的材料的一些例子包括：(1)糖，如乳糖、葡萄糖和蔗糖；(2)淀粉，如玉米淀粉和马铃薯淀粉；(3)纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和乙酸纤维素；(4)粉末状黄蓍胶；(5)麦芽；(6)明胶；(7)滑石粉；(8)可可脂和栓剂蜡；(9)油，如花生油、棉籽油、葵花籽油、芝麻油、橄榄油、玉米油和大豆油；(10)二醇，如丙二醇；(11)多元醇，如甘油、山梨醇、甘露糖醇和聚乙二醇；(12)酯，如油酸乙酯和月桂酸乙酯；(13)琼脂；(14)缓冲剂，如氢氧化镁和氢氧化铝；(15)海藻酸；(16)无热原的水；(17)等渗盐水；(18)林格氏溶液；(19)乙醇；(20)磷酸盐缓冲液；和(21)药物制剂中使用的其他无毒的相容物质。
“施用”表示将剂量药物给患者的方法。在本发明方法中使用的组合物可通过选自但不限于如下途径施用：吸入、眼部、肠胃外、皮肤、经皮、口腔、直肠、舌下、舌周(Perilingual)、鼻、局部给药和口服给药。肠胃外给药包括静脉内、腹膜内、皮下和肌内给药。优选的给药方法可根据多种因素变化，如，被施用的组合物的组分和被治疗的病症的严重度。
以本领域熟练技术人员已知的方式制备本发明的药物，如通过常规的溶解、冻干、混合、制粒或成型方法。制造制剂的本领域熟知方法示于如Remington：The Science andPractice of Pharmacy，第20版，A.R.Gennaro编，2000，Lippincott Williams&Wilkins，Philadelphia，和Encyclopedia of Pharmaceutical Technology(制药技术百科)，J.Swarbrick和J.C.Boylan编，1988-1999，Marcel Dekker，New York。
本领域熟练技术人员可容易地确定在本发明药物中使用的任何试剂的剂量。希望地，本发明药物中的试剂的剂量会足以缓解患者的热性惊厥的症状。可选择性地，所述剂量会足以抑制患者细胞中的miR-148a-3p。
诊断
本发明的“诊断”包括对疾病是否发生的诊断，对疾病发生风险大小的诊断，对疾病复发的诊断，对疾病患者预后的诊断。
下面结合附图和实施例对本发明作进一步详细的说明。以下实施例仅用于说明本发明而不用于限制本发明的范围。实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York：Cold Spring HarborLaboratoryPress，1989)中所述的条件，或按照制造厂商所建议的条件。
实施例1miR-148a-3p在神经系统中的表达
1、大鼠海马组织中miR-148a-3p的表达
1.1FS动物模型构建
选取10只SPF级14日龄正常的SD大鼠，根据经典的热水浴诱导惊厥动物模型的方法进行造模(具体方法参见文献：Jiang W,Duong TM,de Lanerolle NC.Theneuropathology of hyperthermic seizures in the rat.Epilepsia 40,5-19(1999)，连续热水浴7天，按照FS的5级行为学评分标准(评分标准参见文献：Jiang W,Duong TM,deLanerolle NC.The neuropathology of hyperthermic seizures in the rat.Epilepsia40,5-19(1999)，选取行为综合评分最高的3只SD大鼠作为实验组，同时选取3只正常SD大鼠作为对照组。实验动物来源于山东大学实验动物中心，动物合格证编号：SCXK(鲁)20030004。
1.2荧光定量PCR
利用实时荧光定量PCR的方法检测海马组织样本中miR-148a-3p表达情况。
首先，采用TRizolTM法进行总RNA提取，按说明书操作。
(1)加入预冷的TRizolTM试剂lml于玻璃匀浆器中，称取海马组织约100mg，置匀浆器中，在冰上迅速匀浆，研磨组织。
(2)将组织匀浆液转移到1.5ml Ep管中，混匀后室温静置5min。
(3)加入0.4ml氯仿，快速振动30s，冰上静置10rain。
(4)4℃12000rpm离心15min。
(5)小心吸取上层无色水相入新的tube管中。
(6)加入预冷的异丙醇0.5ml/ml Trizol颠倒混匀，20℃静置l0min。
(7)4℃12000rpm离心10min。
(8)弃上清，加预冷的75％无酶水乙醇lml充分振荡洗涤总RNA沉淀。
(9)4℃7500rpm离心5min，尽量吸净上清。
(10)无菌环境中干燥RNA沉淀5min。
(11)取20μl DEPC处理的双蒸水溶解RNA沉淀，55℃水浴助溶5min。
(12)取2μl RNA溶液至新的200μl管中，准备测浓度，其余分装后-80℃保存。
其次，将提取的RNA逆转录成cDNA，具体操作步骤如下
(1)逆转录体系的配制：在20μl的反应体系中，加入lng-5ng的总RNA。体系如下(具体体系依照逆转录试剂盒的说明书而定)：
在0.5ml无RNase的离心管中配制表1中所示的体系A：
表1逆转录体系A
| 组分 | 体积 |
| 总RNA | 2μl |
| RT工作液 | 2μl |
| DEPC水 | 7μl |
(2)短暂离心后振荡混匀，再离心，置70℃孵育l0min，冰上孵育2分钟；而后加入表2中的体系B：
表2逆转录体系B
(3)短暂离心后轻轻混匀，再离心，42℃孵育60min，70℃孵育10min，以灭活反应。
(4)逆转录产物的保存：短期使用，于4℃保存；长期保存，分装后置于-70℃避免反复冻融。
设计引物并检测表达情况；按照天根荧光定量试剂盒标准操作方法操作。
反应体系：
反应程序：
反应程序如下：预变性94℃1min,，94℃变性30sec，60℃退火温度15sec，68℃延伸15sec，40个循环。
选取大鼠U6基因为内参基因。为了分析基因的相对表达量，根据公式2-△△ct＝[ct(target)-ct(actin)]疾病组–[ct(target)-ct(actin)]正常对照组。
选取大鼠U6基因为内参基因，根据2-△△ct法计算基因相对表达量。
引物序列如下所示：
miR-148a-3p引物
上游引物：5’-GGGTCAGTGCACTACAGA-3’(SEQ ID NO.1)，
下游引物：5’-CAGTGCGTGTCGTGGAGT-3’(SEQ ID NO.2)；
U6引物
上游引物：5’-TCGAGTCTACTGGCGTCTT-3’(SEQ ID NO.3)，
下游引物：5’-ATGAGCCCTTCCACGAT-3’(SEQ ID NO.4)。
1.3结果
结果显示，与正常组比较，热性惊厥组miR-148a-3p的表达水平显著高于正常组，差异具有统计学差异(P<0.01)，见图1。
2、H19-7神经元细胞中miR-148a-3p的表达与定位
2.1免疫荧光标记步骤
(1)细胞固定：将H19-7海马神经元细胞爬片用4％多聚甲醛固定20min后，PBS洗三遍备用。
(2)消化：根据固定时间长短(20min)，爬片于修复液中煮沸10分钟，自然冷却。后基因笔画圈，根据不同指标特性，滴加蛋白酶K(20μg/ml)37℃消化30min。纯水冲洗后PBS洗3次×5min。
(3)预杂交：滴加预杂交液，37℃孵育1h。
(4)杂交：倾去预杂交液，滴加含探针miR-148a-3p杂交液，浓度0.5ng/μl，恒温箱42℃杂交过夜，同时加入GFAP/Neun的一抗稀释液(1:100/1:200)。
(5)杂交液和一抗液洗涤：洗去杂交液和一抗液，2×SSC，37℃洗10min，1×SSC，37℃洗2×5min，0.5×SSC室温洗10min。若非特异杂交体较多，可以增加甲酰胺洗涤。
(6)孵育二抗：二抗稀释液以一定的比例加入，与一抗结合，常温孵育50min，PBS洗三遍。
(7)DAPI复染核：切片滴加DAPI染液，避光孵育8min，冲洗后滴加抗荧光淬灭封片剂封片。
(8)镜检拍照：切片于尼康正置荧光显微镜下观察并采集图像。(紫外激发波长330-380nm，发射波长420nm,发蓝光；FAM(488)绿光激发波长465-495nm，发射波长515-555nm，发绿光；CY3红光激发波长510-560，发射波长590nm，发红光；CY5激发激发波长630-650nm，发紫光)；DAPI染核，为蓝光。
2.2结果
利用免疫荧光标记法，检测了miR-148a-3p大鼠海马神经元细胞H19-7中的表达定位情况。实验结果发现miR-148a-3p主要在胞质中表达，见图2。
实施例2miR-148a-3p表达对神经元细胞凋亡的影响
利用海人酸(Kainic acid，KA)诱导神经元细胞惊厥实验。
(1)H19-7神经元细胞培养和凋亡诱导
选取大鼠海马神经元H19-7细胞作为研究对象，传代5次后，以1×105细胞/孔的密度接种于6孔培养板，当细胞生长密度到90％，分别以50μM、100μM、150μM、200μM KA(Kainicacid，海人酸)干预24h。
(2)TUNEL检测神经元细胞凋亡
不同浓度的KA作用24h后，使用原位ApoBrdU DNA片段分析试剂盒(BioVision,Mountain View,CA,USA)按照操作说明进行TUNEL分析，以测量细胞凋亡的程度。用碘化丙啶(PI)染色显示细胞核。每组选取5张玻片，每片任选5个高倍视野(x400)拍摄照片，观察细胞计数，凋亡细胞率按(凋亡海马星形胶质细胞数/海马星形胶质细胞总数)×100％计算。
结果：KA浓度为50μM神经元凋亡程度显著增加，凋亡率呈现剂量依赖性(100μM<150μM<200μM)。KA剂量越高，产生的神经毒性作用越大，可导致神经元大量死亡，见图3。因此，在后续实验中选用100μM的KA干预神经元细胞，模拟体外惊厥细胞模型实验。
(3)流式细胞检测神经元细胞凋亡
首先将处于对数生长期的H19-7细胞，用胰酶消化配置成细胞悬液，均分接种于6孔板中，于37℃、5％CO2培养箱内培养24h。分别转染miR-148a-3p mimics，miR-148a-3pmimics NC，miR-148a-3p inhibitor，miR-148a-3p inhibitor NC(购自广州锐博生物科技有限公司，序列如下)，转染48h后加入KA(100μM)处理24h，收集细胞，用PBS和5μl膜联蛋白V-FITC和5μl丙碘双胍(PI)进行处理，室温黑暗条件下停留30分钟。使用流式细胞仪(BDBiosciences)分析细胞凋亡，评估细胞凋亡率。
结果显示，与转染control组比较，转染miR-148a-3p mimics后海马神经元细胞凋亡率显著升高(P<0.01)；而转染miR-148a-3p inhibitors后，细胞凋亡率显著降低(P<0.01)。与miR-148a-3p mimics组比较，miR-148a-3p mimics NC(miR-148a-3p m NC)组和miR-148a-3p inhibitors组细胞凋亡率显著降低(P<0.01)。与miR-148a-3p inhibitorsNC(miR-148a-3p i NC)组比较，miR-148a-3p inhibitors组细胞凋亡率显著降低(P<0.01)，见图4和图5。
(1)大鼠miR-148a-3p mimic序列为：
5'-UCAGUGCACUACAGAACUUUG-3'(SEQ ID NO.5)，
3'-AGUCACGUGAUGUCUUGAAAC-5'(SEQ ID NO.6)；
(2)大鼠miR-148a-3p mimic NC序列为：
5'-UUUGUACUACACAAAAGUACUG-3'(SEQ ID NO.7)，
3'-AAACAUGAUGUGUUUUCAUGAC-5'(SEQ ID NO.8)；
(3)大鼠miR-148a-3p inhibitor序列为：
5'-CAAAGUUCUGUAGUGCACUGA-3'(SEQ ID NO.9)；
(4)大鼠miR-148a-3p inhibitor NC序列为：
5'-CAGUACUUUUGUGUAGUACAAA-3'(SEQ ID NO.10)。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。
序列表
<110> 潍坊市妇幼保健院（潍坊市妇幼保健计划生育服务中心）
<120> miR-148a-3p作为热性惊厥诊治标志物的应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
<213> 人工序列(Artificial Sequence)
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gggtcagtgc actacaga 18
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cagtgcgtgt cgtggagt 18
<210> 3
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tcgagtctac tggcgtctt 19
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgagccctt ccacgat 17
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<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 5
ucagugcacu acagaacuuu g 21
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<212> RNA
<213> 人工序列(Artificial Sequence)
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agucacguga ugucuugaaa c 21
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<212> RNA
<213> 人工序列(Artificial Sequence)
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uuuguacuac acaaaaguac ug 22
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<212> RNA
<213> 人工序列(Artificial Sequence)
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aaacaugaug uguuuucaug ac 22
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<211> 21
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 9
caaaguucug uagugcacug a 21
<210> 10
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 10
caguacuuuu guguaguaca aa 22
Claims (1)
1.Inhibitor在制备治疗热性惊厥的药物中的应用，其特征在于，所述inhibitor的核苷酸序列如SEQ ID NO.9所示。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012018258A1 (en) * | 2010-08-05 | 2012-02-09 | Umc Utrecht Holding B.V. | Markers of febrile seizures and temporal lobe epilepsy |
| CN108277280A (zh) * | 2018-02-05 | 2018-07-13 | 杭州更蓝生物科技有限公司 | 一种检测试剂盒 |
| CN109337960A (zh) * | 2018-11-14 | 2019-02-15 | 昆明新开源暾秀生物科技有限公司 | 一种儿童癫痫的诊断试剂盒及其检测方法 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012018258A1 (en) * | 2010-08-05 | 2012-02-09 | Umc Utrecht Holding B.V. | Markers of febrile seizures and temporal lobe epilepsy |
| CN108277280A (zh) * | 2018-02-05 | 2018-07-13 | 杭州更蓝生物科技有限公司 | 一种检测试剂盒 |
| CN109337960A (zh) * | 2018-11-14 | 2019-02-15 | 昆明新开源暾秀生物科技有限公司 | 一种儿童癫痫的诊断试剂盒及其检测方法 |
Non-Patent Citations (1)
| Title |
|---|
| 上海市崇明县热性惊厥与SCN1A基因热点多态性变异的相关性分析;叶桂云等;《上海交通大学学报(医学版)》;20161128(第11期);第1643-1647页 * |
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