CN110846319A - 一种人内含子源性27碱基microRNA及在调节血压中应用 - Google Patents
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Abstract
本发明公开了一种人内含子源性27碱基microRNA及在调节血压中应用，该人内含子源性27碱基microRNA是由eNOS第四内含子27碱基重复子重复4次产生，其核苷酸序列如SEQ ID NO.1所示。根据该序列制备其高表达质粒，将其高表达质粒与脂质体按质量比为3：1混合并经生理盐水稀释，经大鼠尾静脉注入SD大鼠体内，证明本发明的人内含子源性27碱基microRNA对大鼠血压具有调节作用，可用于研究高血压疾病，并最终可用于某些降压药物的筛选和制备高血压早期预警芯片，具有潜在的应用前景。
Description
技术领域
本发明属于大鼠尾静脉注射及基因重组技术领域，具体地说，涉及一种人内含子源性27碱基microRNA及在调节血压中应用。
背景技术
高血压是最常见的心血管疾病，常引起严重的心、脑、肾并发症，影响着全球20％～50％的成年人。据统计，目前我国约有2亿高血压患者，每5个成年人中就1人患高血压，约占全球高血压总人数的1/5，全球每年有接近760万的患者死于该病。世界卫生组织(WHO)对高血压定义为：在未使用抗高血压药物的情况下，收缩压≥140mmHg和/或舒张压≥90mmHg。高血压又被分为1、2、3级：1)1级高血压，又称轻度高血压，收缩压140～159mmHg或舒张压90～99mmHg；2)2级高血压，又称中度高血压，收缩压160～179mmHg或舒张压100～109mmHg；3)3级高血压，又称重度高血压，收缩压≥180mmHg或舒张压≥110mmHg。值得关注的是，美国心脏协会在2017年底修改高血压标准为收缩压≥130mmHg和/或舒张压≥80mmHg,显示高血压的防治越来越引起高度重视；按照修改后的高血压标准，全球高血压患者的数目至少将翻一倍以上。
miRNA是一类在进化上高度保守、长约17～25个核苷酸的内源性非编码小分子RNA。迄今为止，在人类基因组中已经鉴定出了超过1000个在各种特定的组织中表达的miRNA，这些miRNA调节人类近一半的蛋白质编码基因；自1993年第一个miRNA(lin-4)被发现以来，迄今已在226种不同生物体中鉴定出28645个miRNA前体发夹结构及35828个成熟的miRNAs。miRNAs通过降解靶基因mRNA或抑制其翻译来调控对蛋白表达，控制细胞分化、增殖和调亡等病生理过程，参与癌症、糖尿病、心血管疾病等人类重大疾病的相关基因表达调节。
目前认为miRNA分为两大类：基因间隔miRNA和基因内含子源性miRNA，后者约占10％，来源于某些编码蛋白基因转录的mRNA前体序列中的内含子。内含子的发现和定义最早可追溯到1977年，几乎所有的真核细胞基因的mRNA前体物中的内含子在mRNA编辑过程中都要被剪切，因而长期以来内含子被认为是“多余的无用的”垃圾RNA序列。尽管也有一些研究发现它的基因调节作用，例如某些核蛋白的结合位点的在内含子部位，在p53依赖性基因激活和转录中起着重要作用，但直到近年来miRNA的发现，这些具有很强基因表达调节功能的内含子序列才被真正重视，并将其归类为内含子源性miRNA；迄今为止，利用生物信息学方法已鉴定出408种内含子源性miRNA，约占人类miRNA总数的40％。
一氧化氮(NO)在心血管调节方面具有十分重要的生物学作用，它能松弛血管平滑肌、维持器官血流量相对稳定、控制各种血管的静息张力，是主要的血压调节因子。NO以L-精氨酸为底物，由三种不同的NO合成酶(NOS)亚型介导产生：神经型(nNOS)、诱导型(iNOS)和内皮型(eNOS)。eNOS基因敲除小鼠表现为全身性高血压，与其在内皮依赖性超极化介导的松弛受损相关。而单次注射裸露人eNOS质粒DNA能使SHR大鼠血压降低5-6周。在受体激动剂的占领，或某些离子通道的开放，导致细胞内钙升高，激活eNOS产生NO。NO-cGMP信号在血管平滑肌细胞舒张和血压调节中的作用在人和动物均得到很好的表征，越来越多的证据表明，NO信号受损参与高血压的发病机制。
miRNAs与高血压的关系。有关研究表明，miRNA参与高血压发生发展的多个环节，主要有以下三个方面：1)miRNAs参与肾素-血管紧张素-醛固酮系统功能调节。肾素-血管紧张素-醛固酮系统是由肾脏和肝脏分泌的一组相互作用又相互调节的激素与受体系统，在高血压的调节中起着关键性作用，肾素-血管紧张素-醛固酮系统的过激反应是高血压的主要病理因素，血管紧张素Ⅱ是RAAS系统的主要生物效应分子，既能使小动脉血管收缩，又能激活交感神经系统，促进醛固酮产生。miR-181a抑制肾交感神经活性和降低肾素的分泌；血管紧张素转换酶(ACE)是miR-143/145的靶基因，miRNA143/145增加导致ACE基因表达减少；miR-421抑制ACE2蛋白转录表达；miR-124和miR-135a可以通过降低盐皮质激素受体蛋白，抑制肾盐潴留；miRNA对RAAS系统产生的这些抑制效应将会降低血压。2)miRNAs影响血管内皮细胞功能。持续的血管腔内高压会导致内皮细胞功能紊乱，内皮功能紊乱时，血管舒张因子分泌减少，血管收缩因子分泌增加，使外周血管强烈收缩，外周阻力明显增加，促进高血压的发生发展。miR-125a/b-5p抑制血管内皮细胞内皮素-1的表达，而miR-155又能引起内皮eNOS转录下调、NO等血管活性因子释放降低，从而影响血压调节。3)miRNAs参与血管平滑肌细胞增殖调节过程。各种因素的刺激使血管平滑肌异常增殖和凋亡，血管的顺应性下降，阻力增加，导致血压升高。miR-122和miR-222参与血管平滑肌的增殖，AngⅡ通过调控两者的转录物lncRNAs(longnoncodingRNAs)调节平滑肌细胞对AngⅡ刺激的反应，进而影响动脉粥样硬化和高血压的形成。miR-145能直接控制平滑肌的增殖、凋亡，miR-143和miR-145能调控多种细胞骨架元件和血清响应因子(serumresponsefactor,SRF)的表达，在血管平滑肌细胞收缩表型和合成表型的转换过程中发挥着至关重要的作用，它们表达水平的高低影响血管的收缩性从而影响动脉血压的变化。
目前高血压的病因不明，其病理发生发展的机制也不清楚，因此缺乏彻底地、高效地根治这一疾病的防治措施。因此，寻找更加高效、无副作用的抗高血压新药是目前心血管领域研究的重要任务。
发明内容
有鉴于此，本发明针对上述的问题，提供了一种人内含子源性27碱基microRNA及在调节血压中应用，可用于研究高血压疾病，并最终可用于某些降压药物的筛选和制备高血压早期预警芯片，具有潜在的应用前景。
为了解决上述技术问题，本发明公开了一种人内含子源性27碱基microRNA，其核苷酸序列如SEQIDNO.1所示。
本发明还公开了一种含有上述的人内含子源性27碱基microRNA的重组表达质粒、表达盒、转基因细胞系或重组菌。
本发明还公开了一种含有上述的人内含子源性27碱基microRNA的重组表达质粒的构建方法，包括以下步骤：通过NCBI中的Blast系统得到人内含子源性27碱基microRNA核苷酸序列，其核苷酸序列如SEQ ID NO.1所示；经PCR扩增目的基因片段，双酶切后线性连接，再应用北京天根生化科技有限公司小量提取试剂盒提取质粒，取阳性克隆菌液送公司进行测序鉴定，得到人内含子源性27碱基microRNA高表达质粒。
可选地，PCR反应体系为：10ng/μL DNA模板1μL，ddH2O 32.5μL，5×PS Buffer 10μL，2.5mM dNTP Mix 4μL，10μM上游扩增引物1μL，10μM下游扩增引物1μL，PrimeSTAR HS DNA聚合酶0.5μL。
可选地，上游扩增引物:5’-GGAAGTCTAGACCTGCTGCA-3’，如SEQ ID NO.2所示；
下游扩增引物:5’-GTCGGTGTCGTGGAGTCG-3’，如SEQ ID NO.3所示。
可选地，PCR反应条件为：95℃5min，1循环；98℃10s，55℃10s，72℃90s，30循环，72℃8min，4℃24h。
本发明还公开了一种上述的人内含子源性27碱基microRNA、人内含子源性27碱基microRNA的重组表达质粒在制备降压药物中的应用。
可选地，药物包括人内含子源性27碱基microRNA的重组表达质粒和脂质体，其质量比为3：1。
本发明还公开了一种上述的人内含子源性27碱基microRNA、人内含子源性27碱基microRNA的重组表达质粒在制备高血压早期预警芯片中的应用。
与现有技术相比，本发明可以获得包括以下技术效果：
本发明提供一种对血压具有调节作用的新型分子，通过应用基因重组技术得到其高表达质粒，可用于高血压的相关基础研究，并最终可用于某些降压药物的筛选和制备高血压早期预警芯片，具有潜在的应用前景。
当然，实施本发明的任一产品并不一定需要同时达到以上所述的所有技术效果。
附图说明
此处所说明的附图用来提供对本发明的进一步理解，构成本发明的一部分，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1是本发明人内含子源性27碱基microRNA高表达质粒构建图；
图2是本发明注射人内含子源性27碱基microRNA高表达质粒后血压的变化；
图3是本发明注射人内含子源性27碱基microRNA高表达质粒大鼠血管壁的荧光图片；其中，a代表27碱基microRNA组，b代表空白质粒组；
图4是本发明注射人内含子源性27碱基microRNA高表达质粒大鼠血管eNOS的免疫组化图；其中，a代表27碱基microRNA组，b代表空白质粒组；
图5是本发明注射人内含子源性27碱基microRNA高表达质粒大鼠血管SP1的免疫组化图；其中，a代表27碱基microRNA组，b代表空白质粒组；
图6是本发明注射人内含子源性27碱基microRNA高表达质粒大鼠血浆eNOS酶的活性变化；
图7是本发明注射人内含子源性27碱基microRNA高表达质粒大鼠血浆NO产物含量的变化。
具体实施方式
以下将配合实施例来详细说明本发明的实施方式，藉此对本发明如何应用技术手段来解决技术问题并达成技术功效的实现过程能充分理解并据以实施。
实施例1人内含子源性27碱基microRNA高表达质粒的构建与检测
通过NCBI中的Blast系统得到人内含子源性27碱基microRNA核苷酸序列，其核苷酸序列如SEQ ID NO.1所示；应用PCR扩增目的基因，在PCR反应体系下得到目的基因片段。经双酶切和线性连接后，利用小量质粒提取试剂盒(北京天根生化科技有限公司)提取质粒，并取阳性克隆菌液送公司进行测序鉴定。验证正确后再进行大量扩增抽提，得到人内含子源性27碱基microRNA高表达质粒(详见图1)。
上游扩增引物:5’-GGAAGTCTAGACCTGCTGCA-3’，
下游扩增引物:5’-GTCGGTGTCGTGGAGTCG-3’；
其中，PCR反应体系为：1μL DNA模板(10ng/μL)，32.5μL ddH2O，10μL 5×PSBuffer，4μL dNTP Mix(2.5mM each)，1μL上游扩增引物(10μM)，1μL下游扩增引物(10μM)，0.5μL PrimeSTAR HS DNA聚合酶；
PCR反应条件为：95℃5min(1循环)，(98℃10s，55℃10s，72℃90s)30循环，72℃8min，4℃24h。
人内含子源性27碱基microRNA来源于内皮型一氧化氮合酶(eNOS)第4内含子中的27碱基重复子RNA序列，eNOS主要存在于血管系统的内皮细胞中。它不仅参与各种细胞生理过程的调节，还参与机体的血压调整、调节血管通透性、防止血小板聚集等。eNOS具有基因多态性，包括点突变和片段缺失。点突变是指eNOS的5’端第786位的胸腺嘧啶突变为胞嘧啶(T-786C，rs2070744)或者是第七外显子上894位的鸟嘌呤突变为胸腺嘧啶(G-894T，rs1799983)。片段缺失则是指eNOS的基因序列中第4内含子内存在一种27个碱基(5’-GAAGTCTAGACCTGCTGCAGGGGTGAG-3’)的数目可变串联重复序列(VariableNumberofTandemRepeats，VNTR)，该VNTR重复4次为a，重复5次为b，则产生aa、ab、bb三种不同基因型。本发明中小分子人内含子源性27碱基microRNA为aa基因型。27碱基microRNA对eNOS的转录和表达具有明显的反馈式调节作用，27碱基microRNA抑制eNOS表达更早在核内RNA转录阶段，并且与和核转录因子SP-1的表达水平密切相关，Sp1是结合于eNOS增强子元件的重要转录因子，可增强eNOS启动子的活性，促进eNOS的转录。eNOS基因表达减少及活性下降对诱导肺动脉高压起到了关键性的作用。
实施例2大鼠主动脉组织人内含子源性27碱基microRNA高表达质粒的转染与转染确定
将动物随机分为两组，每组10只，即27碱基microRNA组和空白质粒组。27碱基microRNA组的大鼠用27-nt-miRNA高表达质粒与脂质体按质量比为3：1混合并经生理盐水稀释，经尾静脉注入体内，空白质粒组的大鼠用空白质粒经尾静脉注入体内。两组用量均为100ng质粒DNA/kg体重，每两天注射一次，连续注射15周，将大鼠置于适宜环境中饲养，期间让老鼠正常进食和饮水。
15周后将动物处死，取大鼠的主动脉组织，经-80℃冰冻后，制作冰冻切片，并于荧光显微镜下观察动脉组织管腔内壁绿色荧光。结果可见，两组大鼠的动脉组织管腔内均可见绿色荧光，而27碱基microRNA组大鼠血管内壁可见显著绿色荧光(如图3)，绿色荧光代表成功转染的质粒。
实施例3大鼠血压的测定
于注射前和注射后，分别应用大鼠尾动脉无创血压测定仪测定各大鼠收缩压(SBP)、舒张压(DBP)、平均压(MBP)和心率(HR)，每只大鼠每次至少连续测量5次血压，取平均值。每周测定两次，连续观察15周。实验过程中，大鼠正常饮食、给水，环境适宜。结果显示，27碱基microRNA组动物SBP、DBP和MBP均有所升高。空白质粒组动物SBP、DBP和MBP出现先稍微升高，第10周后逐渐下降至恢复正常水平(如图2)；说明了人内含子源性27碱基microRNA可使正常大鼠血压升高。
实施例4免疫组化法检测大鼠血管内皮细胞eNOS蛋白和SP1蛋白表达情况
取上述大鼠，应用10％水合氯醛腹腔注射麻醉大鼠，剂量为0.3～0.5ml/100g，腹主动脉采集血液后，开胸收集血管组织，用10％甲醛固定液固定的血管组织，制成组织蜡块，4℃冻存2小时后进行组织切片，进行组织化学染色，具体步骤如下：
(1)室温脱蜡、水化：二甲苯一号10min、二甲苯二号8min、二甲苯三号8min、无水乙醇5min、90％乙醇3min、80％乙醇3min、70％乙醇3min；蒸馏水3min*3次，PBS3min*3次。
(2)热抗原修复，换新的切片架，放入水化后的切片。配置抗原修复液(一包柠檬酸钠粉末配置2LPBS溶液)，用铁饭盒或者锅加热煮沸。温度控制在96-98℃。然后将切片放入热锅中15min，最后自然冷却室温。PBS5min*2次，蒸馏水3min*2次。
(3)免疫组化笔画圈：取出组织，甩干水分，可用吸水纸吸干水珠。用组化笔画圈围住组织，圆圈完整。
(4)灭活内源酶活性：将切片放入湿盒中，盒中加入少量蒸馏水，滴加3％过氧化氢，室温孵育10min。PBSmin*3次，蒸馏水水洗3min。
(5)封闭非特异性位点：甩干水分，吸干水珠，加山羊血清封闭液，放入湿盒内，室温10min。
(6)甩掉封闭液，滴加Ⅰ抗覆盖组织，然后放入湿盒内4℃过夜。
(7)次日，从4℃冰箱取出湿盒，室温复温30min，PBS洗涤3min*3次，蒸馏水洗涤3min。
(8)甩干水分，吸干水珠，滴加Ⅱ抗一滴或者50μL，室温孵育10min。PBS洗涤3min*3次，蒸馏水水洗3min。
(9)每张片子滴加一滴或者50μL链霉菌抗生物素-过氧化物酶溶液，室温孵育10min，PBS洗涤3min*3次，蒸馏水水洗3min。
(10)每张片子滴加两滴或者100μLDAB溶液，显微镜下观察3-10min。染色合适即可终止染色，自来水冲洗。
(11)苏木素复染，1-2分钟，细胞核变蓝色即可终止，自来水冲洗反蓝。
(12)1％盐酸酒精分化3s，自来水冲洗3min。
(13)凉干片子后滴加适量中性树胶封片，盖上盖玻片，小心气泡，载片板上晾干。
(14)通过显微镜观察，观察各组血管内皮细胞eNOS和SP1的蛋白表达情况。结果显示，eNOS蛋白阳性染色呈棕黄色，主要在细胞胞浆表达。SP1蛋白阳性染色呈棕黄色，主要在细胞核表达。干预组大鼠血管内皮细胞棕黄色较对照组弱，提示27碱基microRNA抑制eNOS和SP1蛋白的表达(如图4,图5)。
实施例5大鼠血浆eNOS的活性测定
(1)取样：大鼠腹主动脉采集全血，3000rmp，离心15min后收集上层淡黄色澄清液体，-80℃保存备用。取出样品前用37℃水浴锅溶解，离心。
(2)加样：设置空白孔、标准品孔和样品孔，设置2个复孔于每个孔中，按照标准品浓度梯度往5个标准孔中依次加样50μl，待测样品孔先添加样品稀释液48μl，再滴加2μl待测样品，避免触碰孔壁，微微混匀，待测样品目标稀释倍数为25倍。空白孔与其他孔相同，只是未添加样品和酶标记的试剂。
(3)配液：2ml浓缩洗涤液使用蒸馏水来稀释，稀释至目标30倍后保存待用。
(4)清洗：尽量无损的把封板膜揭开，吸除液体，风干，每孔滴加洗涤液至满，放置30s后吸除，重复5次，拍干。
(5)显色：每孔滴加50μl显色剂A，滴加50μl显色剂B，避光37℃显色10min，液体逐渐变蓝。
(6)终止：后续终止反应实验的操作为添加终止液50μl于每个孔中，至出现肉眼可见孔内液体蓝色转为黄色。
(7)测定，计算浓度：10min内在450nm波长处测定每孔的OD值。结果显示，干预组大鼠血浆中eNOS活性下降(如图6)。
实施例6大鼠血浆NO含量测定
严格按照硝酸还原试剂盒(南京建成)测定手册的操作程序进行血清NO的检测。
(1)取样：取出样品前用37℃水浴锅溶解，离心。
(2)加样：在每个EP管上标记，空白管中加入0.1ml双蒸水，标准管加入0.1ml100μmol/L标准品应用液，样品管分别加入样品0.1ml。
(3)配液：试剂1和2在37℃水浴中溶解，标准品、试剂5、试剂6根据说明书溶解。
(4)加入混合剂0.4ml：试剂1与试剂2按1:1配置组成混合试剂，现配现用，37℃精确水浴60min。
(5)加入试剂3和试剂4：加入试剂30.2ml，试剂40.1ml充分漩涡混匀30s，室温静置40min，3500-4000转，离心10min，取上清液。
(6)加入显色剂：按试剂5:试剂6:试剂7＝2.5：1：1配置显色剂，向上清液中分别加入0.6ml显色剂，混合并在室温下放置10min。
(7)测定并计算浓度：每孔加入15μl至96孔板，双蒸水至零，测量各管在550nm波长处的OD，并根据OD值计算每个样品的NO浓度。结果显示，干预组大鼠血浆中NO含量减少(详见图7)。
利用本发明可得到一种对大鼠血压具有调节作用的新型分子，且通过验证，证明了27碱基microRNA可使正常大鼠血压升高，同时可抑制高血压相关基因eNOS和SP1基因的表达以及血管舒张因子NO的含量；即27碱基microRNA可抑制血管内皮细胞内皮型一氧化氮合酶(eNOS)和SP1的mRNA和蛋白的表达，进而影响血管内皮细胞的增殖。
上述说明示出并描述了发明的若干优选实施例，但如前所述，应当理解发明并非局限于本文所披露的形式，不应看作是对其他实施例的排除，而可用于各种其他组合、修改和环境，并能够在本文所述发明构想范围内，通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离发明的精神和范围，则都应在发明所附权利要求的保护范围内。
序列表
<110> 广西国际壮医医院
<120> 一种人内含子源性27碱基microRNA及在调节血压中应用
<130> 2019
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> RNA
<213> 人体(human)
<400> 1
gaagucuaga ccugcugcag gggugag 27
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 2
ggaagtctag acctgctgca 20
<210> 3
<211> 18
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 3
gtcggtgtcg tggagtcg 18
Claims (9)
1.一种人内含子源性27碱基microRNA，其特征在于，其核苷酸序列如SEQIDNO.1所示。
2.含有权利要求1所述的人内含子源性27碱基microRNA的重组表达质粒、表达盒、转基因细胞系或重组菌。
3.一种含有权利要求1所述的人内含子源性27碱基microRNA的重组表达质粒的构建方法，其特征在于，包括以下步骤：通过NCBI中的Blast系统得到人内含子源性27碱基microRNA核苷酸序列，其核苷酸序列如SEQ ID NO.1所示；经PCR扩增目的基因片段，双酶切后线性连接，再应用北京天根生化科技有限公司小量提取试剂盒提取质粒，取阳性克隆菌液送公司进行测序鉴定，得到人内含子源性27碱基microRNA高表达质粒。
4.根据权利要求3所述的构建方法，其特征在于，PCR反应体系为：10ng/μL DNA模板1μL，ddH2O 32.5μL，5×PS Buffer 10μL，2.5mM dNTP Mix 4μL，10μM上游扩增引物1μL，10μM下游扩增引物1μL，PrimeSTAR HS DNA聚合酶0.5μL。
5.根据权利要求4所述的构建方法，其特征在于，上游扩增引物:5’-GGAAGTCTAGACCTGCTGCA-3’，如SEQ ID NO.2所示；
下游扩增引物:5’-GTCGGTGTCGTGGAGTCG-3’，如SEQ ID NO.3所示。
6.根据权利要求3所述的构建方法，其特征在于，PCR反应条件为：95℃5min，1循环；98℃10s，55℃10s，72℃90s，30循环，72℃8min，4℃24h。
7.权利要求1所述的人内含子源性27碱基microRNA、权利要求2-6所述的人内含子源性27碱基microRNA的重组表达质粒在制备降压药物中的应用。
8.根据权利要求7所述的应用，其特征在于，药物包括人内含子源性27碱基microRNA的重组表达质粒和脂质体，其质量比为3：1。
9.权利要求1所述的人内含子源性27碱基microRNA、权利要求2-6所述的人内含子源性27碱基microRNA在制备高血压早期预警芯片中的应用。
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