CN117143977A - 一种MicroRNA133b的检测方法 - Google Patents
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Abstract
本发明属于医疗技术领域，公开了MicroRNA133b的检测方法，对样品进行采集，释放出MicroRNA；对样品中的MicroRNA进行提取和纯化，并连接PolyA尾巴；使用含有茎环结构的引物进行退火处理，利用连有PolyA尾巴的MicroRNA和退火后的引物进行反转录，生成cDNA；利用荧光定量PCR方法，检测目标MicroRNA133b的表达。本发明的MicroRNA133b检测方法融入了专门针对MicroRNA的细胞破裂、提取和反转录关键步骤，采用专业的细胞溶解剂进行样品处理，可以提高MicroRNA的释放效率，从而提升最终的检测灵敏度。
Description
技术领域
本发明属于医疗技术领域，尤其涉及一种MicroRNA133b的检测方法。
背景技术
脑卒中对中国人群健康的危害性日益突出，我国人口健康领域正面临着卒中带来的严重挑战，解决卒中的防治问题是实现改善我国人口健康水平战略目标的国家重大需求。静脉内注射组织型纤溶酶原激活物(tPA)进行药物溶栓治疗是急性缺血性卒中的一线治疗，但是治疗时间窗局限，血管再通率较低，受益患者人群较少。
MicroRNAs是一类内源性非编码单链小分子RNA，长约20-24个核苷酸，通过与靶基因mRNA分子的非翻译区互补配对，降解mRNA或抑制mRNA翻译，从而参与靶基因转录后的表达调控。一个microRNA通常可调节多个靶基因功能，一个基因也可能受多个microRNA调节。MicroRNA和蛋白质编码基因之间的相互作用组成复杂的调节网络。MicroRNAs与神经细胞分化、轴突形成和突触可塑性有关。
研究发现，microRNAs参与缺血性卒中和脑缺血再灌注损伤的发生和发展。已有报道对大脑中动脉闭塞(MCAO)诱导的急性脑缺血小鼠模型进行microRNAs表达谱分析。例如，在急性缺血性卒中小鼠模型中，给予外源性microRNA-9能显著改善神经功能评分，减少脑梗塞体积和脑水肿。脑梗塞周围区microRNA-424在缺血再灌注1小时和4小时明显上调，24小时明显下调，缺血前脑室给予microRNA-424激动剂对脑缺血再灌注损伤起神经保护作用。但是，MicroRNA133b与脑卒中的关系与作为神经保护的潜在治疗靶点，尚未见明确报道
MicroRNA133b位于14q32染色体位点，已有专利报道提示14q32位点的几个microRNAs在心脑血管发育和重构方面起着一定作用。例如在动物模型上静脉注射应用microRNA-329、microRNA-487b、microRNA-494或者microRNA-495抑制剂能恢复缺血肢体的血流，减少动脉粥样硬化斑块形成，促进血管生成。但是，没有报道干预MicroRNA133b与上述情况的关系。
通过上述分析，现有技术存在的问题及缺陷为：
(1)当前的反转录方法可能对不同的MicroRNA有不同的反应效率，导致测定结果的偏差。
(2)当前用于提取MicroRNA的试剂和方法可能无法完全提取出所有的MicroRNA，或者在提取过程中可能会带入其他RNA和蛋白质等杂质，影响最后的测定结果。
发明内容
针对现有技术存在的问题，本发明提供了一种MicroRNA133b的检测方法。
本发明是这样实现的，一种MicroRNA133b的检测方法，其特征在于，包括：
对样品进行采集，使用RIPA作为细胞溶解剂，对样品中的细胞进行破裂以释放出MicroRNA；
利用针对MicroRNA的专用提取试剂对样品中的MicroRNA进行提取，将所得到的MicroRNA进行纯化，并连接PolyA尾巴；
使用含有茎环结构的引物进行退火处理，然后使用miScript II RTKit作为反转录试剂，利用连有PolyA尾巴的MicroRNA和退火后的引物进行反转录，生成cDNA；
利用荧光定量PCR方法，加入扩增目标MicroRNA133b的引物，以生成的cDNA为模板，检测目标MicroRNA133b的表达。
进一步，所述反转录试剂利用特殊设计的引物和反转录酶对MicroRNA进行反转录，生成cDNA：
使用专门为MicroRNA设计的引物，引物通常包含能和MicroRNA的3'端形成互补配对的序列，保证引物能够和所有的MicroRNA进行配对；将这些引物和MicroRNA一起加入反应液中，通过退火处理，让引物和MicroRNA形成稳定的配对；加入反转录酶和脱氧核苷酸三磷酸，以MicroRNA和引物为模板进行反转录，生成cDNA。
进一步，所述MicroRNA133b具有如SEQ ID No:1和SEQ ID No:2所示的碱基序列。
本发明另一目的在于提供一种由MicroRNA133b制备治疗急性脑缺血和脑缺血再灌注损伤的药物组合物，由NBO与NAC组成。
进一步，NBO的有效剂量范围是95％O2～100％O2。
进一步，NAC的有效剂量是60mg/kg～150mg/kg。
本发明另一目的在于提供所述的药物组合物在制备治疗急性脑缺血和脑缺血再灌注损伤药物中的应用。
本发明另一目的在于提供所述的药物组合物在制备降低缺血再灌注对BBB的破坏和紧密连接蛋白的降解的药物中的应用。
本发明另一目的在于提供所述的药物组合物在制备治疗急性脑缺血后的神经保护药物中的应用。
本发明另一目的在于提供所述的药物组合物在制备治疗急性脑缺血后的血管保护药物中的应用。
本发明另一目的在于提供所述的药物组合物在制备降低氧自由基的产生和/或PARP-1裂解和/或PAR的积累的影响的药物中的应用。
结合上述的技术方案和解决的技术问题，本发明所要保护的技术方案所具备的优点及积极效果为：
本发明的MicroRNA133b检测方法融入了专门针对MicroRNA的细胞破裂、提取和反转录等关键步骤，带来了许多优点和积极效果。首先，采用专业的细胞溶解剂进行样品处理，可以提高MicroRNA的释放效率，从而提升最终的检测灵敏度。其次，使用针对MicroRNA的专用提取试剂可以大幅提高MicroRNA的提取效率和纯度，使得更多的MicroRNA能被有效利用，同时降低可能的杂质对后续实验的影响。采用MicroRNA专用的反转录试剂，可以提高MicroRNA的反转录效率和特异性，确保每一个MicroRNA分子都能准确地被反转录为cDNA，并准确地检测出其表达情况。
附图说明
图1是本发明实施例提供的MicroRNA133b的检测方法流程图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
如图1所示，本发明实施例提供一种MicroRNA133b的检测方法，对样品进行采集，使用RIPA作为细胞溶解剂，对样品中的细胞进行破裂以释放出MicroRNA；
利用针对MicroRNA的专用提取试剂对样品中的MicroRNA进行提取，将所得到的MicroRNA进行纯化，并连接PolyA尾巴；
使用含有茎环结构的引物进行退火处理，然后使用miScript II RT Kit作为反转录试剂，利用连有PolyA尾巴的MicroRNA和退火后的引物进行反转录，生成cDNA；
利用荧光定量PCR方法，加入扩增目标MicroRNA133b的引物，以生成的cDNA为模板，检测目标MicroRNA133b的表达。
本发明实施例提供一种MicroRNA133b，其具有如SEQ ID No:1和SEQ ID No:2所示的碱基序列。
本发明实施例提供一种由MicroRNA133b制备治疗急性脑缺血和脑缺血再灌注损伤的药物组合物，由NBO与NAC组成。
进一步，NBO的有效剂量范围是95％O2～100％O2。
进一步，NAC的有效剂量是60mg/kg～150mg/kg。
本发明实施例提供所述的药物组合物在制备治疗急性脑缺血和脑缺血再灌注损伤药物中的应用。
本发明实施例提供所述的药物组合物在制备降低缺血再灌注对血脑屏障(BBB)的破坏和紧密连接蛋白降解的药物中的应用。
本发明实施例提供所述的药物组合物在制备治疗急性脑缺血后的神经保护药物中的应用。
本发明实施例提供所述的药物组合物在制备治疗急性脑缺血后的血管保护药物中的应用。
本发明实施例提供所述的药物组合物在制备降低氧自由基的产生和/或PARP-1裂解和/或PAR的积累的影响的药物中的应用。
本发明实施例的三个应用案例为：
神经退行性疾病的研究：研究者可以使用新的MicroRNA133b检测方案在阿尔茨海默病或帕金森病等神经退行性疾病模型中研究MicroRNA133b的表达变化，进一步揭示其在这些疾病发生和发展中的作用。
脑缺血性疾病的疗效评估：当使用某种药物或治疗手段治疗脑缺血性疾病时，可以通过新的MicroRNA133b检测方案评估其对MicroRNA133b表达的影响，从而评估其治疗效果。
癌症的早期诊断和预后评估：某些类型的癌症中MicroRNA133b的表达会发生改变。利用新的MicroRNA133b检测方案，可以在癌症患者的血液或者肿瘤组织中检测MicroRNA133b的表达，从而实现癌症的早期诊断或者对疗效和预后的评估。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，都应涵盖在本发明的保护范围之内。
Claims (10)
1.一种MicroRNA133b的检测方法，其特征在于，包括：
步骤一，对样品进行采集，使用RIPA作为细胞溶解剂，对样品中的细胞进行破裂以释放出MicroRNA；
步骤二，利用针对MicroRNA的专用提取试剂对样品中的MicroRNA进行提取，将所得到的MicroRNA进行纯化，并连接PolyA尾巴；
步骤三，使用含有茎环结构的引物进行退火处理，然后使用miScript II RT Kit作为反转录试剂，利用连有PolyA尾巴的MicroRNA和退火后的引物进行反转录，生成cDNA；
步骤四，利用荧光定量PCR方法，加入扩增目标MicroRNA133b的引物，以生成的cDNA为模板，检测目标MicroRNA133b的表达。
2.一种MicroRNA133b的检测方法，其特征在于，所述反转录试剂利用特殊设计的引物和反转录酶对MicroRNA进行反转录，生成cDNA：
使用专门为MicroRNA设计的引物，引物通常包含能和MicroRNA的3'端形成互补配对的序列，保证引物能够和所有的MicroRNA进行配对；将这些引物和MicroRNA一起加入反应液中，通过退火处理，让引物和MicroRNA形成稳定的配对；加入反转录酶和脱氧核苷酸三磷酸，以MicroRNA和引物为模板进行反转录，生成cDNA。
3.一种MicroRNA133b的检测方法，其特征在于，所述MicroRNA133b具有如SEQ ID No:1和SEQ ID No:2所示的碱基序列。
4.一种由MicroRNA133b制备治疗急性脑缺血和脑缺血再灌注损伤的药物组合物，其特征在于，由NBO与NAC组成。
5.根据权利要求4所述的药物组合物，其特征在于，NBO的有效剂量范围是95％O2～100％O2；
NAC的有效剂量是60mg/kg～150mg/kg。
6.根据权利要求3至5任一项所述的药物组合物在制备治疗急性脑缺血和脑缺血再灌注损伤药物中的应用。
7.根据权利要求3至5任一项所述的药物组合物在制备降低缺血再灌注对BBB的破坏和紧密连接蛋白的降解的药物中的应用。
8.根据权利要求3至5任一项所述的药物组合物在制备治疗急性脑缺血后的神经保护药物中的应用。
9.根据权利要求3至5任一项所述的药物组合物在制备治疗急性脑缺血后的血管保护药物中的应用。
10.根据权利要求3至5任一项所述的药物组合物在制备降低氧自由基的产生和/或PARP-1裂解和/或PAR的积累的影响的药物中的应用。
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