CN113151454A - miR-328-3p在制备脑梗死及脑缺血再灌注预后预测试剂中的应用 - Google Patents
miR-328-3p在制备脑梗死及脑缺血再灌注预后预测试剂中的应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，涉及miR‑328‑3p在制备脑梗死及脑缺血再灌注预后预测试剂中的应用。本发明提出血清外泌体miR‑328‑3p在脑梗死及脑缺血再灌注预后预测和治疗中的应用，并通过具体试验结果进行了验证，为脑梗死及脑缺血再灌注预后预测试剂和试剂盒以及预后药物的研发提供新思路。
Description
技术领域
本发明属于生物技术领域，涉及miR-328-3p在制备脑梗死及脑缺血再灌注预后预测试剂中的应用。
背景技术
脑梗死（Cerebral Infarction，CI）是危害人类健康的主要疾病之一，26%-43%的脑梗死有进展性。随着医疗水平的提高，脑梗死后的溶栓治疗等改善了病人的预后，但再灌注会加重脑组织原有的缺血性损伤。早期预测脑梗死预后，减少再灌注损伤，是脑梗死患者治疗的关键。
目前临床上尚无早期预测脑梗死及脑缺血再灌注患者预后预测的有效方法。miRNA的异常表达与脑血管病等多种疾病的发生、发展密切相关。而血液外泌体中存在丰富而稳定的miRNA，由于受到外泌体的保护，miRNA能免受酶降解在体液中远距离运输并被稳定地检出，且其表达谱具有明显的特异性。采用高通量测序筛选miRNA并进行RT-PCR验证，有望找到脑梗死及脑缺血再灌注预后预测的标志物，针对相关miRNA致病机制的研究有助于寻找改善预后的治疗方法。
发明内容
本发明针对目前脑梗死及脑缺血再灌注预后预测还没有一种比较实用可靠的预后试剂或方法的问题提出miR-328-3p在制备脑梗死及脑缺血再灌注预后预测试剂中的应用。
为了达到上述目的，本发明是采用下述的技术方案实现的：
本发明提供miR-328-3p在制备脑梗死及脑缺血再灌注预后预测试剂或试剂盒中的应用，所述miR-328-3p的核苷酸序列为：CUGGCCCUCUCUGCCCUUCCGU。
所述预后预测试剂或试剂盒以miR-328-3p作为预后预测标记物， miR-328-3p水平高表示预后不良。
作为优选，miR-328-3p含量高低通过高通量测序手段和/或RT-PCR进行分析。
作为优选，分析对象为人体外周静脉血血清。
本发明还提出抑制miR-328-3p表达量的物质在制备改善脑梗死及脑缺血再灌注病人预后药物中的应用。
本发明提出miR-328-3p基因在筛选改善脑梗死及脑缺血再灌注病人预后药物中的应用。
特异性识别miR-328-3p的引物在筛选脑梗死及脑缺血再灌注预后预测试剂或试剂盒中的应用，所述引物序列为：正向:5'-TTCGCTTATCTGGCCCTCTCT-3'， 反向: 5'-TATGGTTGTTCACGACTGCTTCAC-3'。
与现有技术相比，本发明的优点和积极效果在于：
本发明提出血清外泌体miR-328-3p在脑梗死及脑缺血再灌注预后预测和治疗中的应用，并通过具体试验结果进行了验证，为脑梗死及脑缺血再灌注预后预测试剂和试剂盒以及治疗药物的研发提供新思路。
附图说明
图1 A为差异miRNA火山图，图1B为差异miRNA聚类图。
图2为血清外泌体miR-328-3p在脑梗死患者中的表达和线栓法小鼠大脑中动脉栓塞再灌注术后评分结果图。
图3为 脑组织TTC染色结果和电镜结果。
图4为miR-328-3p agomir对炎症的影响。
具体实施方式
为了能够更清楚地理解本发明的上述目的、特征和优点，下面结合具体实施例对本发明做进一步说明。需要说明的是，在不冲突的情况下，本申请的实施例及实施例中的特征可以相互组合。
在下面的描述中阐述了很多具体细节以便于充分理解本发明，但是，本发明还可以采用不同于在此描述的其他方式来实施，因此，本发明并不限于下面公开说明书的具体实施例的限制。
实施例1
选择正常对照和CI病人各10例，采集外周静脉血，分离血清后保存。两小时内将样本在４℃下3500r/min转速离心10min，剔除溶血标本，取上层血清再次12000rpm转速离心10min，除上清液中可能残留的血细胞碎片后放到无RNA酶的EP管中，-80℃冻存直至使用。采集病人病史及影像检查等信息。HiSeq/MiSeq高通量测序：将正常对照人群、CI病人在发病第二天（急性期、炎症反应高峰期）和第十天（恢复期）保存的血清，分别等量混合后用RiboTM Exosome Isolation Reagent for plasma or serum试剂盒分离血清中外泌体，采用QIAGEN miRNeasy Micro Kit提取外泌体的microRNA。用高通量测序手段分析筛选差异miRNA。病例的纳入及排除方法：1）符合1995年全国第四届脑血管病学术会议制定的脑血管病诊断标准，并经头颅CT或MRI证实诊断的CI患者。2）首次发病，且发病时间<8h入院。3）入院后完善常规实验室检查。4）入院前1个月未服用过抗血小板药和调脂药。并除外房颤、严重心、肺、肝及肾功能障碍、恶性肿瘤及血小板计数<100×109/L或其他血液系统疾病。
1. 进行CI病人的PCR验证：外泌体提取，外泌体总RNA提取同上。使用RevertAidFirst Strand cDNA Synthesis Kit和light cycler 480 SYBR Green I Master试剂盒按照说明书进行RT-PCR检测microRNA。每个样品在定量实验重复3次。microRNA的相对表达量用2-ΔΔCt法计算。使用3%的琼脂糖凝胶电泳来分析和验证PCR产物的特异性。RT-PCR检测miR-328-3p的表达情况，用Wilcoxon matched-pairs signed rank test筛选有差异的miRNA，分析临床资料，评估microRNA在脑梗死诊断和预后判断中的作用。
2. 使用mir-328-3p agomir（mir-328-3p激动剂）和对照治疗大脑中动脉远端闭塞（MCAO）再灌注诱导的缺血小鼠（每组6只），术前半小时进行颅内注射（每只注射10微升，药物用量为10 OD/每只）。然后同侧进行MCAO实验，MCAO后2小时再灌注。术后进行术后进行Zea Longa评分，进行功能结果评估。24小时后进行mNSS评分，进行功能结果评估。评分后进行多聚甲醛灌注和取材。每组3只进行脑组织的大体TTC染色，另外3只多聚甲醛灌注以后，取梗死区送电镜。
实验结果
1.利用高通量测序手段分析正常对照人群、CI病人在发病第二天（急性期、炎症反应高峰期）和第十天（恢复期）血清外泌体中的miRNA，结果显示筛选到差异miRNA 58个，其中上调和下调的miRNA个数分别为30和28个（图1A）（SE脑梗死第二天，SL脑梗死第十天）。差异miRNA聚类分析见图1B。
图1 A为差异miRNA火山图（横坐标代表miRNA表达倍数变化，纵坐标代表miRNA表达量变化的统计学显著程度，图中的散点代表各个miRNA，虚线下方圆点表示无显著性差异的miRNA，虚线上方右侧圆点表示显著上调的差异miRNA，虚线上方左侧圆点表示显著下调的差异miRNA）。 B.差异miRNA聚类图（原图中红色表示高表达miRNA，蓝色表示低表达miRNA）
2. 图2为血清外泌体miR-328-3p在脑梗死患者中的表达和线栓法小鼠大脑中动脉栓塞再灌注术后评分。从图中可以发现，血清外泌体中RT-PCR验证发现miR-328-3p在脑梗死患者中略低（图2A）。miR-328-3p在梗死体积>=10cm3的患者中显著降低（P=0.01）。对脑梗死患者在发病24h和2周进行NIHSS评分，评估脑梗死两周时的预后，病人病情进展为预后不良，miR-328-3p与脑梗死两周的预后相关（P=0.03）（图2B），病情好转的患者miR-328-3p水平低（P=0.03）。用miR-328-3p水平>1.24来预测患者发病2周的病情重的灵敏度为56%，特异度为87%（AUC=0.70,P=0.03）(图2C)。
3.为了进一步明确miR-328-3p影响预后的机制，我们采用了小鼠脑缺血再灌注模型进行研究。术前半小时用miR-328-3p agomir和对照进行颅内注射。进行线栓法小鼠大脑中动脉栓塞，缺血2h后再灌注。结果发现：
（1）术后进行Zea Longa评分（图2D），进行功能结果评估，miR-328-3p agomir组评分略高于对照组，差异没有统计学意义。术后24小时进行mNSS评分（图2E），进行功能结果评估，miR-328-3p agomir组评分高于对照组(P=0.03)。
（2）术后24h处死小鼠，每组取三只脑组织做TTC染色，结果显示miR-328-3pagomir组脑坏死大于对照组（图3A）。
图3为脑组织TTC染色结果和电镜结果。对照组电镜：神经元细胞呈圆形，核膜双层结构清晰，细胞膜局部面积模糊，胞内细胞器轻微肿胀，细胞质电子密度高；细胞核（N）呈卵圆形，常染色质为主，核膜清晰，核孔（NP）清晰可见；线粒体（M）分布较均匀，但有轻微肿胀扩张，少量伴有嵴断裂、消失；粗面内质网（RER）大多轻度扩张，表面核糖体无明显脱颗粒，滑面内质网（SER）轻微扩张；高尔基体（Go）正常，可能为功能性扩张；胞内未见典型自噬，存在少量次级溶酶体（SL）。miR-328-3p agomir组电镜：细胞形态大致呈卵圆形，细胞膜部分模糊不清楚，细胞器中度水肿，细胞质电子密度降低，胞质轻微水肿；细胞核（N）呈卵圆形，细胞核核皱缩，未见核仁，常染色质为主，核膜部分模糊不清，核孔不可见；线粒体（M）分布均匀，大多中度肿胀，基质略不均，嵴断裂；粗面内质网（RER）轻微扩张，表面核糖体无明显脱颗粒；胞内无自噬，高尔基体（Go）肥大扩张。
（3）动物组织透射电镜结果：两组细胞均呈有不同程度的损伤，但是对照组损伤较miR-328-3p agomir组轻（图3B）。对照组的线粒体损伤较轻，轻微肿胀部分嵴消失外膜模糊，细胞核饱满正常，胞质无水肿。miR-328-3p agomir组的线粒体中度肿胀扩张，基质变浅，嵴断裂。细胞核皱缩，细胞质有低电子密度水肿。激动剂组的细胞核损伤水平也重于对照组。
（4）图4显示了miR-328-3p agomir对炎症的影响。
HE染色显示，miR-328-3p agomir组的中性粒细胞浸润要严重，炎症因子TNFa和IL-6要高于对照组（图4）。
我们认为过高的miR-328-3p水平能够诱导更严重的炎症反应，加重脑细胞损伤，增加缺血再灌注损伤范围，加重临床症状。因此，miR-328-3p在脑梗死和脑缺血再灌注中能够预测预后，并提示降低miR-328-3p水平可能是临床改善脑梗死及脑缺血再灌注病人预后的新方法。
以上所述，仅是本发明的较佳实施例而已，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例应用于其它领域，但是凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。
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<110> 山东大学第二医院
<120> miR-328-3p在制备脑梗死及脑缺血再灌注预后预测试剂中的应用
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Claims (6)
1.miR-328-3p在筛选脑梗死及脑缺血再灌注预后预测试剂或试剂盒中的应用，所述miR-328-3p的核苷酸序列如SEQ ID NO.1。
2.根据权利要求1所述应用，其特征在于，所述预后预测试剂或试剂盒以miR-328-3p作为预后预测标记物，miR-328-3p水平高提示预后不良。
3.根据权利要求2所述应用，其特征在于，采用高通量测序和/或RT-PCR手段进行分析。
4.根据权利要求2所述应用，其特征在于，分析对象为人体外周静脉血血清。
5.抑制miR-328-3p表达水平的物质在制备改善脑梗死及脑缺血再灌注病人预后药物中的应用。
6.特异性识别miR-328-3p的引物在脑梗死及脑缺血再灌注预后预测试剂或试剂盒中的应用，其特征在于，所述引物序列为
正向:5'-TTCGCTTATCTGGCCCTCTCT-3'， 反向: 5'-TATGGTTGTTCACGACTGCTTCAC-3'。
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