CN111705126A - miR-668-3p作为肾纤维化无创诊断标志中及检测试剂盒 - Google Patents
miR-668-3p作为肾纤维化无创诊断标志中及检测试剂盒 Download PDFInfo
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Abstract
本发明涉及miR‑668‑3p作为肾纤维化诊断标志的应用,以及miR‑668‑3p作为肾血管性高血压的应用;还涉及一种用于诊断肾纤维化的试剂盒,包括用于检测miR‑668‑3p表达量的检测剂。通过使用本发明提出的外周血中的miR‑668‑3p表达量作为分子标志,可对肾血管性高血压及其引起的肾脏纤维化损伤进行无创检测,并且对其病变过程进行无创动态监控,具有重要的临床应用价值。
Description
技术领域
本发明属于生物医药领域,更具体地,涉及miR-668-3p作为肾纤维化诊断标志的应用以及基于该诊断标志的检测试剂盒。
背景技术
纤维化是一个渐进的病理过程,可导致器官损伤甚至功能衰竭。肾纤维化的主要特征在于肾小管和肾小管周围毛细血管之间的区域病理性纤维状基质的沉积。在病理条件下,肾小管上皮细胞可分化成间充质细胞和活化的成纤维细胞,最终形成肌成纤维细胞,该过程被称为上皮间质转化(Epithelial–Mesenchymal Transition,EMT)。
肾血管性高血压可导致肾纤维化、甚至肾功能衰竭,而纤维化是一个渐进的病变过程,因此,肾血管性高血压的诊断有利于后期的纤维化控制。欧洲心脏病协会(ESC,2011)制定的外周动脉疾病诊断和治疗指南把肾动脉超声、肾动脉断层成像、核磁共振血管成像和数字减影血管造影列为肾血管性高血压的临床推荐诊断方法。但以上方法或者属于有创诊断,或者有碘造影剂肾病风险,且对于早期纤维化的检测不敏感。肾脏纤维化最可靠的直接诊断法是肾活检,但肾活检属创伤性检查,费用贵,临床应用受限。
因此,为了及早发现肾血管性高血压,有效防控由其引起的肾脏纤维化损伤,对纤维化发病过程进行无创而准确的动态监测,需要一种新的安全精准的肾脏纤维化无创诊断的生物标志成为当今社会亟待解决的课题。
发明内容
为解决以上问题,本发明提供了miR-668-3p作为肾纤维化诊断标志的应用。
本发明还提供了miR-668-3p作为肾血管性高血压诊断标志的应用。
本发明还提供了一种用于诊断肾纤维化的试剂盒,包括用于检测miR-668-3p表达量的检测剂。
在一个优选实施方案中,所述miR-668-3p的序列如SEQ ID NO:1所示。
在一个具体实施方案中,所述试剂盒为荧光定量检测试剂盒。
在一个具体实施方案中,所述试剂盒包括用于检测miR-668-3p表达量的检测引物和荧光标记的探针。
通过使用本发明提出的外周血中的miR-668-3p表达量作为分子标志,可对肾血管性高血压及其引起的肾脏纤维化损伤进行无创检测,并且对其病变过程进行无创动态监控,具有重要的临床应用价值。
附图说明
图1为小鼠肾脏组织的HE染色、马松染色和免疫组化染色照片;
图2为根据免疫组化染色照片计算的Col III、CD31和α-SMA阳性率统计图;
图3是小鼠肾脏组织的免疫印迹照片;
图4是小鼠肾脏组织的免疫印迹照片计算的的Col III、CD31和α-SMA相对表达量统计图。
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,以下结合附图及实施例,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。此外,下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
为了有效的防控肾血管性高血压引起肾脏纤维化损伤,对纤维化发病过程进行无创而准确的动态监测,本发明提供一种无创诊断生物标志miR-668-3p分子,序列为SEQ IDNo.1所示的核酸序列。
1、肾血管性高血压模型的建立(两肾一夹法/2K1C):
C57/BL6小鼠麻醉(sodium pentobarbital,50mg/kg)后于蜡盘固定、备皮、剪开腹部皮肤后钝性分离皮下筋膜及组织,翻出小肠等,用眼科镊仔细钝性分离左肾肾蒂筋膜,在肾静脉后上方小心分离出肾动脉进行缩窄操作(U型0.12mm开口银夹,RenovascularHypertension组,即RH组),检查肾脏缺血情况后轻柔缝合肌肉层和皮肤。对照组(Sham组)的小鼠实行与上述同样的手术流程,只是不进行缩窄操作,对照组肾脏与实验组肾脏均为各小鼠同侧肾脏。术后4周后尾套加压法测量小鼠BP明显升高(>150mmHg),夹侧肾脏明显萎缩,说明小鼠肾血管性高血压模型建立成功。
2、病理学分析
对小鼠模型进行安乐死,分离肾脏,肾脏经多聚甲醛(4%,m/v)固定、包埋、切片(5-7μm)后进行免疫组化染色分析(H&E染色、Masson染色、Collagen III、CD31、α-SMA免疫组化)与转录组测序(RNA-seq,RH组,Sham组,n=12)。
石蜡切片免疫组化分析简要过程如下:①石蜡切片脱蜡至水;②抗原修复;③封闭;④一抗二抗处理;⑤DAB显色;⑥苏木素复染;⑦脱水、透明、中性树胶封片;⑧显微镜镜检,图像采集分析。
Masson染色简要过程如下:①石蜡切片脱蜡至水;②苏木素染核;③丽春红染色;④苯胺蓝染色;⑤脱水、透明、中性树胶封片;⑥显微镜镜检,图像采集分析。
结果如图1和2所示,相对于Sham组,RH组出现了严重的纤维化特征,纤维化面积、Collagen I/III阳性率和α-SMA都大幅提高,CD31的阳性率大幅降低。
3、免疫印迹分析
使用蛋白免疫印迹法Western blot对肾脏组织中的,取适量肾脏组织采用组织匀浆机研磨(添加蛋白酶抑制剂)后加蛋白上样缓冲液煮沸15min,冰上冷却20min,-20℃保存备用。此外,手术过程必需严格无菌操作并注意麻醉深度,避免术后感染、降低术后死亡率。
结果如图3和4所示,与Sham组项目比,Collagen III和α-SMA的表达量大幅提高,CD31的表达量大幅降低。
4、小鼠肾脏转录组测序与结果验证
肾脏miRNA转录组测序与mRNA转录组测序(用Qubit 2.0RNA检测试剂盒对tRNA精确定量,用Qubit 2.0DNA检测试剂盒对回收DNA精确定量)由生工生物工程(上海)股份有限公司完成。miRNA转录组数据的验证同样采用小鼠肾血管性高血压模型(2K1C法),分组同转录组测序(RH组,Sham组,n=12)。在建模成功后高效提取小鼠肾脏miRNAs(HaiGene组织细胞miRNA提取试剂盒,B1802),经反转录获得miRNAs的cDNA(HaiGene一步法miRNAs反转录试剂盒,D1801)后进行实时荧光定量PCR分析。基于miRNA的转录组数据与临床血液样本的miRNA定量PCR数据,在不同群体大小(第一轮:50人/50人,第二轮:300人/300人)的患者外周血中分析筛选出与肾脏miRNA的转录组数据相一致的候选miRNA分子hsa-miR-668-3p,序列为3’-ugucacucggcucggcccacuac-5’(SEQ ID NO:1)。建立ROC分析模型,结果显示,外周血中miR-668-3p相对表达量诊断肾脏纤维化的曲线下面积为:AUC=0.92±0.023,敏感性和特异性都很高,具有非常好的诊断价值。
综上所述,本发明中的生物标志miR-668-3p分子可作为有效的无创诊断生物标志。本发明为防控肾血管性高血压引起的肾脏纤维化损伤和对其病变过程进行无创动态监测具有重要临床应用价值。
本领域的技术人员容易理解,以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,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,均应包含在本发明的保护范围之内。
序列表
<110> 武汉市皮诺飞生物科技有限公司
<120> miR-668-3p作为肾纤维化无创诊断标志中及检测试剂盒
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 1
ugucacucgg cucggcccac uac 23
Claims (6)
1.miR-668-3p作为肾纤维化诊断标志的应用。
2.miR-668-3p作为肾血管性高血压的应用。
3.一种用于诊断肾纤维化的试剂盒,其特征在于,包括用于检测miR-668-3p表达量的检测剂。
4.根据权利要求3所述的用于诊断肾纤维化的试剂盒,其特征在于,所述miR-668-3p的序列如SEQ ID NO:1所示。
5.根据权利要求3所述的用于诊断肾纤维化的试剂盒,其特征在于,所述试剂盒为荧光定量检测试剂盒。
6.根据权利要求5所述的用于诊断肾纤维化的试剂盒,其特征在于,包括用于检测miR-668-3p表达量的检测引物和荧光标记的探针。
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CN110257502A (zh) * | 2019-06-28 | 2019-09-20 | 南京市儿童医院 | 肠无神经节细胞症血浆外泌体诊断标志物及其应用 |
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CN110257502A (zh) * | 2019-06-28 | 2019-09-20 | 南京市儿童医院 | 肠无神经节细胞症血浆外泌体诊断标志物及其应用 |
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