CN109182459B - 一种主动脉夹层fish检测用探针及试剂盒 - Google Patents
一种主动脉夹层fish检测用探针及试剂盒 Download PDFInfo
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Abstract
本发明涉及一种主动脉夹层FISH检测用探针及试剂盒，包括用于检测has‑miR‑4769‑3P、has‑miR‑636、has‑miR‑142‑3p、has‑miR‑425‑3p和has‑miR‑191‑3p的FISH荧光探针，其核苷酸序列依次如SEQ ID No.1‑5所示。本发明的试剂盒可用于大动脉夹层分子诊断、辅助大动脉夹层病因学诊断、区分夹层和非夹层组织用于手术和病理判断病变范围。
Description
技术领域
本发明涉及生物检测技术，特别涉及一种主动脉夹层FISH检测用探针及试剂盒。
背景技术
主动脉夹层(aortic dissection，AD)指各种原因造成的主动脉内膜破裂，血液经内膜撕裂口进入主动脉壁内，并在内膜与中外膜之间形成一个假腔，是一种严重危及生命的主动脉疾病，一旦破裂致死率极高或重要脏器(如：心脏、大脑、肾脏、肠道等)急性缺血引起严重并发症，病情凶险。近年来在我国和世界范围内，AD的发病率和检出率正逐年上升，早期诊断并施以合理的治疗是挽救患者生命的关键。AD是一个复杂的疾病，病因和发病学至今未明。相关的高危因素包括高血压、吸烟、血脂异常、动脉粥样硬化、遗传因素(如马凡综合征、Turner综合征、Noonan综合征和Ehlers-Danlos综合征)、先天性心血管畸形、特发性中膜退行性变、主动脉炎症、外伤、妊娠等因素。急性AD的诊断主要依赖于影像学检查，例如：电子计算机X线断层血管成像、经食道多普勒超声或者核磁共振血管成像，这些检查对急性AD具有高的敏感度和特异性。但是，这些检查是在首诊医生认为该患者有罹患急性AD可能的情况下进行的，且需要接诊医生首先排除具有相似临床表现的、发病率更高的急性冠脉综合征、肺梗死等疾病；加之这些检查的操作和等待过程需要大量时间，常会使患者丧失珍贵的早起诊疗时机。因此，寻找特异性强、敏感度高的生物学标志物并应用于AD的早期诊断，使AD患者能得到及时有效的治疗，降低病死率并改善愈后，是未来的研究方向。
目前已经发现了一些有望早期诊断AD的生物学标志物，包括血清D-二聚体、基质金属蛋白酶、肌球蛋白重链、钙调蛋白结合蛋白、可溶性弹性蛋白碎片、C反应蛋白等，但半衰期短、灵敏度低或特异性差限制了其在临床的广泛应用。理想的早期诊断指标应具备灵敏度高、特异性强、能够反映疾病的病程转归、快捷、易用、价格低廉、具有良好的临床应用前景和广泛的商业供应商，这为AD生物标记物的研究提供了理论标准。
对AD发病机制的研究曾经聚焦在遗传异质性、临床病理、血流动力学以及蛋白质谱的差异表达等。之后，人们又试图从遗传基因、基因转录后表达(如：mRNA或蛋白质)、转录后基因调节等水平(如：sRNAs)发现那些与AD发病学相关生物标志物并研究其对AD早期诊断的价值。
正常主动脉中层主要平滑肌细胞(SMCs)和其间富于弹力纤维的细胞外基质构成，两者相互作用维持着主动脉壁结构和功能的完整性。研究显示，基质金属蛋白酶2(MMP2)和9(MMP9)的过度表达可能诱导细胞外基质降解和SMCs凋亡，SMCs表型从收缩型转化为合成型，这种病理变化可能导致主动脉中层不稳定并发生AD；此外，单基因突变可能使主动脉SMCs功能障碍并导致AD发生。Brahma-related gene 1(BRG1)是一种SWI/SNF复合物ATP酶亚基，在染色质重塑中，参与特异性基因表达转录调控，在SMCs增殖和分化过程中起起重要作用。最近研究发现，BRG1过表达可诱导大鼠间充质干细胞和人类肿瘤细胞凋亡并促进MMP2和MMP9在人类肿瘤细胞中过表达；BRG1在AD的主动脉SMCs中显着上调并可能通过调节MMP2和MMP9过表达诱导SMC凋亡和表型转化从而诱发AD。CD40配体(CD40L)、髓过氧化物酶(MPO)、MMP-1、MMP-2、MMP-9和金属肽酶组织抑制剂1(TIMP-1)等均是炎症相关分子，参与组织损伤和重塑，与AD发生关系密切。在AD发生早期同时检测血液中这些因子的含量有助于AD的早期诊断并可能作为AD诊断的生物标志物。
微小RNA(mircoRNA，miRNA)是一类长度约19-25个核苷酸、高度保守的小分子非编码RNA，在转录后水平负性调节基因表达。mircoRNA心血管系统发育和心血管疾病发展的关键调节剂。MiRNAs参与心脏肥大、心力衰竭、心肌梗死后重塑，与AD发生和发展密切相关。AD发生与高血压密不可分，临床上80％以上的AD患者均有高血压并成为AD的主要诱因之一。研究显示，特定的miRNAs在血管紧张素II受体(AT1R)信号途径中起着积极的作用，特别是在AT1R信号激活后的Gαq信号通路。MiRNAs芯片和定量PCR(qPCR)分析结果显示，miR-132和miR-212在高血压大鼠和缓慢输入AngII的大鼠心肌、主动脉壁和肾脏中异常高表达；另外，激活内皮素受体(另一种是Gαq偶联受体)也可增加这些组织高表达miR-132和miR-212。AT1R阻滞剂可能减少人类血管壁样本中miR-132和miR-212含量。这些结果表明均表明，miR-132和miR-212参与AngII诱发高血压病与AD发生密切相关。
MiRNAs广泛参与心肌肥大、心力衰竭、心律失常、心肌梗死后重塑；但关于miRNA在AD发生和发展中的研究相对较少。目前，用于遗传信息学研究所采用的生物组织样本大多为活组织新鲜样本，但样本采集、保存困难，核酸等物质极易降解，使实验结果难以精确。经福尔马林固定、石蜡包埋组织样本易于获取并可作长时间保存；新鲜组织样本如立即固定，核酸酶等失活，组织内的核酸等遗传物质保持相对稳定。尽管石蜡包埋组织中的RNA会发生一定的降解和片段化(80nt左右)，这用于检测较小的核酸如miRNAs(19-23nt)是可行的，研究证明从石蜡包埋组织中检测的miRNAs表达谱与鲜活组织非常类似；石蜡包埋组织利于做大量回顾性研究。
基于上述，现有技术中虽然披露了大量的早期诊断主动脉夹层的可能性理论研究，但是，目前仍未出现可有效进行主动脉夹层的早期诊断的检测试剂盒。
发明内容
本发明所要解决的技术问题是：提供一种主动脉夹层FISH检测用探针，进一步提供一种包含上述主动脉夹层FISH检测用探针的试剂盒，可有效进行主动脉夹层的早期诊断。
为了解决上述技术问题，本发明采用的技术方案为：
一种主动脉夹层FISH检测用探针，包括用于检测has-miR-4769-3P、has-miR-636、has-miR-142-3p、has-miR-425-3p和has-miR-191-3p的FISH荧光探针，其核苷酸序列依次如SEQ ID No.1-5所示。
一种主动脉夹层FISH检测试剂盒，包括上述的主动脉夹层FISH检测用探针。
本发明的有益效果在于：
针对hsa-miR-636、hsa-miR-142-3p、hsa-miR-425-3p、hsa-miR-191-3p和has-miR-4769-3P五种大动脉夹层病变相对特异性miRNAs设计上述探针，采用miRNAs FISH方法及针对大动脉夹层石蜡包埋组织切片进行检测；可用于大动脉夹层分子诊断、辅助大动脉夹层病因学诊断、区分夹层和非夹层组织用于手术和病理判断病变范围。
附图说明
图1为观察各样品数据分布的整体特征的Box Plot图；
图2为表达基因或者差异基因数据做聚类分析结果图；
图3为主成份分析图结果图；
图4为主动脉中层弹力板自发光现象的FISH检测镜下图；
图5为阳性目标基因的FISH检测镜下图；
图6为显示有阳性表达信号的FISH检测镜下图；
图7为未加探针的阴性对照的FISH检测镜下图；
图8为AD患者主动脉内膜、中层has-miR-4769-3P表达阳性的FISH检测镜下图；
图9为AD患者主动脉内膜、中层及撕裂局部hsa-miR-636表达阳性的FISH检测镜下图；
图10为AD患者主动脉内膜、中层hsa-miR-425-3p表达阳性的FISH检测镜下图；
图11为AD患者主动脉内膜、中层hsa-miR-191-3p表达阳性的FISH检测镜下图；
图12为AD患者主动脉中层、撕裂局部hsa-miR-142-3p表达阳性的FISH检测镜下图；
图13为以hsa-miR-636表达为例，对照组主动脉壁目标基因反应阴性或局部弱阳性的FISH检测镜下图。
具体实施方式
为详细说明本发明的技术内容、所实现目的及效果，以下结合实施方式并配合附图予以说明。
本发明最关键的构思在于:针对上述五种大动脉夹层病变相对特异性miRNAs设计上述探针，采用miRNAs FISH方法及针对大动脉夹层石蜡包埋组织切片进行检测。
一种主动脉夹层FISH检测用探针，包括用于检测has-miR-4769-3P、has-miR-636、has-miR-142-3p、has-miR-425-3p和has-miR-191-3p的FISH荧光探针，其核苷酸序列依次如SEQ ID No.1-5所示。
一种主动脉夹层FISH检测试剂盒，包括上述的主动脉夹层FISH检测用探针。
从上述描述可知，本发明的有益效果在于：
针对hsa-miR-636、hsa-miR-142-3p、hsa-miR-425-3p、hsa-miR-191-3p和has-miR-4769-3P五种大动脉夹层病变相对特异性miRNAs设计上述探针，采用miRNAs FISH方法及针对大动脉夹层石蜡包埋组织切片进行检测；可用于大动脉夹层分子诊断、辅助大动脉夹层病因学诊断、区分夹层和非夹层组织用于手术和病理判断病变范围。
本发明的研发思路如下：
1、首先采用miRNA芯片法建立主动脉夹层病变石蜡包埋组织miRNAs表达文库并观察其差异表达；
2、考虑到差异表达上调的miRNAs对病变标志物的选择和进一步的试验验证更具实际意义，因此，从表达上调的miRNAs中选择数个miRNAs进行遗传信息学GO途径富集和KEGG途径富集分析，确定那些与主动脉夹层发病密切相关的miRNAs组成主动脉夹层诊断靶分子，设计其荧光标记探针；
3、采用miRNAs FISH原位杂交方法对大动脉夹层石蜡包埋组织进行杂交标记验证；
4、观察组织中杂交信号分布、检测荧光强度并分析杂交结果；
5、设计主动脉夹层诊断诊断试剂盒。
实施例
材料与方法
1、实验对象及分组：本项研究搜集深圳市孙逸仙心血管医院2000-2017年以来经临床诊断和病理确诊的AD病变石蜡包埋样本。分实验组(n＝72)：取自AD升主动脉撕裂的内膜和部分中膜组织并除去坏死及血栓；对照组(n＝35)：样本取自上述样本中主动脉弓或以远的、距病变撕裂血管1cm以外的正常(光滑、无粥样硬化病变)血管壁组织，并去除外膜。全部实验样本均来自深圳市孙逸仙心血管医院住院患者，经病史回顾、主动脉CT、超声心动图检查、术中及病理确诊为Stanford A型AD，排除马凡综合征、Ehler-Danlos综合征、家族性胸主动脉瘤以及大动脉炎等其它主动脉疾病。该研究通过伦理委员会批准，所有患者均签署知情同意书。
研究分2个阶段：
1)miRNAs筛查：实验组25例，对照组：16例。分离总RNA，建立主动脉夹层基因文库，采取芯片法筛查样本中miRNAs表达并建立表达谱，获得差异表达miRNAs；
2)荧光原位杂交法(FISH)验证：实验组47例，对照组：19例。选择筛查阶段差异表达并与主动脉夹层发病密切相关的miRNAs设计其荧光标记探针，采用FISH法检测验证分析。
2、miRNAs筛查
2.1主要试剂
miRNeasy试剂盒-Qiagen p/n 217004；
mirVana RNA分离试剂盒–Applied Biosystem p/n AM1560；
mirVanaTM PARISTM(Cat#AM1556,Ambion,Austin,TX,US)；
还原AllTM全核酸分离试剂(Cat#AM1975,Ambion,Austin,TX,US)；
小牛肠碱性磷酸酶(CIP)；
10×小牛肠磷酸酶缓冲液；
T4RNA连接酶；
10×T4RNA连接缓冲液；
二甲亚砜(DMSO)；
去核酸酶酶水；
Cyanine 3-pCp荧光染料；
10×GE终止剂；
2×Hi-RPM杂交缓冲液。
2.2样本miRNAs文库的建立及差异表达检测
利用Agilent公司的miRNA全标记和杂交试剂盒(Complete Labeling andHybKit)进行芯片杂交，方法如下：
2.2.1样本总RNA的提取：用Trizol一步法提取样本中的总RNA，用分光光度计测定RNA的纯度，甲醛变性胶电泳质检RNA的质量。
2.2.2去磷酸化：取50ng/ul的RNA样品2ul，加入2ul去磷酸化混合液(10×反应缓冲液0.4ul，Labeling Spike-In 1.1ul，CIP 0.5ul)，终体积4ul；充分混匀后，37℃保温30分钟。
2.2.3样品变性：每管样品中添加2.8ul 100％DMSO，置于100℃金属浴中加热5-10分钟，然后将样品迅速转入冰水浴中冷却。
2.2.4标记反应：配制4.5ul连接反应混合液(10×T4RNA连接缓冲液(LigaseBuffer)1.0ul，Cyanine3-pCp 3.0ul，T4RNALigase 0.5ul)，取上述混合液至样品管中，混匀离心，16℃温育2小时。反应结束后，将样品置于真空浓缩仪中完全抽干备用。用Labelingspike-in)RNA和Hyb spike-in)RNA作为整个实验过程的质控。
2.2.5芯片杂交、清洗及扫描：将抽干的样品重新溶解在17μl无核酸酶(nuclease-free)水中，每个样品加入1.0μl Hyb Spike-In solution(3rd dilution)，4.5μl配制好的10×GE Blocking Agent，22.5μl 2×Hi-RPM杂交缓冲液，轻微涡旋混匀。将上述反应混合液放置在100℃金属浴中加热5分钟。反应结束后，迅速将其转至冰水浴中冷却5分钟。离心收集反应液。用移液器缓慢地将约45μl反应液吸至盖片上。将芯片点样面(朝下)缓慢放置在盖片上。组装SureHybchamber，并拧紧。平衡放置在杂交炉中，55℃，20rpm，杂交20小时。Agilent人miRNA芯片V21.0数据库来源于microRNA数据库(miRBase)21.0版本，覆盖2549个人类相关的miRNA。杂交结束后用基因表达洗涤缓冲液(Gene Expression Wash Buffer)洗涤芯片。洗涤后用Agilent微芯片扫描仪(Microarray Scanner)进行扫描。
2.2.6数据提取及分析，使用Agilent Feature Extraction(AFE)softwareversion 10.7.1.1软件对杂交图片进行分析并提取数据；原始数据经归一化后,贝叶斯检验筛选差异的miRNA。挑选差异倍数在2倍以上且统计学上差异有显著性(P小于0.05)的miRNAs进行荧光定量PCR验证。
3、FISH原位杂交验证
3.1材料：PCR仪(用于探针变性，ABI)、原位杂交仪(用于标本杂交，广州市外显子生物技术有限公司)、荧光显微镜(日本Nikon80I)。
3.2实验分组：根据筛查实验结果，共选出5个上调的miRNAs，它们是：hsa-miR-636、hsa-miR-142-3p、hsa-miR-425-3p、hsa-miR-191-3p和has-miR-4769-3P(增高7倍以上)，作为一组备选靶标志物，设计其相应探针进行荧光标记的原位杂交(FISH)。引物采用Primer Express 3.0.1软件设计，由生工生物工程(上海)股份有限公司合成，探针序列及相关信息见下表1。
表1
| 名称 | 规格 | 单位 | 序列(5'-3') | 序号 |
| has-miR-4769-3P | 50ul | 支 | GTAGGGGAGGGAGGATGGCAGA | SEQ ID No.1 |
| has-miR-636 | 50ul | 支 | TGCGGGCGGGACGAGCAAGCACA | SEQ ID No.2 |
| has-miR-142-3p | 50ul | 支 | TCCATAAAGTAGGAAACACTACA | SEQ ID No.3 |
| has-miR-425-3p | 50ul | 支 | GGGCGGACACGACATTCCCGAT | SEQ ID No.4 |
| has-miR-191-3p | 50ul | 支 | GGGGACGAAATCCAAGCGCAGC | SEQ ID No.5 |
3.3原位杂交实验流程：烤片65℃、30min，二甲苯脱蜡，5min/次，3次，无水乙醇、85％、70％乙醇梯度脱水，5min/次。然后再放入水中2分钟，使用PBS(DEPC水配)洗5分钟，2次，蛋白抽提：使用0.2mol/LHCL浸泡标本20min(将核酸序列进行一定程度的水解，增加杂交检测的性噪比)，蛋白消化：20ug/ml蛋白酶K滴入标本30min。加入PBS(DEPC水配)5min/次，2次，3％H2O2浸泡15min，去除内源性的过氧化物酶，预杂交：在标本上滴加100-200uLmiRNA杂交液，置于湿盒中，恒温箱55℃，2hour，防止较高的背景染色，准备探针：探针与RNA杂交液按1:50稀释，88℃变成3min，37℃平衡2min。探针滴于标本上，盖上盖玻片，放入湿盒中37℃杂交16-72小时，12.2×SSC洗标本，5min/次，3次，BSA孵育，37℃,30min。Anti-DigHRP(二抗):1％BSA＝1:100稀释，放入湿盒中37℃1h，PBS洗10min，2次，显色：TSA(红色荧光)：0.15％H2O2：TSA稀释液＝1:1:100，避光显色15min，PBS洗5min，3次，70％乙醇、85％无水乙醇、无水乙醇脱水依次1分钟，DAPI封片。避光15分钟，荧光显微镜观察结果。实验数据使用Image-Pro Plus 6.0软件分析结果。下表2为不同甲酰胺浓度的杂交液配制表。
表2
二、结果
1、入选患者临床特征：共72例Stanford A型主动脉夹层患者参与了这项研发。患者的临床特征见表2(Clinical characteristics of aortic dissection patients(n＝133))。可见患者大多数为男性，女性占一定比例。大多数患者急诊入院，病史短，最短为2h。年龄分布从35岁-74岁，高血压史是此类患者的主要特征之一，急诊入院时血压最高可达220mmHg，甚至发生高血压危象。吸烟患者近1/3。
表3
表3中缩写说明：
CH：病史；HT：高血压；DM II：糖尿病II；HL：高脂血症。
2、大动脉夹层主动脉壁病理改变：手术切除送检主动脉壁主要由内膜和部分中膜构成。内皮细胞局部缺失，内膜不规则增厚或局部变薄，部分可见泡沫样细胞散在或堆积，可见典型的粥样斑块，中央呈粥样坏死伴局部钙化，病变内膜表面可见厚薄不同的纤维帽，周边部位可见大量纤维组织增生、玻变，不同程度的炎性浸润，伴有缺血坏死时，局部可见较多中性粒细胞浸润。动脉中膜弹力层减少，弹力纤维变性、断裂、坏死，局部溶解缺失，由于动脉滋养血管断裂，可见动脉中层条带状坏死、撕裂。在中层变性、坏死的同时，可见局部纤维母细胞、小血管长入。严重的动脉粥样硬化时，病灶基底部中膜弹力板明显萎缩、减少、缺失。可见动脉中层细胞间基质不同程度增多、堆积，局部囊性变和粘液湖形成。α-actin免疫组化染色可见平滑肌细胞明显减少或大量缺失。动脉撕裂局部可见中层坏死，大量中性白细胞浸润等炎症反应，夹层或撕裂间可见混合性血栓，病程较长的样本局部可见肉芽组织增生和血栓机化。
3、大动脉壁MiRNAs检测筛查数据分析结果
3.1箱线图(Box Plot)：
将原始数据归一化后，对归一化后的数据用Box Plot图来观察各样品数据分布的整体特征。结果详见图1：
图1中，横坐标为样品名，纵坐标为探针的信号值取了log2之后的值。矩形盒对应数据的上下四分位数(Q1和Q3)，矩形盒内部中位线为中位数，上划线为Q1－1.5IQR处，下划线为Q3+1.5IQR，其中IQR为四分位间距，IQR＝Q3-Q1。
3.2聚类图(hclust)
为了全面和直观展示样品之间的关系及差异情况，将表达基因或者差异基因数据做聚类分析(表达基因或者差异基因数据做聚类分析结果见图2)。同一类样品聚类后出现在同一个簇(cluster)中，聚在同一个簇的基因可能具有类似的生物学功能。可见对照组分布相对集中，聚在同一个簇中，与实验组可明显分开。
3.3主成份分析图(PCA图)
PCA(Principal Component Analysis)的原理是找到数据方差最大的两个或者三个主成分(就是向量)，将数据投影在这些主成分上，以达到降维的目的，通过图像上的点之间的相互距离来显示样品之间的相似度。PCA通常用来评价样品的分组情况，验证实验设计的合理性，生物学重复样品的均一性等(至少2组数据)。主成份分析图(PCA图)结果见图3：
3.4差异基因筛选
原始数据经归一化后进行差异miRNAs筛选。所用的miRNAs定量为相对表达量。实验设定与对照组相比miRNAs表达量的倍数变化≥2且P值＜0.05(方差分析比较)为差异有显著性。
结果显示，22个miRNAs表达上调；49个miRNAs下调。考虑到表达上调的miRNAs对未来作为病变标志物的选择和进一步的试验验证更具实际意义，并根据GO和KEGG途径富集分析，与大动脉夹层发病密切程度，本研究从表达上调的miRNAs中选择5个miRNAs包括：hsa-miR-636、hsa-miR-142-3p、hsa-miR-425-3p、hsa-miR-191-3p和has-miR-4769-3P(表达增高7倍以上)作为一组备选靶标志物，设计其相应探针进行荧光标记的原位杂交(FISH)。
4、FISH原位杂交验证结果
通过miRNAFISH检测样本表达情况，检测的目的基因为红色，并测量平均光密度。结果可见，使用Image-Pro Plus 6.0软件分析，实验组每一个标本都有阳性结果，同一标本使用不同探针，其表达量不同，结果见图4-13。
图4-13均为FISH×100镜下视图。图4中绿色背景显示自发光，可见主动脉中层弹力板自发光现象。图5中红色点状为阳性目标基因。图6中蓝色箭头所指的是蓝色的细胞核，绿色箭头所指即是阳性表达信号。图7为未加探针的阴性对照片，没有杂交信号显示。图8中AD患者主动脉内膜、中层has-miR-4769-3P表达阳性。图9中AD患者主动脉内膜、中层及撕裂局部。hsa-miR-636表达阳性。图10中AD患者主动脉内膜、中层hsa-miR-425-3p表达阳性。图11中AD患者主动脉内膜、中层hsa-miR-191-3p表达阳性。图12中AD患者主动脉中层、撕裂局部hsa-miR-142-3p表达阳性。图13中以hsa-miR-636表达为例，对照组主动脉壁目标基因反应阴性或局部弱阳性。
根据上述，本发明针对上述5种大动脉夹层(Stanford A型)病变相对特异性miRNAs包括：hsa-miR-636、hsa-miR-142-3p、hsa-miR-425-3p、hsa-miR-191-3p和has-miR-4769-3P设计、合成及荧光标记其探针；采用miRNAs FISH方法及针对大动脉夹层石蜡包埋组织切片进行检测，可用于大动脉夹层分子诊断、辅助大动脉夹层病因学诊断、区分夹层和非夹层组织用于手术和病理判断病变范围等。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等同变换，或直接或间接运用在相关的技术领域，均同理包括在本发明的专利保护范围内。
SEQUENCE LISTING
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Claims (4)
1.一种主动脉夹层FISH检测用探针在制备主动脉夹层FISH检测试剂盒的应用，其特征在于，所述主动脉夹层FISH检测用探针为检测has-miR-4769-3P、has-miR-636、has-miR-142-3p、has-miR-425-3p和has-miR-191-3p的FISH荧光探针，其核苷酸序列依次如SEQ IDNo .1-5所示。
2.根据权利要求1所述的主动脉夹层FISH检测用探针在制备主动脉夹层FISH检测试剂盒的应用，其特征在于，所述主动脉夹层FISH检测试剂盒包括杂交液和染色液。
3.根据权利要求2所述的主动脉夹层FISH检测用探针在制备主动脉夹层FISH检测试剂盒的应用，其特征在于，所述杂交液包括甲酰胺、NaCl、Tri-HCl、SDS和水。
4.根据权利要求3所述的主动脉夹层FISH检测用探针在制备主动脉夹层FISH检测试剂盒的应用，其特征在于，所述甲酰胺的浓度为20-55%，体积为80-220mL；所述NaCl的浓度为5M，体积为68-75mL；所述Tri-HCl的浓度为1M，体积为6-10mL；所述SDS的浓度为9-12%，体积为0.28-0.52mL。
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