CN111100928A - 动脉粥样硬化生物标志物及动脉粥样硬化诊断试剂盒 - Google Patents
动脉粥样硬化生物标志物及动脉粥样硬化诊断试剂盒 Download PDFInfo
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Abstract
本发明提出了一种动脉粥样硬化生物标志物,其核苷酸序列如SEQ ID NO.1所示,该动脉粥样硬化生物标志物在动脉粥样硬化患者体内表达量明显低于正常健康人。本发明还提供了一种动脉粥样硬化诊断试剂盒,包括检测该动脉粥样硬化生物标志物表达量的试剂,具体为RNA提取试剂、逆转录试剂和PCR扩增试剂,本发明试剂盒还提供了检测CX3CL1表达水平的试剂和检测Cx43表达情况的试剂,利用本发明试剂盒检测动脉粥样硬化,操作方便易行,结果更全面可靠,重复性好,为动脉粥样硬化早期诊断提供可靠依据。
Description
技术领域
本发明涉及生物医学领域,尤其涉及一种动脉粥样硬化生物标志物及动脉粥样硬化诊断试剂盒。
背景技术
动脉粥样硬化(atherosclerosis,AS)是各种心血管疾病的主要病理基础,严重危害人类身体健康和生存质量,已成为心血管领域的关注热点。其主要病因是由于过多的脂质沉积在血管内壁,导致血管壁增厚,同时激活炎症反应,造成血管壁损伤,导致动脉粥样硬化。缝隙连接重构(gap junction remodeling)是参与动脉粥样硬化发生、发展的病理基础之一,缝隙连接蛋白43(connexin43,Cx43)则是组成缝隙连接的基本结构单位,是构成心肌细胞间缝隙连接的主要结构基础。有研究发现,早期内皮细胞缝隙连接蛋白表达排列变化,介导血管细胞慢性炎症诱导趋化因子表达、白细胞向内皮细胞聚集及浸透入其下,形成动脉粥样硬化斑块,甚至心肌梗死。其中,不规则趋化因子Fractalkine(CX3CL1)已被证实参与了动脉粥硬化的炎症反应过程。
microRNAs(miRNAs)是一类在植物、动物和部分病毒中发现的单链非编码小RNA分子(含有大约20~25个核苷酸),它通过与靶基因mRNA的3’-非编码区(3'-UTR)结合,降解或抑制其翻译,并在基因表达转录后起调节作用。近年来,miRNA参与动脉粥样硬化的作用机制研究已成为研究热点,miRNA在AS炎症反应的发展过程中发挥着重要的调控作用。因miRNA稳定存在于人的体液及血液系统中,具有可重复性,能够从外周血中稳定检测,可以作为疾病的生物标志物。发现特异性表达的miRNA对于动脉粥样硬化的早期诊断和治疗具有非常重要的意义。
目前,动脉粥样硬化的识别诊断主要借助于影像学检查,如OCT、MRI、PET等,由于血管不断移动,成像质量不稳定,会影响诊断结果的准确性。并且动脉粥样硬化的症状主要决定于血管病变及受累器官的缺血程度,早期诊断困难,冠状动脉粥样硬化管径狭窄达75%以上,则可发生心绞痛、心肌梗塞、心律失常,甚至猝死,发病后极为凶险,预后不良。因此,现在亟待找寻一种具有特异性且准确的动脉粥样硬化早期临床诊断方法。
发明内容
有鉴于此,本发明提出了一种动脉粥样硬化生物标志物以及包括该生物标志物检测试剂的动脉粥样硬化诊断试剂盒,通过检测受试者血液中该生物标志物的浓度判断其是否患有动脉粥样硬化,其结果准确可靠,更具有早期诊断的应用价值。
本发明的技术方案是这样实现的:
第一方面,本发明提供了一种动脉粥样硬化生物标志物,所述动脉粥样硬化生物标志物为miRNA,其核苷酸序列如SEQ ID NO.1所示。
第二方面,本发明提供了第一方面所述的动脉粥样硬化生物标志物在制备动脉粥样硬化诊断或检测产品中的应用。
本申请发明人在长期研究中发现,miR-15a-5p(序列如SEQ ID NO.1所示)在动脉粥样硬化病人血液中的表达量明显低于正常健康人血液中的表达量(P值<0.05),能较好地反映动脉粥样硬化的进展状况,因此可以作为动脉粥样硬化的临床诊断分子生物标志物,并能应用于制备动脉粥样硬化诊断或检测的相关产品,提供了新的动脉粥样硬化诊断和检测手段。
第三方面,本发明提供了一种动脉粥样硬化诊断试剂盒,包括检测本发明第一方面所述的动脉粥样硬化生物标志物表达水平的试剂。
在以上技术方案的基础上,优选的,检测所述动脉粥样硬化生物标志物表达水平的试剂包括RNA提取试剂、逆转录试剂和PCR扩增试剂。
在以上技术方案的基础上,优选的,检测所述动脉粥样硬化生物标志物表达水平的试剂还包括内参。
进一步,优选的,所述内参为U6。
在以上技术方案的基础上,优选的,所述动脉粥样硬化诊断试剂盒还包括检测CX3CL1表达水平的试剂。
进一步,优选的,所述检测CX3CL1表达水平的试剂为ELISA检测方法用试剂。
在以上技术方案的基础上,优选的,所述动脉粥样硬化诊断试剂盒还包括检测Cx43表达情况的试剂。
进一步,优选的,所述检测Cx43表达情况的试剂为活体分子成像方法用试剂,其包括Cx43靶向性分子探针。
本发明的动脉粥样硬化生物标志物及动脉粥样硬化诊断试剂盒相对于现有技术具有以下有益效果:
(1)本发明提供了一种新的动脉粥样硬化的miRNA生物标志物miR-15a-5p,提取待测者血液中RNA后通过qRT-PCR检测miR-15a-5p表达量,特异性好,灵敏度高,重复性好,可用于动脉粥样硬化的辅助诊断;
(2)本发明应用miR-15a-5p qRT-PCR检测协同CX3CL1 ELISA检测和Cx43活体分子成像作为检测动脉粥样硬化是否发病的依据,以细胞因子组合缝隙连接蛋白的浓度值构建预测模型,以预测早期动脉粥样硬化发病的易感人群,其检测更全面,结果更可靠,为进一步研究动脉粥样硬化的发病机理,探索其防治药物提供新的理论依据和方向;
(3)利用本发明试剂盒对动脉粥样硬化进行诊断,相比传统仅利用影像学诊断方法,更具有早期诊断的应用价值,有助于早期发现治未病,减少疾病发展的医疗成本,降低急重动脉粥样硬化发生的几率,改善病人生存质量。
具体实施方式
下面将结合本发明实施方式,对本发明实施方式中的技术方案进行清楚、完整地描述,显然,所描述的实施方式仅仅是本发明一部分实施方式,而不是全部的实施方式。基于本发明中的实施方式,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施方式,都属于本发明保护的范围。
实施例1、检测对比动脉粥样硬化患者与正常健康人血液样本中miR-15a-5p和CX3CL1的表达水平
S1、动脉粥样硬化患者与正常健康人血液样本收集
分别选取100例确诊动脉粥样硬化患者和100例正常健康人作为受试者,取所有患者及正常健康人对应的血清样本,并于4℃放置备用。
S2、miRNA提取
利用TIANGEN公司的miRcute miRNA提取分离试剂盒(离心柱型)(DP501)按照其操作说明书提取各血清样本中miRNA。
利用NanDrop 2000检测血清样本miRNA的含量和质量,在NanDrop 2000分光光度计上测定RNA在260nm和280nm波长下的OD值,并得出A260/A280的比值,保留A260/A280值在1.8-2.0之间的样本miRNA。
S3、qRT-PCR检测miR-15a-5p浓度
利用广州锐博生物公司的Bulge-LoopTM miRNAqRT-PCR Starter Kit检测样本中miR-15a-5p浓度。由广州锐博生物公司设计并合成miR-15a-5p的特异性茎环反转录引物、特异性正向引物和与茎环序列相匹配的反向引物,反向引物为通用引物。按照试剂盒使用说明书,以受试者血清样本中miRNA为模板,利用5M特异性茎环反转录引物进行反转录,进而利用特异性miRNA-qPCR反应体系进行实验。选择U6作为内参统计数据分析,其中U6扩增的正向引物为5’-CTCGCTTCGGCAGCACA-3’,U6扩增的反向引物为5’-AACGCTTCACGAATTTGCGT-3’。
S4、检测CX3CL1浓度
利用R&D Systems公司的Human CX3CL1/Fractalkine Quantikine ELISA Kit(DCX310,96assays Version:02)检测样本中CX3CL1浓度。
按照试剂盒操作说明书配制标准品,将标准品与待测样本(受试者血清样本,50μL/孔)经过酶联免疫吸附实验步骤,终止反应后使用酶标仪测量450nm的吸光值。利用标准曲线的吸光值绘制标准曲线,根据标准曲线算出CX3CL1的最终浓度。
S5、结果分析
动脉粥样硬化患者与正常健康人血液样本中miR-15a-5p和CX3CL1的表达水平如表1所示,所有数据以均数±标准差表示。
表1受试者血液样本中miR-15a-5p和CX3CL1的表达水平
组别 | miR-15a-5p相对表达量 | CX3CL1相对表达量 |
动脉粥样硬化患者组 | 12.53±4.21 | 18.261±5.455 |
正常健康组 | 30.42±3.61 | 4.307±4.122 |
由表1结果显示,动脉粥样硬化患者血液中miR-15a-5p相对表达量明显低于正常健康人,miR-15a-5p对CX3CL1的转录后调控作用减弱,CX3CL1相对表达量高于正常健康人水平。由此说明,检测血液中miR-15a-5p表达水平的降低和CX3CL1表达水平的升高可以作为诊断动脉粥样硬化的指标。若待测者血液miR-15a-5p相对表达含量与正常人之比低于0.412,CX3CL1相对表达含量与正常人之比高于4.24,则可判断其患有动脉粥样硬化。
实施例2、Cx43活体分子成像检测动脉粥样硬化
本发明试剂盒提供Cx43靶向性分子探针,由以下三个部分组成:
(1)信号组分:是探针可供影像学设备检测的部分,可以为放射性同位素(PET和SPECT成像)、荧光染料和量子点(光学成像)、顺磁性材料和超顺磁性材料和磁性纳米粒子(磁共振成像)、超声微泡(超声成像)、各种光声纳米颗粒(光声成像)及以上各种组分进行组合而成的多模式成像技术检测的影像学材料。
(2)靶向亲和组分:是探针与成像的分子靶点特异性结合的部分,二者之间的结合具有高度特异性和高亲和力,相当于“钥匙和锁”的关系。Cx43的羧基末端(C末端)有一个门控颗粒状结构,功能相当于特异性的受体结构域,是Cx43靶向性结合肽(Cx43SP)的主要结合靶点,是Cx43靶向成像的靶点。
(3)连接体:将信号组分和靶向亲和组分连接起来的部分。
本实施例提供近红外荧光染料Cy5.5标记的Cx43SP1作为Cx43靶向性分子探针,Cx43SP1的结构为Gly-Ala-Pro-Gly-4Hyp-Pro-Tyr,又称为:AAP10,分子式:C26H37N7O8,分子量:575.6。
对小鼠动脉粥样硬化ApoE-/-模型注射Cy5.5-Cx43SP1一小时后,处死小鼠,进行近红外荧光成像,统计血管壁Cx43阳性面积。成像显示,阳性面积/外弹力膜横截面积×100%≥60%时,可判断早期动脉粥样硬化斑块形成。
以上所述仅为本发明的较佳实施方式而已,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、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 武汉科技大学
<120> 动脉粥样硬化生物标志物及动脉粥样硬化诊断试剂盒
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> RNA
<213> 人(Homo sapiens)
<400> 1
guguuuggua auacacgacg au 22
<210> 2
<211> 17
<212> DNA
<213> (人工序列)
<400> 2
ctcgcttcgg cagcaca 17
<210> 3
<211> 20
<212> DNA
<213> (人工序列)
<400> 3
aacgcttcac gaatttgcgt 20
Claims (10)
1.一种动脉粥样硬化生物标志物,其特征在于:所述动脉粥样硬化生物标志物为miRNA,其核苷酸序列如SEQ ID NO.1所示。
2.权利要求1所述的动脉粥样硬化生物标志物在制备动脉粥样硬化诊断或检测产品中的应用。
3.一种动脉粥样硬化诊断试剂盒,其特征在于:包括检测权利要求1所述的动脉粥样硬化生物标志物表达水平的试剂。
4.如权利要求3所述的动脉粥样硬化诊断试剂盒,其特征在于:检测权利要求1所述的动脉粥样硬化生物标志物表达水平的试剂包括RNA提取试剂、逆转录试剂和PCR扩增试剂。
5.如权利要求3所述的动脉粥样硬化诊断试剂盒,其特征在于:检测权利要求1所述的动脉粥样硬化生物标志物表达水平的试剂还包括内参。
6.如权利要求5所述的动脉粥样硬化诊断试剂盒,其特征在于:所述内参为U6。
7.如权利要求3所述的动脉粥样硬化诊断试剂盒,其特征在于:还包括检测CX3CL1表达水平的试剂。
8.如权利要求7所述的动脉粥样硬化诊断试剂盒,其特征在于:所述检测CX3CL1表达水平的试剂为ELISA检测方法用试剂。
9.如权利要求3所述的动脉粥样硬化诊断试剂盒,其特征在于:还包括检测Cx43表达情况的试剂。
10.如权利要求9所述的动脉粥样硬化诊断试剂盒,其特征在于:所述检测Cx43表达情况的试剂为活体分子成像方法用试剂,其包括Cx43靶向性分子探针。
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