摘要
近年来,随着对基质金属蛋白酶与疾病关系的研究逐渐深入,越来越多的研究发现基质金属蛋白酶与多种疾病的严重程度、诊断和预后等密切相关。因此基质金属蛋白酶作为一种非常有潜力的生物标志物得到了越来越多的关注。本文总结了基质金属蛋白酶在肿瘤、心血管疾病、炎症性疾病和神经退行性疾病等疾病诊断中的应用,并对基于RNA水平、蛋白质水平和MMPs的水解酶活性的3类检测方法进行了介绍,以期为基质金属蛋白酶检测的临床应用提供理论参考。
细胞外基质(ECM)是组织的细胞外组分,能够起到为细胞提供支持、储存生长因子、调节细胞运动、细胞间相互作用和细胞间通讯的作用。ECM由多种基质大分子组成,这些基质大分子的具体组成和结构因组织而异。ECM的主要成分包括胶原蛋白、弹性蛋白、纤连蛋白、层粘连蛋白、糖蛋白、蛋白聚糖和糖胺聚糖
目前,在脊椎动物中共鉴定出24种已知的MMPs,其中23种被发现在人体内有表达。MMPs使用数字进行编号,从MMP-1到MMP-28。但是由于有不同团队同时发现同一种MMP的情况出现,所以MMPs的编号中不包括MMP-4、MMP-5、MMP-6和MMP-2
大多数MMPs不是组成型表达的,并且大多数细胞需要被激活以表达MMPs。MMPs的活性在转录和翻译后水平以及细胞定位中受到严格调
本文主要总结MMPs作为生物标志物在各种疾病诊断中的应用以及检测方法,以期对MMPs的临床应用的进一步发展提供参考。
MMPs参与到人体许多生理功能中,例如细胞增殖、分化、凋亡、免疫功能、组织愈合以及血管生成等。并且MMPs的活性在转录、翻译、酶促激活和调节蛋白的抑制等方面都受到严密的调控。MMPs也参与到许多病理过程
ECM是肿瘤微环境基质的非细胞成分,在肿瘤中形成支架,主要负责促进肿瘤恶性表
2017年,Peisker
另外,MMPs在实体瘤中呈现特异性的时空分布,针对过表达MMPs设计荧光探针可以对肿瘤进行生物成像,有助于肿瘤的精确诊断和治疗。Cheng
正常心肌拥有许多种ECM蛋白,包括胶原蛋白、层粘连蛋白、纤连蛋白和低水平的基质细胞蛋白。这些蛋白都在心脏的生理功能中发挥作用,其中最丰富的是胶原蛋白,它能够形成一个复杂的网络,为心肌提供三维结构和拉伸强
Kormi
MMPs可以在各种水平上协调炎症功能。它们可以调节炎症细胞从脉管系统向组织中炎症部位的迁移,还通过加工ECM成分、生长因子、细胞因子和趋化因子来调节炎症细胞的募集和向炎症部位的流
Zhou
神经退行性疾病,包括阿尔茨海默病、帕金森病、多发性硬化、肌萎缩性脊髓侧索硬化症和亨廷顿舞蹈病等,是一种以神经元结构和功能逐渐丧失为特征的神经系统疾病。中枢神经系统(CNS)中的ECM主要由蛋白多糖组成,对神经细胞的发育、存活和活动至关重要。MMPs主要通过以下方式参与调节神经系统病变常见的神经炎症过
Valado
由于MMPs对人体许多病理过程的广泛参与,还有其他许多疾病也可以利用MMPs作为诊断或预后的检测指标。Zhou
在MMPs的表达过程中,MMPs先通过转录、翻译的过程以酶原的形式产生,随后通过蛋白水解或化学激活,才能够最终生成具有活性的MMPs。根据在这一过程中检测目标物的不同,可以将MMPs的检测方法分为3类:(1)基于RNA水平的检测;(2)基于蛋白水平的检测;(3)基于MMPs的水解酶活性的检测。
MMPs的合成需要经过mRNA的转录。通过检测mRNA的水平,就能够研究MMPs的转录表达情况。常用的检测mRNA的方法主要包括反转录-聚合酶链式反应、核酸探针和Northern印迹杂交。
RT-PCR是先将RNA反转录成为互补DNA(cDNA)后,再以cDNA作为模板通过PCR进行扩增的一种PCR技术。通过使用荧光监测扩增反应可以将实时荧光定量PCR与RT-PCR结合,对特定RNA序列进行快速灵敏的定量检测,该技术被广泛用于研究和临床环境中的基因表达分析和病毒RNA定量。Lee
核酸探针是指将已知碱基序列的核酸在体内或体外进行标记,使其可以通过碱基互补配对特异性检测组织或细胞中目标DNA或RNA,直接、简便地研究目的基因表达和定位的一种技术。根据核酸探针的来源和性质可以将其分为cDNA探针、基因组DNA探针、寡核苷酸探针、RNA探针等。Shiozawa
Northern印迹杂交是通过电泳分离不同相对分子质量的RNA后将其转印到印迹膜上,并使用与部分或全部靶序列互补的杂交探针进行检测的技术。Northern印迹检测的优势包括可以检测RNA大小,观察剪接产物等,并且转印完成的印迹膜可以进行长期储存和重复检测。与RT-PCR相比,Northern印迹法灵敏度较低,但特异性高,可以减少检测中的假阳性结果。Li
基于蛋白质水平的检测即利用免疫手段对MMPs进行检测,是通过抗原抗体的特异性结合对MMPs进行识别。这些方法需要使用到价格较为昂贵的抗MMPs抗体,可以识别酶原形式的MMPs和活性MMPs。
Western印迹杂交是分子生物学及生物化学等多种学科中广泛使用的分析技术,用于定性和半定量检测单个蛋白质和蛋白修饰,特异性强、灵敏度高,但操作繁琐,成本较高。2019年,Ando
ELISA是一种固相酶免疫测定(EIA),在各行业中都被广泛使用。通常形式的ELISA测定是将具有未知抗原量的样品特异性或非特异性地固定在固体支持物表面,然后添加检测抗体与抗原形成复合物。检测抗体可以使用与酶连接的二抗进行检测或直接使用酶标记的抗体作为检测抗体。最后添加酶促底物产生可见信号,通常使用呈色反应进行检测。Farkas
免疫组化是利用特异性抗体选择识别组织切片细胞中抗原的一种免疫染色技术。抗体通常与酶或荧光基团连接进行可视化检测。免疫组化技术已经被广泛应用于诊断异常细胞以及对特定分子标志物的检测。通过使用这一检测方法,可以了解生物标志物和差异表达蛋白在生物组织不同部位的分布和定位。Roy
免疫荧光是通过抗原抗体特异性结合来对细胞内特定生物分子靶标进行定位的免疫染色技术。免疫荧光可以观察到目标分子或结构在样品中的分布,并进行半定量分析。通过与具有不同功能的显微镜结合,可以达到超高分辨率成像、实时跟踪、分层扫描等效果。Zhou
磁分离酶联免疫测定(magnetic affinity immunoassay,MAIA)是一种使用磁珠代替传统的固相载体对样品中抗原进行分离的免疫测定方法。通过使用抗体标记的磁珠与目标抗原结合,利用磁力分离代测样品中的目标抗原。MAIA的方法和原理与ELISA和Western blot非常相似,但可以在液体介质中进行检测,具有高灵敏度、高特异性、操作简便、样品用量少等特点。将MAIA与电化学检测技术相结合可以用来开发电化学磁免疫传感器(electrochemical magneto-immunosensors),大幅度提高检测灵敏度和通量,简化检测步骤。2018年,Ruiz-Vega
MMPs是一种锌依赖型的内切酶。根据酶对底物的特异性,可以使用MMPs的底物(通常是明胶)或特定的氨基酸序列对MMPs进行识别,并使用不同方式产生可检测的信号。这些方法可以只检测具有活性的MMPs的水平,直接反应样品中MMPs的活性变化。
明胶酶谱法是以明胶作为底物,对明胶敏感的MMPs(MMP-2和MMP-9)进行测定的一种酶谱检测。根据检测部位不同可以分为凝胶酶谱、原位酶谱和体内酶谱。由于MMP-2和MMP-9对明胶的特异性,明胶酶谱法作为一种高灵敏度、低成本的检测方法被广泛应用于MMP-2和MMP-9的检测中。Fietz
使用可以被MMPs特异性切割的敏感肽与不同检测基团结合可以制备MMPs特异性探针。由于MMPs敏感肽连接方便、特异性高,可以作为一种通用的MMPs识别方式与多种不同的信号检测方式进行组合,得到广泛应用。
MMPs敏感肽最常见的应用方式是与各种荧光基团连接,利用荧光成像进行检测和定位。例如在荧光探针中,可以通过荧光共振能量转移(Förster resonance energy transfer,FRET)使荧光基团淬灭。当敏感肽被MMPs特异性切割时,荧光基团释放恢复荧光,产生可检测的荧光信号。
Li
化学交换饱和转移(chemical exchange saturation transfer,CEST)是一种核磁共振成像新方法。它是通过使用特定脉冲对特定物质(比如蛋白质、葡萄糖、黏多糖等生物大分子)中的氢质子进行饱和,饱和氢质子通过化学交换,影响水分子信号的变化,通过检测水的信号,可以间接反应目标物质的含量。使用镧系金属螯合物可以制成顺磁性CEST对比剂,以加强CEST效应。2017年,Ferrauto
(3)电化学生物传感器 使用电化学传感器检测MMPs除了使用特异性抗体修饰的免疫传感器,还可以使用可被酶降解的天然或合成底物包被在电极表面,制成活性传感器。通过电极将目标分子的浓度信息转换成电势、电流、电阻或电容等可测量的响应信号,从而进行定性或定量检测,该方法具有灵敏度高、易微型化等特点。Biela
随着对MMPs作为生物标志物的进一步研究,其检测技术也得到了快速发展。传统的实验室方法繁琐耗时,已经不能满足临床疾病诊断和跟踪评估的要求,人们对更加快速、灵敏、简单、高通量检测方法的需求日益增加。近年来,随着电化学检测技术、测流色谱技
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