基质金属蛋白酶（MMPs）家族蛋白作为癫痫诊断及预后判断生物标志物研究

Epilepsy is one of the most common chronic neurological disorders. Biomarkers are urgently needed for the diagnosis and monitoring of disease progression in epilepsy disease. Matrix metalloproteinases (MMPs) constitute a family of over 20 Zn2+-dependent proteolytic enzymes that are able to cleave all structural components in the extracellular environment and many biologically active molecules involved in signal transduction. MMP-2 and MMP-9 are suggested to be important in a variety of developmental processes within the nervous system. MMPs and tissue inhibitors of metalloproteinases (TIMPs) remodel the pericellular environment by regulating the cleavage of extracellular matrix proteins, cell surface components, neurotransmitter receptors, and growth factors, which together regulate cell adhesion, synaptogenesis, synaptic plasticity, and long-term potentiation. Recent studies have suggested that increased MMP activity and dysregulation of the balance between MMPs and TIMPs could be implicated in the process of epileptogenesis. Activation of MMP-2 and MMP-9 occurs in the brain in the kainic acid rat seizure model. Several studies have shown robust activation of MMP-9 by seizure-evoking stimuli. In addition, supported by the National Natural Science Fund (“screening and functional analysis of key proteins involved in refractory epilepsy”), with state-of-the-art proteomics, our experiments have identified many more novel proteins that have not yet been associated with but probably involved in pathogenesis of medically refractory epilepsy. If validated, these proteins may provide serve as new diagnostic biomarkers for epilepsy. Our proteomics study suggested that MMPs increased significantly in CSF of epilepsy. Therefore, based on our proteomic results, we would like to further explore the serum/plasma/CSF biomarkers for epilepsy diagnosis and progression. MMPs which were identified as differentially expressed proteins in our proteomics study have been chosen as candidate biomarkers. Sensitive and quantitative liquid chip assays will be established. Concentrations of candidate biomarkers in serum/plasma/CSF of patients with epilepsy and healthy individuals will be analyzed using newly developed, highly sensitive liquid chip technology while controlling for several major confounders. After scientific statistical analysis, this study could identify several biomarkers for epilepsy diagnosis and progression, which may be very helpful in prognosis prediction and treatment.

癫痫是神经系统常见疾病，目前尚没有有效的生物标记物，大量病人难以确诊，癫痫生物标志物研究迫在眉睫。诸多研究显示，基质金属蛋白酶（MMPs）家族蛋白在神经系统发育及突触可塑性方面发挥重要作用，与癫痫发生的关系极为密切。另外，既往的报道和我们以往的研究均显示，癫痫患者脑组织及脑脊液中MMPs含量明显增高。进而，我们前驱小样本实验也提示血清中MMP2水平在癫痫患者和正常对照之间存在显著性差异。因此，我们推测MMPs极有可能成为癫痫的诊断或预后判断生物标志物。综上，鉴于癫痫生物标志物研发的迫切性和MMPs家族蛋白在该领域的潜力，在以往工作的基础上，发挥本课题组优势，本课题拟利用先进的液相芯片技术，围绕MMPs家族系列蛋白，通过对癫痫患者以及正常人群的血液标本、脑脊液等标本进行大规模检测；从而筛选出有效的癫痫生物标记物，为该疾患的鉴别诊断及早期诊断及预后判断提供客观指标，有利于该疾病的防治。

癫痫是一类反复发作性神经系统疾病，临床上采用多种方法对癫痫进行诊断，包括病史、神经影像学、视频脑电图等，但有时患者不能提供可靠地病史并且检查结果如脑电图为阴性，而在发作间期患者可以表现为完全正常，因此有时对癫痫的诊断仍存在困难。本研究目的在于通过构建超敏的检测方法，对癫痫患者以及正常人群的血清标本中基质金属蛋白酶家族(matrix metalloproteinases, MMPs)即MMP-2、MMP-3、MMP-9和金属蛋白酶组织抑制因子-1(TIMP-1)浓度进行检测，从而筛选出癫痫血液诊断分子标志物（biomarker）。.本研究共纳入233名癫痫患者和97名健康志愿者分别作为试验组和对照组。所有纳入者采用Luminex技术对血清中MMP-2、MMP-3、MMP-9和TIMP-1水平进行检测。通过对比分析显示，癫痫患者血清MMP-2，MMP-3水平比正常对照组血清水平明显下降（P<0.05），血清TIMP-1水平在癫痫患者中明显比正常对照组升高（P<0.05），而血清MMP-9水平在两组之间无明显差别（P>0.05）。另外女性血清MMP-3水平较男性明显减低，差异具有统计学意义（P<0.05）；血清MMP-2水平随年龄增加逐渐减低，而血清MMP-3水平随年龄增加而增加（P<0.05）。当浓度取175.40ng/ml时，血清MMP-2作为癫痫与正常对照诊断指标的灵敏度为71.13%，特异度为62.66%。由于血清MMP-3水平受性别和年龄的影响，分别对不同年龄段的癫痫患者和正常对照作ROC曲线，在20-40岁年龄组，当男性和女性的血清MMP-3浓度分别为23.87 ng/ml和 12.31 ng/ml时，其诊断敏感度分别为72.22%和45%，特异度分别为76.67%和94.12%；在大于40岁年龄组，当男性和女性的血清MMP-3浓度分别为20.70 ng/ml和 10.92 ng/ml时，其诊断敏感度分别为85.71%和85.62%，特异度分别为47.62%和100%。血清TIMP-1浓度取74.35ng/ml时，TIMP-1作为癫痫与正常对照诊断指标的灵敏度为66.52%，特异度为59.80%，AUC 0.594。.血清MMP-2、TIMP-1有可能作为癫痫诊断的生物标记物，在研究血清MMP-3作为可能的癫痫诊断标记物时，必须把性别和年龄两个因素考虑在内。