CN111812331A - 一种用于检测高原缺氧的生物标志物及其应用 - Google Patents
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Abstract
本发明公开了一种用于检测高原缺氧损伤的生物标志物及其应用。所述生物标志物为神经元特异性烯醇化酶(NSE)或其特异性抗体，用于制备高原缺氧的检测试剂或试剂盒。本发明提供的生物标志物可用于血清标志物检测，判断机体高原缺氧程度，有助于检测或辅助检测缺氧状态，从而有助于尽早采取相应治疗措施。利用该标志物检测方便快速，容易为病人接受，更便于动态监察高原人群的缺氧损伤进展。
Description
技术领域
本发明属于检测高原缺氧技术领域，具体涉及一种用于检测高原缺氧的生物标志物及其应用。
背景技术
高原有着特殊的自然环境,其特点是低压、低氧、气候干燥寒冷、风速大、太阳辐射和紫外线照射量明显增大。在高原环境下,随着海拔的升高,空气中的氧分压不断降低,人如果长期处在这种缺氧环境中,严重者可出现低氧血症。由于人的神经组织对内外环境变化最为敏感,因此在缺氧条件下,脑功能损害发生的最早,损害程度也比较严重,且暴露时间越长,损害越严重,特别是对感觉、记忆、思维和注意力等认知功能的影响显著而持久。
如果缺氧严重或持续，将不可避免的导致多个器官的血样饱和度下降，最终导致多器官功能障碍综合症。在动物模型中，模拟的高原缺氧(0.9％持续24小时)能够导致大鼠肺水肿、炎症和出血，并伴有脑水肿、海马氧化应激和认知功能障碍。随着越来越多的人群工作、旅行和生活在高海拔地区，如何有效预防缺氧损伤越来越受到关注。急性高原病，特别是高原肺水肿的临床诊断主要通过病人体征检查、胸部X线、CT、磁共振等影像学手段，这些手段都依赖于临床医师的经验。目前仍缺少比较成熟的可量化的客观灵敏的临床诊断指标。
生物标志物指具有特异性生物学特性、生物化学特征或者方面的分子指示物，其可用于确定存在或不存在特定疾病或状况和/或特定疾病或状况的严重程度。若筛选出急性高原病的诊断标志物，并研制相应的诊断试剂盒，采用生物标志物诊断急性高原病必将改善高原病的诊断现状。
神经元特异性烯醇化酶(Neuron-specific enolase,NSE)是参与糖酵解途径的烯醇化酶中的一种,主要存在于神经组织和神经内分泌组织中,在脑组织细胞中的活性最高。在脑外伤、高血压性脑出血、动脉瘤性蛛网膜下腔出血等多种急性脑损伤性疾病中,NSE被释放并通过脑脊液进入外周血液中，因此，外周血中NSE水平会有明显地增高。NSE是目前反映脑损伤程度的重要指标。
遗传和蛋白质组学研究表明某些标志物可能参与了急性高原病的发展。筛选和发现新的急性高原病的生物标志物，特别是可作为即时检验的生化标志物，具有广泛的临床应用。
发明内容
本发明的目的在于提供一种用于检测高原缺氧的生物标志物及其应用。
在本发明的第一方面，提供了一种用于检测高原缺氧的生物标志物，所述生物标志物为神经元特异性烯醇化酶(NSE)或其特异性抗体。
在本发明的第二方面，提供了所述生物标志物在制备高原缺氧检测产品中的应用。
优选地，所述高原缺氧检测为血清检测，使用ELISA检测法。
优选地，所述产品包括试剂或试剂盒。
在本发明的第三方面，提供了一种用于检测高原缺氧的产品，所述产品含有所述的神经元特异性烯醇化酶或其特异性抗体。
优选地，所述神经元特异性烯醇化酶或其特异性抗体偶联有或带有可检测标记。
优选地，所述可检测标记包括生色团、化学发光基团、荧光团、同位素或酶。
在本发明的第四方面，提供了一种用于检测高原缺氧的试剂盒，所述试剂盒包含一个容器以及一份标签或说明书，所述容器中含有神经元特异性烯醇化酶或其特异性抗体，所述标签或说明书注明所述试剂盒用于高原缺氧检测的使用方法和注意事项。
优选地，所述神经元特异性烯醇化酶或其特异性抗体偶联有或带有可检测标记。
本发明的有益效果：本发明提供的生物标志物可用于血清标志物检测，判断机体高原缺氧程度，有助于检测或辅助检测缺氧状态，从而有助于尽早采取相应治疗措施。利用该标志物检测，方便快速，容易为病人接受，更便于动态监察高原人群的缺氧损伤进展。
附图说明
图1是常氧组和模拟高原缺氧组的大鼠血清中的NSE含量比较；其中横坐标是常氧组和模拟高原缺氧组，纵坐标是NSE含量(ng/mL),n＝4,P＜0.01。
具体实施方式
下面结合附图，对本发明的具体实施方式进行详细描述，但应当理解本发明的保护范围并不受具体实施方式的限制。
实施例1动物模拟高原缺氧暴露
1.选用合格实验动物SD大鼠，雄性，购自北京维通利华实验动物技术有限公司，饲养于恒温(23±2℃)、恒湿动物室，每天12/12h昼夜循环，可自由进食进水。模拟高原环境的低压低氧动物实验舱购自贵州风雷航空军械有限责任公司，型号：DYC-DWI。该舱连接有两个抽气泵，可以将舱内气体不断抽出舱外，从而模拟某一海拔高度的低压低氧环境；同时接有电子显示屏和氧传感探头(实时显示舱内所处的海拔高度及氧浓度)。
2.将12只大鼠随机分为三组，常氧组，LPS(4mg/kg,腹腔注射)+模拟高原海拔3000米组，LPS(4mg/kg,腹腔注射)+模拟高原海拔5000米组，每组各4只。在放置于低压低氧舱之前30分钟，腹腔注射LPS，然后将大鼠放置于低压低氧舱中，并将参数设置为模拟高原海拔3000米(或5000米)，上升速度为30m/s，处理时间为24小时。
3.将大鼠戊巴比妥钠麻醉处死，肝素抗凝管取全血，然后3000rpm，离心30min，取上清即血清进行NSE含量测定。
实施例2神经元特异性烯醇化酶含量检测
血清内神经元特异性烯醇化酶的检测方法按照如下步骤进行：
(1)从室温平衡20min后的铝箔袋中取出所需板条，剩余板条用自封袋密封放回4℃；
(2)设置标准品孔(浓度依次为0,1,2,4,8,16ng/mL)和样本孔，标准品孔各加不同浓度的标准品50μl；
(3)样本孔先加待测样本10μl，再加样本稀释液40μl；空白孔不加；
(4)除空白孔外，标准品孔和样本孔中每孔加入辣根过氧化物酶(HRP)标记的检测抗体100μl，用封板膜封住反应孔，37℃恒温孵育60min；
(5)弃掉液体，用吸水纸拍干，每孔加满洗涤液，静置1min，甩去洗涤液，吸水纸上拍干，如此重复洗板5次；
(6)每孔加入有A液和B液组成的显色液各50μl(现用现配)，37℃恒温孵育15min；所述底物A为过氧化氢(H2O2)、B为四甲基联苯胺(TMB)。
(7)每孔加入终止液50μl，于15min内在450nm波长处测定各孔的吸光值(OD)；所述终止液为硫酸溶液(H2SO4)。
(8)绘制标准曲线，在Excel工作表中，以标准品浓度作横坐标，对应OD值作纵坐标，绘制出标准品线性回归曲线，按曲线方程计算各样本孔的浓度值。
实验结果如图1所示，高原低氧损伤后，大鼠血清内NSE含量随高原海拔的增加而增加，模拟海拔3000米和5000米的血清NSE浓度从4.59ng/mL分别上升为6.11ng/mL和7.06ng/mL，与常氧组相比具有统计学差异。
通过上述实验证实，血清中神经元特异性烯醇化酶含量可作为高原低氧损伤的标志物，利用该标志物进行检测高原缺氧的检测方法简单易操作。
以上公开的仅为本发明的具体实施例，但是，本发明并非局限于此，任何本领域的技术人员能思之的变化都应落入本发明的保护范围。
Claims (9)
1.一种用于检测高原缺氧的生物标志物，其特征在于，所述生物标志物为神经元特异性烯醇化酶或其特异性抗体。
2.权利要求1所述的生物标志物在制备高原缺氧检测产品中的应用。
3.根据权利要求2所述的应用，其特征在于，所述高原缺氧检测为血清检测，使用ELISA检测法。
4.根据权利要求2所述的应用，其特征在于，所述产品包括试剂或试剂盒。
5.一种用于检测高原缺氧的产品，其特征在于，所述产品含有权利要求1所述的神经元特异性烯醇化酶或其特异性抗体。
6.根据权利要求5所述的产品，其特征在于，所述神经元特异性烯醇化酶或其特异性抗体偶联有或带有可检测标记。
7.根据权利要求6所述的产品，其特征在于，所述可检测标记包括生色团、化学发光基团、荧光团、同位素或酶。
8.一种用于检测高原缺氧的试剂盒，其特征在于，所述试剂盒包含一个容器以及一份标签或说明书，所述容器中含有神经元特异性烯醇化酶或其特异性抗体，所述标签或说明书注明所述试剂盒用于高原缺氧检测的使用方法和注意事项。
9.根据权利要求8所述的一种用于检测高原缺氧的试剂盒，其特征在于，所述神经元特异性烯醇化酶或其特异性抗体偶联有或带有可检测标记。
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