CN109212226B - 预测急性高山病发病风险的血浆蛋白标志物及其在制备诊断ams易感性试剂盒中的应用 - Google Patents

预测急性高山病发病风险的血浆蛋白标志物及其在制备诊断ams易感性试剂盒中的应用 Download PDF

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CN109212226B
CN109212226B CN201811038384.0A CN201811038384A CN109212226B CN 109212226 B CN109212226 B CN 109212226B CN 201811038384 A CN201811038384 A CN 201811038384A CN 109212226 B CN109212226 B CN 109212226B
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鹿辉
王荣
熊娟
贾正平
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904th Hospital of the Joint Logistics Support Force of PLA
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Abstract

本发明涉及医学生物检测技术领域，提供了预测急性高山病发病风险的血浆蛋白标志物以及该血浆蛋白标志物在制备急性高山病易感性诊断试剂或诊断试剂盒中的应用。本发明的血浆蛋白标志物为血浆蛋白ACADL、ACADM、ASS1、ATP11C、CANX、DDX3X、HMGB1、HSP90AA1、PSMC2以及RAN的组合。本发明进一步提供了一种利用急性高山病易感性诊断的试剂盒和检测方法，本发明的试剂盒和检测方法简便，可靠，周期短，特异性高，易于临床推广。

Description

预测急性高山病发病风险的血浆蛋白标志物及其在制备诊断 AMS易感性试剂盒中的应用
技术领域
本发明涉及医学生物检测技术领域，涉及预测急性高山病发病风险的血浆蛋白标志物，以及该血浆蛋白标志物在制备急性高山病易感性诊断试剂或试剂盒中的应用，通过检测血浆蛋白标志物的表达量来预测急性高山病发病风险，从而用于评定人体急性高山病的易感性。
背景技术
平原人群快速进入高海拔地区(≥2500m)时，容易患急性高山病(acute mountainsickness,AMS)。AMS是一种以头痛为主要症状并伴有肠胃不适，眩晕，疲劳和失眠等症状的自限性疾病，其发病率远较高原肺水肿及高原脑水肿高。研究表明，快速上升(1000m/d-1600m/d)到海拔5000m以上的地区时，AMS的发病率将达到60％-75％。AMS会严重影响人体在高海拔地区的活动能力，如果AMS患者得不到及时恰当的治疗措施，症状可能进一步加重并诱发高原脑水肿，造成生命危险。
目前，预防急性高山病的主要药物为乙酰唑胺和地塞米松，但这两种药物均有较显著的毒副作用。急性高山病的发生具有个体差异性，人群分为易感和耐受人群，但目前对于急性高山病的预防是急进高原人群无差别的服用药物，这就有可能造成易感人群预防措施不充分，而耐受人群会受到不必要的药物毒副作用。如果不对人群进行AMS易感性的区分，均服用药物进行预防，将对AMS耐受人群造成不必要的毒副作用。因此迫切需要对急进高原的人群进行AMS易感性的提前检测，筛查出AMS易感人群，提前做好保护措施。
研究表明，急性高山病易感及耐受人群的外周血蛋白质组急进高原前后均有显著性差异，提示可以利用外周血的差异蛋白作为生物标记物来区分易感和耐受人群。尽管国内外在寻找AMS的生物标记物方面作了很多尝试和努力，但与AMS相关性高并可以作为准确预测AMS易感性的生物标记物还未被发现，这阻碍了临床对AMS预防和治疗工作的开展以及相关药物的研发。
目前筛选AMS标记物的普遍方法是先需要通过高原环境的刺激，再对AMS易感人群和易感人群的血样进行分析，筛选有差异的生物表记物。高原刺激后进行标记物筛选的方法筛选到了一些有意义的生物分子，极大地促进了AMS生物标记物的研究。但是此方法需要将受试人群暴露于高原实地或模拟氧仓，低氧会诱导产生大量蛋白，有可能会掩盖丰度低急性高山病生物标记物；其次，此方法受平台条件的限制，操作不便，不利于推广应用。
因此，在非低氧诱导的正常环境下寻找急性高山病易发性标志物，对于急性高山病的针对性预防具有重要意义。目前尚无应用于急性高山病发病风险诊断的标志物的文献报道。
发明内容
本发明的目的在于提供急性高山病发病风险的诊断标志物，本发明的目的也在于提供诊断标志物的新用途，即在制备急性高山病易感性诊断试剂盒中的应用，该试剂盒用于平原人群急进高原前AMS易感性的筛选，使AMS的防治更具有针对性。
本发明的第一方面，提供了预测急性高山病发病风险的血浆蛋白标志物，该血浆蛋白标志物为血浆蛋白ACADL、ACADM、ASS1、ATP11C、CANX、DDX3X、HMGB1、HSP90AA1、PSMC2以及RAN的组合。
血浆蛋白ACADL、ACADM、ASS1、ATP11C、CANX、DDX3X、HMGB1、HSP90AA1、PSMC2以及RAN的氨基酸序列分别如SEQ ID NO.1～SEQ ID NO.10所示。
关于上述血浆蛋白标志物的筛选，发明人通过两部分实验筛选得到：第一部分实验是利用isobaric tags for relative and absolute quantitation(iTRAQ)蛋白质定量技术对小样本进行初筛，在平原地区招募23名志愿者，利用路易斯湖评分标准进行AMS诊断，获得7名AMS易感者和7名AMS耐受者，并剔除介于易感者和耐受者之间的属性模糊者。利用iTRAQ技术对两组患者的血浆蛋白进行分析，结果显示，与AMS耐受者相比，AMS敏感者有326个蛋白下调，81个蛋白上调，得到AMS易感性初筛标志物。
第二部分实验是另外招募120名志愿者，采用大样本以及利用SPRi技术对初筛生物标记物进行验证。利用同样的AMS诊断标准，从120名志愿者中获得22名AMS易感者，21名AMS耐受者，并剔除介于易感者和耐受者之间的属性模糊者。利用表面等离子共振免疫分析(Surface Plasmon Resonance immunoassay，SPRi)技术分析志愿者血浆中的初筛标志物蛋白，结果显示，与AMS耐受者相比，AMS敏感者中有九个下调蛋白，一个上调蛋白。
该九个下调蛋白为血浆蛋白ACADL、ACADM、ASS1、ATP11C、DDX3X、HMGB1、HSP90AA1、PSMC2以及RAN；一个上调蛋白为血浆蛋白CANX。
本发明的第二方面，提供了上述预测急性高山病发病风险的血浆蛋白标志物在制备急性高山病易感性诊断试剂或诊断试剂盒中的应用。
本发明所述的预测急性高山病发病风险的血浆蛋白标志物在制备急性高山病易感性诊断试剂或诊断试剂盒中的应用，诊断试剂为检测血液生物样品中血浆蛋白ACADL、ACADM、ASS1、ATP11C、CANX、DDX3X、HMGB1、HSP90AA1、PSMC2以及RAN表达量的试剂。
诊断试剂盒包含了检测血液生物样品中血浆蛋白ACADL、ACADM、ASS1、ATP11C、CANX、DDX3X、HMGB1、HSP90AA1、PSMC2以及RAN表达量的试剂。
所述的血液生物样品选自：获自对象的外周血。
本发明的第三方面，提供了一种急性高山病易感性的诊断试剂盒，该试剂盒包含了检测血液生物样品中血浆蛋白ACADL、ACADM、ASS1、ATP11C、CANX、DDX3X、HMGB1、HSP90AA1、PSMC2以及RAN表达量的试剂。
优选的，该诊断试剂盒包括样品处理和总蛋白质提取试剂、多肽分离试剂、多肽串联质谱合并HPLC分析试剂以及表面等离子共振检测试剂。
优选的，该诊断试剂盒中还附有试剂盒的使用说明书，其中记载了如何采用试剂盒进行检测，如何利用检测结果对急性高山病发病风险进行判断、对预防方案进行选择。
本发明的第四方面，提供了一种利用上述诊断试剂盒进行急性高山病易感性检测的方法，如下：
A.将待检血液样本常温离心，获取血浆，利用高峰度蛋白去除试剂盒处理血浆，去除其中的高丰度蛋白，而后提取血浆中的上清蛋白，并测定上清蛋白浓度；
B.对步骤A中的上清蛋白依次进行iTRAQ标记、SCX分离、LC-ESI-MS分析以及表面等离子共振技术芯片分析，对ACADL、ACADM、ASS1、ATP11C、CANX、DDX3X、HMGB1、HSP90AA1、PSMC2以及RAN进行定量检测。
数据以平均值±标准差的形式表示。AMS耐受人群与敏感人群血浆中蛋白质生物标记物的表达差异统计使用t检验的方法，所有p值均为双侧，p<0.05被认为有统计学意义。
本发明的有益保障及效果如下：
本发明通过实验筛选到了十种能够对急性高山病易感性进行诊断的血浆蛋白标志物，在平原人群进入高海拔地区前，通过对上述十种血浆蛋白标志物的表达量进行检测，能够实现对待检者的急性高山病易感性进行预测判定，并针对易感人群提前实施针对性预防，既避免了易感人群预防措施不充分，又避免了耐受人群受到不必要的药物毒副作用。
就技术而言，血浆蛋白标志物的检测本质上是一种血液中血浆蛋白的定量检测，具有操作简便、检测灵敏、特异性好、重复性高等特点，现今已越来越多地被应用于临床检验技术中。
此外，本发明所涉及的十种血浆蛋白标志物在AMS易感者以及AMS耐受者中的表达存在显著差异，且差异具有统计学意义(P<0.05)，其临床参考价值和可信度较高，对急性高山病发病风险的预测准确率高，对于急性高山病发病风险的临床诊断有着重大的意义，而且只需要采集检测者的血液即可获得检测结果，接受程度高。
附图说明
图1是本发明中十种血浆蛋白生物标记物ACADL、ACADM、ASS1、ATP11C、CANX、DDX3X、HMGB1、HSP90AA1、PSMC2和RAN在AMS耐受人群和AMS敏感人群的表达情况。
具体实施方式
现结合实施例和附图，对本发明作详细描述，但本发明的实施不仅限于此。
本发明所用试剂和原料均市售可得或可按文献方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按体积计算。
一、材料与标本收集描述
为了验证血浆蛋白质分子与AMS的关联性，本实验招募两批实验者进行试验。第一批招募23名平原受试者，AMS易感者血浆标本收集自急进高原人群中的AMS患者进入高原前，合计7例；正常人群的血浆标本收集自急进高原人群中的正常健康人群进入高原前，合计7例。
第二批招募120名平原受试者，AMS易感者血浆标本收集自急进高原人群中的AMS患者进入高原前，合计21例；正常人群的血浆标本收集自急进高原人群中的正常健康人群进入高原前，合计22例。
AMS的诊断是通过国际通用诊断标准-路易斯湖评分来确认的。所有人群在取血之前没有服用任何预防性药物，每个样本用EDTA-Na抗凝管合计收集2mL血液。
实验步骤为利用EDTA包被的采血管采集23名拟急进高原人群的外周血，离心取上层血浆，并0.2mL每份分装于-80℃下保存备用，通过isobaric tags for relative andabsolute quantitation(iTRAQ)蛋白质组分析技术对血浆中蛋白质组表达水平进行检测。待人群进入高原后，根据AMS路易斯湖评分诊断系统区分AMS患者和健康人群，比较AMS患者和健康人群蛋白质表达谱，筛选AMS易感性相关的蛋白质分子，作为候选AMS生物标记物。
随后用EDTA包被的采血管采集120名拟急进高原人群的外周血，离心取上层血浆，并0.2mL每份分装于-80℃下保存备用，通过表面等离子共振免疫分析(Surface PlasmonResonance immunoassay，SPRi)对血浆中蛋白质候选AMS生物标记物组达水平进行检测。待人群进入高原后，根据AMS路易斯湖评分诊断系统区分AMS患者和健康人群，比较AMS患者和健康人群蛋白质表达谱，筛选出上述十个蛋白质在AMS易感者和健康者中存在表达差异。
二、样品处理和蛋白质提取
为了降低血浆中高丰度蛋白对实验结果的干扰，血浆样品利用高丰度蛋白去除试剂盒ProteoMiner TM Kits(bio-Rad laboratories,Hercyles,CA,USA)处理。利用裂解缓冲液冲洗样品，并用10mM DTT在56℃条件下处理1h，随后利用55mM IAM在暗室中处理1h，经还原和烷基化处理的蛋白质加入4倍体积的预冷的的丙酮，在-20℃条件下过夜生成沉淀；在4℃、30000g的条件下离心，生成的沉淀溶解于0.5M的TEAB中，并在冰浴的条件下进行超声处理，而后再次在4℃、30000g的条件下离心，测定上清蛋白质浓度，并将其分装储存于-80℃备用。
三、iTRAQ标记和SCX分离
每个待分析样本取出总蛋白100μg，并用Trypsin Gold(Promega,Madison,WI,USA)蛋白酶以蛋白：蛋白酶＝30:1的比例，将蛋白在37℃条件下消化处理16h，随后利用真空离心对样品进行干燥；而后将多肽重溶于0.5M的TEAB中，同时多肽被同位素iTRAQ标签标记，在室温条件下孵育2h，收集标记的多肽混合物并利用真空离心干燥。
利用LC-20ABHPLC分析仪进行SCX色谱分析，iTRAQ标记的多肽混合物重溶于4mL缓冲液A(25mmol/LNaH2PO4于25％ACN中，pH 2.7)并上样于4.6×250mm含有5μm颗粒的SCX色谱柱。依次采用缓冲液A以流速1mL/min的速度冲洗10min，用5％-60％的缓冲液B(25mmol/LNaH2PO4和1mol/L KCl于25％ACN，pH 2.7)冲洗27min，用60％-100％的缓冲液B冲洗1min，经色谱柱分离的多肽分为20份，用C18色谱柱脱盐并真空干燥。
四、LC-ESI-MS/MS分析
蛋白样品重悬于缓冲液A(2％ACN和0.1％FA混合液)中，并在20000g的条件下离心10min，最后调整溶液浓度为0.5μg/μL。10μL上清液上样于2cm的C18分析柱并用LC-20ADnanoHPLC进行分析，而后多肽用10cm C18柱进行洗脱，上样速率为8μL/min，持续4min，随后依次利用2％-35％的缓冲液B(98％ACN和0.1％FA混合液)以300nL/min的速度梯度洗脱44min，利用80％的缓冲液B线性洗脱2min，最后用5％的缓冲液B洗脱1min。多肽溶液经纳米电喷雾电离处理后经串联质谱(Thermo Fisher Scientific,San Jose,CA)合并HPLC进行分析。
五、表面等离子共振免疫分析(Surface Plasmon Resonance immunoassay，SPRi)技术芯片点样
在3D Dextron芯片上打印肝脏靶标蛋白分子库的31个样品，严格参照美国Plexera公司标准打印流程执行。芯片上打印的阴性、阳性、空白对照样品用于对照数据质量，标准品(系统对照)用于测定芯片质量。利用处理好的芯片通过SPR V3版本的Plexera
Figure BDA0001791473350000061
HT生物分子相互作用分析仪器对样品进行实时监测分析。
六、相互作用测试
分别将血清用PBS缓冲液(Thermo Fisher)进行1:10稀释、1:50稀释、1:100稀释、1:200稀释、1:300稀释作为流通样品。所有样品按照浓度从低至高依次流通经过芯片表面，起始测试时以PBS缓冲液作为参考进行基线调零，PBS在整个实验中均作为流动相基质。
在芯片表面的反应池中流通分析物(血清)，流速为2μL·s-1，结合时间为300s，解离时间为300s，解离后用Glycine-HCl溶液作为重生液，流速为32μL·s-1，流动时间为150s。每样品测试全程12000秒，每张芯片测试1个血清样品，每个血清样品平行测试2次。
七、统计分析
数据以平均值±标准差的形式表示。AMS耐受人群与敏感人群血浆中蛋白质生物标记物的表达差异统计使用t检验的方法，所有p值均为双侧，p<0.05被认为有统计学意义。
结果如图1所示，与AMS耐受组人群相比，AMS敏感人群血浆中蛋白质生物标记物ACADL、ACADM、ASS1、ATP11C、DDX3X、HMGB1、HSP90AA1、PSMC2和RAN的表达是显著下调的，而蛋白质生物标记物CANX是显著上调的，表明这10种生物标志物可被用于预测AMS的发病风险。
实际检测时，直接采取待检者的外周血，然后根据第二至第六部分对血液样品进行处理以及对样品中ACADL、ACADM、ASS1、ATP11C、DDX3X、HMGB1、HSP90AA1、PSMC2和RAN的相对表达量进行测试。
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可作出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
序列表
<110> 中国人民解放军兰州军区兰州总医院
<120> 预测急性高山病发病风险的血浆蛋白标志物及其在制备诊断AMS易感性试剂盒中的应用
<130> 权利要求书 说明书
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Phe Tyr Lys Asn Leu Cys Phe Ile Leu Pro Gln Phe Leu Tyr Gln Phe
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Arg Ile Val Asp Asp Trp Ala Asn Asp Gly Trp Gly Leu Lys Lys Ala
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Asp Ile Leu Glu Lys Lys Val Glu Lys Val Val Val Ser Asn Arg Leu
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Val Thr Ser Pro Cys Cys Ile Val Thr Ser Thr Tyr Gly Trp Thr Ala
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Asn Met Glu Arg Ile Met Lys Ala Gln Ala Leu Arg Asp Asn Ser Thr
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Ser Ile Ile Glu Thr Leu Arg Gln Lys Ala Glu Ala Asp Lys Asn Asp
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Ser Ser Gly Phe Ser Leu Glu Asp Pro Gln Thr His Ala Asn Arg Ile
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Tyr Arg Met Ile Lys Leu Gly Leu Gly Ile Asp Glu Asp Asp Pro Thr
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Val Asp Asp Thr Ser Ala Ala Val Thr Glu Glu Met Pro Pro Leu Glu
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Gly Asp Asp Asp Thr Ser Arg Met Glu Glu Val Asp
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<212> PRT
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Lys Asp Asp Lys Pro Ile Arg Ala Leu Asp Glu Gly Asp Ile Ala Leu
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Leu Lys Thr Tyr Gly Gln Ser Thr Tyr Ser Arg Gln Ile Lys Gln Val
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Glu Asp Asp Ile Gln Gln Leu Leu Lys Lys Ile Asn Glu Leu Thr Gly
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Ile Lys Glu Ser Asp Thr Gly Leu Ala Pro Pro Ala Leu Trp Asp Leu
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Ala Ala Asp Lys Gln Thr Leu Gln Ser Glu Gln Pro Leu Gln Val Ala
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Arg Cys Thr Lys Ile Ile Asn Ala Asp Ser Glu Asp Pro Lys Tyr Ile
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Ile Asn Val Lys Gln Phe Ala Lys Phe Val Val Asp Leu Ser Asp Gln
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Val Ala Pro Thr Asp Ile Glu Glu Gly Met Arg Val Gly Val Asp Arg
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Asn Lys Tyr Gln Ile His Ile Pro Leu Pro Pro Lys Ile Asp Pro Thr
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Val Thr Met Met Gln Val Glu Glu Lys Pro Asp Val Thr Tyr Ser Asp
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Thr Pro Leu Leu His Pro Glu Arg Phe Val Asn Leu Gly Ile Glu Pro
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Pro Lys Gly Val Leu Leu Phe Gly Pro Pro Gly Thr Gly Lys Thr Leu
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Ile Gly Ser Glu Leu Val Gln Lys Tyr Val Gly Glu Gly Ala Arg Met
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Val Arg Glu Leu Phe Glu Met Ala Arg Thr Lys Lys Ala Cys Leu Ile
260 265 270
Phe Phe Asp Glu Ile Asp Ala Ile Gly Gly Ala Arg Phe Asp Asp Gly
275 280 285
Ala Gly Gly Asp Asn Glu Val Gln Arg Thr Met Leu Glu Leu Ile Asn
290 295 300
Gln Leu Asp Gly Phe Asp Pro Arg Gly Asn Ile Lys Val Leu Met Ala
305 310 315 320
Thr Asn Arg Pro Asp Thr Leu Asp Pro Ala Leu Met Arg Pro Gly Arg
325 330 335
Leu Asp Arg Lys Ile Glu Phe Ser Leu Pro Asp Leu Glu Gly Arg Thr
340 345 350
His Ile Phe Lys Ile His Ala Arg Ser Met Ser Val Glu Arg Asp Ile
355 360 365
Arg Phe Glu Leu Leu Ala Arg Leu Cys Pro Asn Ser Thr Gly Ala Glu
370 375 380
Ile Arg Ser Val Cys Thr Glu Ala Gly Met Phe Ala Ile Arg Ala Arg
385 390 395 400
Arg Lys Ile Ala Thr Glu Lys Asp Phe Leu Glu Ala Val Asn Lys Val
405 410 415
Ile Lys Ser Tyr Ala Lys Phe Ser Ala Thr Pro Arg Tyr Met Thr Tyr
420 425 430
Asn
<210> 10
<211> 179
<212> PRT
<213> RAN
<400> 10
Met Ala Ala Gln Gly Glu Pro Gln Val Gln Phe Lys Leu Val Phe His
1 5 10 15
Thr Asn Arg Gly Pro Ile Lys Phe Asn Val Trp Asp Thr Ala Gly Gln
20 25 30
Glu Lys Phe Gly Gly Leu Arg Asp Gly Tyr Tyr Ile Gln Ala Gln Cys
35 40 45
Ala Ile Ile Met Phe Asp Val Thr Ser Arg Val Thr Tyr Lys Asn Val
50 55 60
Pro Asn Trp His Arg Asp Leu Val Arg Val Cys Glu Asn Ile Pro Ile
65 70 75 80
Val Leu Cys Gly Asn Lys Val Asp Ile Lys Asp Arg Lys Val Lys Ala
85 90 95
Lys Ser Ile Val Phe His Arg Lys Lys Asn Leu Gln Tyr Tyr Asp Ile
100 105 110
Ser Ala Lys Ser Asn Tyr Asn Phe Glu Lys Pro Phe Leu Trp Leu Ala
115 120 125
Arg Lys Leu Ile Gly Asp Pro Asn Leu Glu Phe Val Ala Met Pro Ala
130 135 140
Leu Ala Pro Pro Glu Val Val Met Asp Pro Ala Leu Ala Ala Gln Tyr
145 150 155 160
Glu His Asp Leu Glu Val Ala Gln Thr Thr Ala Leu Pro Asp Glu Asp
165 170 175
Asp Asp Leu

Claims (6)

1.预测急性高山病发病风险的血浆蛋白标志物，其特征在于，该血浆蛋白标志物为血浆蛋白ACADL、ACADM、ASS1、ATP11C、CANX、DDX3X、HMGB1、HSP90AA1、PSMC2以及RAN的组合。
2.根据权利要求1所述的预测急性高山病发病风险的血浆蛋白标志物，其特征在于，血浆蛋白ACADL、ACADM、ASS1、ATP11C、CANX、DDX3X、HMGB1、HSP90AA1、PSMC2以及RAN的氨基酸序列分别如SEQ ID NO.1～SEQ ID NO.10所示。
3.权利要求1或2所述的预测急性高山病发病风险的血浆蛋白标志物在制备急性高山病易感性诊断试剂或诊断试剂盒中的应用。
4.根据权利要求3所述的预测急性高山病发病风险的血浆蛋白标志物在制备急性高山病易感性诊断试剂或诊断试剂盒中的应用，其特征在于，所述的诊断试剂为检测血液生物样品中血浆蛋白ACADL、ACADM、ASS1、ATP11C、CANX、DDX3X、HMGB1、HSP90AA1、PSMC2以及RAN表达量的试剂。
5.根据权利要求3所述的预测急性高山病发病风险的血浆蛋白标志物在制备急性高山病易感性诊断试剂或诊断试剂盒中的应用，其特征在于，所述的诊断试剂盒包含了检测血液生物样品中血浆蛋白ACADL、ACADM、ASS1、ATP11C、CANX、DDX3X、HMGB1、HSP90AA1、PSMC2以及RAN表达量的试剂。
6.根据权利要求4或5所述的预测急性高山病发病风险的血浆蛋白标志物在制备急性高山病易感性诊断试剂或诊断试剂盒中的应用，其特征在于，所述的血液生物样品获自对象的外周血。
CN201811038384.0A 2018-09-06 2018-09-06 预测急性高山病发病风险的血浆蛋白标志物及其在制备诊断ams易感性试剂盒中的应用 Active CN109212226B (zh)

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