CN114113631B - 脓毒症的检测试剂盒 - Google Patents
脓毒症的检测试剂盒 Download PDFInfo
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Abstract
本公开涉及一种脓毒症的检测试剂盒,用于脓毒症的预后,包括以下试剂中的至少一种:用于检测总蛋白乳酸化水平的试剂;用于检测组蛋白H3K18乳酸化水平的试剂;用于检测细胞因子的试剂;用于检测精氨酸酶‑1的信使核糖核酸水平的试剂。本公开提出的脓毒症的检测试剂盒能通过不同种标志物检测脓毒症。
Description
技术领域
本发明属于医药生物技术领域,具体涉及一种脓毒症的检测试剂盒。
背景技术
脓毒症是由感染引起的全身炎症反应,可发展为脓毒性休克,甚至危及生命的器官功能障碍。由于其不同的病因和表现,它仍然是一个具有挑战性的临床问题。
发明内容
有鉴于此,本公开提出了一种脓毒症的检测试剂盒,用于脓毒症的预后,包括以下试剂中的至少一种:
用于检测总蛋白乳酸化水平的试剂;
用于检测组蛋白H3K18乳酸化水平的试剂;
用于检测细胞因子的试剂;
用于检测精氨酸酶-1的信使核糖核酸水平的试剂。
在本公开实施例中,用于检测总蛋白乳酸化水平的试剂包括:裂解缓冲液、十二烷基硫酸钠聚丙烯酰胺凝胶、上样缓冲液、十二烷基硫酸钠、预染蛋白、电泳缓冲液、电转缓冲液、考马斯亮蓝、赖氨酸残基乳酸化抗体、山羊抗兔二抗、封闭牛奶、TBST缓冲液和SuperECLPlus超敏发光液。
在本公开实施例中,裂解缓冲液包括:质量分数均为1%的十二烷基硫酸钠和蛋白酶抑制剂、3μM组蛋白去乙酰化酶抑制剂和50mM烟酰胺。
在本公开实施例中,用于检测组蛋白H3K18乳酸化水平的试剂包括:组蛋白提取试剂组合、十二烷基硫酸钠聚丙烯酰胺凝胶、上样缓冲液、十二烷基硫酸钠、预染蛋白、电泳缓冲液、电转缓冲液、组蛋白H3K18乳酸化抗体、组蛋白H3抗体、山羊抗兔二抗、封闭牛奶、TBST缓冲液和SuperECLPlus超敏发光液。
在本公开实施例中,细胞因子包括IL-1β、IL-2、IL-4、IL-5、IL-6、IL-8、IL-10、IL-12p70、IL-17、IFN-α、IFN-γ、TNF-α中的一种或多种;其中,所述用于检测细胞因子的试剂包括用于检测IL-10水平的试剂。
在本公开实施例中,用于检测细胞因子的试剂包括捕获微球抗体、检测抗体、SA-PE和洗涤液。
在本公开实施例中,用于检测精氨酸酶-1的信使核糖核酸水平的试剂包括:反转录试剂、DNA酶、引物和实时荧光定量PCR混合物。
在本公开实施例中,引物包括以下引物中的一种或多种:
5’-GGGTTGACTGACTGGAGAGC-3’/5’-CGTGGCTGTCCCTTTGAGAA-3’,
5’-TGACTTCAACAGCGACACCCA-3’/5’-CACCCTGTTGCTGTAGCCAAA3’,
5’-GCTGTGGATGGAAATTCGCC-3’/5’-CTTCTGGCAGTGTGGGTCAT-3’。
在本公开实施例中,实时荧光定量PCR混合物包括:5微升的2×PCR混合物、0.5微升的引物F、0.5微升的引物R、1微升的模板和3微升的双蒸水。
在本公开实施例中,检测试剂盒还包括:磷酸盐缓冲盐溶液、淋巴细胞分离液、红细胞裂解液、胎牛血清和二甲基亚砜。
本公开的脓毒症的检测试剂盒能通过不同标志物检测脓毒症。
更具体的,本发明涉及一种血清学检测标志物在制备检测脓毒症预后的检测试剂盒中的应用,所述的检测标志物为:乳酸化的组蛋白H3K18。
所述的检测脓毒症预后指:
(1)判断因脓毒症导致的脓毒症休克和非脓毒症休克,所述的非脓毒症休克包括但不限于:失血性休克、心源性休克和梗阻性休克;
(2)预测脓毒症休克的风险。
进一步地,
判断因脓毒症导致的脓毒症休克和非脓毒症休克时,
当所述的乳酸化的组蛋白H3K18的血清学浓度高于0.45时,判断患者为脓毒症休克;
预测脓毒症休克的风险时,
当所述的乳酸化的组蛋白H3K18的血清学浓度高于0.56时,判断患者为脓毒症休克高危。
附图说明
图1A-1C是本公开实施例的不同组总蛋白乳酸化水平的示意图。
图2是本公开实施例的重症患者样本和健康者样本中H3K18la水平的示意图。
图3是本公开实施例的H3K18la与血清乳酸的关系示意图。
图4是本公开实施例的休克例样本与非休克例样本中H3K18la水平的示意图。
图5是本公开实施例的重症例样本中不同H3K18la水平的分类示意图。
图6是本公开实施例的重症例样本中的H3K18la与精氨酸酶-1的关系示意图。
具体实施方式
脓毒症是由感染引起的全身炎症反应,可发展为脓毒性休克,甚至危及生命的器官功能障碍。脓毒症是重症监护的一个主要问题,发病率高达每10万人年535例,并且还在上升。此外,住院死亡率仍然很高,为25-30%,脓毒性休克的住院死亡率为40-60%。总体而言,脓毒症和脓毒症休克患者在ICU期间的健康相关生活质量急剧下降,在长期护理中死亡率增加。尽管我们在脓毒症的诊断和管理方面有了显著的改进,但由于其不同的病因和表现,它仍然是一个具有挑战性的临床实体,通常只有在住院期间临床恶化后才有诊断记录。当患者为循环休克患者,特别是当伴有如心脏损伤、低血容量和外伤等其他情况时,或在如婴幼儿、老年人和免疫功能低下者中没有感染的典型体征时,鉴别诊断同样也很困难。
脓毒症会引发复杂的免疫反应,导致促炎和抗炎之间的稳态平衡失调,使得有相似损伤的患者其预后可能有很大不同。遗传调控可能在此发挥核心作用。事实上,广泛的基因重编程,如DNA甲基化、组蛋白修饰和非编码RNA的转录调节,可导致细胞周期破坏、内皮功能障碍、线粒体损伤、代谢紊乱、免疫功能衰竭和心血管衰竭。组蛋白可进行多种共价修饰,包括甲基化、瓜氨酸化、乙酰化和磷酸化,这些修饰改变了它们彼此之间和与DNA的关系。局部和全身促炎细胞因子的衰减、远处器官损伤的保护、细菌清除和吞噬作用的增强以及抑制免疫细胞凋亡与生存率改善相关。组蛋白乙酰化和瓜氨酸化修饰可以显著提高生存率,减弱“细胞因子风暴”和脓毒症相关的凝血功能障碍,并在致死性小鼠脓毒症模型中减少骨髓萎缩。
巨噬细胞在缺氧、脂多糖和细菌(如大肠杆菌、鲍曼不动杆菌、铜绿假单胞菌等)刺激下诱导了一种新的翻译后修饰(PTM),即组蛋白乳酸化。当M1巨噬细胞暴露于缺氧、脂多糖、IFN-γ或细菌下时,组蛋白发生乳酸化。升高的组蛋白H3赖氨酸18乳酸化(H3K18la)诱导了参与愈合的稳态基因的表达,包括精氨酸酶-1。p300(也称为赖氨酸-乙酰基转移酶(KAT3B))可特异性乙酰化组蛋白H3K18和H3K27。在细菌和腺病毒感染期间,通过SIRT2和CBP/p300通路H3K18的乙酰化显著降低。因此,在感染中发生的H3K18乙酰化的减少可能会通过p300使H3K18的乳酸化上升(相同位点下修饰),这两种修饰都可能与脓毒症和脓毒性休克密切相关。
基线信息和统计分析
本公开实施例采用不同临床血样本进行试剂盒检测,样本来自于24名不同类型的休克患者(包括13名脓毒性休克、7名失血性休克、2名阻塞性休克和2名心源性休克)、11名无休克的危重病人和13名健康志愿者。本公开收集样本对象的以下基线信息:年龄、性别、基础疾病、序贯器官衰竭估计评分(SOFA)(ICU入院第1天至第3天)、24小时内急性生理学与慢性健康状况评分Ⅱ(APACHEⅡ)、机械通气时间、ICU住院时间、总住院时间、28天死亡率以及同一采集日期下的实验室指标:血清乳酸、白细胞计数(WBC)、中性粒细胞计数、中性粒细胞百分比、淋巴细胞计数、淋巴细胞百分比、单核细胞计数、单核细胞百分比、降钙素原水平(PCT)和c反应蛋白(CRP)。本公开对正态分布的数据采用学生t检验或单因素方差分析(One-way ANOVA)进行比较,结果以平均值±SD表示;相关性分析采用Pearson相关性检验。非正态分布的数据使用非参数曼-惠特尼U检验进行分析,结果以中位数和四分位数范围(IQR)表示;相关性分析采用应用Spearman相关性检验。分类变量的比较采用卡方或费希尔精确检验,结果以数字和百分比表示。P<0.05被认为具有统计学意义。样本基线信息可见下表1。
表1:样本基线信息
由表1可知,休克组的平均年龄和男性比例分别为69.00岁和58.33%,无休克的危重病人组为64.27岁和45.5%,而健康志愿者组为26.00岁和38.5%。有休克的危重病人和无休克的危重病人在性别和年龄上没有显著差异(P=0.436;P=0.716)。休克组和无休克的危重病人之间在基础疾病方面也没有显著差异。然而,与无休克的重症患者组相比,休克组患者的SOFA和APACHEⅡ评分更高(P=0.003;P=0.000),在ICU住院时间和机械通气时间更长(P=0.001;P=0.005)。相反,两组之间的总住院时间没有显著差异(P=0.346)。研究期间有三名患者死亡,均为休克组,休克组的28天死亡率为12.5%。其中,脓毒性休克是指尽管进行了充分的液体复苏,但仍需要血管加压药维持,平均动脉压(MAP)≥65mmHg和血清乳酸水平≥2mmol/L。非脓毒性休克包括失血性休克、心源性休克和阻塞性休克(肺栓塞)。休克是指包括有低灌注和低血压症状,组织灌注不足表现如下:1、皮肤冷、湿、发蓝、苍白或变色;2、精神状态的改变,表现为迟钝、迷失方向和困惑;3、尿量减少:<0.5mL/kg/h。低血压定义为收缩压(SBP)<90mmHg,平均动脉压(MAP)<65mmHg,或较基线降低>40mmHg。无休克是指危重症患者不符合休克标准,但进入ICU进行重症监护,如经历重大手术和老年共病患者。
血液样本的外周血单个核细胞分离和血清分离
基于此,本公开使用一种脓毒症的检测试剂盒,用于检测脓毒症的预后,包括以下试剂中的至少一种:用于检测总蛋白乳酸化水平的试剂、用于检测组蛋白H3K18乳酸化水平的试剂、用于检测细胞因子的试剂、用于检测精氨酸酶-1的信使核糖核酸水平的试剂。此外,本公开检测试剂盒还可包括BCA蛋白浓度测定试剂盒、组蛋白提取试剂盒、SDS-PAGE凝胶制备试剂盒。
具体地,首先采集血液样本,然后将采集到的血液样本分别进行外周血单个核细胞分离和血清分离。采用含乙二胺四乙酸(EDTA)采血管和血清分离采血管采集血液样本(5~10ml),分别进行外周血单个核细胞分离和血清分离。其中,外周血单个核细胞分离可经pH-7.2的磷酸盐缓冲盐溶液以1:1稀释;稀释后的血液样本置于15ml的淋巴细胞分离液(STEMCELL Technologies Cat#07851)上,在500xg和20℃的条件下离心20分钟;上层的大部分被吸出,在间期留下白色的浅黄色被毛(单个核细胞);将单个核细胞分离出并充满磷酸盐缓冲盐溶液,混合后在500x g下20℃离心7分钟;完全清除上清,若管底沉淀物中有红色杂质,加入红细胞缓冲液(Solarbio Cat#R1010)5分钟;加入足够量的磷酸盐缓冲盐溶液,在20℃和500xg的条件下离心7分钟;去除上清后,收集外周血单个核细胞,用2ml冷冻保护剂重悬(胎牛血清:二甲基亚砜=9:1),获取到的外周血单个核细胞于-80℃保存。血清分离可在无菌条件下,将血液标本在4℃下以3000rpm离心10分钟;上清吸入并低温保存,将获取到的血清于-80℃保存。
总蛋白乳酸化水平
本公开采用检测总蛋白乳酸化水平的试剂包括:裂解缓冲液、十二烷基硫酸钠聚丙烯酰胺凝胶、上样缓冲液、十二烷基硫酸钠、预染蛋白、电泳缓冲液、电转缓冲液、考马斯亮蓝、赖氨酸残基乳酸化抗体、山羊抗兔二抗、封闭牛奶、TBST缓冲液和SuperECLPlus超敏发光液。其中,裂解缓冲液包括:质量分数均为1%的十二烷基硫酸钠和蛋白酶抑制剂、3μM组蛋白去乙酰化酶抑制剂和50mM烟酰胺。
本公开采用裂解缓冲液和超声处理裂解细胞,将已提取的外周血单个核细胞加入裂解液超声处理,离心后去除细胞碎片(4℃下以12000xg离心10分钟)。使用BCA蛋白浓度检测试剂盒测定蛋白质的浓度。根据蛋白浓度的结果,将等量的15μg总蛋白加入5μl 4×装载缓冲液和2%的十二烷基硫酸钠,使其最终体积为20μl。将一微升预染蛋白和20μl样品加入到十二烷基硫酸钠-12%的聚丙烯酰胺凝胶。15mA/gel,大约15分钟,使样品显示为一条线。和35mA/gel直到样品被触及底部。染色可用考马斯亮蓝(R-250)在室温下染色2小时。然后对凝胶进行脱色,直到凝胶的背景颜色几乎为无色。本实施例可通过西方印迹法测总蛋白乳酸化水平。其中,一抗可采用赖氨酸残基乳酸化抗体(PTM-1401RM;批号:K111421;在LifeTechnologiesTM抗体稀释试剂溶液中1:1000稀释,cat#003218),在4℃下应用过夜。二级抗体可是山羊抗兔IgG(H+L)(Thermo Pierce,过氧化物酶结合的,31460),在TBST缓冲液中以1:10000的比例稀释,加5%的封闭牛奶,在室温下放置2小时。使用VILBER Fusion Solo S对条带进行定量检测,获得泛乳酸化修饰水平。
为了获得样本中乳酸化的总体情况,本公开实施例收集了4名非脓毒性休克患者、6名脓毒性休克患者和8名健康志愿者的临床资料和血液样本,并通过Western blotting测定外周血单个核细胞中全蛋白乳酸化的水平,如图1A和图1B所示,乳酸化是在健康和休克患者中发现的一种全蛋白翻译后修饰,两组之间的表达差异可以清楚地检测到,如相应条带密度的差异所示。请参见图1C,在全蛋白范围内,休克患者样本的乳酸化水平高于健康者样本的水平(t=2.172,P=0.045),而脓毒性和非脓毒性休克患者之间的差异并不明显(z=-1.066,P=0.286)。在总蛋白乳酸化修饰水平检测中内部参考蛋白不适用,因为它们也可以自身可被修饰。在本公开中,每个泳道中加入了等量的15μg蛋白质,因此,样本之间的WB结果是可比较的。
多重微球免疫荧光分析
本公开采用检测细胞因子的试剂可检测IL-1β、IL-2、IL-4、IL-5、IL-6、IL-8、IL-10、IL-12p70、IL-17、IFN-α、IFN-γ、TNF-α中的一种或多种。本公开采用检测细胞因子的试剂可包括捕获微球抗体、检测抗体、SA-PE和洗涤液。
本公开采用流式细胞术检测分离出的血清中的细胞因子,包括:IL-1β、IL-2、IL-4、IL-5、IL-6、IL-8、IL-10、IL-12p70、IL-17、IFN-α、IFN-γ和TNF-α。校准管中加入25μl校准产物样品,基质B中加入25μl血清样品缓冲液。样品与25μl捕获微球抗体充分混合;所有试管中加入检测抗体25μl,室温避光,振荡400-500r/min孵育。2小时后,在所有试管中加入25μl SA-PE,室温避光,400-5500r/min摇瓶孵育。半小时后,加入500μl 1×洗涤液,旋转数秒,500xg离心5分钟。在去除上清液后,向试管中加入300μl 1×洗涤液,旋转几秒钟。采用流式细胞仪对标本进行检测,可检测到12种细胞因子,包括IL-1β、IL-2、IL-4、IL-5、IL-6、IL-8、IL-10、IL-12p70、IL-17、IFN-α、IFN-γ和TNF-α。为保证数据的准确性,每个样品至少采集1100个微球。
RNA提取和qRT-PCR
本公开采用检测精氨酸酶-1的信使核糖核酸水平的试剂包括:反转录试剂、DNA酶、引物和实时荧光定量PCR混合物。其中,实时荧光定量PCR混合物包括:5微升的2×PCR混合物、0.5微升的引物F(10uM)、0.5微升的引物R(10uM)、1微升的模板和3微升的双蒸水。引物包括以下引物中的一种或多种:
5’-GGGTTGACTGACTGGAGAGC-3’/5’-CGTGGCTGTCCCTTTGAGAA-3’(精氨酸酶-1),
5’-TGACTTCAACAGCGACACCCA-3’/5’-CACCCTGTTGCTGTAGCCAAA3’(GAPDH),
5’-GCTGTGGATGGAAATTCGCC-3’/5’-CTTCTGGCAGTGTGGGTCAT-3’(KLF4)。
本公开实施例的总RNA采用异硫氰酸胍-苯酚-氯仿法提取(RNAiso Plus,TaKaRaBio Code No.9109)。用Thermo nanodrop 2000C(A260/A280)测定RNA产量。用琼脂糖凝胶电泳检测RNA的质量。将合格样品在65℃变性5分钟,用反转录试剂对cDNA进行反转录。采用DNA酶步骤去除残留的基因组DNA。利用Primer5.0设计引物,组装目的基因的上下游区域。cDNA和引物加入到qRT-PCR系统(Tip Green qPCR SuperMix,AQ141-02;ThermoFisher Scientific)。实时荧光定量PCR混合物包含5μl 2×PCR混合物、0.5μl引物F(10uM)、0.5μl引物R(10uM)、1μl模板和3μl ddH2O,最终体积为10μl。在LightCycler480 II(Roche Diagnostics)中进行反应,条件如下:95℃处理5分钟、95℃变性10秒、60℃退火30秒、72℃10秒共45个PCR扩增周期;熔化曲线包括95℃5秒,65℃1分钟和97℃1秒。最后,在4℃冷却30秒和荧光测量。采用GAPDH作为内部参照。用delta-delta CT方法将表达数据归一化为GAPDH。
组蛋白提取和H3K18乳酸化水平
本公开采用检测组蛋白H3K18乳酸化水平的试剂包括:组蛋白提取试剂盒、十二烷基硫酸钠聚丙烯酰胺凝胶、上样缓冲液、十二烷基硫酸钠、预染蛋白、电泳缓冲液、电转缓冲液、组蛋白H3K18乳酸化抗体、组蛋白H3抗体、山羊抗兔二抗、封闭牛奶、TBST缓冲液和SuperECLPlus超敏发光液。
本公开将分离出的外周血单个核细胞在4℃下以1000rpm离心5分钟,然后以107个细胞/毫升重悬于稀释的1X预溶解缓冲液中。将试管在冰上保持10分钟,轻轻搅拌,并在4℃下以10,000rpm离心1分钟。在去除上清液后,将细胞重新悬浮在3倍体积(约200微升/107个细胞)的裂解液中并在冰上孵育30分钟。在4℃下以12,000rpm离心5分钟后,向上清液部分(含有酸溶性蛋白质)加入0.3倍体积的平衡-DTT缓冲液,测定蛋白浓度。分离出的组蛋白在-80℃下保存。本公开采用BCA蛋白检测试剂盒检测从外周血单个核细胞中提取的组蛋白浓度,根据蛋白浓度的结果,向等量的15μg组蛋白中加入5μl 5×加载缓冲液和2%十二烷基硫酸钠,使其最终体积为20μl。将5微升预染蛋白和20μl样品不加到5%层析胶+15%分离胶中。80V/gel,使样品显示为一条线,120V/gel,直到样品被触及到底部。进行湿法转移,在300mA下将蛋白质转移到0.2μm的PVDF膜(ImmobilonTM-PSQ膜)上3小时。使用的主要抗体是抗乳酸组蛋白H3(Lys18)的兔mAb(PTM-1406RM;Lot:K111421;1:1000稀释在LifeTechnologiesTM Antibody Diluent Reagent Solution cat#003218中)和抗组蛋白H3抗体Nuclear Marker and ChIP Grade(ab1791)(1:1000稀释在Life TechnologiesTM AntibodyDiluent Reagent Solution中),在4℃下应用过夜。二级抗体是山羊抗兔IgG H&L(HRP)(ab6721),在含有5%封闭牛奶的TBS-T缓冲液中以1:3000稀释,室温下2小时。使用VILBERFusion Solo S对检测到的条带进行定量分析,获得组蛋白H3K18乳酸化修饰水平。
重症患者样本中H3K18乳酸化水平
请参见图2,H3K18la在所有受试者样本中都有表达,包括健康志愿者样本。将受试者按严重程度分为3组(休克病人、无休克的危重病人和健康志愿者)后,休克组样本的平均相对密度为0.56,是三组中最高的(与无休克的危重病人相比P=0.002;与健康志愿者相比P=0.000)。无休克的危重病人组样本的平均相对密度为0.32,健康志愿者组样本为0.21,两组之间没有差异(P=0.308)。
H3K18乳酸化水平与疾病严重程度关系
结果显示,H3K18la与APACHE II评分、第1天SOFA评分、ICU住院时间和机械通气时间呈正相关(Spearman相关系数分别为0.42、0.63、0.39、0.55;P=0.012、0.000、0.019和0.003),H3K18la可反映危重病的严重程度,详见下表2。
表2:H3K18la与疾病严重程度关系
疾病严重尘毒 | Pearson/Spearman R(with H3K18la) | P value |
APACHE II评分 | 0.42 | 0.012 |
第一天SOFA评分 | 0.63 | 0.000 |
第二天SOFA评分 | 0.61 | 0.000 |
第三天SOFA评分 | 0.62 | 0.000 |
ICU住院时间(天) | 0.39 | 0.019 |
总住院时间(天) | -0.04 | 0.827 |
机械通气时间 | 0.55 | 0.003 |
如图3所示,H3K18la与血清乳酸呈正相关(Spearman相关系数0.48;P=0.003)。
H3K18乳酸化水平与感染的关系
为了进一步说明H3K18la的表达与脓毒症的关系,本公开将休克患者样本分为两组:脓毒性休克和非脓毒性休克(失血性、阻塞性和心源性)。脓毒性休克组和非脓毒性休克组的H3K18la的平均相对密度分别为0.65和0.45;其中,0.65和0.45是H3K18la与H3的条带灰度值的比值。在所有ICU患者样本中,H3K18la在脓毒性休克患者样本中最高(与非脓毒性休克患者样本相比,P=0.033;与无休克的重症患者样本相比,P=0.000)。非脓毒性休克患者样本的H3K18la相对密度也比健康志愿者高(P=0.005)。非脓毒性休克和无休克的危重病人(P=0.265)或无休克的危重病人和健康志愿者(P=0.390)样本之间没有发现明显差异,具体可见图4所示。
然而,由于H3K18la的水平与疾病严重程度相关,如表3所示,临床指标如APACHEⅡ、第1天的SOFA评分和ICU住院时间在脓毒症休克和非脓毒症休克患者之间有明显差异。
表3:
根据严重程度(APACHE、SOFA、ICU住院时间和乳酸),本公开对脓毒性休克和非脓毒性休克(失血性休克、心源性休克和梗阻性休克)患者样本进行匹配,考虑到上述参数,对调整后的中位数/平均值进行了配对比较。在去除3名重度脓毒性休克患者和1名非脓毒性休克患者样本后,脓毒性患者和非脓毒性患者样本在APACHEⅡ、SOFA评分第1天、ICU住院时间或血清乳酸方面没有显著差异(P=0.141,0.052,0.052和0.353,具体请见表4),重新测试的脓毒症和非脓毒症休克患者样本的H3K18la水平结果仍显示有显著差异(t=-2.208,P=0.040),表明H3K18la与感染有关。
表4:
H3K18乳酸化水平与炎症生物指标的关系
本公开进一步分析了H3K18la表达与炎症参数之间的联系,包括降钙素(PCT)、C反应蛋白(CRP)、白细胞计数(WBC)、中性粒细胞计数、中性粒细胞百分比、淋巴细胞计数、淋巴细胞百分比、单核细胞计数和单核细胞百分比。请参见表5,H3K18la与PCT呈正相关(Spearman相关系数=0.71,P=0.010),但与单核细胞百分比呈负相关(Pearson相关系数=-0.36,P=0.041)。
表5:H3K18la与实验室炎症指标的关系
Laboratory indicators | Pearson/Spearman R(with H3K18la) | P value |
PCT | 0.71 | 0.010 |
CRP | 0.35 | 0.078 |
WBC | 0.04 | 0.845 |
中性粒细胞计数 | 0.09 | 0.633 |
中性粒细胞比例 | 0.28 | 0.116 |
淋巴细胞计数 | -0.01 | 0.973 |
淋巴细胞比例 | -0.22 | 0.227 |
单核细胞计数t | -0.22 | 0.219 |
单核细胞比例 | -0.36 | 0.041 |
H3K18乳酸化水平与炎症因子的关系
如图5所示,为了探讨H3K18la和炎症相关细胞因子之间的关系,本公开实施例选择了H3K18la相对密度超过75%的患者样本,并且其相对密度低于所有ICU患者样本相对密度的25%,来说明H3K18la与感染的相关性。如表6所示,高H3K18la的患者样本比低H3K18la的患者样本有更高的IL-10水平(Z=-2.455;P=0.012)。
表6:H3K18la与炎症因子的关系
H3K18乳酸化水平与巨噬细胞功能学指标的关系
如图6所示,H3K18la与精氨酸酶-1呈正相关(Spearman相关系数=0.561,P=0.005)。
乳酸化是一种蛋白质翻译后修饰,在健康志愿者和危重病人的外周血样本中存在差异。本公开实施例表明H3K18la与危重病人的严重程度和预后明显相关,并能区分脓毒性休克患者和非脓毒性休克患者样本。乳酸是碳水化合物和非必需氨基酸代谢的最关键的中间产物之一,长期以来被误认为是代谢废物。但是,根据本公开实施例,乳酸更可能是一种″压力信号″,是一种具有自分泌、旁分泌和内分泌因素。在酸中毒的情况下,外源性乳酸盐的输注具有碱化作用。作为主要的葡萄糖生成前体,乳酸在脑外伤、急性胰腺炎、肝炎、心肌梗塞、心脏手术、急性心力衰竭、烧伤和脓毒症中作为燃料和抗炎剂。此外,乳酸的积累有助于癌细胞逃避免疫,对免疫杀伤有抑制作用。巨噬细胞在生理和病理生理条件下都会吸收细胞外乳酸,促进自身蛋白乳酸化,这进一步解释了乳酸在分子水平上的免疫功能。
在本公开实施例中,乳酸化是一种全蛋白翻译后修饰,存在于健康和疾病患者血液样本中。而且,乳酸化普遍存在于人类血液和几乎所有大小/不同的蛋白大小中,而不仅仅是组蛋白的范围。患者和健康志愿者血液样本之间有明显的可检测的差异。在暴露于缺氧、脂多糖/IFN-γ或细菌的M1巨噬细胞中发生组蛋白乳酸化。组蛋白乳酸化(H3K18la)的增加诱导了参与愈合的同源基因的表达,并与M2样表型有关,包括精氨酸酶-1。本公开实施例选择H3K18作为修饰位点来检测所有参与者的乳酸化水平。如图2所示,休克组患者样本的H3K18la水平最高,H3K18la与APACHEⅡ评分、第1天的SOFA评分、ICU住院时间和机械通气时间表现出正相关,提示H3K18la反映了危重病的严重程度。H3K18乳酸化是一个突出的独立生物标志物,反映了危重病的严重程度。
在本公开实施例中,乳酸化与脓毒症期间炎症因子的释放相关。p300(又称KAT3B)是一种典型的乙酰化转移酶,能特异性地乙酰化组蛋白H3K18和H3K27,也能催化蛋白质的乳酸化,在细菌和腺病毒感染期间,H3K18的乙酰化通过SIRT2和CBP/p300明显下降。即,感染中H3K18乙酰化的减少可能反过来通过p300促进H3K18的乳酸化(同一部位的修饰)。为了进一步说明H3K18la在检测脓毒症中的作用,本公开实施例将休克患者样本分为脓毒性休克和非脓毒性休克两个亚组,结果可见图4所示,前者的H3K18la水平最高。由于脓毒性休克组患者的临床预后比非脓毒性休克组患者差(详见表3),本公开实施例进一步将APACHEⅡ评分、SOFA评分、ICU住院时间和乳酸水平,对调整后的中位数/平均值进行配对比较(详见表2)。经重新评估脓毒症和非脓毒症休克患者样本的H3K18la水平,结果仍显示有明显差异(t=-2.208,P=0.040),说明H3K18la参与了脓毒症引起的休克的病理生理过程。
为了进一步证实H3K18la与感染的关系,本公开实施例比较了H3K18la的表达与感染的临床生物标志物,包括WBC、中性粒细胞计数、中性粒细胞百分比、淋巴细胞计数、淋巴细胞百分比、单核细胞计数、单核细胞百分比、PCT和CRP。结果显示,H3K18la与PCT密切相关(Spearman相关系数=0.711,P=0.010)。血清中的PCT水平随着细菌感染而增加,但在非感染状态下,PCT水平保持不变或仅适度增加,表明H3K18与感染有重要关系。
关于H3K18la在感染中的机制,本公开实施例评估了感染中关键的细胞因子和基因的表达。在M1极化过程中,H3K18la增加的1,223个特异性标记的基因更有可能在后期时间点中(16或24小时)被激活或重新激活,这与这些后期时间点的细胞内乳酸和组蛋白赖氨酸残基乳酸化水平的诱导有很大关联。请参见表6,H3K18la与炎症相关的细胞因子相关,H3K18la高的患者比H3K18la低的患者样本中的IL-10水平高。IL-10信号激活Jak-STAT途径和PI3K-Akt-GSK途径,这一过程抑制了各种炎症基因的表达。结果显示,IL-10驱动了在晚期脓毒症期间增强免疫抑制的分子路径,这与感染患者的死亡率相关。对患者循环细胞因子的分析结果显示,除了促炎症细胞因子外,强效抗炎细胞因子IL-10的浓度也有所增加。结合与IL-10的显著关系,H3K18la不仅可区分脓毒症患者,而且还反映了脓毒症的预后。另一方面,IL-10与IL-4协同增强了M2巨噬细胞的表型,从而诱导了抗炎基因的表达,包括精氨酸酶-1。
在第二类免疫反应和伤口修复期间,巨噬细胞中的精氨酸酶-1和KLF4水平增加。受干扰素-γ(IFN-γ)以及白细胞介素-4(IL-4)和IL-10刺激的巨噬细胞诱导精氨酸酶-1产生更多的iNOS,抑制了细菌的有效清除。在感染后的后期,H3K18la介导的抗炎症(如IL-10和精氨酸酶-1)的过度表达与后期死亡有关。本公开的结果与目前细胞和分子研究结果的进展一致。
总之,乳酸化是一种全蛋白翻译后修饰,存在于健康和患病的病人中。H3K18la可能反映了危重病的严重程度以及感染的存在。在感染后的后期,H3K18la介导的抗炎作用,如IL-10的过度表达,可能在巨噬细胞的抗炎功能以及脓毒症的精氨酸酶-1表达中发挥重要作用。
本公开检测所有蛋白的乳酸化作用,并进一步评估了H3K18la在外周血单个核细胞中的表达,以评估其在脓毒性休克和非脓毒性休克中的作用。通过比较同一目标对象的炎症细胞因子和巨噬细胞功能生物标志物,进一步检测其潜在的机制和生理相关性。本公开采用含EDTA和血清分离采血管采集血样,分别进行外周血单个核细胞分离和血清分离。Western blotting检测各组蛋白和H3K18的乳酸化水平。流式细胞术检测血清炎症因子IL-1β、IL-2、IL-4、IL-5、IL-6、IL-8、IL-10、IL-12p70、IL-17、IFN-α、IFN-γ、TNF-α水平。采用实时定量PCR(qRT-PCR)检测精氨酸酶-1和Krüppel-like factor-4(Klf4)的信使核糖核酸的表达。乳酸化是一种全蛋白翻译后修饰,在健康和危重患者的外周血单个核细胞中均可检测到,而休克患者的样本中相对密度明显较高(t=2.172,P=0.045)。H3K18la在包括志愿者在内的所有入组患者中均有表达,其中休克患者表达水平最高(与未休克的危重患者和健康志愿者相比P=0.002,P=0.000)。H3K18la蛋白表达与APACHEⅡ评分、第1天SOFA评分、ICU住院时间、机械通气时间和血清乳酸水平呈正相关(ρ=0.42、0.63、0.39、0.55、0.48;P=0.012,0.000,0.019,0.003,0.003)。当将脓毒性休克患者与非脓毒性休克患者按严重程度进行配对时,脓毒性休克组H3K18la水平较高(t=-2.208,P=0.040)。H3K18la与降钙素原(PCT)水平密切相关(ρ=0.711,P=0.010)。H3K18la高表达患者IL-10水平升高(Z=-2.455;P=0.012)。H3K18la表达与精氨酸酶-1mRNA水平呈正相关(ρ=0.561,P=0.005)。H3K18la可能介导IL-10和精氨酸酶-1过表达,刺激巨噬细胞的抗炎功能。
最后需要说明的是,以上实施例仅用作帮助本领域技术人员理解本发明的实质,不对本发明的保护范围加以限定。
SEQUENCE LISTING
<110> 北京医院
<120> 脓毒症的检测试剂盒
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Claims (8)
1.用于检测组蛋白H3K18乳酸化水平的试剂在制备脓毒症的检测试剂盒的应用,用于脓毒症的预后,
其中,所述预后包括:判断因脓毒症导致的脓毒症休克和非脓毒症休克,及预测脓毒症休克的风险。
2.根据权利要求1所述的应用,其特征在于,所述检测试剂盒还包括用于检测IL-10水平的试剂和/或用于检测精氨酸酶-1的信使核糖核酸水平的试剂。
3.根据权利要求1所述的应用,其特征在于,所述用于检测组蛋白H3K18乳酸化水平的试剂包括:组蛋白提取试剂组合、十二烷基硫酸钠聚丙烯酰胺凝胶、上样缓冲液、十二烷基硫酸钠、预染蛋白、电泳缓冲液、电转缓冲液、组蛋白H3K18乳酸化抗体、组蛋白H3抗体、山羊抗兔二抗、封闭牛奶、TBST缓冲液和SuperECLPlus超敏发光液。
4.根据权利要求2所述的应用,其特征在于,所述用于检测IL-10水平的试剂包括捕获微球抗体、检测抗体、SA-PE和洗涤液。
5.根据权利要求2所述的应用,其特征在于,所述用于检测精氨酸酶-1的信使核糖核酸水平的试剂包括:反转录试剂、DNA酶、引物和实时荧光定量PCR混合物。
6.根据权利要求5所述的应用,其特征在于,所述引物包括以下引物中的一种或多种:
5’-GGGTTGACTGACTGGAGAGC-3’/5’-CGTGGCTGTCCCTTTGAGAA-3’,
5’-TGACTTCAACAGCGACACCCA-3’/5’-CACCCTGTTGCTGTAGCCAAA3’,
5’-GCTGTGGATGGAAATTCGCC-3’/5’-CTTCTGGCAGTGTGGGTCAT-3’。
7.根据权利要求5所述的应用,其特征在于,所述实时荧光定量PCR混合物包括:5微升的2×PCR混合物、0.5微升的引物F、0.5微升的引物R、1微升的模板和3微升的双蒸水。
8.根据权利要求1所述的应用,其特征在于,所述检测试剂盒还包括:磷酸盐缓冲盐溶液、淋巴细胞分离液、红细胞裂解液、胎牛血清和二甲基亚砜。
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