CN116875674A - 一组预测慢加急性肝功能衰竭预后的标志物及其应用 - Google Patents
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Abstract
本发明公开了一组预测慢加急性肝功能衰竭预后的标志物及其应用,所述标志物包含CHGB、COL1A2、ENTPD1、ENTPD3、ENTPD8、LAMB1、LAMC1、LRP1、LYVE1、NID1、SPARCL1、TNC、COMP、FGFR1、GRN、LAMA2、MMP2、THBS4、CD44、COL1A1、FSTL3、DDX19B、LGALS1、APP、CTSL、CSPG4、VCAM1、CCN2、AXL、PECAM1中的至少一种,与ACLF‑2级病人中短期预后相关,通过检测标志物的表达水平可以有效评估患者的预后情况,具有很高的应用价值。
Description
本申请是申请日为2021年06月04日、申请号为202110627166.6、发明名称为“一组预测慢加急性肝功能衰竭预后的标志物及其应用”的中国专利申请的分案申请。
技术领域
本发明属于医药技术领域,具体涉及一组预测慢加急性肝功能衰竭预后的标志物及其应用。
背景技术
慢加急性肝功能衰竭(ACLF)是一种复杂的综合征,在肝硬化患者中发展,其特征是急性代偿失调,器官衰竭和短期死亡率高(Singal,A.K.,et al.,ACG ClinicalGuideline:Alcoholic Liver Disease.Am J Gastroenterol,2018.113(2):p.175-194.)。EASL定义要求发生急性失代偿事件(肝性脑病,胃肠道(GI)出血,腹水或细菌感染),然后发展为一个或多个器官衰竭(OF)以指定ACLF的严重程度。有六种潜在的器官衰竭-肝,肾,脑,凝血,循环和呼吸-根据现有器官衰竭的数量和类型,将患者的评分从EASL ACLF 0级(无ACLF)提高到3级(严重ACLF))。ACLF的病理生理学与持续性炎症,最初免疫广泛激活的免疫失调,系统性炎症反应综合征的状态以及随后由于免疫抑制引起的败血症有关。可以通过肝和肝外器官衰竭来预测疾病的严重程度和短期预后。
目前,由于对慢加急性肝功能衰竭教育的普及不充分,全球的慢加急性肝功能衰竭发病率仍呈上升趋势,尤其在中国更是进入了增长期。尽快阻止慢加急性肝功能衰竭在我国的发展已成了一件刻不容缓的大事。因此,继续扩大我们对慢加急性肝功能衰竭致病机制的认识,开发出更有效,更经济的检测手段来预知ACLF患者病情的发展,仍是我们能否治疗ACLF的关键。
发明内容
本发明的目的在于提出一种肝衰竭相关标志物及其应用。
本发明所采取的技术方案是:
本发明的第一个方面,提供定量检测标志物的试剂在制备肝衰竭诊断和/或预后评估产品中的应用,所述标志物包括以下至少一种:CHGB、COL1A2、ENTPD1、ENTPD3、ENTPD8、LAMB1、LAMC1、LRP1、LYVE1、NID1、SPARCL1、TNC、COMP、FGFR1、GRN、LAMA2、MMP2、THBS4、CD44、COL1A1、FSTL3、DDX19B、LGALS1、APP、CTSL、CSPG4、VCAM1、CCN2、AXL、PECAM1。
在本发明的一些实施方式中,所述定量检测标志物的试剂为检测标志物基因表达水平的试剂。
在本发明的一些实施方式中,所述定量检测标志物基因表达水平的试剂包括通过测序技术、核酸杂交技术、核酸扩增技术或免疫测定的方法检测标志物基因表达水平的试剂。
在本发明的一些实施方式中,所述检测标志物基因表达水平的试剂包括引物、探针、基因芯片。
在本发明的一些实施方式中,所述检测标志物的试剂为定量检测标志物蛋白质表达水平的试剂。
在本发明的一些实施方式中,所述检测标志物蛋白质表达水平的试剂包括免疫组化、Western-Blot、蛋白免疫印迹、酶联免疫吸附(ELISA)、流式细胞术、紫外分光光度法-近红外光谱法、高效液相色谱法、比色法或质谱鉴定的方法检测蛋白表达水平的试剂。
在本发明的一些优选实施方式中,所述检测标志物蛋白质表达水平的试剂为高效液相色谱和/或质谱鉴定检测方法检测蛋白表达水平的试剂。
在本发明的一些实施方式中,所述检测标志物基因编码的蛋白质表达水平的试剂包括抗体、配体。
在本发明的一些实施方式中,所述肝衰竭为慢加急性肝功能衰竭(ACLF)。
在本发明的一些实施方式中,所述标志物的表达水平上调预示预后较差。
在本发明的一些实施方式中,所述预后具体为ACLF-2级病人中短期预后。
在本发明的一些优选实施方式中,所述预后具体为ACLF-2级病人优选为28天的预后。
本发明的第二个方面,提供一种检测试剂,所述检测试剂包含本发明第一方面所述的定量检测标志物的试剂。
本发明的第三个方面,提供一种试剂盒,所述试剂盒包含本发明的第二个方面所述检测试剂。
本发明的第四个方面,提供一种评估肝衰竭预后风险的方法,包括以下步骤:
(1)检测受试者本发明第一方面或第二方面或第三方面所述标志物的表达量;
(2)根据步骤(1)所得表达量,预测肝衰竭预后风险。
在本发明的一些实施方式中,所述肝衰竭为慢加急性肝功能衰竭(ACLF)。
在本发明的一些实施方式中,所述标志物的表达水平上调预示预后较差。
在本发明的一些实施方式中,所述预后具体为ACLF-2级病人中短期预后。
在本发明的一些优选实施方式中,所述预后具体为ACLF-2级病人优选为28天的预后。
本发明的第五个方面,提供一种筛选药物的方法,通过检测给药前后本发明第一方面或第二方面或第三方面所述标志物的表达量。
本发明的有益效果是:
发明人运用蛋白质组学技术,发现有30个标志物(CHGB、COL1A2、ENTPD1、ENTPD3、ENTPD8、LAMB1、LAMC1、LRP1、LYVE1、NID1、SPARCL1、TNC、COMP、FGFR1、GRN、LAMA2、MMP2、THBS4、CD44、COL1A1、FSTL3、DDX19B、LGALS1、APP、CTSL、CSPG4、VCAM1、CCN2、AXL、PECAM1)与ACLF-2级病人中短期预后相关,其病情严重程度与靶标表达量上升数值呈现相关联的趋势,进而说明患者肝细胞的受损程度可以通过发明中的靶标的表达水平变化反映出来,可以有效评估患者的预后情况。为进一步的检测手段研发提供的强有力的理论基础和实践基础,具有重要的研发价值和开发意义。
附图说明
图1为入组流程图。
图2为蛋白筛选流程。
图3为标志物(CHGB、COL1A2、ENTPD1、ENTPD3、ENTPD8、LAMB1)与肝衰竭患者病情发展情况的关系。
图4为标志物(LAMC1、LRP1、LYVE1、NID1、TNC、SPARCL1)与肝衰竭患者病情发展情况的关系。
图5为标志物(COMP、FGFR1、GRN、LAMA2、MMP2、THBS4)与肝衰竭患者病情发展情况的关系。
图6为标志物(CD44、COL1A1、FSTL3、DDX19B、LGALS1、APP)与肝衰竭患者病情发展情况的关系。
图7为标志物(CTSL、CSPG4、VCAM1、CCN2、AXL、PECAM1)与肝衰竭患者病情发展情况的关系。
图8为标志物(CHGB、COL1A2、ENTPD1、ENTPD3、ENTPD8、LAMB1)的ROC曲线图。
图9为标志物(LAMC1、LRP1、LYVE1、NID1、TNC、SPARCL1)的ROC曲线图。
图10为标志物(COMP、FGFR1、GRN、LAMA2、MMP2、THBS4)的ROC曲线图。
图11为标志物(CD44、COL1A1、FSTL3、DDX19B、LGALS1、APP)的ROC曲线图。
图12为标志物(CTSL、CSPG4、VCAM1、CCN2、AXL、PECAM1)的ROC曲线图。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本发明的目的、特征和效果。显然,所描述的实施例只是本发明的一部分实施例,而不是全部实施例,基于本发明的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本发明保护的范围。
实施例1
(1)实验方法
1.材料与试剂
蛋白酶抑制剂购自Calbiochem、胰酶(trypsin)购自Promega、乙腈(acetonitrile)购自Fisher Chemical、三氟乙酸(trifluoroacetic acid)购自Sigma-Aldrich、甲酸(formic acid)购自Fluka、碘代乙酰胺(iodoacetamide)、二硫苏糖醇(dithiothreitol)、尿素(urea)、三乙基碳酸氢铵(TEAB)购自Sigma、超纯水(H2O)购自Fisher Chemical。
2.蛋白提取
血浆项目除高丰度法:样品从-80℃取出,4℃,12000g离心10分钟,去除细胞碎片,上清液转移至新的离心管,用Thermo公司生产的试剂盒参照PierceTMTop 12AbundantProtein Depletion Spin Columns Kit说明书去除高丰度蛋白。利用BCA试剂盒进行蛋白浓度测定。
3.胰酶酶解
蛋白溶液中加入二硫苏糖醇使其终浓度为5mM,56℃还原30min。之后加入碘代乙酰胺使其终浓度为11mM,室温避光孵育15min。最后将样品的尿素浓度稀释至低于2M。以1:50的质量比例(胰酶:蛋白)加入胰酶,37℃酶解过夜。再以1:100的质量比例(胰酶:蛋白)加入胰酶,继续酶解4h。
4.TMT标记
胰酶酶解的肽段用Strata X C18(Phenomenex)除盐后真空冷冻干燥。以0.5MTEAB溶解肽段,根据TMT试剂盒操作说明标记肽段。简单的操作如下:标记试剂解冻后用乙腈溶解,与肽段混合后室温孵育2h,标记后的肽段混合后除盐,真空冷冻干燥。
5.HPLC分级
肽段用高pH反向HPLC分级,色谱柱为Agilent 300Extend C18(5μm粒径,4.6mm内径,250mm长)。操作如下:肽段分级梯度为8%-32%乙腈、pH9,60min时间分离60个组分,随后肽段合并为18个组分,合并后的组分经真空冷冻干燥后进行后续操作。
6.液相色谱-质谱联用分析
肽段用液相色谱流动相A相(0.1%(v/v)甲酸水溶液)溶解后使用EASY-nLC 1000超高效液相系统进行分离。流动相A为含0.1%甲酸和2%乙腈的水溶液;流动相B为含0.1%甲酸和90%乙腈的水溶液。液相梯度设置:0-26min,9%~23%B;26-34min,23%~35%B;34-37min,35%~80%B;37-40min,80%B,流速维持在300nL/min。
肽段经由超高效液相系统分离后被注入NSI离子源中进行电离然后进OrbitrapFusion质谱进行分析。离子源电压设置为2.0kV,肽段母离子及其二级碎片都使用高分辨的Orbitrap进行检测和分析。一级质谱扫描范围设置为350-1550m/z,扫描分辨率设置为60,000;二级质谱扫描范围则固定起点为100m/z,二级扫描分辨率设置为30,000。数据采集模式使用数据依赖型扫描(DDA)程序,即在一级扫描后选择信号强度最高的前20肽段母离子依次进入HCD碰撞池使用35%的碎裂能量进行碎裂,同样依次进行二级质谱分析。为了提高质谱的有效利用率,自动增益控制(AGC)设置为5E4,信号阈值设置为5000ions/s,最大注入时间设置为100ms,串联质谱扫描的动态排除时间设置为30秒避免母离子的重复扫描。
7.数据库搜索
二级质谱数据使用Maxquant(v1.5.2.8)进行检索。检索参数设置:数据库为Human_SwissPort(20387条序列),添加了反库以计算随机匹配造成的假阳性率(FDR),并且在数据库中加入了常见的污染库,用于消除鉴定结果中污染蛋白的影响;酶切方式设置为Trypsin/P;漏切位点数设为2;肽段最小长度设置为7个氨基酸残基;肽段最大修饰数设为5;First search和Main search的一级母离子质量误差容忍度分别设为20ppm和5ppm,二级碎片离子的质量误差容忍度为0.02Da。将半胱氨酸烷基化设置为固定修饰,可变修饰为甲硫氨酸的氧化,蛋白N端的乙酰化,脱酰胺化(NQ)。定量方法设置为TMT-11plex,蛋白鉴定、PSM鉴定的FDR都设置为1%。
(2)实验实施流程
以EASL-ACLF为诊断标准,选取ACLF-2级病人,将病人分为两组,一组为血小板功能极差,临床表现为血栓弹力图检测中,ADP抑制率(ADP inhibition rate)大于70%的且28天因肝衰竭病死的患者,一组为血小板功能正常,临床表现为血栓弹力图检测中,ADP抑制率小于30%的且28天内存活的患者;收集10例确诊慢加急性肝衰竭患者高ADP抑制率的患者、10例确诊慢加急性肝衰竭患者低ADP抑制率的患者的外周血样品,各份血液样品收集在EDTA抗凝血管中,体积为5ml左右,5000rpm离心10min,吸取上层澄清的血浆。通过蛋白质组学技术检测血浆中的各类蛋白浓度,蛋白质组学技术由景杰生物提供,运用非靶标蛋白组学方法,通过质谱分析、差异表达蛋白功能富集分析最终得到多个非特异蛋白;其次扩大样本量以后,其中高抑制率患者42例,低抑制率患者45例,运用靶标蛋白组学方法对非特异蛋白进行定量确认,最终确定特异性差异蛋白,其中由于上质谱前样本处理步骤较为繁琐,样本消耗掉较多,质谱仪本身仪器性能目前还不能检测到上机的所有肽段等,从而导致不能一次检测到每个样本里面的所有的蛋白;因此,在检测COL1A1/LGALS1/APP/CCN2这四个蛋白时,在28天内死亡的这一组患者中,样本量为32例,在28天内存活的这一组患者中,样本量为36例。MELD、总胆红素及肌酐浓度由病人病史资料得到。
最终,CHGB、COL1A2、ENTPD1、ENTPD3、ENTPD8、LAMB1、LAMC1、LRP1、LYVE1、NID1、TNC、SPARCL1、COMP、FGFR1、GRN、LAMA2、MMP2、THBS4这几个蛋白的样本量是28天内存活组和28天内死亡组各10例;CD44、FSTL3、DDX19B、CTSL、CSPG4、VCAM1、AXL、PECAM1这几个蛋白的样本量是28天内死亡组42例,28天内存活组45例;COL1A1、LGALS1、APP、CCN2这几个蛋白的样本量是28天内死亡组32例,28天内存活组36例。
利用GraphPad Prism 6.0软件进行ROC曲线绘制,对组间比较用Mann-Whitney检验,对相关性分析用Spearman相关性检验。P<0.05认为有显著差异。
(3)实验结果及分析
具体纳排标准见表1,入组流程见图1。
表1纳排标准
蛋白质筛选结果见图2~图7,其中从图2中左图代表探索过程的三个阶段,第一阶段为非靶标蛋白质组学(TMT)探索阶段,第二阶段为靶标蛋白质组学(PRM)验证阶段,第三阶段为实验验证阶段,包括但不局限于酶联免疫吸附试验(ELISA)或者免疫印迹实验(WB);中图代表每个阶段需要的样本数量;右图代表蛋白质筛选的流程,本次实验中,在TMT过程中定量到了1271个蛋白,后再内部验证阶段选择了50个蛋白作为候选指标,然后扩大样本量进行检测后,最终选定了30个差异蛋白。
图3为标志物(CHGB、COL1A2、ENTPD1、ENTPD3、ENTPD8、LAMB1)与肝衰竭患者病情发展情况的关系。其中图3中A图为CHGB与肝衰竭患者病情发展情况的关系。图3中B图为COL1A2与肝衰竭患者病情发展情况的关系。图3中C图为ENTPD1与肝衰竭患者病情发展情况的关系。图3中D图为ENTPD3与肝衰竭患者病情发展情况的关系。图3中E图为ENTPD8与肝衰竭患者病情发展情况的关系。图3中F图为LAMB1与肝衰竭患者病情发展情况的关系。
图4为标志物(LAMC1、LRP1、LYVE1、NID1、TNC、SPARCL1)与肝衰竭患者病情发展情况的关系。其中图4中A图为LAMC1与肝衰竭患者病情发展情况的关系。图4中B图为LRP1与肝衰竭患者病情发展情况的关系。图4中C图为LYVE1与肝衰竭患者病情发展情况的关系。图4中D图为NID1与肝衰竭患者病情发展情况的关系。图4中E图为TNC与肝衰竭患者病情发展情况的关系。图4中F图为SPARCL1与肝衰竭患者病情发展情况的关系。
图5为标志物(COMP、FGFR1、GRN、LAMA2、MMP2、THBS4)与肝衰竭患者病情发展情况的关系。其中图5中A图为COMP与肝衰竭患者病情发展情况的关系。图5中B图为FGFR1与肝衰竭患者病情发展情况的关系。图5中C图为GRN与肝衰竭患者病情发展情况的关系。图5中D图为LAMA2与肝衰竭患者病情发展情况的关系。图5中E图为MMP2与肝衰竭患者病情发展情况的关系。图5中F图为THBS4与肝衰竭患者病情发展情况的关系。
图6为标志物(CD44、COL1A1、FSTL3、DDX19B、LGALS1、APP)与肝衰竭患者病情发展情况的关系。其中图6中A图为CD44与肝衰竭患者病情发展情况的关系。图6中B图为COL1A1与肝衰竭患者病情发展情况的关系。图6中C图为FSTL3与肝衰竭患者病情发展情况的关系。图6中D图为DDX19B与肝衰竭患者病情发展情况的关系。图6中E图为LGALS1与肝衰竭患者病情发展情况的关系。图6中F图为APP与肝衰竭患者病情发展情况的关系。
图7为标志物(CTSL、CSPG4、VCAM1、CCN2、AXL、PECAM1)与肝衰竭患者病情发展情况的关系。其中图7中A图为CTSL与肝衰竭患者病情发展情况的关系。图7中B图为CSPG4与肝衰竭患者病情发展情况的关系。图7中C图为VCAM1与肝衰竭患者病情发展情况的关系。图7中D图为CCN2与肝衰竭患者病情发展情况的关系。图7中E图为AXL与肝衰竭患者病情发展情况的关系。图7中F图为PECAM1与肝衰竭患者病情发展情况的关系。
p值越小,差异越明显。由图3~图7可知,慢加急性肝衰竭患者血浆中高ADP抑制率患者中CHGB、COL1A2、ENTPD1、ENTPD3、ENTPD8、LAMB1、LAMC1、LRP1、LYVE1、NID1、SPARCL1、TNC、COMP、FGFR1、GRN、LAMA2、MMP2、THBS4、CD44、COL1A1、FSTL3、DDX19B、LGALS1、APP、CTSL、CSPG4、VCAM1、CCN2、AXL、PECAM1这30种蛋白水平显著高于低ADP抑制率的患者(P<0.01),提示血清中上述30种蛋白的水平可以作为血浆标志物,用于监测慢加急性肝衰竭患者病情进展和预后。
血浆中CHGB、COL1A2、ENTPD1、ENTPD3、ENTPD8、LAMB1、LAMC1、LRP1、LYVE1、NID1、SPARCL1、TNC、COMP、FGFR1、GRN、LAMA2、MMP2、THBS4、CD44、COL1A1、FSTL3、DDX19B、LGALS1、APP、CTSL、CSPG4、VCAM1、CCN2、AXL、PECAM1在慢加急性肝衰竭患者血浆中高ADP抑制率组中28天生存和死亡患者比较中有显著差异(P<0.01)。
进一步的,提供了30个蛋白的ROC曲线下面积(areaunder ROC:AUROC),结果见图8~图12。
图8为标志物(CHGB、COL1A2、ENTPD1、ENTPD3、ENTPD8、LAMB1)的ROC曲线图。其中图8中A图为CHGB的ROC曲线图。图8中B图为COL1A2的ROC曲线图。
图8中C图为ENTPD1的ROC曲线图。图8中D图为ENTPD3的ROC曲线图。图8中E图为ENTPD8的ROC曲线图。图8中F图为LAMB1的ROC曲线图。
图9为标志物(LAMC1、LRP1、LYVE1、NID1、TNC、SPARCL1)的ROC曲线图。
其中图9中A图为LAMC1的ROC曲线图。图9中B图为LRP1的ROC曲线图。图9中C图为LYVE1的ROC曲线图。图9中D图为NID1的ROC曲线图。图9中E图为TNC的ROC曲线图。图9中F图为SPARCL1的ROC曲线图。
图10为标志物(COMP、FGFR1、GRN、LAMA2、MMP2、THBS4)的ROC曲线图。
其中图10中A图为COMP的ROC曲线图。图10中B图为FGFR1的ROC曲线图。图10中C图为GRN的ROC曲线图。图10中D图为LAMA2的ROC曲线图。图10中E图为MMP2的ROC曲线图。图10中F图为THBS4的ROC曲线图。
图11为标志物(CD44、COL1A1、FSTL3、DDX19B、LGALS1、APP)的ROC曲线图。
其中图11中A图为CD44的ROC曲线图。图11中B图为COL1A1的ROC曲线图。图11中C图为FSTL3的ROC曲线图。图11中D图为DDX19B的ROC曲线图。图11中E图为LGALS1的ROC曲线图。图11中F图为APP的ROC曲线图。
图12为标志物(CTSL、CSPG4、VCAM1、CCN2、AXL、PECAM1)的ROC曲线图。
其中图12中A图为CTSL的ROC曲线图。图12中B图为CSPG4的ROC曲线图。图12中C图为VCAM1的ROC曲线图。图12中D图为CCN2的ROC曲线图。图12中E图为AXL的ROC曲线图。图12中F图为PECAM1的ROC曲线图。
ROC曲线是100%-特异度为横轴,敏感度为纵轴所组成的坐标图;AUROC代表曲线下面积大小,面积越大,诊断能力越强;灵敏度,又称真阳性率,即实际死亡,并且按照该诊断试验的标准被正确地判为死亡的百分比。它反映了诊断试验发现病人的能力。特异度,又称真阴性率,即实际没死亡,同时被诊断试验正确地判为没死亡的百分比。
从图8~图12可知,标志物CHGB的灵敏度为70%,特异度为90%;标志物COL1A2的灵敏度为60%,特异度为90%;标志物ENTPD1的灵敏度为80%,特异度为90%;标志物ENTPD3的灵敏度为80%,特异度为90%;标志物ENTPD8的灵敏度为90%,特异度为90%;标志物LAMB1的灵敏度为70%,特异度为90%;标志物LAMC1的灵敏度为80%,特异度为90%;标志物LRP1的灵敏度为50%,特异度为90%;标志物LYVE1的灵敏度为80%,特异度为90%;标志物NID1的灵敏度为100%,特异度为60%;标志物TNC的灵敏度为80%,特异度为70%;标志物SPARCL1的灵敏度为80%,特异度为90%;标志物COMP的灵敏度为50%,特异度为90%;标志物FGFR1的灵敏度为60%,特异度为90%;标志物GRN的灵敏度为100%,特异度为70%;标志物LAMA2的灵敏度为70%,特异度为80%;标志物MMP2的灵敏度为60%,特异度为90%;标志物THBS4的灵敏度为50%,特异度为90%;标志物CD44的灵敏度为71%,特异度为79%;标志物COL1A1的灵敏度为64%,特异度为91%;标志物FSTL3的灵敏度为7%,特异度为98%;标志物DDX19B的灵敏度为31%,特异度为95.2%;标志物LGALS1的灵敏度为16.7%,特异度为93.8%;标志物APP的灵敏度为64%,特异度为78.1%;标志物CTSL的灵敏度为18%,特异度为85.7%;标志物CSPG4的灵敏度为18%,特异度为90.5%;标志物VCAM1的灵敏度为33.1%,特异度为81%;标志物CCN2的灵敏度为44.4%,特异度为84.4%;标志物AXL的灵敏度为33.3%,特异度为81%;标志物PECAM1的灵敏度为42.2%,特异度为71.4%。
因此,血浆中CHGB、COL1A2、ENTPD1、ENTPD3、ENTPD8、LAMB1、LAMC1、LRP1、LYVE1、NID1、SPARCL1、TNC、COMP、FGFR1、GRN、LAMA2、MMP2、THBS4、CD44、COL1A1、FSTL3、DDX19B、LGALS1、APP、CTSL、CSPG4、VCAM1、CCN2、AXL、PECAM1的浓度差异可预测ACLF-2级患者的短期预后。
上述具体实施方式对本发明作了详细说明,但是本发明不限于上述实施例,在所属技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。此外,在不冲突的情况下,本发明的实施例及实施例中的特征可以相互组合。
Claims (6)
1.定量检测标志物ENTPD1的试剂在制备慢加急性肝功能衰竭诊断和/或预后评估产品中的应用,其特征在于,所述慢加急性肝功能衰竭为ACLF-2级,所述预后为ACLF-2级病人中短期预后。
2.根据权利要求1所述的应用,其特征在于,所述定量检测标志物的试剂为检测标志物基因表达水平的试剂。
3.根据权利要求1所述的应用,其特征在于,所述定量检测标志物的试剂为检测标志物蛋白质表达水平的试剂。
4.根据权利要求3所述的应用,其特征在于,所述定量检测标志物蛋白质表达水平的试剂包括免疫组化、Western-Blot、蛋白免疫印迹、酶联免疫吸附(ELISA)、流式细胞术、紫外分光光度法-近红外光谱法、高效液相色谱法、比色法或质谱鉴定的方法检测标志物蛋白表达水平的试剂。
5.根据权利要求4所述的应用,其特征在于,所述定量检测标志物蛋白质表达水平的试剂为质谱鉴定的方法检测标志物蛋白表达水平的试剂。
6.根据权利要求5所述的应用,其特征在于,所述标志物的表达水平上调预示预后较差。
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