CN116515982A - Ets2分子标志物在制备判断肝衰竭危险分层试剂的用途及试剂盒及应用 - Google Patents
Ets2分子标志物在制备判断肝衰竭危险分层试剂的用途及试剂盒及应用 Download PDFInfo
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Abstract
本发明公开了一种ETS2分子标志物在制备判断肝衰竭危险分层试剂的用途及试剂盒及应用。本发明通过临床样本转录组测序分析筛选得到ETS2基因,其表达水平与肝衰竭疾病的严重程度相关,并通过qRT‑PCR实验验证这一结果,公开了ETS2分子标志物在制备判断肝衰竭危险分层试剂的用途及检测肝衰竭和判断肝衰竭危险分层的试剂盒,试剂盒包含用于检测ETS2表达量的试剂。本发明发现ETS2在肝衰竭患者各类样本中呈特征性高表达,与疾病严重程度有很好的相关性,稳定可靠。ETS2mRNA检测试剂盒能够准确检测ETS2mRNA含量,有利于肝衰竭的早期诊断和治疗,具有重要临床意义。
Description
技术领域
本发明涉及临床医学、生物医学和医学检验技术领域,具体地说,是ETS2分子标志物在制备判断肝衰竭危险分层试剂的用途及试剂盒及应用。
背景技术
肝功能衰竭是各种原因导致肝功能严重损伤,伴有肝内和肝外(凝血,肾,脑,循环,呼吸)多器官衰竭的一种复杂的临床综合征。肝衰竭病情凶险,预后不佳,常规内科综合治疗病死率高达50%-90%,除肝移植外没有特效的治疗方法,严重威胁人类健康。早发现,早诊断,早治疗,对改善肝衰竭患者的预后,提高生存率具有重要作用。目前在临床上,肝衰竭的诊断主要基于传统的临床指标如胆红素、凝血功能指标INR等。然而,这些指标具有滞后性,患者往往已经出现明显临床症状,伴有肝细胞坏死和肝外器官衰竭。因此,寻找新的敏感度更高的特异性指标辅助肝衰竭诊断具有重要的临床价值。
mRNA动态检测是一种在无症状患者检测效率非常高的新型技术手段,可以辅助疾病的诊断和预后评估。其在心血管疾病、肿瘤,胃肠道疾病等多种类型的疾病中均展示了良好的应用价值。在器官受到感染或产生病变时,循环系统可以起到信息传递的作用,募集免疫细胞启动防御机制。通过检测外周血中白细胞mRNA的改变,可以同时监测机体不同组织、器官的疾病风险,作为组织活检的辅助手段应用前景广阔。
ETS2是ETS转录因子家族成员之一,参与了细胞增殖、分化、凋亡等一系列细胞功能。本课题组研究发现ETS2的mRNA表达水平与肝衰竭的发生、发展和转归密切相关。ETS2与肝衰竭患者固有免疫激活,适应性免疫抑制的机制相关,参与了巨噬细胞炎症等生物学功能。因此,ETS2可以作为一种早期诊断肝衰竭的指标,具有潜在的应用价值。
发明内容
本发明公开了ETS2分子标志物在制作判断肝衰竭危险分层试剂的用途及试剂盒及应用,第一目旨在提供一种ETS2分子的检测在检测肝衰竭疾病和判断肝衰竭危险分层中的应用,第二目的旨在提供一种用于检测ETS2 mRNA表达量的检测试剂盒。
为实现上述第一目的,本发明通过临床样本转录组测序分析筛选得到ETS2基因,其表达水平与肝衰竭疾病的严重程度相关,并通过qRT-PCR实验验证这一结果。
为实现上述第二目的,本发明采取的技术方案是:
本发明公开了ETS2分子标志物在制备判断肝衰竭危险分层试剂的用途。
作为进一步地改进,本发明用途包括用于区分肝衰竭人群中短期预后不佳的高危人群和低危人群。
作为进一步地改进,本发明所述的试剂通过检测细胞、组织或体液临床样本中ETS2的表达量,来判断肝衰竭危险分层。
作为进一步地改进,本发明所述的试剂通过测序、qRT-PCR的方法检测ETS2在细胞、组织或体液样本中的表达量。
本发明公开了一种检测肝衰竭和判断肝衰竭危险分层的试剂盒,试剂盒包含用于检测ETS2表达量的试剂。
作为进一步地改进,本发明所述的试剂为荧光定量PCR引物,如SEQ ID NO.1和SEQID NO.2所示的ETS2基因引物对,ETS2表达量检测阈值为1.35。
作为进一步地改进,本发明所述的试剂盒判断肝衰竭危险分层的步骤如下:1)试剂盒检测临床样本中ETS2分子表达量;2)当检测到的ETS2分子表达量大于1.35时,患者被预测为肝衰竭人群中短期预后不佳的高危人群。
作为进一步地改进,本发明检测试剂包含如下组分:RNA抽提试剂;RNA逆转录体系和实时荧光定量PCR反应体系;如SEQ ID NO.7和SEQ ID NO.8所示的内参GAPDH基因引物对和如SEQ ID NO.1和SEQ ID NO.2所示的ETS2基因引物对。
本发明还公开了一种用于判断肝衰竭危险分层试剂盒的应用方法,具体步骤为:
1)收集检测样本,提取RNA;
2)将RNA逆转录合成cDNA;
3)对cDNA进行实时荧光定量PCR扩增检测ETS2基因相对表达量,辅助肝衰竭的检测和危险分层。
作为进一步地改进,本发明所述的PCR扩增的条件为:将RNA样本1ng-100μg与逆转录酶0.01-10μL混合后进行反转录,所得产物cDNA与qPCR混合液0.01-100μL充分混匀后进行PCR扩增,疾病的检测样本来自肝衰竭患者或肝衰竭高危人群,检测细胞样本为外周血单个核细胞样本,组织样本为肝组织样本,体液样本为血液样本。
本发明优点在于:
1、本发明通过转录组mRNA测序分析联合qRT-PCR验证,发现ETS2在肝衰竭患者各类样本中呈特征性高表达,与疾病严重程度有很好的相关性,稳定可靠。
2、本发明的ETS2 mRNA检测试剂盒可采用外周血单个核细胞进行检测,样本容易获得,对患者创伤性小,可操作性强。
3、本发明的ETS2 mRNA检测试剂盒步骤简便,具有相关专业背景的工作人员都能轻松掌握该试剂盒的使用。
4、本发明的ETS2 mRNA检测试剂盒能够准确检测ETS2 mRNA含量,有利于肝衰竭的早期诊断和治疗,具有重要临床意义。
附图说明
图1是本发明实施案例1中肝衰竭组(n=50)、肝损伤组(n=19)和健康对照组(n=14)的外周血单个核细胞测序数据ETS2表达量对照图表;
图2是本发明实施案例1中肝衰竭I级(n=30)和肝衰竭II/III级(n=20)的肝衰竭患者外周血单个核细胞测序数据ETS2表达量的对照图表;
图3是本发明实施案例1中28天和90天不同预后的衰竭患者外周血单个核细胞测序数据ETS2表达量结果对照图,以及以ETS2表达量预测生死的AUROC曲线图;
图4是本发明实施案例2中28天和90天不同预后的衰竭患者外周血单个核细胞qRT-PCR验证ETS2相对表达量结果对照图,以及以ETS2表达量预测生死的AUROC曲线图。
图5是本发明实施案例2中肝衰竭不同转归(死亡、存活)患者ETS2mRNA外周血单个核细胞qRT-PCR数据的分布图,表明ETS2 mRNA表达水平大于1.35时,患者被预测为短期预后不佳的高危人群。
具体实施方式
下面结合具体实施方式,进一步阐述本发明。尽管为说明目的公开了本发明的实施例,但是本领域的技术人员可以理解:在不脱离本发明及所附权利要求的精神和范围内,各种替换、变化和修改都是可能的,因此,本发明的范围不局限于实施例和附图所公开的内容。
本发明实验方法包括:
1、基于肝衰竭多中心、前瞻性队列,前期收集慢加急性肝衰竭、慢性肝损伤(包括肝硬化、慢性乙型肝炎)和健康对照组患者外周血单个核细胞,通过高通量转录组测序结合生物信息学分析发现在肝衰竭患者中高表达,且与患者危险分层相关的mRNA——ETS2。
2、对慢加急性肝衰竭患者外周血单个核细胞的ETS2 mRNA表达水平进行qRT-PCR验证,证实ETS2 mRNA表达量对预测肝衰竭患者不良预后有良好的敏感新和特异性。
3、ETS2 mRNA检测试剂盒的制作工艺和操作流程主要是基于PCR技术。通过实验1和实验2步骤筛选出ETS2 mRNA作为检测肝衰竭患者疾病和判断肝衰竭患者危险分层的指标。所述ETS2扩增引物对序列如SEQ ID NO.1正义引物和SEQ ID NO.2反义引物所示的引物对,内参引物对如SEQ ID.NO.7正义引物和SEQ ID NO.8反义引物的内参引物对所示。
下面通过具体实施例对本发明的技术方案作进一步地说明:
实施例一
实验方法
一、外周血单个核细胞分离
1.抽取5mL患者全血,静置约30分钟,离心机常规预冷至4℃。
2.采用密度梯度离心法,在4℃,3500转/分钟条件下离心10分钟。分离血清血浆,用5mL无菌0.1MPBS缓冲液将血细胞沉淀充分重悬混匀。在新的离心管中加入5ml Ficoll-paque TM PLUS medium淋巴细胞分离液,缓慢将等体积的血细胞混合液铺在分离液上,离心使其自然形成分层。设置离心机温度20℃,转速为2500转/分钟,升速9,降速1,离心20分钟。
3.用1ml移液枪吸取中间白膜层细胞至新的离心管中,加入PBS缓冲液10mL,吹打混匀,4℃1400转/分钟离心4分钟后弃上清。该步骤重复3次。
4.吸干残留PBS缓冲液,加入1mL TRIzol细胞裂解液使细胞完全裂解。
二、总mRNA抽提
1.将PBMCs-TRIzol样本于4℃3000转/分钟离心2分钟后取出,加入200μL三氯甲烷,剧烈摇荡混匀后静置5分钟使其自然分层。
2.将上述样本于4℃12000g/min条件下离心15分钟,离心后管中液体分为三层,即上层为水相、中层为蛋白层、下层为有机层,RNA溶解于上层水相中,吸取上层水相约400μL左右,转移至另一清洁1.5mL离心管中。加入500μL异丙醇,剧烈摇荡混匀后静置5分钟。
3.将上述样本于4℃12000g/min条件下离心15分钟后取出,弃去上清。加入1mL75%乙醇溶液,上下颠倒2次,用手指拨弹试管底部使白色羽毛状沉淀物漂起。
4.将上述样本于4℃9000g/min条件下离心5分钟后取出,弃去上清。
5.将上述样本于4℃9000g/min条件下离心2分钟后取出,用移液枪吸干残留液体。
6.加入30μLDEPC水溶解mRNA。
三、mRNA测序
1.RNA质检:取2μLRNA溶液于Nanodrop仪器定量。
2.根据Nanodrop仪器定量结果,1.5%琼脂糖凝胶电泳检测。
3.依次进行dscDNA合成、末端补平、加A、加接头、PCR富集和Qubit定量
4.簇生成:稀释至10nM后取4μL10nM文库,加1μL2NNaOH,加15μLTris.Cl,混匀,室温放置5min;取6μL上述溶液,加994μL冷的杂交buffer;取140μL上述溶液,放在8连管中,置于cBot的template栏中;启动cBot仪器,开始簇生成。
5.IlluminaHiseq2500测序
图1是本发明实施案例1中肝衰竭组(n=50)、肝损伤组(n=19)和健康对照组(n=14)的外周血单个核细胞测序数据对照图表;结果筛选出具有特征性表达的mRNA,经分析发现ETS2 mRNA在肝衰竭、慢性肝损伤和健康对照组的外周血单个核细胞中呈特征性表达,肝衰竭组的ETS2 mRNA表达量显著高于其他各组。
图2是图2是本发明实施案例1中肝衰竭I级(n=30)和肝衰竭II/III级(n=20)的肝衰竭患者ETS2表达量的对照图表。经分析发现ETS2 mRNA表达量在肝衰竭II/III级级患者高表达。
图3是本发明实施案例1中28天和90天不同预后的衰竭患者外周血单个核细胞测序数据ETS2表达量结果对照图,以及以ETS2表达量预测生死的AUROC曲线图。经分析发现ETS2 mRNA表达量在28天和90天预后不良的患者中高表达,且预测效果较好。
实施例中的其中的细胞样本,也可以为组织或体液样本。
实施例二
实时荧光定量PCR(qRT-PCR)检测肝衰竭、肝衰竭高危人群、肝硬化、慢性乙型肝炎和健康对照组ETS2 mRNA表达情况。
试剂盒包含:ETS2扩增引物SEQ ID NO.1正义引物和SEQ ID NO.2反义引物所示引物对或SEQ ID NO.3正义引物和SEQ ID NO.4反义引物所示的引物对或SEQ ID NO.5正义引物和SEQ ID NO.6反义引物所示的引物对。根据各引物对序列的特异性、灵敏度、实验重复性、观察扩增曲线、观察溶解曲线等,筛选出SEQ ID NO.1前引物和SEQ ID NO.2后引物所示的引物对为ETS2最优扩增引物序列对。以SEQ ID NO.1正义引物和SEQ ID NO.2反义引物所示的用于扩增ETS2的引物对为具体实施例;SEQ ID.NO.7正义引物和SEQ ID NO.8反义引物所示的内参引物对、4×gDNA去除混合液、5×逆转录酶混合液、DEPC水、2×qPCR混合液、裂解液、75%乙醇、氯仿、DEPC水、异丙醇。
实验步骤
qRT-PCR采用两步法进行实验操作。同实施例一方法进行患者外周血单个核细胞分离、总RNA抽提,mRNA经浓度测试后进行下一步实验操作。
1.反转录:反转录采用诺唯赞公司的HiScript III RT SuperMix for qPCR(+gDNA wiper)试剂盒,按照试剂盒说明进行操作,得到cDNA。
2.取上述cDNA模板进行PCR扩增:PCR试剂采用诺唯赞公司Taq Pro UniversalSYBR qPCR Master Mix试剂,操作按照试剂盒说明。
3.所述的PCR扩增的条件为:将RNA样本1ng-100μg与逆转录酶0.01-10μL混合后进行反转录,所得产物cDNA与qPCR混合液0.01-100μL充分混匀后进行PCR扩增。
图4是本发明实施案例2中28天和90天不同预后的衰竭患者外周血单个核细胞qRT-PCR验证ETS2相对表达量结果对照图,以及以ETS2表达量预测生死的AUROC曲线图。证实ETS2 mRNA表达量在不良预后的肝衰竭患者中高表达,且预测效果较好。
图5是本发明实施案例2中肝衰竭不同转归(死亡、存活)患者ETS2 mRNA外周血单个核细胞qRT-PCR数据的分布图,表明ETS2 mRNA表达水平大于1.35时,患者被预测为短期预后不佳的高危人群。
上述实验结果表明:ETS2 mRNA在健康人群、慢性肝病人群和慢加急性肝衰竭患者中特异性表达,且对预测肝衰竭患者不良预后有良好的敏感新和特异性。通过检测ETS2的表达水平可预警预测肝衰竭高危患者,从而能帮助该类患者及时进行治疗,具有很好的临床应用前景。本发明开发的ETS2mRNA检测肝衰竭试剂盒能够提供准确的评估结果,且能够及时准确反映肝衰竭患者的疾病状态,便于临床医生及时诊断,并采取及时有效的治疗。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明原理的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
h ETS2-F(ETS2正义引物):CCCCTGTGGCTAACAGTTACA(SEQ ID NO.1);
h ETS2-R(ETS2反义引物):AGGTAGCTTTTAAGGCTTGACTC(SEQ ID NO.2)
h ETS2-F(ETS2正义引物):CTGGGCATTCCAAAGAACCC(SEQ ID NO.3);
h ETS2-R(ETS2反义引物):CCAGACTGAACTCATTGGTGG(SEQ ID NO.4)
h ETS2-F(ETS2正义引物):CAGTCTGGTGAACGTGAATCTG(SEQ ID NO.5);
h ETS2-R(ETS2反义引物):CGGAGGTGAGGTGTGAATTTT(SEQ ID NO.6)
h GAPDH-F(GAPDH正义引物):GGAGCGAGATCCCTCCAAAAT(SEQ ID NO.7);
h GAPDH-R(GAPDH反义引物):GGCTGTTGTCATACTTCTCATGG(SEQ ID NO.8)
Claims (10)
1.ETS2分子标志物在制备判断肝衰竭危险分层试剂的用途。
2.根据权利要求1所述的用途,其特征在于,所述用途包括用于区分肝衰竭人群中短期预后不佳的高危人群和低危人群。
3.根据权利要求1所述的用途,其特征在于,所述的试剂通过检测细胞、组织或体液临床样本中ETS2的表达量,来判断肝衰竭危险分层。
4.根据权利要求2和3所述的用途,其特征在于,所述试剂通过测序、qRT-PCR的方法检测ETS2在细胞、组织或体液样本中的表达量。
5.一种检测肝衰竭和判断肝衰竭危险分层的试剂盒,其特征在于,所述试剂盒包含用于检测ETS2表达量的试剂。
6.根据权利要求5所述的试剂盒,其特征在于,所述试剂为荧光定量PCR引物,如SEQ IDNO.1和SEQ ID NO.2所示的ETS2基因引物对,ETS2表达量检测阈值为1.35。
7.根据权利要求5所述的试剂盒,其特征在于,所述的试剂盒判断肝衰竭危险分层的步骤如下:1)试剂盒检测临床样本中ETS2分子表达量;2)当检测到的ETS2分子表达量大于1.35时,患者被预测为肝衰竭人群中短期预后不佳的高危人群。
8.根据权利要求5所述的试剂盒,其特征在于,所述检测试剂包含如下组分:RNA抽提试剂;RNA逆转录体系和实时荧光定量PCR反应体系;如SEQ ID NO.9和SEQ ID NO.10所示的内参GAPDH基因引物对和如SEQ ID NO.1和SEQ ID NO.2所示的ETS2基因引物对。
9.一种用于判断肝衰竭危险分层试剂盒的应用方法,其特征在于,具体步骤为:
1)收集检测样本,提取RNA;
2)将RNA逆转录合成cDNA;
3)对cDNA进行实时荧光定量PCR扩增检测ETS2基因相对表达量,辅助肝衰竭的危险分层。
10.根据权利要求9所述的应用方法,其特征在于,所述的PCR扩增的条件为:将RNA样本1ng-100μg与逆转录酶0.01-10μL混合后进行反转录,所得产物cDNA与qPCR混合液0.01-100μL充分混匀后进行PCR扩增,所述疾病的检测样本来自肝衰竭患者或肝衰竭高危人群,所述检测细胞样本为外周血单个核细胞样本。
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