CN114958859A - 一种用于诊断急性呼吸窘迫综合征的circRNA标志物及诊断试剂 - Google Patents
一种用于诊断急性呼吸窘迫综合征的circRNA标志物及诊断试剂 Download PDFInfo
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Abstract
本发明提供了一种circRNA,其核苷酸序列如SEQ ID NO.1所示。本发明提供了一种用于诊断急性呼吸窘迫综合征的circRNA标志物,所述circRNA标志物为hsa_circRNA_042882,所述的hsa_circRNA_042882核苷酸序列如SEQ ID NO.1所示。本发明还提供了circRNA标志物hsa_circRNA_042882在制备作为急性呼吸窘迫综合征检测标志物中的应用。本发明还提供了检测circRNA标志物hsa_circRNA_042882的试剂在制备用于诊断急性呼吸窘迫综合征的试剂盒中的应用。本发明发现ARDS的血浆和肺泡灌洗液外泌体hsa_circRNA_042882,可用来区分ARDS病人和正常人。
Description
技术领域
本发明属于生物检测领域,涉及急性呼吸窘迫综合征,具体来说是一种用于诊断急性呼吸窘迫综合征的circRNA标志物及诊断试剂。
背景技术
急性肺损伤(Acute Lung Injury,ALI)及其更为严重阶段的急性呼吸窘迫综合征(Acute Respiratory Distress Syndrome,ARDS)是由多种病因引起的肺部急性弥漫性炎性损伤,是全球范围内严重威胁人类健康的高病死率急危重症;近期一项覆盖全球50个国家的大型流行病学调查数据显示,ARDS在ICU的发病率超过10%,死亡率高达34.9-46.1%。目前针对ALI/ARDS的治疗仍然集中在控制诱发ARDS的基础疾病及旨在改善气体交换和预防并发症为主的支持治疗。迄今尚无任何药物可以改善其预后,使ALI/ARDS的治疗面临巨大挑战。
外泌体介导的信息传递是细胞间通讯的新机制。外泌体是由多种细胞分泌的大小介于40-150nm的膜性囊泡,内含包括核酸(如circRNA、LncRNA、miRNA、DNA)、脂质、蛋白信号分子等多种成分,这些分子主要分为结构分子和亲代细胞特异性分子两大类。结构分子通常作为外泌体鉴定的分子标志物;亲代细胞特异性分子与外泌体来源的母细胞密切相关,在维持外泌体的独特生物功能方面发挥重要作用。不同细胞衍生的外泌体通过携带细胞活性成分到靶细胞调控其功能。
circRNA是具有反向剪接位点的闭合长链非编码RNA,近期研究表明circRNA在疾病进展中发挥重要的基因调节功能。与线性RNA分子相比,circRNA特有的闭合环状结构使其不易被RNA外切酶剪切,因而高度稳定,便于样本的保存;其次circRNA可以通过外泌体进入多种体液和分泌液,便于取样;circRNA长度多在200-400碱基之间,可用qPCR定量检测,可操作性强。因此,将circRNA用作疾病诊断的生物分子标志物具有明确的优势,近期在肝癌、胃癌等恶性消化系统肿瘤中相继发现了具有早期诊断价值的circRNA,但是在ARDS领域尚未见circRNA相关报道。
发明内容
针对现有技术中的上述技术问题,本发明提供了一种用于诊断急性呼吸窘迫综合征的circRNA标志物及诊断试剂,所述的这种用于诊断急性呼吸窘迫综合征的circRNA标志物及诊断试剂要解决现有技术中诊断急性呼吸窘迫综合征困难的技术问题。
本发明提供了一种circRNA,其核苷酸序列如SEQ ID NO.1所示。
本发明提供了一种用于诊断急性呼吸窘迫综合征的circRNA标志物,所述circRNA标志物为hsa_circRNA_042882,所述的hsa_circRNA_042882核苷酸序列如SEQ ID NO.1所示。
本发明还提供了circRNA标志物hsa_circRNA_042882在制备作为急性呼吸窘迫综合征检测标志物中的应用,所述的hsa_circRNA_042882核苷酸序列如SEQ ID NO.1所示。
本发明还提供了检测circRNA标志物hsa_circRNA_042882的试剂在制备用于诊断急性呼吸窘迫综合征的试剂盒中的应用,所述的hsa_circRNA_042882核苷酸序列如SEQ IDNO.1所示。
本发明还提供了一种试剂盒,包括检测circRNA标志物hsa_circRNA_042882的试剂,所述的hsa_circRNA_042882的核苷酸序列如SEQ ID NO.1所示。
进一步的,所述的试剂包括SEQ ID NO.2~23所示的引物。
本发明采用circRNA芯片检测以获取异常表达的肺泡灌洗液外泌体circRNA表达谱,并应用qPCR的方法进行验证。本发明发现ARDS的肺泡灌洗液外泌体hsa_circRNA_042882,可用来区分ARDS病人和正常人。扩大样本量验证hsa_circRNA_042882在患者BALF外泌体中的表达差异,发现在ARDS急性期,hsa_circRNA_042882在肺泡灌洗液外泌体的表达明显减低,ROC曲线下的面积分别是0.8050。
本发明发现血浆hsa_circRNA_042882,可用来区分ARDS病人和正常人。本发明发现在ARDS急性期,hsa_circRNA_042882在血浆中的表达明显减低,ROC曲线下的面积是0.835,敏感性为76.32%,特异性为78.95%。血浆hsa_circRNA_042882是对ARDS的诊断具有较高的特异性和敏感度。
本发明和已有技术相比,其技术进步是显著的。本发明为ARDS的病情判断提供参考依据。本发明所提供的circRNA生物标志物对于人ARDS具有很高的灵敏度和特异性,可以作为新型的生物标志物用于ARDS的检测。本发明为临床医生快速判断ARDS患者的病情,为提高这类患者的临床治疗效果奠定基础,为发现具有潜在治疗价值的新型小分子药物靶标提供帮助,具有较好的应用价值和前景。
附图说明
图1:肺泡灌洗液外泌体分离流程图。
图2、肺泡灌洗液外泌体的TEM电镜图像。
图3、肺泡灌洗液外泌体NTA粒径分布图。
图4、外泌体标志蛋白Westernblot分析。
图5、ARDS肺泡灌洗液外泌体差异表达circRNA聚类分析。
图6、芯片筛选的差异表达显著的环状RNA的qPCR验证。
图7、hsa_circRNA_042882在正常人和ARDS患者肺泡灌洗液外泌体中的表达差异和受试者工作曲线。
图8、本发明与ARDS相关的血浆circRNA标志物检测方法的实验流程图。
图9、hsa_circRNA_042882在正常人和ARDS患者血浆中的表达差异(A)和受试者工作曲线(B)。
具体实施方式
实施例1
1.1患者样本收集
选取2020年5月至2021年5月期间在上海东方医院呼吸重症监护室(RICU)住院的重症肺炎致ARDS患者作为病例组。这些患者根据2012年发表的柏林定义诊断为ARDS,同时招募健康志愿者和诊断为肺部小结节的体检者作为对照组,年龄18-75岁。排除所有无法耐受支气管镜检查或存在支气管镜检查禁忌症的受试者。ARDS患者在诊断后48h内进行支气管镜肺泡灌洗。
1.2肺泡灌洗液收集
参照《肺部感染性疾病支气管肺泡灌洗病原体检测中国专家共识》(2017年版)及《支气管肺泡灌洗细胞学在间质性肺病的临床应用指南》(ATS 2012年版)要求采集支气管肺泡盥洗液(Bronchoalveolar Lavage Fluid,BALF)。所有患者排除禁忌后在静脉复合麻醉或2%利多卡因表面麻醉下行支气管镜检查,选择目标段支气管分次注入等量生理盐水后负压抽吸采集盥洗液20ml-80℃保存备用。
1.3肺泡灌洗液外泌体的分离
用超速离心法分离BALF外泌体。取BALF 20ml 500g离心5min,去除细胞。将上清液转移至新离心管2000g离心10分钟,10,000g离心30分钟,用0.22um滤膜过滤大囊泡,取1ml上清液100,000g离心70min取沉淀。PBS 100ul重悬外泌体。(肺泡灌洗液外泌体的分离流程见图1)
1.4外泌体的鉴定
使用Hitachi 7650透射电子显微镜(TEM)来鉴定分离的外泌体,应用纳米颗粒跟踪分析(NanoSight NS300)前述外泌体的粒径和浓度。用RIPA裂解肺泡灌洗液外泌体分离蛋白。BCA法对蛋白进行定量。SDS-PAGE跑胶,5%BSA封闭,转移至PVDF膜,一抗(CD9#13403,CD63ab134045,CD86#91882)4℃孵育过夜。辣根过氧化物酶HRP标记的二抗37℃孵育1h。应用ECL试剂盒进行检测。IMAGE J软件进行图像分析。
1.5外泌体总RNA的提取
利用Trizol LS从肺泡灌洗液外泌体中抽提总RNA,用Nanodrop对RNA进行定量。
1.6CircRNA芯片高通量筛选
用RNase去除线性RNA,以富集环RNA。circRNAs经过扩增并转录成cy3标记的cRNA(Arraystar Super RNA labeling Kit,Arraystar Inc.美国)。然后使用Arraystar HumancircRNA microarray进行杂交,并使用Agilent Scanner G2505C(Jamul,CA,USA)进行数据提取。
1.7逆转录反应
按照下述的逆转录反应组分进行逆转录反应
上述混合液在65℃水浴5min,冰上放置2min,短暂离心后,在下述的反应体系中继续反应。
按照上述反应体系混合后于37℃,恒温1分钟;移液枪轻轻吸打几次混合均匀,50℃温育60分钟,70℃温育15分钟使酶失活。将逆转录的cDNA置冰浴待用或-20℃保存。
1.8定量PCR扩增
定量PCR所使用的CircRNA引物序列见下表1:按照下述的反应体系实施定量PCR反应
将上述384-PCR板置于Realtime PCR仪上进行PCR反应。所有的指标均按以下程序进行:应用-ViiA 7Real-time PCR System,95℃,10min;40个PCR循环(95℃,10秒;60℃,60秒,收集荧光)。为了建立PCR产物的熔解曲线,扩增反应结束后,按(95℃,10秒;60℃,60秒;95℃,15秒);并从60℃缓慢加热到99℃(仪器自动进行-Ramp Rate为0.05℃/秒)。
表1:定量PCR使用的CircRNA引物序列
2.9结果分析
使用公式2-△△CT计算circRNA表达的差异变化,student t-test计算p value,p值<0.05认为有统计学差异。
实验结果:
1、外泌体鉴定
透射电镜观察提取的外泌体:
TEM观察细胞外囊泡形态,外泌体呈茶盘状结构,与外泌体形态特征一致。(肺泡灌洗液外泌体TEM电镜图像见图2)
外泌体粒径分析:
NTA分析显示,分离的BALF外体的粒径分布在50至150nm之间(外泌体粒径分析结果详见图3)。
Western blot鉴定外泌体特异性分子标志物
此外,利用western blotting通过表达CD9和TSG101鉴定了这些细胞外囊泡的性质,结果也证明了这些细胞外囊泡是外来体。随后,用抗CD86抗体检查了这些外来体的可能细胞来源,结果表明肺泡巨噬细胞可能是这些外来体的主要来源(Western blot鉴定外泌体特异性分子标志物结果详见图4)。
2、肺泡灌洗液外泌体circRNA芯片结果
circRNA芯片共检测到13,228个circRNA,其中629个circRNA在ARDS患者和健康受试者的BALF外体中差异表达(fold change>2,p<0.05),其中430个上调,199个下调。聚类热图均直观地显示了这些差异表达的circRNAs(circRNA芯片聚类图结果详见图5),红色表示高表达,绿色表示低表达。随后根据差异倍数>4、p值<0.005、原始信号值>1000等标准,筛选出前9位环状RNA进行RT-qPCR验证(芯片筛选的差异表达显著的环状RNA的qPCR验证结果详见图6),验证结果显示有7个circRNA指标与芯片结果相符。
3、肺泡灌洗液外泌体circ042882生物标志物的鉴定和检测
对筛选出在病例及对照组BALF外泌体中表达差异显著的环状RNA circ042882进一步扩大样本量进行验证。
SEQ ID NO.1:CircRNA序列
>hsa_circRNA_042882
cttccaataaaaacaggacagcagaacacacataccaaagtcagtactgagcacaacaaggaatgtctaatcaatatttccaaatacaagttttctttggttataagcggcctcactactattttaaagaatgttaacaatatgagaatatttggagaagctgctgaaaaaaatttatatctctctcagttgattatattggatacactggaaaaatgtcttgctgggcaaccaaaggacacaatgagattagatgaaacgatgctggtcaaacagttgctgccagaaatctgccattttcttcacacctgtcgtgaaggaaaccagcatgcagctgaacttcggaattctgcctctggggttttattttctctcagctgcaacaacttcaatgcagtctttagtcgcatttctaccaggttacaggaattaactgtttgttcagaagacaatgttgatgttcatgatatagaattgttacagtatatcaatgtggattgtgcaaaattaaaacgactcctgaaggaaacagcatttaaatttaaagccctaaagaaggttgcgcagttagcagttataaatagcctggaaaaggcattttggaactgggtagaaaattatccagatgaatttacaaaactgtaccagatcccacagactgatatggctgaatgtgcagaaaagctatttgacttggtggatggttttgctgaaagcaccaaacgtaaagcagcagtttggccactacaaatcattctccttatcttgtgtccagaaataatccaggatatatccaaagacgtggttgatgaaaacaacatgaataagaagttatttctggacagtctacgaaaagctcttgctggccatggaggaagtaggcagctgacagaaagtgctgcaattgcctgtgtcaaactgtgtaaagcaagtacttacatcaattgggaagataactctgtcattttcctacttgttcagtccatggtggttgatcttaagaacctgctttttaatccaagtaagccattctcaagaggcagtcagcctgcagatgtggatctaatgattgactgccttgtttcttgctttcgtataagccctcacaacaaccaacactttaagatctgcctggctcagaattcaccttctacatttcactatgtgctggtaaattcactccatcgaatcatcaccaattccgcattggattggtggcctaagattgatgctgtgtattgtcactcggttgaacttcgaaatatgtttggtgaaacacttcataaagcagtgcaaggttgtggagcacacccagcaatacgaatggcaccg
应用RT-qPCR方法分别在ARDS病例组和对照组肺泡灌洗液外泌体中检测hsa_circRNA_042882的表达差异(ARDS患者的人口学特征详见表2),结果显示hsa_circRNA_042882在ARDS患者肺泡灌洗液外泌体中的表达明显低于正常组,ROC曲线下面积为0.8050,诊断ARDS的敏感度是90%,特异度是65%(p<0.001)(circ042882在正常人和ARDS患者肺泡灌洗液外泌体中的表达差异和受试者工作曲线结果详见图7)。
表2 急性呼吸窘迫综合征(ARDS)患者的人口学和临床特征。
实施例2
2.1实验流程及样本收集
本发明与ARDS相关的血浆circRNA标志物检测方法的实验流程图详见图8。研究样本收集参考实施例1。
2.2血浆的采集与制备
用EDTA抗凝管收集ARDS及正常人的全血3mL。采血后立即颠倒5次使得抗凝剂和血液充分混匀;血样采集后室温放置30min后,2000rpm离心10min,上清液即为血浆;保存于-80℃冰箱。
2.3血浆circRNA的提取
利用Invitrogen公司的Trizol LS从中抽提血浆总RNA,洗脱后-80℃保存备用。RNA的纯度和浓度用Nanodrop-1000进行检测。
2.4逆转录反应
参照实施例1的逆转录反应体系进行逆转录反应,上述混合液在65℃水浴5min,冰上放置2min,短暂离心后,继续参照实施例1中的反应体系中继续反应。
按照上述反应体系混合后于37℃,恒温1分钟;移液枪轻轻吸打几次混合均匀,50℃温育60分钟,70℃温育15分钟使酶失活。将逆转录的cDNA置冰浴待用或-20℃保存。
2.5定量PCR扩增
参照实施例1的反应体系实施定量PCR反应,将上述384-PCR板置于Realtime PCR仪上进行PCR反应。所有的指标均按以下程序进行:应用-ViiA 7Real-time PCR System,95℃,10min;40个PCR循环(95℃,10秒;60℃,60秒,收集荧光)。
为了建立PCR产物的熔解曲线,扩增反应结束后,按(95℃,10秒;60℃,60秒;95℃,15秒);并从60℃缓慢加热到99℃(仪器自动进行-Ramp Rate为0.05℃/秒)。定量PCR所使用的CircRNA引物序列参照实施例1中表1:
结果分析:
使用公式2-△△CT计算circRNA表达的差异变化,student t-test计算p value,p值<0.05认为有统计学差异。
实验结果:
环状RNAhsa_circRNA_042882生物标志物的鉴定和检测:在病例及对照组血浆中检测hsa_circRNA_042882的表达差异,应用RT-qPCR方法分别在ARDS病例组和对照组血浆中检测hsa_circRNA_042882的表达差异(ARDS患者的人口学特征参见实施例1中表2),结果显示hsa_circRNA_042882在ARDS患者血浆中的表达明显低于正常组,ROC曲线下的面积是0.835,敏感性为76.32%,特异性为78.95%(hsa_circRNA_042882在正常人和ARDS患者血浆中的表达差异和受试者工作曲线结果详见图9,p<0.001)。
序列表
<110> 上海市东方医院(同济大学附属东方医院)
<120> 一种用于诊断急性呼吸窘迫综合征的circRNA标志物及诊断试剂
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attttaaaga atgttaacaa tatgagaata tttggagaag ctgctgaaaa aaatttatat 180
ctctctcagt tgattatatt ggatacactg gaaaaatgtc ttgctgggca accaaaggac 240
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tggaactggg tagaaaatta tccagatgaa tttacaaaac tgtaccagat cccacagact 660
gatatggctg aatgtgcaga aaagctattt gacttggtgg atggttttgc tgaaagcacc 720
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aattcacctt ctacatttca ctatgtgctg gtaaattcac tccatcgaat catcaccaat 1200
tccgcattgg attggtggcc taagattgat gctgtgtatt gtcactcggt tgaacttcga 1260
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Claims (7)
1.一种circRNA,其特征在于,其核苷酸序列如SEQ ID NO.1所示。
2.一种用于诊断急性呼吸窘迫综合征的circRNA标志物,其特征在于,所述circRNA标志物为hsa_circRNA_042882,所述的hsa_circRNA_042882核苷酸序列如SEQ ID NO.1所示。
3.circRNA标志物hsa_circRNA_042882在制备作为急性呼吸窘迫综合征检测标志物中的应用,所述的hsa_circRNA_042882核苷酸序列如SEQ ID NO.1所示。
4.检测circRNA标志物hsa_circRNA_042882的试剂在制备用于诊断急性呼吸窘迫综合征的试剂盒中的应用,所述的hsa_circRNA_042882核苷酸序列如SEQ ID NO.1所示。
5.根据权利要求4所述的应用,其特征在于,所述的试剂包括SEQ ID NO.2~23所示的引物。
6.一种试剂盒,其特征在于,包括检测circRNA标志物hsa_circRNA_042882的试剂,所述的hsa_circRNA_042882的核苷酸序列如SEQ ID NO.1所示。
7.根据权利要求6所述的一种试剂盒,其特征在于,所述的试剂包括SEQ ID NO.2~23所示的引物。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110241199A (zh) * | 2019-06-05 | 2019-09-17 | 杭州市西溪医院 | miR-584-5p在作为急性呼吸窘迫综合征生物标记物中的应用 |
CN111378742A (zh) * | 2020-04-16 | 2020-07-07 | 嘉兴程瑞医药科技有限公司 | microRNA生物标志物及其在制备自身免疫疾病检测试剂盒中的应用 |
CN113999909A (zh) * | 2021-11-17 | 2022-02-01 | 江苏大学 | 一种用于胃癌诊断的血清外泌体标志物及其应用、扩增引物对和诊断试剂盒 |
CN114182009A (zh) * | 2021-12-30 | 2022-03-15 | 暨南大学附属第一医院(广州华侨医院) | 一种血浆外泌体CircOGDH作为急性缺血性卒中诊断性生物标志物的应用 |
-
2022
- 2022-06-30 CN CN202210770002.3A patent/CN114958859B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110241199A (zh) * | 2019-06-05 | 2019-09-17 | 杭州市西溪医院 | miR-584-5p在作为急性呼吸窘迫综合征生物标记物中的应用 |
CN111378742A (zh) * | 2020-04-16 | 2020-07-07 | 嘉兴程瑞医药科技有限公司 | microRNA生物标志物及其在制备自身免疫疾病检测试剂盒中的应用 |
CN113999909A (zh) * | 2021-11-17 | 2022-02-01 | 江苏大学 | 一种用于胃癌诊断的血清外泌体标志物及其应用、扩增引物对和诊断试剂盒 |
CN114182009A (zh) * | 2021-12-30 | 2022-03-15 | 暨南大学附属第一医院(广州华侨医院) | 一种血浆外泌体CircOGDH作为急性缺血性卒中诊断性生物标志物的应用 |
Non-Patent Citations (1)
Title |
---|
AGNIESZKA RYBAK-WOLF: "Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed", MOLECULAR CELL, vol. 58, no. 5, pages 1 - 18 * |
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