WO2020216385A2 - 血清外泌体has_circ_0004771在制备酒精依赖综合征诊断试剂中的应用 - Google Patents

血清外泌体has_circ_0004771在制备酒精依赖综合征诊断试剂中的应用 Download PDF

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WO2020216385A2
WO2020216385A2 PCT/CN2020/097041 CN2020097041W WO2020216385A2 WO 2020216385 A2 WO2020216385 A2 WO 2020216385A2 CN 2020097041 W CN2020097041 W CN 2020097041W WO 2020216385 A2 WO2020216385 A2 WO 2020216385A2
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alcohol dependence
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彭英
刘云云
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中山大学孙逸仙纪念医院
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  • the present invention belongs to the field of biotechnology, and specifically relates to a serum exosomal circular biomarker has_circ_0004771 used for the diagnosis and severity assessment of alcohol dependence syndrome and its detection primer, a kit for detecting the marker, and the detection primer , The application of a kit or marker for detecting the marker in the diagnosis and severity assessment of alcohol dependence syndrome.
  • AD Alcohol Dependence Syndrome
  • Exosomes 30-100nm, spherical vesicles that transport functional proteins or nucleic acids to recipient cells are considered to be an important part of intercellular communication.
  • exosomes may be produced by a wide range of cell types in the central nervous system, including oligodendrocytes, Schwann cells, microglia and astrocytes.
  • Exosomes are actively involved in synaptic plasticity, signal transduction, neuroinflammation and degeneration, involving a wide range of normal and pathological processes. This is consistent with their role in the occurrence and development of many central nervous system diseases such as infection, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and stroke.
  • research on the application of exosomes to identify a new circulating biomarker for the detection of alcohol dependence syndrome is still lacking.
  • CircRNAs are a new type of RNA that are widely present in eukaryotes from Caenorhabditis elegans to humans, and have the characteristics of evolutionary conservation. circRNAs are produced in a covalent closed loop structure, which does not have a 5'cap or 3'polyadenylic acid tail. circRNAs can be formed from candidate gene sites through a special splicing event called "reverse splicing", including coding and non-coding exons (ecircRNAs), introns (ciRNAs), exons and introns (EIciRNAs) or transcribed antisense to 5'and 3'UTRs.
  • ecircRNAs coding and non-coding exons
  • ciRNAs introns
  • EIciRNAs exons and introns
  • circRNAs are highly abundant in organisms and expressed in cell type, tissue type and stage-specific patterns. Although circRNAs have been detected in various tissues, they have proven to be more abundant in the brain. circRNAs play a key role in the proliferation and differentiation of neurons.
  • circRNAs may be biomarkers and therapeutic targets for AD, and further research is needed to fill this gap. This study aims to evaluate the changes in the expression of circRNAs and to find a new cyclic biomarker in serum exosomes for AD detection.
  • the first object of the present invention is to provide a specific amplification primer for detecting the serum exosomal biomarker has_circ_0004771, the primer includes:
  • the second objective of the present invention is to provide a serum exosomal biomarker has_circ_0004771, and the nucleotide sequence of the biomarker has_circ_0004771 is shown in SEQ ID NO.1.
  • the third purpose of the present invention is to provide the application of the above-mentioned specific amplification primers for detecting the serum exosomal biomarker has_circ_0004771 in preparing reagents for diagnosis and severity assessment of alcohol dependence syndrome.
  • the fourth objective of the present invention is to provide the above-mentioned serum exosomal biomarker has_circ_0004771 as a biomarker in the preparation of reagents for the diagnosis and severity assessment of alcohol dependence syndrome.
  • the fifth objective of the present invention is to provide the application of a preparation for detecting the content of the biomarker has_circ_0004771 in serum exosomes in the preparation of a kit for diagnosis and severity assessment of alcohol dependence syndrome.
  • the sixth object of the present invention is to provide a kit for the diagnosis and severity assessment of alcohol dependence syndrome, which contains the above-mentioned specific amplification for detecting the serum exosomal biomarker has_circ_0004771 Primer.
  • the kit also contains all the reagents for extracting exosomes from serum, extracting RNA from exosomes and performing reverse transcription and fluorescent quantitative PCR.
  • the beneficial effect of the present invention is that the circular RNA in serum exosomes of hsa_circ_0004771 was discovered for the first time, and the expression levels of hsa_circ_0004771 in serum exosomes of healthy volunteers and alcohol dependence syndrome (AD) patients were detected by QRT-PCR, and the results showed The expression level of serum exosomes hsa_circ_0004771 in AD patients was significantly higher than that of healthy volunteers (p ⁇ 0.001), suggesting that hsa_circ_0004771 exosomes have a higher diagnostic value for alcohol dependence syndrome.
  • the biomarkers can distinguish AD patients from non-AD patients.
  • serum circRNA markers and diagnostic kits the diagnosis of AD can be made more convenient and easy, for clinicians to quickly and accurately grasp the condition of patients, lay the foundation for improving clinical treatment effects, and for discovering potential treatments Provide help with valuable new small molecule drug targets.
  • Figure 1 shows the results of using transmission electron microscopy (TEM), NTA and Western blot techniques to identify circulating serum exosomes.
  • B is a Western blot to detect the expression levels of CD63, TSG101, and HSP90B1 in exosomes;
  • C is the result of NTA measurement and analysis of the number and size distribution of exosomes.
  • TEM transmission electron microscopy
  • Figure 2 shows the expression levels of serum exosomes hsa_circ_0004771 in healthy volunteer controls (Healthy controls) and alcohol dependence syndrome (AD) patients.
  • Figure 3 is the analysis result of the correlation between the expression level of serum exosomes hsa_circ_0004771 and the severity of alcohol dependence syndrome (AD).
  • A is the correlation between the expression level of serum exosomes hsa_circ_0004771 and SADQ score;
  • B is the correlation between the expression level of serum exosomes hsa_circ_0004771 and ADS score;
  • C is the area under the curve (AUC) of serum exosomes hsa_circ_0004771 in ROC analysis.
  • the subjects of the study were either abstinents or social drinkers, and their daily intake of alcohol did not exceed the 25 grams recommended by the 2016 Chinese Dietary Guidelines. And most importantly, they must be non-alcohol dependent and their AUDIT-C score is less than 5 (33).
  • the AUD subjects and the control group meet one or more of the following criteria, they will be excluded from the study: 1. Drug dependence other than nicotine. 2. In addition to mild anxiety or depression, comorbidities are accompanied by activities or previous mental illnesses (ie, schizophrenia, bipolar disorder). 3. Comorbidities with neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease). 4.
  • NanoSight NS300 instrument (NanoSight Ltd., Amesbury, UK) was used to detect the size distribution and number of exosomes. Dilute the exosomes with particle-free PBS and transfer to the NanoSight sample chamber. Use NTA software (version 2.3; NanoSight Co., Ltd.).
  • Reverse transcription reaction Set the conditions for reverse transcription as follows: 37°C (15min) ⁇ 85°C (5s) when the reaction is over, save the cDNA at -80°C or immediately perform PCR quantitative detection.
  • the content of clinical data analysis includes case number, name, gender, age, drinking time, drinking dose, SADQ score, ADS score, clinical data are shown in Table 5.
  • hsa_circ_0004771 was a sensitive biomarker that can distinguish AD patients from non-AD patients.

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Abstract

一种血清外泌体has_circ_0004771在制备酒精依赖综合征诊断试剂中的应用。通过研究酒精依赖综合征(AD)患者血清外泌体中的circRNA表达的改变，筛选出最具显著意义的circRNA并应用于临床检测AD具有明确的有效性。所述的外泌体hsa_circ_0004771对AD具有较高的诊断价值，其表达水平与AD的严重程度有关，可作为标志物用于AD诊断及其严重程度评估。通过血清circRNA标志物和诊断试剂盒的研制和应用，可以使得AD的诊断更加方便易行，为临床医生快速准确掌握患者病情，为提高临床治疗效果奠定基础，并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。

Description

血清外泌体has_circ_0004771在制备酒精依赖综合征诊断试剂中的应用 技术领域
本发明属于生物技术领域，具体涉及一种用于酒精依赖综合征诊断和严重程度评估的血清外泌体环状生物标志物has_circ_0004771及其检测引物、检测该标志物的试剂盒，以及该检测引物、检测该标志物的试剂盒或标志物在酒精依赖综合征诊断和严重程度评估中的应用。
背景技术
酒精依赖综合征(AD)定义为患者不能自我控制的饮酒，对身心健康、人际关系有负面影响。AD在发达国家和发展中国家非常普遍，有大约10％的全球人口患病率。尽管已经做出了精心的努力来开发基于DSM-IV/v的访谈格式用于筛查AD，但据报道接受诊断和治疗的患者不到15％。因此，探索一种新的生物标志物作为AD检测的辅助手段，引起了神经科学家的极大兴趣。
外泌体，是30-100nm，球形包围的囊泡，通过运输功能蛋白质或核酸给受体细胞，被认为是细胞间通信的一个重要组成部分。最新证据显示外泌体可能由中枢神经系统广泛的细胞类型产生，包括少突细胞，许旺细胞，小胶质细胞和星形胶质细胞。外泌体积极参与突触可塑性、信号转导、神经炎症和变性，涉及广泛的正常和病理过程。这与它们在感染、帕金森病、阿尔茨海默病、肌萎缩侧索硬化症、中风等许多中枢神经系统疾病的发生和发展中的作用是一致的。然而，应用外泌体来识别一种用于检测酒精依赖综合征的新型循环生物标志物的研究仍然缺乏。
环状RNA(circRNAs)是一类新型RNA，广泛存在于从秀丽隐杆线虫到人类的真核生物 中，具有进化守恒的特征。circRNAs是在共价闭环结构中产生的，它不具有5'帽或3'聚腺苷酸尾巴。circRNAs可以通过一种称为“反向剪接”的特殊剪接事件从备选基因位点形成，包括编码和非编码外显子(ecircRNAs)、内含子(ciRNAs)、外显子和内含子(EIciRNAs)或转录反义到5’和3’UTRs。circRNAs在生物体中高度丰富，以细胞型、组织型和阶段特异性模式表达。虽然circRNAs已经在各种组织中被检测到，但它们在大脑中被证明更丰富。circRNAs在神经元的增殖和分化过程中起着关键作用。
综上所述，中枢神经系统中circRNAs的失调可能最终导致各种疾病。此外，其他神经心理疾病，如重度抑郁症、精神分裂症、杜氏肌营养不良症和神经胶质瘤也有与circRNAs相关病理的研究报道。circRNAs可能是AD的生物标志物和治疗靶点，需要进一步的研究来填补这一空白。本研究旨在评价circRNAs表达的变化，并在血清外泌体中寻找一种新的环状生物标志物用于AD的检测。
发明内容
本发明的第一个目的是提供一种用于检测血清外泌体生物标志物has_circ_0004771的特异性扩增引物，所述的引物包括：
has_circ_0004771-F：5'-CTCCGGATGACATCAGAGCT-3'
has_circ_0004771-R：5'-TCTGGCTGTGTTTCTCCCAA-3'。
本发明的第二个目的是提供一种血清外泌体生物标志物has_circ_0004771，所述的生物标志物has_circ_0004771的核苷酸序列如SEQ ID NO.1所示。
本发明的第三个目的是提供上述的用于检测血清外泌体生物标志物has_circ_0004771的特异性扩增引物在制备酒精依赖综合征诊断和严重程度评估的试剂中的应用。
本发明的第四个目的是提供上述的血清外泌体生物标志物has_circ_0004771作为生物标志物在制备酒精依赖综合征诊断和严重程度评估的试剂中的应用。
本发明的第五个目的是提供用于检测血清外泌体中生物标志物has_circ_0004771的含量的制剂在制备酒精依赖综合征诊断和严重程度评估的试剂盒中的应用。
本发明的第六个目的是提供一种用于酒精依赖综合征诊断和严重程度评估的试剂盒，所述的试剂盒含有上述的用于检测血清外泌体生物标志物has_circ_0004771的特异性扩增引物。
进一步的，所述的试剂盒还含有从血清中提取外泌体、由外泌体中提取RNA并进行逆转录及荧光定量PCR的所有试剂。
本发明的有益效果是：首次发现hsa_circ_0004771这种血清外泌体中的环状RNA，利用QRT-PCR分别检测健康志愿者和酒精依赖综合征(AD)患者血清外泌体hsa_circ_0004771表达水平，结果显示，AD患者血清外泌体hsa_circ_0004771表达水平显著高于健康志愿者对照组(p<0.001)，提示外泌体hsa_circ_0004771对酒精依赖综合征具有较高的诊断价值。另外，通过分析hsa_circ_0004771水平与SADQ和ADS评分的相关性，发现外泌体hsa_circ_0004771水平与两种评分(r＝0.8484和0.8616)呈正相关，提示hsa_circ_0004771与AD的严重程度有关，hsa_circ_0004771可能是一种敏感的生物标志物，可以区分AD患者和非AD患者。进一步的，通过血清circRNA标志物和诊断试剂盒的研制和应用，可以使得AD的诊断更加方便易行，为临床医生快速准确掌握患者病情，为提高临床治疗效果奠定基础，并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。
附图说明
图1为采用透射电镜(TEM)、NTA和Western blot技术对循环血清外泌体进行鉴定的结果。A为外泌体的代表性TEM图像(bar＝100nm)；B为Western blot检测外泌体中CD63、TSG101、HSP90B1的表达水平；C为NTA测量分析外泌体的数量和大小分布结果。
图2为健康志愿者对照组(Healthy controls)和酒精依赖综合征(AD)患者血清外泌体hsa_circ_0004771的表达水平。
图3为血清外泌体hsa_circ_0004771的表达水平与酒精依赖综合征(AD)严重程度相关性的分析结果。A为血清外泌体hsa_circ_0004771表达水平与SADQ评分的相关性；B为血清外泌体hsa_circ_0004771表达水平与ADS评分的相关性；C为ROC分析血清外泌体hsa_circ_0004771的曲线下面积(AUC)。
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1
1、入组选择
本研究纳入37名年龄匹配的健康志愿者(非AD对照组)和60名AD患者。所有患者于2016年12月至2018年12月连续入住中山大学中山纪念医院神经内科。AD患者的纳入标准为:1.年龄从18岁到80岁不等。2.目前AD的诊断符合DSM-IV(精神疾病诊断与统计手册)标准。3.入院前一周内仍在饮酒。4.有足够的认知水平完成基本的研究访谈(MMSE评分>10)。5.自愿为外泌体和环状RNA分析提供血液样本。年龄配对的非AD对照组是来自中山纪念医院附近社区的健康志愿者，他们通过广告招募。为了满足作为对照的标准，研究对象要么是戒酒者，要么是社交饮酒者，每天摄入的酒精不超过2016年《中国膳食指 南》推荐的25克。而且最重要的是，他们必须是非酒精依赖的，他们的AUDIT-C分数小于5(33)。为了尽量减少未知混杂物对我们结果的影响，如果AUD受试者和对照组符合以下一个或多个标准，他们将被排除在研究之外：1.尼古丁以外的药物依赖。2.共病除轻度焦虑或抑郁外，伴有活动或先前的精神疾病(即精神分裂症、双相情感障碍)。3.伴有神经退行性疾病的共病(如帕金森病、阿尔茨海默病)。4.重大身体疾病(如糖尿病、肾功能不全、梗塞、肝硬化、严重传染性疾病、癌症)。5.艾滋病毒感染。6.孕妇。7.拒绝提供血样及/或书面知情同意书。本研究经中山大学中山纪念医院伦理委员会批准(编号:SYSEC-KY-KS-2019007)，所有参与者均提供书面知情同意书。所有连续数据都表示为中值(范围)。分类变量表示为值(百分比)。
2、临床标本的提取
临床提取健康志愿者和AD患者10mL血液，进行外泌体的提取，为了分离外泌体，将血清在4℃条件下以3000g离心15分钟以除去凋亡小体，得到上清液。
3、血清外泌体的提取
将上清液转移到新管中，并根据方案用ExoQuick溶液(01.SBI.EXOQ5A-1，Sigma，USA)进行外泌体的提取。具体的步骤如下：
(1)吸取已分离好的上清液500μL至一新的Ep管内，3000rcf离心15分钟，以去除细胞或细胞碎片，将上清液转移至另一高压灭菌的Ep管中。
(2)使用250μL的凝血酶与(1)中上清液充分混匀，37℃孵育15分钟，然后使用超速离心机10000rpm室温下离心15分钟，使上清和沉淀完全分离。
(3)去除沉淀，保存上清，即得到“血清样”液体，取600μL“血清样”液体转移至 另一离心管内，加入150μL Exoquick试剂后充分混匀，在4℃孵育30分钟，室温下1500rcf离心30分钟，管底可见有淡黄色或白色沉淀。
(4)去除上清，1500rcf继续离心5分钟后，尽量去除上层的液体组分，加入适量的PBS，重悬沉淀，若沉淀难以溶解，可酌量加至完全溶解，将所得溶解有外泌体的溶液储存于-80℃冰箱待用。
4、外泌体的鉴定
(1)透射电镜(TEM)
使用日立HT7700透射电镜观察外泌体的形态；将20-40μL解决液放置到碳涂层formvar网格10分钟，沾染磷钨酸(pH值6.8)5分钟。将样本在透射电镜下拍摄。
(2)Nanoparticle-tracking分析(NTA)
用NanoSight NS300仪器(NanoSight Ltd.，Amesbury,UK)检测外泌体的大小分布和数量。用无颗粒PBS对外泌体进行稀释，然后转移到NanoSight样品室。使用NTA软件(version 2.3；NanoSight有限公司)。
(3)免疫印迹分析
Western blot检测外泌体标记物。用RIPA裂解缓冲液(Pierce,Rockford,IL,USA)裂解外泌体蛋白，12％十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离。转入PVDF膜后，用5％牛血清白蛋白封闭膜。这些墨迹是分开孵化与鼠标反CD63应承担(Abcam 1:400稀释)，anti-TSG101(Abcam 1:1000稀释)，兔多克隆反HSP90B1(细胞信号技术，5:10000稀释)，或用鼠标单克隆反β-actin(Abcam，2.5:10000稀释)。用增强化学发光法(Pierce,Rockford,IL,USA)分析蛋白。
采用透射电镜(TEM)、NTA和Western blot技术对循环血清外泌体进行鉴定的结果见图1。透射电镜观察到外泌体为不规则球体，双层膜结构，大小范围为50-100nm(图1A)。利用纳米视觉系统分析外泌体的数量和大小分布。通过NTA测量，我们观察到外泌体的颗粒大小峰值约为105nm，分散性较低(图1B)。Western blot检测鉴定外泌体标记物CD63、TSG101、HSP90B1的表达结果显示：外泌体中CD63和TSG101的存在，以及HSP90B1的缺失，证实了外泌体的纯化(图1C)。以上结果表明，从血清中提取的外泌体具有外泌体的特征。
5、外泌体RNA提取
(1)收集上述步骤提取的AD患者(或健康志愿者)血浆外泌体200μL，加入1mL的TRIZOL试剂反复吹打，使外泌体裂解，匀浆后将样品于15-30℃孵育5分钟，以便核酸蛋白复合体完全解离。
(2)每1mL TRIZOL试剂匀浆样品中加0.2mL的氯仿，盖紧管盖，手动剧烈振荡管体15秒后，15-30℃孵育2-3分钟，然后在4℃下12000rcf离心15分钟，离心后的混合液体分为下层的红色酚氯仿相，中间层和上层的无色水相。
(3)将水相转移到新离心管中，加入0.5mL异丙醇，混匀后15-30℃孵育10分钟，于4℃下12000rcf离心10分钟，移去上清液，加入至少1mL 75％乙醇水溶液清洗RNA沉淀，振荡后，4℃下7500rcf离心5分钟；去除乙醇水溶液，空气中干燥RNA沉淀5-10分钟，加入50μL的DEPC水溶解RNA，保存RNA用于后期的实验。
(4)使用Nano
Figure PCTCN2020097041-appb-000001
ND-2000测定RNA浓度和纯度。
6、外泌体cDNA合成
(1)融解circRNA反转录所需的试剂，轻微颠倒混匀，短暂离心后置冰上待用；
(2)circRNA反转录反应液的配制：往冰上预冷的RNase free反应管内加入试剂至总体积20μL(反应体系见表1)
表1 RT-PCR反应体系组成
Figure PCTCN2020097041-appb-000002
(3)反转录反应：设置反转录的条件为：37℃(15min)→85℃(5s)反应结束，-80℃保存cDNA或者立刻进行PCR定量检测。
7、实时荧光定量PCR反应
(1)引物设计生成与检验
hsa_circ_0004771及内参基因GAPDH的引物序列见表2。
表2 引物序列
Figure PCTCN2020097041-appb-000003
(2)选择SYBR Premix Ex Taq TMII Tli RNase H plus
(RR820A)试剂盒，溶解试剂：按表3中的比例在冰上进行PCR反应液的配制。
表3 PCR反应体系
Figure PCTCN2020097041-appb-000004
(3)充分混匀PCR反应液，添加至96-PCR板对应孔中，贴上封口膜，2000rcf离心2分钟，混匀。
(4)qRT-PCR反应，按照表4的PCR反应循环条件进行检测。
表4 PCR反应循环条件
Figure PCTCN2020097041-appb-000005
(5)反应结束后，按试剂相关要求立即进行溶解曲线分析，对得到的Ct值运用2 －△△ Ct法计算目的基因的相对表达量，△Ct＝目的基因的Ct值-内参基因的Ct值，△△Ct＝实验样本的△Ct值-对照样本的△Ct值。基因的相对表达量(Relative Quantification，RQ)＝2 －△ △Ct，表示某个circRNA在实验样本中的表达相对于对照样本中的倍数。RQ>1表示目的circ RNA表达上调，RQ<1则表达下调。一般认为，在运用2 －△△Ct法计算相对表达量的时候，相对表达量在2倍以上或0.5倍以下可以认为是有统计学意义。
qRT-PCR检测健康志愿者对照组(Healthy controls)和酒精依赖综合征(AD)患者血清外泌体hsa_circ_0004771的表达水平结果见图2。由图2可知，AD患者血清外泌体 hsa_circ_0004771表达水平显著高于健康志愿者对照组(p<0.001)，提示外泌体hsa_circ_0004771对酒精依赖综合征具有较高的诊断价值。
8、临床病例的数据统计
(1)对临床酒精依赖综合征患者进行临床资料的分析
临床资料分析内容包括病案号，姓名，性别，年龄，饮酒时间，饮酒剂量，SADQ评分，ADS评分，临床资料见表5。
表5 AD患者和健康志愿者的临床资料
Figure PCTCN2020097041-appb-000006
(2)临床资料和上述检测的hsa_circ_0004771表达水平的相关性进行分析
为了评价血清外泌体hsa_circ_0004771与AD严重程度的相关性，我们分析了hsa_circ_0004771水平与SADQ和ADS评分的相关性。采用qRT-PCR检测AD患者血清外泌体中hsa_circ_0004771的表达。评估AD的严重程度由SADQ和ADS量表决定。我们发现外泌体hsa_circ_0004771水平与两种评分(r＝0.8484和0.8616，图3A和B)呈正相关，提示hsa_circ_0004771与AD的严重程度有关。为了进一步评价hsa_circ_0004771在AD患者中的诊断价值，我们对60例AD患者和37例正常样本进行了ROC分析。血清外泌体 hsa_circ_0004771曲线下面积(AUC)为0.964(95％CI:0.932-0.997,p<0.001，图3C)。这一发现提示hsa_circ_0004771可能是一种敏感的生物标志物，可以区分AD患者和非AD患者。
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Figure PCTCN2020097041-appb-000007

Claims (7)

  1. 一种用于检测血清外泌体生物标志物has_circ_0004771的特异性扩增引物，其特征在于，所述的引物包括：has_circ_0004771-F：5'-CTCCGGATGACATCAGAGCT-3'，
    has_circ_0004771-R：5'-TCTGGCTGTGTTTCTCCCAA-3'。
  2. 一种血清外泌体生物标志物has_circ_0004771，其特征在于，所述的生物标志物has_circ_0004771的核苷酸序列如SEQ ID NO.1所示。
  3. 权利要求1所述的用于检测血清外泌体生物标志物has_circ_0004771的特异性扩增引物在制备酒精依赖综合征诊断和严重程度评估的试剂中的应用。
  4. 权利要求2所述的血清外泌体生物标志物has_circ_0004771作为生物标志物在制备酒精依赖综合征诊断和严重程度评估的试剂中的应用。
  5. 根据权利要求4所述的应用，其特征在于，检测血清外泌体中生物标志物has_circ_0004771的含量的制剂在制备酒精依赖综合征诊断和严重程度评估的试剂盒中的应用。
  6. 一种用于酒精依赖综合征诊断和严重程度评估的试剂盒，其特征在于，包含有权利要求1所述的用于检测血清外泌体生物标志物has_circ_0004771的特异性扩增引物。
  7. 根据权利要求6所述的试剂盒，其特征在于，还包含有从血清中提取外泌体、由外泌体中提取RNA并进行逆转录及荧光定量PCR的所有试剂。
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