CN116606927A - 血清来源外泌体标志物miR-511-5p在制备抑郁症血清诊断产品中的应用 - Google Patents
血清来源外泌体标志物miR-511-5p在制备抑郁症血清诊断产品中的应用 Download PDFInfo
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Abstract
本发明公开了血清来源外泌体标志物miR‑511‑5p在制备抑郁症血清诊断产品中的应用,属于生物医药技术领域。本发明通过高通量RNA测序联合qRT‑PCR个例验证技术对抑郁症混合血清样本和个例血清样本进行了检测和验证,发现miR‑511‑5p在抑郁症血清外泌体中呈现特异性高表达,miR‑511‑5p可以作为诊断抑郁症的血清外泌体标记物,本发明填补了在临床诊断抑郁症的血液指标上的空白。
Description
技术领域
本发明属于生物医药技术领域,具体涉及血清来源外泌体标志物miR-511-5p在制备抑郁症血清诊断产品中的应用。
背景技术
抑郁症(Depressive Disorder,DD)是一种常见的心理障碍,是全球主要的精神疾病之一,其流行病学数据显示:全球范围内,全世界约有5%的人口患有抑郁症,女性患病率高于男性。抑郁症的发病年龄多在20-40岁之间,但在老年人和儿童青少年中也十分常见。目前临床上抑郁症的诊断依据主要包括以下几个方面:1.心理评估:包括抑郁症状自评量表、抑郁症状病史问卷等。2.临床表现:包括情绪低落、失眠、食欲减退、疲劳等。3.生理检查:包括血液、尿液等常规检查,以排除其他疾病的可能。4.精神病理学评估:包括对患者的思维、情感、行为等方面的评估,以确定是否符合抑郁症的诊断标准。抑郁症的精神检查往往取决于临床医师的诊断水平,具有较大的主观性,亟需找到一种使用特定生物标志物或一组能够反映患者状态和治疗效果的生物标志物来定量评估抑郁症的方法。
外泌体(Exosome)是一种细胞来源的囊泡(30-150nm大小)。外泌体可以从各种细胞类型中释放,存在于许多生理液体中。外泌体的具体功能主要取决于其内部功能成分,包括RNA、蛋白质、脂质、糖结构、代谢物和其他物质。此外,外泌体在其表面携带表征分子,从而有可能对囊泡类型进行分类并丰富来自组织特异性起源的特征。外泌体是细胞间通讯系统的一部分,癌细胞经常将它们用作生物信使以促进其生长。由于外泌体是疾病过程的一部分,因此它们在生物标志物研究中引起了极大的兴趣。外泌体在血浆和尿液等生物体液中非常稳定,即使在疾病的早期阶段也可以分离出来用于临床评估。因此,通过高通量RNA测序技术对抑郁症患者血清外泌体miRNA进行差异表达分析,再联合qRT-PCR技术进行临床个例验证,筛选并分析出敏感性和特异性较好的抑郁症血清来源外泌体标志物,能够填补抑郁症临床血液诊断指标的空白。
miR-511-5p在以往的报道中发现其与胃癌、结直肠癌等密切相关。例如,Yong等通过研究发现miR-511-5p通过靶向活化激酶2(PAK2)抑制胃癌中的细胞迁移、侵袭和上皮-间质转化;Wang等通过研究发现miR-511-5p通过直接靶向G蛋白偶联受体116(GPR116)作为肿瘤抑制因子和预测结直肠癌的预后;miR-511-5p也能够参与前脑发育,发育生长的调节,中枢神经系统神经元发育,分支结构的形态发生,树突形态发生,轴突发生,轴突发育,中枢神经系统神经元分化等多项生物过程。但是,目前尚未有关于miR-511-5p在抑郁症诊断方面的相关报道。
发明内容
本发明的目的在于提供一种血清来源外泌体标志物miR-511-5p在制备抑郁症血清诊断产品中的应用。
为了达到上述目的,本发明采用以下技术方案予以实现:
本发明公开了一种血清来源外泌体标志物miR-511-5p在制备抑郁症血清诊断产品中的应用。
优选地,所述产品为诊断试剂或诊断试剂盒。
优选地,通过高通量RNA测序、qRT-PCR技术对血清来源外泌体miR-511-5p表达进行检测。
优选地,所述血清来源外泌体标志物miR-511-5p在抑郁症血清中显著高表达。
本发明还公开了用于检测血清来源外泌体标志物miR-511-5p的试剂在制备用于抑郁症血清诊断产品中的应用。
优选地,所述产品为诊断试剂或诊断试剂盒。
优选地,通过高通量RNA测序、qRT-PCR技术对血清来源外泌体miR-511-5p表达进行检测。
优选地,所述血清来源外泌体标志物miR-511-5p在抑郁症血清中显著高表达
本发明还公开了一种抑郁症血清诊断试剂盒,该试剂盒中包括用于检测待测抑郁症的血清样品中外泌体标志物miR-511-5p的试剂。
与现有技术相比,本发明具有以下有益效果:
本发明应用高通量RNA测序筛选出抑郁症组vs健康对照组差异表达的miRNA即miR-511-5p,并首次揭示了抑郁症血清来源外泌体标志物miR-511-5p在制备抑郁症诊断产品中的新用途,通过高通量RNA测序联合qRT-PCR个例验证技术对抑郁症混合血清样本和个例血清样本进行了检测和验证,结果显示miR-511-5p在抑郁症人群中显著高表达(p<0.001)。ROC曲线分析显示miR-511-5p的AUC值为0.859,最大约登指数为0.66,95%可信区间为(0.782,0.936),最佳临界值为1.7507,灵敏度为86.00%,特异度为80.00%,说明miR-511-5p在抑郁症血清外泌体中呈现特异性高表达,基于以上分析,血清外泌体标志物miR-511-5p可以作为诊断抑郁症的血清标记物在临床中予以应用,本发明填补了在诊断抑郁症的临床血液指标上的空白。
附图说明
图1为抑郁症组外泌体透射电镜成像(1μm);
图2为抑郁症组外泌体透射电镜成像(100nm);
图3为健康对照组外泌体透射电镜成像(1μm);
图4为健康对照组外泌体透射电镜成像(100nm);
图5为抑郁症组血清外泌体浓度示意图;
图6为抑郁症组血清外泌体粒径示意图;
图7为健康对照组血清外泌体浓度示意图;
图8为健康对照组血清外泌体粒径示意图;
图9为抑郁症组纳米流式荧光检测示意图;
图10为健康对照组纳米流式荧光检测示意图;
图11为3例抑郁症组血清混合样本与3例健康对照组血清混合样本的血清外泌体高通量RNA测序分析结果(差异表达miRNA火山图);
图12为3例抑郁症组血清混合样本与3例健康对照组血清混合样本的血清外泌体高通量RNA测序分析结果(差异表达miRNA聚类图);
图13为qRT-PCR验证血清外泌体差异miR-511-5p的表达水平盒须图。qRT-PCR检测在50例抑郁症患者和50例健康对照者中进行,Y轴将miRNA表达水平显示为log102-ΔCt,圆点表示异常值,***代表P<0.001;
图14为ROC曲线分析miR-511-5p在抑郁症中的诊断价值。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分的实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本发明保护的范围。
需要说明的是,本发明的说明书和权利要求书及上述附图中的术语“第一”、“第二”等是用于区别类似的对象,而不必用于描述特定的顺序或先后次序。应该理解这样使用的数据在适当情况下可以互换,以便这里描述的本发明的实施例能够以除了在这里图示或描述的那些以外的顺序实施。此外,术语“包括”和“具有”以及他们的任何变形,意图在于覆盖不排他的包含,例如,包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元,而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
下面结合附图对本发明做进一步详细描述:
1、血清样本的采集和分组
样本采集自延安大学咸阳医院精神科(2020年9月至2022年3月)的60例(男性32例;女性28例)抑郁症患者,60例正常健康人群(男性34例;女性26例)。样本考虑年龄、性别、采集时间、储存条件是否一致以及有无基础疾病等因素。被采集者晨起空腹采血,用真空采血管(黄帽、有隔离胶)采集全血5mL,室温静置30min;室温离心5min(3000g),将上层血清分装成100μL/管,立即保存于-80℃,避免反复冻融。将抑郁症组和健康对照组血清样本按组每20例等量(每例25μL)混合,分为抑郁症组1-3(B1、B2、B3)和健康对照组1-3(H1、H2、H3)共6个混合样,用于高通量RNA测序。其余样本单独保存,用于个例验证。
2、筛选抑郁症组与健康对照组血清外泌体差异表达的miRNAs
2.1、血清外泌体提取
应用基于聚合物沉淀法的ExoQuick试剂盒(Exosome Precipitation Solution,System Biosciences,SBI)分离外泌体:
1)取0.5mL血清样本,加入0.5mL含有蛋白酶抑制剂和磷酸酶抑制剂,无钙和镁的DBS-2缓冲液混匀,4℃下1500g离心20min。
2)取上清液,加252μL ExoQuick试剂,4℃下孵育1h;
3)4℃下1500g离心30min,充分去除上清液,保留沉淀即为外泌体微粒。沉淀中加入150μL DBS-2缓冲液重悬,直接使用或液氮冻存。
2.2、血清外泌体鉴定
(1)透射电镜观察:将外泌体混合液吸取10μL滴加于铜网上沉淀1min,滤纸吸去浮液。醋酸双氧铀10μL滴加于铜网上沉淀1min,滤纸吸去浮液,常温干燥数分钟。100kv进行电镜检测成像,获得透射电镜成像结果。透射电子显微镜下,两组外泌体均呈“茶杯”状结构(图1-4)。
(2)粒径分析:将外泌体混合液取出5μL稀释到30μL。先用标准品进行仪器性能测试,合格后进行外泌体样品上样,需进行梯度稀释避免样本堵塞进样针。待样本完成检测即可获得粒径分析仪(NanoFCM,N30E)检测外泌体的粒径和浓度信息。结果显示:抑郁症组外泌体提取的粒径浓度为2.01E+10Particles/mL(图5),直径主要分布在30-150nm(平均80.71nm),与外泌体大小一致(图6);健康对照组外泌体提取的粒径浓度为2.01E+10Particles/mL(图7),直径主要分布在30-150nm(平均83.07nm),与外泌体大小一致(图8);
(3)外泌体样品荧光标记及纳米流式检测
将20μL外泌体稀释至60μL,取30μL稀释外泌体分别加入20μL荧光标记的抗体CD9(FITC MouseAnti-Human CD9、BD:555371)、CD81(FITC MouseAnti-Human CD81、BD:551108),混匀,避光37℃孵育30min,进行梯度稀释后上机,纳米颗粒跟踪分析仪(PARTICLEMETRIX、ZetaVIEW)获得仪器检测蛋白指标结果(表1、图9-10)。
表1外泌体表面蛋白表达阳性率
2.3外泌体RNA提取
取200μL外泌体混合液,利用EXOsome RNAisolation kit(Norgen)试剂盒提取外泌体总RNA。包括以下步骤:
1)将300μL的lysis buffer A与37.5μL的lysis buffer B添加到含有纯化外泌体的200μL重悬液中;
2)充分混合,室温孵育10min;
3)加入500μL的100%乙醇,涡旋振荡10s;
4)将以上液体转移至Mini Spin Columns中,6000rpm常温离心1min;
5)重复步骤四,直至所有液体都转移至吸附柱中;
6)将600μL的洗脱液A加入到吸附柱中,6000rpm常温离心30s;
7)重复步骤六,共洗两次;
8)空转吸附柱,14000rpm常温离心60s;并将其转移到另一个洗脱管中,添加洗脱液50μL,先后以1800rpm和8000rpm的速度分别常温离心60s和120s;
9)为获得最大回收效率,将洗脱的缓冲液转移回吸附柱,室温静置2min,2000rpm常温离心1min,8000rpm常温离心2min。
2.4筛选差异表达miRNAs
对抑郁症组1-3(B1、B2、B3)和健康对照组1-3(H1、H2、H3)共6个混合样的血清外泌体进行高通量RNA测序,分析抑郁症组与健康对照组血清外泌体miRNAs表达谱的变化,从而筛选出在抑郁症患者外泌体中差异性表达的miRNA共146个,其中上调表达为89个,下调表达为57个(图11-12)。按照差异性最显著且与神经元功能相关的原则,选取其中miR-511-5p作为候选标志物,经临床样本验证,与测序结果一致。
3、验证miR-511-5p在抑郁症组和健康对照组临床样本中的表达情况
3.1逆转录
采用逆转录试剂盒SweScript RT I First Strand cDNASynthesis Kit(G3330)将RNA逆转录为cDNA。包括以下步骤:
(1)逆转录反应体系配制(20μL,见表2,采用茎环法逆转录)。
表2 20μL逆转录反应体系
(2)轻轻混匀并离心。
(3)逆转录程序设置(表3),于普通PCR仪上完成逆转录。
表3逆转录程序设置
3.2实时荧光定量PCR
采用荧光定量试剂盒2×SYBR Green qPCR Master Mix(None ROX)(G3320)在PCR仪上进行定量表达检测,设置3个复孔。2-ΔΔCt法分析miRNA表达量变化(U6为内参)。在miRBase数据库(https://www.mirbase.org)中获得hsa-miR-511-5p的成熟序列(MIMAT0002808):5’-GUGUCUUUUGCUCUG CAGUCA-3’。设计miRNA引物序列见表4。
表4.miRNA引物序列
(1)取0.1ml PCR反应板,配制如下反应体系(15μL,见表5),每个反转录产物配制3管。点完样用PCR封板膜配合封膜仪完成封膜。
表5实时荧光定量PCR反应体系
(2)PCR扩增,于荧光定量PCR仪完成,程序设置(见表6)。
表6PCR扩增程序设置
3.3结果
根据收集的抑郁症组和健康对照组血清外泌体miR-511-5p含量,对筛选出的这一与抑郁症密切相关的miRNA的预测能力进行评估。ROC曲线分析结果显示(图14),在独立的血清样本中,血清外泌体miR-511-5p的曲线下面积(AUC)值为0.859,最大约登指数为0.66,95%可信区间为(0.782,0.936),最佳临界值为1.7507,灵敏度为86.00%,特异度为80.00%,这表明以上检测该miRNA的相对表达量的方法,对抑郁症表现出良好的判断能力和拟合性能。
综上,本发明通过高通量RNA测序联合qRT-PCR个例验证技术对抑郁症混合血清样本和个例血清样本进行了检测和验证,发现miR-511-5p在抑郁症血清外泌体中呈现特异性高表达,因此,以miR-511-5p作为诊断抑郁症的血清外泌体标记物,可以为抑郁症的早期诊断提供重要参考。
以上内容仅为说明本发明的技术思想,不能以此限定本发明的保护范围,凡是按照本发明提出的技术思想,在技术方案基础上所做的任何改动,均落入本发明权利要求书的保护范围之内。
Claims (9)
1.一种血清来源外泌体标志物miR-511-5p在制备抑郁症血清诊断产品中的应用。
2.如权利要求1所述的应用,其特征在于,所述产品为诊断试剂或诊断试剂盒。
3.如权利要求1所述的应用,其特征在于,通过高通量RNA测序、qRT-PCR技术对血清来源外泌体miR-511-5p表达进行检测。
4.如权利要求1所述的应用,其特征在于,所述血清来源外泌体标志物miR-511-5p在抑郁症血清中显著高表达。
5.用于检测血清来源外泌体标志物miR-511-5p的试剂在制备用于抑郁症血清诊断产品中的应用。
6.如权利要求5所述的应用,其特征在于,所述产品为诊断试剂或诊断试剂盒。
7.如权利要求5所述的应用,其特征在于,通过高通量RNA测序、qRT-PCR技术对血清来源外泌体miR-511-5p表达进行检测。
8.如权利要求5所述的应用,其特征在于,所述血清来源外泌体标志物miR-511-5p在抑郁症血清中显著高表达。
9.一种抑郁症血清诊断试剂盒,其特征在于,该试剂盒中包括用于检测待测抑郁症的血清样品中外泌体标志物miR-511-5p的试剂。
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