CN113755570B - 用于预测不明原因复发性流产的生物标志物及应用 - Google Patents
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Abstract
本发明提出了一种用于预测不明原因复发性流产的生物标志物及应用。具体来说，通过对不明原因复发性流产患者未流产时的血清外泌体中miR‑185‑5p的表达情况进行预测或辅助预测，诊断或辅助诊断不明原因复发性流产患者在胚胎停止发育前的不良结局。相应的还提出了利用该生物标志物表达水平的物质在制备不明原因复发性流产检测产品以及利用该生物标志物表达水平的物质制造的用于检测不明原因复发性流产的设备的应用。
Description
技术领域
本发明属于分子诊断和分子生物学技术领域，具体涉及一种以血清外泌体miRNA作为预测不明原因复发性流产的生物标志物及应用。
背景技术
自然流产是指妊娠28周以内、胎儿体重小于1kg的自然状态下发生的胚胎/胎儿丢失，自然流产的发生率可达20％，有一半以上的自然流产是由胚胎染色体异常造成的。然而对于剩下一部分胚胎染色体正常的患者，流产的发生往往是毫无预兆的。经历流产的女性与伴侣经常会经历悲伤的过程，伴有焦虑与抑郁的状态，尤其是对于不孕接受多次辅助生殖技术助孕后妊娠的患者与多次流产的患者。因此，倘若可以提前预测此次流产的发生并及时进行干预可能会减少一部分正常染色体胚胎的丢失。
目前临床上主要通过超声指标以及血清生化标志物水平来预测流产的发生，然而血清hCG与孕酮水平用来预测流产的效果不佳，有文献提出血清CA125是用来预测先兆流产最可靠的标志物，其敏感性为90％，特异性为88％，但血清CA125的水平与多种疾病相关，其预测流产的特异性不高，特别是对于子宫内膜异位症的患者、辅助生殖助孕的患者、肥胖的患者以及存在卵巢囊肿的患者预测效能。也有研究发现超声指标中胎儿心动过缓是预测流产最重要的标志物，其敏感性可达84.18％，然而其对早期流产预测的灵敏性欠佳。对于辅助生殖助孕且超声指标未见异常的患者，目前尚缺乏有效的预测指标。
目前已发表的用于预测流产的技术主要包括：有研究将生化指标(孕激素与β-hCG)与临床症状(腹部疼痛和阴道出血)以及病史(既往妊娠次数)进行联合分析，建立预测模型，该模型对复发性流产的预测的AUC最高达0.81，然而该模型的假阴性率高达90％，并不能给予临床较好的指导意义(DOI：10.1177/0300060520911829)。也有研究发现血浆miR-127-3p和miR-486-5p联合对复发性流产进行预测，可达50％的敏感性以及75.3％的特异性(DOI:10.1186/s12967-018-1556-x)，但是血浆中的miRNA在提取过程中，易被环境中RNA酶所降解，从而造成结果的误差。
发明内容
本发明为了解决现有技术主要通过超声指标以及血清生化标志物水平来预测流产的发生，然而血清hCG与孕酮水平用来预测流产的效果不佳和超声指标中胎儿心动过缓对早期流产预测的灵敏性欠佳，且对于辅助生殖助孕且超声指标未见异常的患者，目前尚缺乏有效的预测指标的问题，提供一种血清外泌体miRNA用于预测不明原因复发性流产的生物标志物及应用。
本发明的具体技术解决方案如下：
一种用于预测不明原因复发性流产的生物标志物，其特殊之处在于：所述生物标志物为miR-185-5p。
该用于预测不明原因复发性流产的生物标志物miR-185-5p来自不明原因的偶发性流产患者，或者复发性流产患者未流产时的血清中。
该用于预测不明原因复发性流产的生物标志物miR-185-5p以来自不明原因的偶发性流产患者，或者复发性流产患者未流产时的血清外泌体中为佳，其准确率和稳定性更高。
相对于健康人群，不明原因复发性流产患者中miR-185-5p表达为上调。
该标志物尤其适用于预测或辅助预测，诊断或辅助诊断不明原因的偶发性流产患者、复发性流产患者在胚胎停止发育前的不良结局。
所述生物标志物表达水平的物质在制备不明原因复发性流产检测产品中的应用时，可以包括基于高通量测序方法和/或基于定量PCR方法和/或基于探针杂交方法检测上述生物标志物表达水平的物质。例如检测装置、试剂盒和设备，这类装置例如：如寡核苷酸探针或其集成、芯片基片或检测基板上的高通量miRNA检测芯片、以及微流控检测芯片等。
术语“探针”是指通常用于检测与探针的序列互补的靶RNA和/或RNA序列的单链寡核苷酸。探针与核苷酸序列由于探针与靶序列之间的互补性而允许核苷酸配对的单链核酸(DNA或RNA)杂交。探针的长度取决于预期用途以及所需的探针特异性。
术语“试剂盒”是指单独提供或提供在单个容器内的上述组分的集合。
利用上述物质制造的用于检测不明原因复发性流产的设备，包含：
1)分析单元，所述分析单元包含：用于确定对象的样品中生物标志物表达水平的检测剂；
2)包含有数据处理器的评估单元，所述数据处理器有形地嵌入有用于将通过所述分析单元确定的量与参考进行比较的算法，并且能够生成包含基于所述比较建立的诊断结果的输出文件。
术语“设备”涉及至少包含有效地互相联系以允许进行诊断的上述装置的装置系统。例如，在应用用于自动化确定基因产物的甲基化状态或量的装置的情况下，可以通过例如计算机程序来处理由所述自动化操作装置获得的数据以建立诊断。
本发明的优点在于：
1、本研究通过对偶发性流产患者、复发性流产患者未流产时的血清外泌体与继续妊娠患者早孕期血清外泌体miRNAs转录组学测序，筛选出具有差异性表达的miRNAs，并进行验证，证明血清外泌体中miRNAs较血清中miRNAs更稳定，更适合作为预测分子标志物；
2、本研究收集的标本为未发生流产时的母体外周血，能更好的在胚胎停止发育前预测不良结局，及时的进行干预与治疗，有望减少不必要的胚胎丢失从而改善妊娠结局；
3、本研究发现以外泌体miR-185-5p作为载体，包裹含有miR-185-5p的抑制剂可能能够对复发性流产患者进行治疗；
4、本研究发现外泌体miR-185-5p可能与流产的发生机制相关，为今后的临床治疗与干预提供了新的思路与证据支持。
附图说明
图1为血清外泌体电镜鉴定图(bar＝100nm)；
图2为血清外泌体粒径分析图；
图1、图2中：A为OP组外泌体；B为SSA组外泌体，C为RSA组外泌体；
图3为血清外泌体Western-blot鉴定图；
图4为差异miRNAs聚类性热图；
图4中：A为OP组vs RSA组，B为OP组vs SSA组，C为OP组vs SSA组vs RSA组；
图5为差异miRNAs的qRT-PCR验证图；
图6为miR-185-5p在OP组(n＝45)与SSA组(n＝20)和RSA组(n＝15)中的表达图(与NC组相比，***P<0.001，****P<0.0001)；
图7为miR-185-5p在流产组组与继续妊娠组中的表达图(**P<0.01)；
图8为ROC曲线评估外泌体miR-185-5p的预测效能图；
图8中：A为SSA的预测分子，B为RSA的预测分子，C为SA的预测分子。
具体实施方式
首先需要说明的是，以下详细说明都是例示性的，旨在对本发明提供进一步的说明。除非另有指明，本发明使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。
技术人员理解本发明中使用的术语例如“miRNA”和“miR”的变化形式，并且其涉及见于真核生物细胞和后生动物生物体的体液中的短核糖核酸(RNA)分子。miRNA包括人miRNA、成熟单链miRNA、前体miRNA(pre-miR)、及其变体，其可以是天然存在的。在一些情况下，术语“miRNA”还包括初级miRNA转录物(pri-miRNA)和双链体miRNA。除非另外指出，否则当在本发明中使用时，特定miRNA的名称是指成熟miRNA。miRNA前体可以由25至数千个核苷酸，通常40至130、50至120、或60至110个核苷酸组成。通常，成熟miRNA由5至100个核苷酸，通常10至50、12至40、或18至26个核苷酸组成。术语miRNA还包括最终进入RNA诱导沉默复合体(RNA-induced silencing complex，RISC)的“引导”链以及与其互补的“过客”链。
术语指标的水平“下调”、“降低”或“下降”是指与参考或参考样品相比，样品中这样的指标的水平降低。术语指标的水平“上调”、“升高”或“提高”是指与参考或参考样品相比，样品中这样的指标的水平更高。属于RIF是指反复种植失败，NC是指自然受孕。
以下结合实施例进行详述：
在本实施例中，收集辅助生殖助孕治疗并成功临床妊娠患者的孕42天、56天、70天血清，根据患者的病史以及妊娠进展情况将患者分为继续妊娠(ongoing pregnancy，OP)组、不明原因偶发性流产(sporadic spontaneous abortion,SSA)组以及不明原因复发性流产(repeated spontaneous abortion,RSA)组，流产组标本取自患者B超第一次显示胚胎停止发育或难免流产前一次(胚胎发育正常状态下)抽取的外周血标本，OP组标本随机筛选相同孕周的外周血标本。提取各组血清外泌体，通过miRNA高通量学测序技术对三组血清外泌体miRNAs进行测序，筛选出差异表达的外泌体miRNAs，并对差异性表达的外泌体miRNAs进行qRT-PCR验证，之后扩大样本量并用ROC曲线评估差异性表达的外泌体miRNA对于RSA患者再次流产的预测价值。
(1)对血清外泌体进行提取与鉴定：所有参与者留取怀孕42天、56天、70天静脉血5mL于无添加剂采血管中，常温静置1小时，然后4℃，3000rpm，离心10分钟，将上清转移至1.5mL无菌离心管中，4℃，3000g，离心10分钟，转移上清于新1.5mL无菌离心管，将血清储存于-80℃备用。根据患者的病史以及妊娠进展情况将患者分为OP组、SSA组以及RSA组，流产组标本取自患者B超第一次显示胚胎停止发育或难免流产前一次(胚胎发育正常状态下)抽取的外周血标本，OP组标本随机筛选相同孕周的外周血标本。根据外泌体提取试剂盒(加拿大，Norgen，57400)说明书进行操作。提取好的外泌体通过电镜鉴定、粒径分析以及Wester-blot鉴定。
结果：实验所得颗粒经鉴定为外泌体。
(2)对血清外泌体进行RNA提取与测序：根据外泌体RNA提取试剂盒(加拿大，Norgen，58000)说明书进行操作。外泌体miRNA测序主要包括制备文库与测序实验，小RNA测序文库的制备采用TruSeq Small RNA Sample Prep Kits(Illumina,San Diego,USA)试剂盒，测序实验使用llumina Hiseq 2000/2500，测序所读长度为单端1×50bp。数据分析使用联川生物公司自主研发的分析软件ACGT101-miR(LC Sciences,Houston,Texas,USA)，将原始数据经过质控处理，得到clean reads。clean reads去除3’接头后进行筛选，保留碱基长度为18-26nt的序列。再将剩余序列与各种RNA数据库序列进行比对(不包括miRNA数据库)，并进行过滤，最后获得的数据即为有效数据，用于后续miRNA数据分析。
结果：将RSA组与OP组组进行比较，选择P<0.05为差异性miRNAs，结果显示有25种差异表达的miRNAs，其中有16种miRNAs上调，7种miRNAs下调；将SSA组与OP组进行比较，选取差异倍数∣log2(Foldchange)∣≥1且P<0.05为差异性miRNAs，结果显示：有11种miRNAs上调，14种miRNAs下调；将OP组、SSA组、RSA组进行比较，选取P<0.05为差异性miRNAs，结果显示有44种差异表达的miRNAs，筛选表达量为中高等水平(平均表达量＞100)，结果显示有17种差异表达的miRNAs。
(3)miRNA反转录与实时荧光定量PCR：外泌体miRNA逆转录根据miRNA逆转录试剂盒(德国，Qiagen，218161)说明书进行操作。外泌体miRNA实时荧光定量PCR根据miRNA定量PCR试剂盒说明书(德国，Qiagen，218073)进行操作，以hsa-miR-26a-5p作为参照，比较三组血清外泌体miRNAs的表达水平。
结果：根据差异miRNAs筛选结果以及文献查阅，选择了5种可能与流产相关的miRNAs进行qRT-PCR验证，包括miR-22-3p、miR-185-5p、miR-335-3p、miR-362-5p、miR-378a-3p。结果显示：OP组、SSA组、RSA组三组进行比较时，miR-185-5p在三组间比较差异有统计学意义(P<0.05)，miR-185-5p在RSA组中的表达显著高于OP组以及SSA组。其余四种miRNAs(miR-22-3p、miR-335-3p、miR-362-5p、miR-378a-3p)在三组中表达无显著性差异(P<0.05)。对miR-185-5p进一步扩大样本量进行验证，结果显示：miR-185-5p在OP组(45例)、SSA组(20例)、继发性流产组(15例)中的表达量具有显著性差异(P<0.05)。再将RSA组与SSA组合并为流产组，与OP组进行比较，结果显示：miR-185-5p在流产组(35例)的表达量明显高于OP组(45例)，差异具有统计学意义(P<0.05)。
(4)分别绘制外泌体miR-185-5p用于预测SSA、RSA以及流产的ROC曲线并计算AUC。结果显示：外泌体miR-185-5p用于预测SSA的AUC为0.670(95％CI：0.500-0.839，P＝0.031)，cutoff值为1.355，敏感性为55％，特异性为90.7％。外泌体miR-185-5p用于预测RSA的AUC为0.925(95％CI：0.807-1.000，P<0.0001)，cutoff值为1.911，敏感性为92.9％，特异性为97.7％。外泌体miR-185-5p用于预测流产的AUC为0.775(95％CI：0.654-0.896，P<0.0001)，cutoff值为1.355，敏感性为70.6％，特异性为90.7％。认为早孕期母体血清外泌体miR-185-5p对于复发性流产患者再次流产的发生具有较高的预测价值。
表1研究样本的基本信息(筛查)
表2研究样本基线资料(验证)
表3 RSA组与OP组差异表达的miRNAs
在本实施例中，选外泌体的动机是：外泌体是一种纳米级别的细胞外囊泡，包裹多种活性分子包括蛋白质、miRNA等，参与细胞间通讯并发挥生物学功能。miRNA属于非编码RNA的子集，是一类长度约为22个核苷酸的内源性的具有调控功能的小RNA，miRNA可以作为无创性分子标志物对疾病进行预测与诊断。近年来，研究发现外泌体中miRNA较血液中miRNA更稳定，外泌体miRNA将是临床诊断中最理想的生物标志物。
本实施例通过对偶发性流产患者、复发性流产患者未流产时的血清外泌体与继续妊娠患者早孕期血清外泌体miRNAs转录组学测序，筛选出具有差异性表达的miRNAs，并进行验证。
本实施例的优点在于血清外泌体中miRNAs较血清中miRNAs更稳定，更适合作为预测分子标志物。
本实施例收集的标本为未发生流产时的母体外周血，能更好的在胚胎停止发育前预测不良结局，及时的进行干预与治疗，有望减少不必要的胚胎丢失从而改善妊娠结局。
并且，本实施例发现外泌体miR-185-5p可能与流产的发生机制相关，为今后的临床治疗与干预提供了新的思路与证据支持。以外泌体作为载体，包裹含有miR-185-5p的抑制剂可能能够对复发性流产患者进行治疗。
Claims (1)
1.检测来源于未发生流产时的母体外周血血清外泌体中的miR-185-5p的物质在制备不明原因复发性流产检测产品中的应用。
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