CN114739890B - 人MDSCs在鉴别CAA和AAA中的应用及鉴别试剂盒 - Google Patents
人MDSCs在鉴别CAA和AAA中的应用及鉴别试剂盒 Download PDFInfo
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Abstract
本发明提供了MDSCs作为生物标志物在鉴别先天性再生障碍性贫血和获得性再生障碍性贫血中的应用。发明人首次发现先天性再生障碍性贫血和获得性再生障碍性贫血患者的外周血中MDSCs的含量存在显著差异,表明MDSCs可作为特异性生物标志物用于鉴别先天性再生障碍性贫血和获得性再生障碍性贫血,因此,检测MDSCs在待测者外周血中含量的试剂即可作为鉴别CAA和AAA的试剂。MDSCs可简单、快速、高效地对先天性再生障碍性贫血和获得性再生障碍性贫血进行鉴别,从而避免因误诊耽误患者的治疗,具有无创、实用性强和易于推广等优点。
Description
技术领域
本发明属于医学检验技术领域,具体涉及人MDSCs在鉴别CAA和AAA中的应用及鉴别试剂盒。
背景技术
髓系抑制性细胞(Myeloid-derived suppressor cells,MDSCs)是一群由转录活性,分化状态各异的不成熟髓系细胞(immature myeloid cell,IMC)组成的异质性群体,其来源于骨髓祖细胞和未成熟髓细胞。正常情况下,IMC是树突状细胞(DC)、巨噬细胞和粒细胞的前体,能迅速地分化为成熟的粒细胞、DCs和巨噬细胞,并进入相应的器官、组织,发挥正常免疫功能。但是在某些病理条件下,受细胞因子的调控,这些髓系来源的前体细胞成熟受阻,因而停留在各个分化阶段,成为具有免疫抑制功能的MDSCs。MDSCs通过不同机制抑制T细胞、B细胞和NK细胞介导的免疫反应。
再生障碍性贫血(aplastic anemia,AA)是一种骨髓造血衰竭(bone marrowfailure,BMF)综合征,临床表现为贫血、出血和/或感染。AA分为先天性再生障碍性贫血(congenital aplastic anemia,CAA)及获得性再生障碍性贫血(acquired aplasticanemia,AAA)。目前认为T淋巴细胞异常活化、功能亢进造成骨髓损伤在原发性获得性AA发病机制中占主要地位,新近研究显示遗传背景在AA发病及进展中也可能发挥一定作用【再生障碍性贫血诊断与治疗中国专家共识(2017年版)】。很多胚系突变性疾病也表现为端粒缩短和类AA的全血细胞减少,包括GATA2谱系障碍,Diamond Blackfan贫血,先天性角化不良症(Dyskeratosis Congenita)、Fanconi贫血、Shwachman-Diamond综合征等,很多患者无特征性的临床表现及家族遗传背景,只有在基因检测时才能发现,需要特别注意与AA相鉴别【Bluteau O,Sebert M,Leblanc T,et al.A landscape of germ line mutations in acohort of inherited bone marrow failure patients[J]Blood.2018;131(7):717–732.doi:10.1182/blood-2017-09-806489.】。
随着全外显子二代测序的检测率提高,越来越多的“特发性再生障碍性贫血”的患者被发现有胚系来源的基因突变,但通过二代测序发现的基因突变往往为意义未明的突变。即使这些突变在clinvar(https://www.ncbi.nlm.nih.gov/clinvar/)等网站显示为既往曾在再生障碍性贫血患者发现,但是并不能仅根据病例上传记录确定为致病基因。而且BMF的发生往往是多基因联合致病,因此仅仅根据二代测序的结果无法判定是否为遗传性再生障碍性贫血。更有甚者,由于经济原因无法进行二代次序,长期按照AAA治疗而病情加重。
总之,在当前认为的CAA和AAA之间存在一个灰色地带,申请人称之为“有基因背景的再障”。这部分患者无特征性的临床表现及家族遗传背景,按照诊断标准不足以诊断为先天性再生障碍性贫血。因此,可能会按照特发性再障进行含或不含ATG的免疫抑制治疗。
但是治疗反应上,这些实际上携带了有临床意义突变的再生障碍性贫血患者对免疫抑制治疗反应差,并且在强化免疫抑制治疗后长达数月的观察过程中可能因感染使病情加重,甚至失去异基因造血干细胞移植的机会。而且,部分有范可尼贫血相关基因及影响端粒的TERT等基因突变的患者可能存在染色体不稳定,在ATG治疗后发生克隆性造血及实体瘤的风险更高【Calado RT,Cooper JN,Padilla-Nash HM,et al.Short telomeres resultin chromosomal instability in hematopoietic cells and precede malignantevolution in human aplastic anemia[J]Leukemia.2012;26(4):700–707.】。因此,这些“有基因背景的再障”本质上应属于CAA。如何更明确的地对CAA和AAA进行鉴别诊断,是AA诊断的一个难点,并有重要的临床应用价值。此前未见将MDSCs用于鉴别CAA和AAA的报道。
发明内容
基于此,本发明的目的在于提供一种有效鉴别CAA和AAA的生物标志物以及其在鉴别CAA和AAA中的应用,所述生物标志物为人MDSCs。发明人经过研究发现,人MDSCs在CAA和AAA患者外周血中的含量存在显著差异,可用于特异性地鉴别CAA和AAA。
实现上述目的的具体技术方案如下。
本发明提供了人MDSCs在制备鉴别先天性再生障碍性贫血和获得性再生障碍性贫血的试剂中的应用。
本发明还提供了检测人MDSCs在待测者生物样本中的含量的试剂在制备鉴别先天性再生障碍性贫血和获得性再生障碍性贫血试剂盒中的应用。
本发明还提供了人MDSCs在制备检测先天性再生障碍性贫血的试剂中的应用。
本发明还提供了检测人MDSCs在待测者生物样本中的含量的试剂在制备检测先天性再生障碍性贫血的试剂盒中的应用。
在其中一些实施例中,所述人MDSCs的流式细胞术分析标记为HLA-DR-/lowCD11b+CD33+。其中,HLA-DR-/lowCD11b+CD33+代表HLA-DR阴性或低表达、CD11b阳性和CD33阳性。
在其中一些实施例中,所述生物样本为外周血。
本发明还提供了一种鉴别先天性再生障碍性贫血和获得性再生障碍性贫血的试剂盒,所述试剂盒包含检测人MDSCs在待测者生物样本中的含量的试剂。
在其中一些实施例中,所述试剂为检测人MDSCs流式细胞术分析标记的抗体。
在其中一些实施例中,所述试剂包括修饰有不同荧光基团的以下抗体:人抗HLA-DR抗体、人抗CD11b抗体和人抗CD33抗体。
在其中一些实施例中,所述人抗HLA-DR抗体、人抗CD11b抗体和人抗CD33抗体修饰的荧光基团分别为:BV421、B510和PE。
在其中一些实施例中,所述试剂盒还包括修饰有不同荧光基团的针对上述抗体的同型对照抗体。
在其中一些实施例中,所述试剂盒还包括红细胞裂解液和PBS缓冲液。
在其中一些实施例中,所述试剂盒还包括人淋巴细胞分离液体。
本发明经过研究首次发现,CAA和AAA患者的外周血中MDSCs的含量存在显著差异:与健康志愿者相比,CAA患者外周血中MDSCs的比例明显升高,而AAA患者外周血中MDSCs的比例明显下降,差异具有统计学意义。进一步进行ROC曲线分析,结果显示MDSCs在用于鉴别CAA和AAA时,AUC为0.9583,灵敏度为83.33%,特异性为100%,说明MDSCs可作为特异性生物标志物用于鉴别先天性再生障碍性贫血和获得性再生障碍性贫血,具有很高的诊断准确性。因此,检测MDSCs在待测者外周血中含量的试剂即可作为鉴别CAA和AAA的试剂。本发明发现MDSCs可简单、快速、高效地对先天性再生障碍性贫血和获得性再生障碍性贫血进行鉴别,从而避免因误诊耽误患者的治疗,具有无创、实用性强和易于推广等优点。
本发明还发现MDSCs在用于诊断CAA时AUC高达0.9053,也具有很高的灵敏度和特异性。
附图说明
图1为样本的流式检测和统计分析结果图;其中,Control代表健康志愿者;CAA代表先天性再生障碍性贫血;AAA代表获得性再生障碍性贫血。
图2为MDSCs在用于鉴别CAA和AAA患者时的ROC曲线分析图;其中,CAA代表先天性再生障碍性贫血;AAA代表获得性再生障碍性贫血。
图3为MDSCs在用于诊断CAA患者时的ROC曲线分析图;其中,Control代表健康志愿者;CAA代表先天性再生障碍性贫血。
具体实施方式
本发明下列实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂,均为市售产品。
除非另有定义,本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不用于限制本发明。
以下结合具体实施例对本发明进行说明。
实施例1
1、病例信息
纳入标准:根据《再生障碍性贫血诊断与治疗中国专家共识(2017年版)》达到再生障碍性贫血诊断标准,并排除:(1)其他血液病及继发性血细胞减少;(2)检测前三天未使用短效升白针,检测前7天未使用长效升白针;(3)除外发热、急性感染及过敏反应状态。具体诊断标准如下:
再生障碍性贫血诊断标准:
(1)血常规检查:
全血细胞(包括网织红细胞)减少,淋巴细胞比例增高。至少符合以下三项中两项:HGB<100g/L;PLT<50×109/L;中性粒细胞绝对值(ANC)<1.5×109/L。
(2)骨髓穿刺:
多部位(不同平面)骨髓增生减低或重度减低;小粒空虚,非造血细胞(淋巴细胞、网状细胞、浆细胞、肥大细胞等)比例增高;巨核细胞明显减少或缺如;红系、粒系细胞均明显减少。
(3)骨髓活检(髂骨):
全切片增生减低,造血组织减少,脂肪组织和(或)非造血细胞增多,网硬蛋白不增加,无异常细胞。
(4)除外检查:
除外全血细胞减少和骨髓增生减低的其他疾病。
先天性再生障碍性贫血诊断标准:
达到再生障碍性贫血的患者,有范可尼贫血(FA)、先天性角化不良(DKC)、先天性纯红细胞再生障碍(DBA)、Shwachmann-Diamond综合征(SDS)等基因异常,且为纯合突变或多基因杂合突变,和/或端粒极短,和/或丝裂霉素断裂试验阳性,和/或有根据ACMG指南判断为致病性的I级突变。
获得性再生障碍性贫血诊断标准:
符合再生障碍性贫血诊断标准,除外先天性再生障碍性贫血、理化因素所致骨髓衰竭症。
根据上述纳入标准,AAA共纳入23例,CAA共纳入6例,同时,纳入19例健康志愿者作为健康对照。具体病例信息如表1和表2所示:
表1 CAA病例信息
表2 AAA病例信息
2、实验方法
(1)健康志愿者和患者的外周血单个核细胞(PBMC)分离
取外周血2~5ml,按以下步骤分离得到单个核细胞:
1)用含EDTA抗凝采血管收集待测者外周血,然后加入等3倍体积的PBS溶液,轻轻混匀,此时体积是2~5ml*3=6~15ml;
2)向50ml无菌离心管中加入与1)中液体等体积的淋巴细胞分离液,然后用吸管将血稀释液沿着管壁缓慢的加至淋巴细胞分离液的上层,于18℃下600g(升5,降1),离心22min;
3)离心后,用移液枪将中间白膜层吸出,放于干净的15ml离心管中,加入10ml预冷的PBS溶液,混匀;室温下,2000rpm/min离心5min,弃尽上清;
4)加入3ml红细胞裂解液(ACK)进行红细胞裂解,室温裂解3~5min后,加入10ml预冷的PBS进行终止裂解;室温下,2000rpm/min离心5min,弃尽上清;
5)用5ml PBS进行重悬细胞沉淀,进行细胞计数,放冰上备用。
(2)对步骤(1)获得的PBMC进行流式细胞术染色分析
1)取1million的PBMC单细胞悬液,加入5ml PBS混匀,2000rpm/min离心5min,弃尽上清;
2)用于分析人MDSCs的流式抗体染色组合为:HLA-DR-BV421、CD11b-B510、CD33-PE。
3)按照抗体说明书,抗体以1:200的比例使用PBS稀释,每个样本用100μl抗体稀释液进行染色,染色方法如下:将流式管于振荡器上振荡,充分混匀;于4℃冰箱避光染色30min。加入4ml PBS,2000rpm/min离心5min,弃去上清;加入300μl PBS重悬细胞后,使用流式分析仪(BD Canto II,USA)获取数据,并用FlowJo10软件进行分析,利用GraphPad 9.2.0计算各组数据中位数和计算P值,P<0.05视为差异具有统计学意义。
3、实验结果
结果如图1所示,与健康志愿者(Control)相比,先天性再生障碍性贫血(CAA)患者外周血中MDSCs的比例明显升高,而获得性再生障碍性贫血患者(AAA)外周血中MDSCs的比例明显下降,差异具有统计学意义。提示MDSCs可作为特异性生物标志物用于鉴别CAA和AAA,检测MDSCs在待测者外周血中含量的试剂即可作为鉴别CAA和AAA的试剂。
为了评价MDSCs在鉴别CAA和AAA中的诊断性能,进一步进行了ROC曲线分析,结果显示,AUC为0.9583,灵敏度为83.33%,特异性为100%(图2),说明MDSCs在用于鉴别CAA和AAA时具有很高的准确性。同时,MDSCs在用于诊断CAA时也具有很高的准确性,AUC可高达0.9053(图3)。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对以上实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (2)
1.检测人MDSCs在待测者生物样本中的含量的试剂在制备鉴别先天性再生障碍性贫血和获得性再生障碍性贫血试剂盒中的应用;所述人MDSCs为HLA-DR-/lowCD11b+CD33+的人MDSCs。
2.如权利要求1所述的应用,其特征在于,所述HLA-DR-/lowCD11b+CD33+的人MDSCs采用流式细胞术进行检测。
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