CN116479107A - 一种活动性肺结核病标志物及其应用 - Google Patents
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Abstract
本发明公开了一种活动性肺结核病标志物及其应用。属于检测标志物技术领域。本发明发现circ_0074371在结核病患者外周血单核细胞中显着下调，治疗好转后得到显著上调；并且circ_0074371可促进巨噬细胞向M1型极化并抑制结核菌在巨噬细胞内的存活，可为结核病临床诊断及治疗提供一种新的策略。
Description
技术领域
本发明涉及检测标志物技术领域，更具体的说是涉及一种活动性肺结核病标志物及其应用。
背景技术
结核病(tuberculosis，TB)一直是全球最紧迫的公共卫生问题之一。TB是一种慢性传染病，由结核分枝杆菌(M.tb)感染引起，其主要感染分布在肺部的先天免疫细胞。在先天免疫反应中发挥重要作用的巨噬细胞是控制结核病感染的关键。活化的巨噬细胞可通过溶酶体杀死M.tb，同时通过分泌促炎细胞因子如IFN-γ和TNF-α来诱导Th1细胞免疫反应清除M.tb。
近年来，随着临床结核病耐药及HIV感染等情况越发严重，潜伏感染者诊断越发困难。未来的目标应该集中在降低TB的发病率，而不仅仅是新药物开发。早期准确诊断和即时治疗是结核病控制的关键。目前，痰标本涂片镜检和痰培养仍是临床诊断TB使用的最广泛的工具，但是它的缺点也是非常明显的，痰标本涂片镜检敏感性太低，容易漏检；痰培养耗时太长，很难用于TB的筛检。因此，寻找并建立一种快速，灵敏和有效的肺结核病诊断和鉴定方法，是防止结核病快速传播的最有效措施。
根据目前关于circRNA的知识，circRNA具有几个显着特征，使其成为人类疾病的潜在生物标记。稳定性：由于缺乏游离5'和3'末端共价闭环结构，circRNA分子是高度抗核酸外切酶的RNaseR，这使得它们相比线性的RNA更稳定。circRNA在血浆中的平均半衰期超过48小时，比mRNA平均值的10小时长得多。通用性：circRNA被认为是分布在人类细胞中的最通用的分子，在某些情况下，circRNA比其线性同工型更为丰富。循环中的circRNA数量是非常普遍的，这归因于其结构的高度稳定性。特异性：circRNA以组织特异性和发育阶段特异性的方式表达，这使其成为特定疾病的潜在生物标记。特别是，许多研究表明，circRNA在癌性组织和非癌性组织之间都有明显的表达。而且在不同类型的癌细胞中circRNA的存在和丰度也很独特。保守性：在不同物种中发现circRNAs在进化上是保守的，这意味着以鼠类模式鉴定的某些circRNA生物标记物具有转化为人类临床应用的潜力。circRNA是具有高度稳定性、物种保守性以及细胞、组织特异性的非编码RNA，与其他RNA类型相比，这些特性使circRNA有可能被用作更理想的生物标志物和更合适的潜在治疗靶点。circRNA不仅在免疫细胞中丰富表达，还在感染及免疫调节中扮演着举足轻重的作用，可通过与miRNA相互作用参与调节巨噬细胞的极化作用，也可以诱导先天免疫基因表达，从而保护宿主免受病毒感染。
综上，如何提供一种辅助诊断肺结核病的circRNA标志物是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一种活动性肺结核病标志物及其应用。
本发明中，我们从活动性肺结核(Active pulmonary tuberculosis，APTB)患者和健康志愿者(HV)中分离出外周血单个核细胞(PBMC)，然后提取PBMCs中Total RNA。本发明中首次通过比较HV和APTB患者PBMCs的circ_0074371表达水平，研究了circ_0074371作为APTB早期诊断的潜在生物标志物的可能性。发现circ_0074371在APTB患者PBMC和结核菌感染的人单核巨噬细胞系THP-1中显著下调。此外，我们证明了circ_0074371可促进巨噬细胞向M1极化，抑制结核菌在巨噬细胞胞内的存活。本发明表明circ_0074371的表达下调相对于健康人具有特异性，其可促进巨噬细胞向M1极化，抑制结核菌在巨噬细胞胞内的存活，可作为一种新的临床诊疗标志物。
为了实现上述目的，本发明采用如下技术方案：
一种活动性肺结核病标志物，所述标志物为circ_0074371，其序列如SEQ ID NO：1所示；
ACCCCAAGACTGCTTCTGAGACAGAAACAGATATCTGTGCTGAATGGGAGATAAAGACCATCACTAGTGCTCTGAAGACCTACCTAAGAATGCTTCCAGGACCACTCATGATGTACCAGTTTCAAAGAAGTTTCATCAAAGCAGCAAAACTGGAGAACCAGGAGTCTCGGGTCTCTGAAATCCACAGCCTTGTTCATCGGCTCCCAGAGAAAAATCGGCAGATGTTACAGCTGCTCATGAACCACTTGGCAAA，SEQ ID NO：1。
进一步的，所述circ_0074371在活动性肺结核病患者的外周血单个核细胞表达下调。
进一步的，所述circ_0074371指示早期活动性肺结核病。
circ_0074371在制备活动性肺结核病特异性诊断试剂中的应用，circ_0074371的序列如SEQ ID NO：1所示。
一种活动性肺结核病诊断试剂盒，所述试剂盒包括能够对circ_0074371的表达量进行定量的试剂，circ_0074371的序列如SEQ ID NO：1所示。
进一步的，所述能够对circ_0074371的表达量进行定量的试剂包括RT-qPCR引物对，所述RT-qPCR引物对的序列如SEQ ID NO：2、SEQ ID NO：3所示；
TCCACAGCCTTGTTCATC，SEQ ID NO：2；
GTCTTTATCTCCCATTCAGC，SEQ ID NO：3。
circ_0074371在制备治疗活动性肺结核病药物中的应用，circ_0074371的序列如SEQ ID NO：1所示。
进一步的，circ_0074371可促进巨噬细胞向M1极化，可促进巨噬细胞的抗结核活性。
经由上述的技术方案可知，与现有技术相比，本发明取得的有益效果为：(1)外周血标本采集方便，标本容易处理。(2)检测耗时短，能够快速得出结果，利于疾病的快速诊断。(3)检测费用相对较低。(4)作为潜在的治疗靶点，有利于新药的开发。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为circ_0074371在APTB患者外周血PBMC中的表达检测，其中，A为circ_0074371在PBMC中的相对表达量差异；B为APTB患者治疗前后circ_0074371的相对表达量差异；C为ROC曲线下面积分析circ_0074371在APTB患者的诊断特异性和敏感性；
图2为胞内感染M.tb的单核/巨噬细胞中circ_0074371的表达检测，其中，A为人单核细胞中circ_0074371的相对表达量差异；B为胞内感染H37Rv的巨噬细胞中circ_0074371的相对表达量差异；C为胞内感染BCG的巨噬细胞中circ_0074371的相对表达量差异；
图3为扩增曲线及溶解曲线；
图4为circ_0074371的结构及反向接头序列验证，其中，A为circ_0074371的分子结构图；B为circ_0074371的反向接头系列示意图；C为RNaseR酶消化前后circ_0074371的表达差异；D为RNaseR酶消化前后线性RNAactin的表达差异；
图5为感染结核菌的巨噬细胞极化因子mRNA表达检测，其中，A为过表达circ_0074371的巨噬细胞感染结核菌后MI型极化因子的相对表达量；B为过表达circ_0074371的巨噬细胞感染结核菌后M2型极化因子的相对表达量；
图6为siRNA敲低circ_0074371促进GFP-BCG的胞内存活，其中，A为THP-1诱导的巨噬细胞感染GFP-BCG后流式细胞术检测散点图；B为比较GFP-BCG+巨噬细胞比例；C为比较GFP-BCG+巨噬细胞的平均荧光强度；D为circ_0074371的相对表达量差异；E为Actin mRNA的相对表达量差异。
具体实施方式
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，都属于本发明保护的范围。
本发明所需药剂为常规实验药剂，采购自市售渠道；未提及的实验方法为常规实验方法，在此不再一一赘述。
实施例1临床样本的收集、PBMCs的提取及单核细胞分选
1.临床样本的收集
分别收集31例活动性肺结核(APTB)患者和31例健康志愿者(HV)外周血样本，作为实验组和健康对照组，其人口学特征和临床基线特征如表1。两组间年龄和性别比较差异无统计学差异(P>0.05)。
表1 人口学特征和临床基线特征
| 分组 | APTB(n＝31) | HV(n＝31) |
| 年龄(岁) | (20～60) | (20～60) |
| 年龄(Mean±SEM) | 40.09±13.76 | 38.64±11.70 |
| 性别(男/女) | 15/16 | 15/16 |
2.外周血单个核细胞(PBMCs)的提取
PBMCs的提取主要参考文献(Zhang and Liu et al,2016)的步骤处理。
收集每个受试者外周血5ml于含EDTA抗凝血液收集管中，通过标准Ficoll(GEHealthcare，Little Chalfont，英国)密度梯度离心法分离新鲜血液中PBMCs。用台盼蓝排斥实验(所有实验>95％)测定细胞活力，然后将PBMCs保存于-80℃中便于下述的实验。
3.单核细胞的分选
根据EasySepTM Human CD14 Positive Selection Kit(Stemcell tech，cat#：18058)操作说明书进行，具体步骤如下：
①样本准备：保持每mL样本中细胞数为108(1×108细胞/mL)，细胞数低于107的情况用0.1mL缓冲液重悬，重悬后转移入5mL流式管中；
②每1×108细胞样本中加入100μL(100μL/mL样本)CD14+ Cocktail，混匀后室温孵育15min；
③加入Magnetic Particles(100μL/mL样本)，移液枪上下吹打混匀5次，室温孵育10min；
④加入缓冲液至总体积为2.5mL，移液枪温和混匀2～3次，然后放入磁力架孵育5min；
⑤拿起磁力架，沿着一个方向倾倒流式管内液体于另一流式管中，挂在管口的液体用Tips吸净，拿下流式管，管内即为分离的目的CD14+细胞；
⑥重复④，⑤一次；
⑦细胞计数后用含10％胎牛血清的RPMI1640重悬备用。
实施例2结核感染人单核细胞系THP-1模型构建
1.人THP-1源巨噬细胞的诱导贴壁
准备THP-1细胞，根据实验需要用适当的细胞铺板，过夜。第二天加入PMA(联科生物)使其终浓度为100ng/mL，处理24h后诱导成THP-1源巨噬细胞。用37℃温预的PBS洗三次，进行后续实验。
2.M.tb感染人单核细胞系THP-1模型构建
诱导贴壁的THP-1源巨噬细胞，分别用10个BCG(MOI＝10)和1个H37Rv(MOI＝1)感染巨噬细胞，感染6h后，PBS洗除胞外未被吞噬的M.tb，添加新鲜培养基根据需要继续培养一定时间。
实施例3circ_0074371的qRT-PCR验证表达差异
采用qRT-PCR检测实施例1中31例APTB患者和31例HV样品中外周血PBMC中circ_0074371的表达，用淋巴细胞分离液分离得到PBMC，TRIzol充分裂解细胞后，氯仿抽提总RNA，定量后每个样本均取450ngRNA进行逆转录构建DNA文库，再进行qRT-PCR检测circRNAcirc_0074371的相对表达量。
我们发现circ_0074371在APTB患者中显著下调(图1A)，说明circ_0074371的表达下调可作为APTB患者早期诊断的生物标志物。
通过对同一个患者在临床抗结核治疗一个月后的样本检测发现，在治疗一个月好转后，circ_0074371的相对表达量得到显著的上调(图1B)，说明circ_0074371的表达与结核病愈后相关，是疾病转归的诊断标志物。
通过ROC曲线下分析，发现APTB与HV相比具有显著的特异性(图1C)，是APTB诊断的标志物。
通过磁珠分选单核细胞并对circ_0074371的相对表达量进行检测发现，在该群细胞中circ_0074371在结核菌感染的病人中同样是显著下调的(图2A)。说明单核细胞是circ_0074371表达下调的特异性细胞群。
我们进一步用实施例2构建的M.tb感染THP-1巨噬细胞模型进行验证，结果表明，circ_0074371在M.tb感染的巨噬细胞中也下调(图2B、图2C)。
通过设计接头引物，进行定量PCR，其扩增曲线及溶解曲线如图3所示，显示所设计的circ_0074371引物(F:TCCACAGCCTTGTTCATC，SEQ ID NO：2；R:GTCTTTATCTCCCATTCAGC，SEQ ID NO：3)可成功扩增该环状分子且具有特异性。
实施例4circ_0074371的生物结构特性
circ_0074371属于环形RNA，为母基因ARHGAP26的15、16、17号外显子反向剪接而成的闭环单链RNA；具有耐核酸酶的生物学特性。根据这些特性，我们对其反向剪接位点进行了测序和耐核酸酶特性的验证，结果均符合circ_0074371的结构和生物学特性。见图4。图4A为circ_0074371的基因结构图；图4B显示PCR产物测序结果与circ_0074371基因序列比对分析是一致的；图4C显示RNA经过RNA酶R处理之后，环状RNA circ_0074371可抵抗RNA酶R的降解，而图4D验证了RNA酶R可显著降解线性RAN Actin。
实施例5circ_0074371促进巨噬细胞的抗结核活性
通过构建慢病毒表达载体，并成功表达成环的circ_0074371，进一步用慢病毒感染THP-1细胞，通过流式分选筛选到高表达circ_0074371的细胞株，经过连续传代均证实可以稳定高表达circ_0074371。通过体外结核菌BCG感染THP-1来源的巨噬细胞，qRT-PCR检测证实高表达circ_0074371的巨噬细胞向M1极化(图5)。
利用带荧光的M.tb感染，通过siRNA干扰下调circ_0074371的表达后可显著抑制结核菌在巨噬细胞胞内的存活(图6)。流式细胞术检测巨噬细胞内荧光M.tb的表达，发现下调circ_0074371的表达后(Si：敲低组；NC：对照组)，胞内M.tb的含量上调。流式细胞散点图(图6A)、柱状统计图(图6B)、统计巨噬细胞胞内平均荧光值(图6C)均提示下调circ_0074371的表达可促进结核菌的胞内存活。同时通过qRT-PCR验证了下调巨噬细胞中circ_0074371的表达情况，在靶向circ_0074371反向接头的siRNA干扰后可显著下调circ_0074371的表达(图6D)；并在同时做了阳性对照组，该体系可显著下调内参基因的表达(图6E)。
小结：
本发明证明了PBMCscirc_0074371在APTB患者中显著性的下调。下调的circ_0074371对APTB患者早期诊断具有一定的意义。因此，利用qRT-PCR方法检测circ_0074371在APTB患者中的表达情况可以对疾病进行诊断。
circ_0074371可促进巨噬细胞向M1极化，可促进巨噬细胞的抗结核活性，利于新药的开发。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (8)
1.一种活动性肺结核病标志物，其特征在于，所述标志物为circ_0074371，其序列如SEQ ID NO：1所示。
2.如权利要求1所述的标志物，其特征在于，所述circ_0074371在活动性肺结核病患者的外周血单个核细胞表达下调。
3.如权利要求1所述的标志物，其特征在于，所述circ_0074371指示早期活动性肺结核病。
4.circ_0074371在制备活动性肺结核病特异性诊断试剂中的应用，其特征在于，circ_0074371的序列如SEQ ID NO：1所示。
5.一种活动性肺结核病诊断试剂盒，其特征在于，所述试剂盒包括能够对circ_0074371的表达量进行定量的试剂，circ_0074371的序列如SEQ ID NO：1所示。
6.如权利要求5所述的诊断试剂盒，其特征在于，所述能够对circ_0074371的表达量进行定量的试剂包括RT-qPCR引物对，所述RT-qPCR引物对的序列如SEQ ID NO：2、SEQ ID NO：3所示。
7.circ_0074371在制备治疗活动性肺结核病药物中的应用，其特征在于，circ_0074371的序列如SEQ ID NO：1所示。
8.如权利要求7所述的应用，其特征在于，circ_0074371可促进巨噬细胞向M1极化，可促进巨噬细胞的抗结核活性。
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