CN113025730B - 一种与肝硬化相关的肝内菌群标志物及其应用 - Google Patents
一种与肝硬化相关的肝内菌群标志物及其应用 Download PDFInfo
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Abstract
本发明公开了一种与肝硬化相关的肝内菌群标志物及其应用，所述肝内菌群标志物为Stenotrophomonas maltophilia(S.maltophilia，嗜麦芽窄食单胞菌)。发明人通过收集肝硬化患者与正常人群的组织样本，由样本中的微生物出发，筛选出其中与肝硬化相关性较高的肝内菌群，利用此类菌群作为肝硬化生物标志物，可以有效用于肝硬化患者的诊断。
Description
技术领域
本发明属于微生物组学和医学分子生物学技术领域，具体涉及一种与肝硬化相关的肝内菌群标志物及其应用。
背景技术
肝硬化是临床常见的慢性进行性肝病，由一种或多种病因长期或反复作用形成的弥漫性肝损害。在我国大多数为肝炎后肝硬化，少部分为酒精性肝硬化。肝硬化是肝细胞发生坏死及炎症刺激时，肝脏内纤维结缔组织异常增生的病理过程；过度纤维化使肝脏萎缩变硬，最终引起肝硬化失代偿或肝功能衰竭而导致患者死亡。尽管有肝移植手术能够从根本治疗终末期肝硬化及其并发症，但由于发病率高，加之供肝匮乏、围术期风险高，肝移植仅使微乎其微的患者受益，大部分患者还是死于其并发症。
最近，人们发现肿瘤组织中而不是肠道或口腔中的菌群与肿瘤的发展密切相关。肿瘤内细菌类型、菌群在特定肿瘤类型以及肿瘤亚型中的预测功能与患者的吸烟状况、肿瘤免疫疗法敏感性之间存在相关性。但目前，其他疾病的组织内相关菌群尚未有报道。
发明内容
本发明的目的之一在于提供了一种与肝硬化相关的肝内菌群标志物。本发明首次提出肝内菌群嗜麦芽窄食单胞菌的丰度与肝硬化有正相关性、嗜麦芽窄食单胞菌的丰度可作为肝硬化生物标志物，并且揭示嗜麦芽窄食单胞菌在肝硬化疾病中的促进作用，为肝硬化的治疗提供基础。
本发明的目的之二在于提供了一种用于诊断肝硬化的产品。
本发明的目的之三在于提供了一种用于诊断肝硬化的系统。
本发明的第一方面提供了一种诊断肝硬化的肝内菌群标志物，所述肝内菌群标志物为Stenotrophomonas maltophilia(S.maltophilia，嗜麦芽窄食单胞菌)。
本发明的第二方面提供了本发明的第一方面所述的肝内菌群标志物在制备用于诊断肝硬化的产品中的应用，所述产品包括检测肝内菌群标志物的试剂。
进一步，所述试剂是对所述肝内菌群标志物具有特异性的探针、引物或抗体。
进一步，所述引物是特异性扩增所述肝内菌群标志物的16SrRNA的引物。
本发明的第三方面提供了本发明的第一方面所述的肝内菌群标志物诊断肝硬化的系统，包括：
核酸样本分离单元，用于从检测对象中分离肝内菌群核酸样本；
测序单元，用于对分离的肠道菌群核酸样本进行测序，以获得测序结果；
数据处理单元，用于根据测序结果，对肠道菌群中的所述微生物标志物的相对丰度进行检测，将所得到的相对丰度值进行分析，得出上述微生物标志物的临界值；
结果判定单元，用于将数据处理单元得到的微生物标志物的临界值与设定诊断值进行比较。
利用该系统，可以检测出被发明的标志物在肝内菌群中的相对丰度。因此，可以通过得到的相对丰度与预定的临界值进行比较，从而确定对象个体是否为肝硬化患者以及疾病进程。
本发明的第四方面提供了一种诊断肝硬化的试剂盒，包括检测本发明的第一方面所述的肝内菌群标志物丰度的试剂。
进一步，所述试剂包括检测所述肝内菌群标志物的特异性的探针、引物或抗体。
进一步，所述特异性的引物是能够检测所述肝内菌群标志物16SrRNA的引物。
进一步，所述产品还包括提取微生物基因组DNA等的试剂。
本发明的第五方面提供了本发明的第一方面所述的肝内菌群标志物在构建预测肝硬化的计算模型中的应用。
本发明的第六方面提供了本发明的第一方面所述的肝内菌群标志物在制备治疗肝硬化的药物组合物中的应用。
与现有技术比较，本发明的有益效果：
本发明首次发现Stenotrophomonas maltophili与肝硬化相关，其丰度在肝硬化患者与健康人群中呈现显著性差异，ROC曲线分析其作为检测变量具有较高的特异性和灵敏度，因此，可以将Stenotrophomonas maltophilia作为检测标志物应用于肝硬化患者的诊断。
附图说明
图1显示肝内菌群嗜麦芽窄食单胞菌在正常肝组织和肝硬化组织中的丰度的差异情况；
图2显示嗜麦芽窄食单胞菌丰富作为检测变量的ROC曲线；
图3显示嗜麦芽窄食单胞菌对肝硬化的作用。
具体实施方式
下面结合具体的实施例进一步说明本发明，本发明的实施例仅限于解释本发明，本发明的保护内容不局限于以下实例。
下述实施例中所使用的的实验方法如无特殊说明，均为常规方法。
下述实例中所使用的的试剂等，如无特殊说明，均可从商业途径得到。
实施例1与肝硬化相关的肝内菌群筛选
1、样本获取：分别收集33例正常肝组织，记为N组、22例肝硬化组织样本，记为C组。所有样本在无菌条件下采集样品，在-80℃冷冻保存，准备进行后续测序。上述所有样本的取得均通过伦理委员会同意，数据收集遵循《赫尔辛基宣言》所概述的原则。
2、16S rRNA测序
我们委托天津诺华基因生物信息技术有限公司进行16S rRNA测序。
3、数据分析
数据预处理
将IonS5TMXL下机数据导出fastq文件。根据barcode序列区分各个样本的数据。测序得到的原始数据(Raw Data)，存在一定比例的干扰数据(Dirty Data)，为了使信息分析的结果更加准确、可靠，首先对原始数据进行拼接、过滤，得到有效数据(Clean Data)。为研究各样本的物种组成，对所有样本的Effective Tags，以97％的一致性(Identity)进行OTUs(Operational Taxonomic Units)聚类，然后对OTUs的代表序列进行物种注释。根据OTUs聚类结果，一方面对每个OTU的序列做物种注释，得到对应的物种信息和基于物种的丰度分布情况。同时，对OTUs进行丰度、Alpha多样性计算、Venn图和花瓣图等分析，以得到样本内物种丰富度和均匀度信息、不同样本或分组间的共有和特有OTUs信息等。
2菌群物种差异分析
根据所有样本(分组)在属水平的物种注释及丰度信息，采用最大值排序法，选取丰度排名前35的属(分组的丰度为组内所有样本的平均丰度)，根据其在每个样本中的丰度信息，从物种层面进行聚类，使用R软件pheatmap包绘制成热图，便于发现哪些物种在哪些样本中聚集较多或含量较低。为避免丰度过低造成影响，在丰度排名前十的物种内做组间的T-test检验，找出差异显著(p值<0.05)的物种。
3结果
结合临床数据，我们发现差异菌中嗜麦芽窄食单胞菌与肝硬化之间存在较高的相关性，物种差异性分析结果显示，肝硬化的病人组织内嗜麦芽窄食单胞菌丰度明显高于没有肝硬化的病人(见图1)。提示，嗜麦芽窄食单胞菌可作为检测标志物应用于肝硬化的诊断。
实施例2与肝硬化的相关性
1、将实施例1的收集的病人临床信息与测序所得结果进行分析
2、结果
将嗜麦芽窄食单胞菌作为检测变量，分析得到ROC曲线(见图2)。ROC曲线结果显示，AUCZ值为0.782，提示将嗜麦芽窄食单胞菌应用于肝硬化的诊断具有较高的诊断效能。
实施例3研究嗜麦芽窄食单胞菌对肝硬化细胞功能的影响
1、嗜麦芽窄食单胞菌培养
嗜麦芽窄食单胞菌的标准菌株购自中国北京BeNaCultureCollection(BNCC)。用哥伦比亚血琼脂平板培养细菌。取单菌落在脑心浸液肉汤(BHI)中搅拌，于37℃培养箱培养。
2、细胞培养
LX-2细胞购自美国模式培养物集存库(ATCC)，用含10％胎牛血清的DMEM高汤培养基，于37℃、5％CO2的培养箱中培养。
3、细胞刺激
将LX-2分为四组，分别为空白对照组、BHI对照组、培养基对照组和嗜麦芽窄食单胞菌组，分别在LX2培养基中不加入任何物质、加入BHI、加入培养基和加入嗜麦芽窄食单胞菌。培养48h后收集各组细胞于1.5ml离心管中，加入1mlTrizol，混匀，室温静置5min，储存于-80℃冰箱保存。
4.、细胞总RNA提取：利用异硫氰酸胍苯酚抽提法提取总RNA，具体步骤如下：
1)取出冻存的细胞样本放置在碎冰上解冻，解冻完加入0.2ml氯仿，震荡15s，静置2min；
2)使用离心机进行离心，离心程序为4℃、12000g、15min，离心结束，取上清放入新的离心管；
3)加入0.5ml异丙醇，将管中的液体轻轻混匀，室温静置10min；
4)使用离心机进行离心，离心程序为4℃、12000g、10min，弃掉上清；
5)加入1ml 75％的乙醇溶液，轻轻洗涤沉淀；
6)使用离心机进行离心，离心程序为4℃、7500g、5min，弃上清；
7)晾干，加入适量的DEPC水溶解。
5、RNA样本的质量分析
使用NanoDrop1000分光光度计检测RNA样品，确保OD260/OD280为1.8-2.0。
6、逆转录
使用TaKaRa公司的PrimeScriptTM RT Master Mix试剂盒，具体步骤如下：按下列组分配置逆转录反应液
轻柔混匀后进行逆转录反应，条件如下：
| 温度 | 时间 |
| 37℃ | 15min |
| 85℃ | 5sec |
| 4℃ | 10min |
得到的反应液放至4℃保存
7、qPCR
1)引物设计
| Gene | Forward(5′-3′) | Reverse(5′-3′) |
| Acta2 | ACTGAGCGTGGCTATTCCTCCGTT | GCAGTGGCCATCTCATTTTCA |
| Tgfb1 | CGACTACTACGCCAAGGA | GAGAGCAACACGGGTTCA |
| Timp1 | GCACATCACTACCTGCAGTC | GAAACAAGCCCACGATTTAG |
| Col1a1 | GGAACACCTCGCTCTCCA | GGGATTCCCTGGACCTAAAG |
| GAPDH | GCCCAATACGACCAAATCC | AGCCACATCGCTCAGACAC |
2)配置PCR反应体系：
使用TaKaRa公司的TaKaRa TB GreenTM Premix Ex TaqTM II试剂盒，按照下表配置反应液：
| 试剂 | 使用量 |
| TB GreenPremix Ex Taq II(TliRNaseH Plus 2× | 10ul |
| PCR Forward Primer10μM | 0.8ul |
| PCR Reverse Primer10μM | 0.8ul |
| ROX Reference Dye or Dye II(50×)*2 | 0.4ul |
| RT反应液(cDNA溶液))*3 | 2ul |
| 灭菌水 | 6ul |
3)PCR反应条件：：95℃30sec，(95℃5sec，60℃30sec)×40个循环。在荧光定量PCR仪上进行PCR反应，以GAPDH作为参照基因，通过ΔΔCT法进行相对定量。
8、结果
嗜麦芽窄食单胞菌组的肝星状细胞活化相关基因表达相对于其他对照组显著增高(见图3)，表明嗜麦芽窄食单胞菌可促进肝星状细胞活化，同时表明嗜麦芽窄食单胞菌可存进肝纤维化，从而导致和加快肝硬化进程。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。
序列表
<110> 江苏省肿瘤防治研究所（江苏省肿瘤医院）
<120> 一种与肝硬化相关的肝内菌群标志物及其应用
<141> 2021-01-14
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Artificial Sequence
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actgagcgtg gctattcctc cgtt 24
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<212> DNA
<213> Artificial Sequence
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gcagtggcca tctcattttc a 21
<210> 3
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<212> DNA
<213> Artificial Sequence
<400> 3
cgactactac gccaagga 18
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<212> DNA
<213> Artificial Sequence
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gagagcaaca cgggttca 18
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<211> 20
<212> DNA
<213> Artificial Sequence
<400> 5
gcacatcact acctgcagtc 20
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<212> DNA
<213> Artificial Sequence
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gaaacaagcc cacgatttag 20
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<212> DNA
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ggaacacctc gctctcca 18
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gggattccct ggacctaaag 20
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gcccaatacg accaaatcc 19
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<213> Artificial Sequence
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agccacatcg ctcagacac 19
Claims (4)
1.一种肝内菌群标志物在制备用于肝硬化诊断的产品中的应用，其特征在于，所述肝内菌群标志物为Stenotrophomonas maltophilia 嗜麦芽窄食单胞菌。
2.根据权利要求1 所述的应用，其特征在于，所述产品包含检测肝内菌群标志物的试剂。
3.根据权利要求2 所述的应用，其特征在于，所述试剂是对所述肝内菌群标志物具有特异性的探针或引物。
4.一种肝内菌群标志物在构建预测肝硬化的计算模型中的应用，其特征在于，所述肝内菌群标志物为如权利要求1 中所述的Stenotrophomonas maltophilia 嗜麦芽窄食单胞菌。
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