CN114689747A - 咪唑丙酸作为生物标志物用于预测溃疡性结肠炎的应用以及成套用具 - Google Patents
咪唑丙酸作为生物标志物用于预测溃疡性结肠炎的应用以及成套用具 Download PDFInfo
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Abstract
本发明提供了咪唑丙酸作为生物标志物在制备用于预测溃疡性结肠炎的试剂盒中的应用以及相关的检测试剂盒。所述生物标志物能够有效地作为预测溃疡性结肠炎的早期诊断标记物，具有准确、早期并且精确度高的优点。
Description
技术领域
本发明涉及诊断领域，更具体地涉及咪唑丙酸作为生物标志物用于预测溃疡性结肠炎的应用，以及包含咪唑丙酸作为生物标志物的成套用具，例如检测试剂盒。
背景技术
溃疡性结肠炎(UC)是一种局限于结肠和直肠粘膜及黏膜下层的慢性炎症性疾病，其主要症状包括腹痛、腹泻、便血、发热、以及体重下降，具有病程长，症状反复难愈的特点。长期患有UC是导致结肠癌症的危险因素之一。根据中国疾病预防控制中心报告的数据，从2005年到2014年，中国已有35万肠炎患者被确诊。到2025年，预计中国肠炎病例将达到150万。
UC的发病机制目前尚不十分清楚，但大量的研究表明肉食性饮食的增加与更高的溃疡性结肠炎风险相关。以肉类为主的西式饮食逐渐替代了中国居民以植物来源为主的传统饮食，这可能是导致中国UC患者逐年增加的关键因素之一。然而，肉类食物引起UC的机制目前还不十分清楚。
人体肠道中定植着大量的微生物，它们参与食物中氨基酸等营养物质的代谢与吸收。其代谢产生的小分子物质通过吸收进入宿主体内，与多种信号通路相互作用，影响肠道生理状态的稳态。组氨酸是大量存在于肉类食物中的人体必须氨基酸，其被大肠中丰富的肠道菌消化代谢，产生一种独特的肠菌代谢物—咪唑丙酸(Imidazole propionate，IMP)。已有报道指出，咪唑丙酸在人体血液中含量升高与饮食引起的代谢性疾病的发生密切相关(例如，参见非专利文献1)。然而，目前尚未有研究报道咪唑丙酸对UC的发生和发展有何影响。
现有技术文献
非专利文献1：Koh,A.,et al.,Microbially Produced Imidazole PropionateImpairs Insulin Signaling through mTORC1.Cell,2018.175(4):p.947-961.
发明内容
针对上述问题，本申请人进行了深入研究，结果发现，通过检测UC患者肠镜活检组织中咪唑丙酸的含量，发现相较于非溃疡肠组织，溃疡肠组织中咪唑丙酸含量显著升高。进一步的动物实验证实，咪唑丙酸可引起小鼠体重减轻、疾病活动指数(disease activityindex，DAI)增加、炎症因子表达增加、粘膜组织损伤、肠屏障通透性增加等结肠炎症状，提示IMP含量增加可能是引发溃疡性结肠炎的危险因素，为开发IMP作为UC的一个早期诊断标记物提供科学依据，以便可以提前进行干预和治疗。
本发明提供以下方面的内容：
1.咪唑丙酸在制备用于预测溃疡性结肠炎的生物标志物中的应用。
2.一种用于预测溃疡性结肠炎的成套用具，包含：咪唑丙酸、用于提取样品的提取溶剂、用于高效液相色谱-串联质谱检测的流动相、以及用于高效液相色谱-串联质谱检测的色谱柱。
3.根据上述项目2所述的成套用具，其特征在于所述成套用具包括诊断试剂盒。
4.根据上述项目2或3所述的成套用具，其特征在于，所述提取溶剂包括乙腈。
5.根据上述项目2或3所述的成套用具，其特征在于，所述流动相包含体积比为5-95:5-95的水和乙腈。
6.根据上述项目2或3所述的成套用具，其特征在于，所述色谱柱选自氨基色谱柱和Biphenyl色谱柱中的至少一种。
7.咪唑丙酸作为生物标志物在制备用于预测溃疡性结肠炎的试剂盒中的应用。
8.根据上述项目7所述的应用，其特征在于，所述试剂盒还包含用于提取样品的提取溶剂、用于高效液相色谱-串联质谱检测的流动相和用于高效液相色谱-串联质谱检测的色谱柱。
9.根据上述项目8所述的应用，其特征在于，所述提取溶剂包括乙腈。
10.根据上述项目8所述的应用，其特征在于，所述流动相包含体积比为5-95:5-95的水和乙腈，所述色谱柱选自氨基色谱柱和Biphenyl色谱柱中的至少一种。
附图说明
图1为显示UC患者的非溃疡结肠组织和溃疡结肠组织中的IMP含量。图中，*表示与非溃疡结肠组织相比，p<0.05。
图2A至图2E是显示IMP对C57BL/6小鼠结肠的影响的图。其中，图2A显示的是IMP处理对小鼠体重和DAI(疾病活动指数)的影响；图2B显示的是IMP处理对结肠通透性的影响；图2C显示的是IMP处理对结肠粘膜结构的影响；图2D显示的是IMP处理后结肠紧密连接蛋白表达的变化；图2E显示的是IMP处理后结肠组织中炎症因子表达变化。*表示与正常组相比，p<0.05；**表示与正常组相比，p<0.01；***表示与正常组相比，p<0.001。
具体实施方式
下面结合具体实施例对本发明所述的技术方案和有益效果做进一步的说明。
患者的选择
对于2020年5月至2021年5月内吉林大学第一医院消化科收治的溃疡性结肠炎患者6例，收集肠镜活检组织(包括溃疡结肠组织和非溃疡结肠组织)。本研究由天津中医药大学伦理委员会批准，并在研究开始前获得所有受试者的知情同意书。
需要说明的是，参照《炎症性肠病诊断与治疗的共识意见(2018，北京)》溃疡性结肠炎部分作为临床诊断依据。结合患者临床表现、结肠镜检查和黏膜活检结果综合诊断为溃疡性结肠炎。
1.UC患者肠镜活检组织中IMP含量检测
参照下述文献[1]，对UC患者肠镜活检组织中的IMP进行提取和检测，具体方法为：取组织样本，加入3倍体积冰乙腈，涡旋震荡5分钟，4℃静置30分钟，12000g离心5分钟，取上清液，过滤。取过滤后的上清液100μl，加入400μl双蒸水稀释，然后直接进样。
IMP标准品(购自Bachem AG,Bubendorf,Switzerland)使用甲醇/水(v/v＝1:1)充分溶解，配置成浓度为1mg/mL的母液。使用甲醇/水(v/v＝1:1)逐次稀释成一系列的浓度：0.5、1、2、5、10、20、50、100ng/ml，并制作标准曲线。采用与前述的样品相同的处理方式离心及过滤后进样。
采用质谱多反应监测技术(Multiple Reaction Monitoring,MRM)检测结肠组织中IMP含量。
参考文献[1]：Koh,A.,et al.,Microbially Produced Imidazole PropionateImpairs Insulin Signaling through mTORC1.Cell,2018.175(4):p.947-961.
色谱条件如下：
色谱柱：Kinetex Bi-phenyl 100A column(3×50mm with 2.6μm particles,Phenomenex)
流动相：A：水(0.1％甲酸)，流动相B：乙腈(0.1％甲酸)
流速：0.5ml/min
柱温：40℃
进样量：5μl
洗脱程序：
质谱条件：
数据采集和处理软件：Analyst TF 1.7；MasterView 1.0
数据定量软件：MultiQuant 3.0.2
离子源条件：ESI+
MS/MS检测方法建立：将优化好的特征母离子、碎片子离子、轰击电压，碰撞口入口电压、轰击电压、碰撞口出口电压，保留时间信息输入到Analyst软件中，通过多反应检测模式对代谢产物进行正负离子转换同时扫描，获得相应的色谱图；
数据处理分析：通过MultiQuant分析软件对MS/MS扫描所得色谱峰进行面积积分，通过添加的内标化合物的峰面积，对各个样品进行归一化，利用偏最小二乘分析对各样品中的差异进行分型，通过VIP分析找出各组间有显著性差异的化合物，进行代谢通路分析。
实验结果如图1所示。图1结果显示，UC患者溃疡结肠组织中的IMP含量显著高于非溃疡肠组织。
2.IMP对C57BL/6小鼠结肠的影响
(i)动物饲养及处理
20只健康雄性C57BL/6小鼠(北京维通利华实验动物技术有限公司)适应性喂养一周后，随机分为正常组和IMP给药组，每组10只。对于IMP给药组小鼠，采用异氟烷吸入的方式麻醉，用石蜡油润滑灌注针，由肛门插入约2-3cm，缓慢推入100μl IMP(100mg/kg)溶液。每日早晚各给药一次，持续5天。第6天，避光灌胃给予小鼠以FD-4(600mg/kg)，4h后，眼内眦取血，颈椎脱臼处死小鼠，分离结肠，用生理盐水冲洗后于液氮速冻，-80℃冰箱保存。
实验期间每日称量记录小鼠体重，观察小鼠活动情况、精神状态等，记录小鼠粪便隐血以及粪便形态，统计小鼠疾病活动指数。小鼠血清于激发波长480nm、发射波长520nm下使用SpectraMax M5进行荧光测定。取近盲肠端1cm结肠组织，生理盐水洗净后于4％多聚甲醛中固定，石蜡包埋，参照文献[2]报道的方法进行H&E染色及AB-PAS染色。
所有动物实验都得到了天津中医药大学科学技术委员会和动物使用与护理委员会的批准。
参考文献[2]：Y Zhao,Luan H,Gao H,et al.Gegen Qinlian decoctionmaintains colonic mucosal homeostasis in acute/chronic ulcerative colitis viabidirectionally modulating dysregulated Notch signaling[J].Phytomedicine,2020,68:153182.
(ii)q-PCR检测结肠组织中炎症因子表达
参照文献[3,4]报道的方法，对结肠组织中的炎症因子进行检测，具体方法为：称取30mg结肠组织，加入1ml TRIzol试剂(北京全式金生物技术股份有限公司)，提取总RNA，用NanoDrop 2000测定RNA浓度。采用High-Capacity cDNA Reverse Transcription Kit试剂盒(康为世纪生物科技股份有限公司)进行逆转录，合成cDNA。采用SYBR Green PCRMaster Mix试剂盒(康为世纪生物科技股份有限公司)及Applied Biosystems 7500Real-time PCR System进行PCR反应扩增，以GAPDH作为内参。
采用的引物序列为
NF-κB:Forward CCTCTCTCGTCTTCCTCCAC；Reverse GTTGCGGAAGGATGTCTCC；
iNOS:Forward GGGTCACAACTTTACAGGGAGT；Reverse GAGTGAACAAGACCCAAGCG；
IL6:Forward GTCCTTCCTACCCCAATTTCCA；Reverse TAACGCACTAGGTTTGCCGA；
GAPDH:Forward GGTGAAGGTCGGTGTGAACG；Reverse CTCGCTCCTGGAAGATGGTG。
反应条件为：95℃预变性10min，95℃变性15s，60℃退火60s，72℃延伸5min，共40个循环。应用2-ΔΔCT方法计算各基因相对含量，每组实验n＝6。
参考文献[3]：Vivinus-Nébot M,Frin-Mathy G,Bzioueche H et al.Functionalbowel symptoms in quiescent inflammatory bowel diseases:role of epithelialbarrier disruption and low-grade inflammation.[J].Gut,2014,63:744-52.
参考文献[4]：Gu Guangli,Lv Xiaodan,Liu Gengfeng et al.Tnfaip6 Secretedby Bone Marrow-Derived Mesenchymal Stem Cells Attenuates TNBS-Induced Colitisby Modulating Follicular Helper T Cells and Follicular Regulatory T CellsBalance in Mice.[J].Front Pharmacol,2021,12:734040.
(iii)Western blot检测结肠组织中紧密连接蛋白表达
参照文献[5]报道的方法，对结肠组织中紧密连接蛋白进行检测，具体方法为：称取30mg结肠组织，加入300μl RIPA蛋白裂解液，提取蛋白。采用BCA(Bicinchoninic Acid)蛋白定量法检测样品蛋白含量。采用SDS-PAGE电泳法，按照预定程序(80V，50min；110V，50min)进行凝胶电泳，电泳结束后，将蛋白转移至PVDF膜。5％脱脂奶粉封闭后，于4℃孵育一抗过夜(Occludin 1:5000；ZO-11:1000；β-actin 1:1000)。二抗(HRP山羊抗兔多克隆抗体1:10000)孵育1h后，用化学发光法检测目标蛋白的表达。
参考文献[5]：Yang Mingyue,Jia Wenxiu,Wang Dong et al.Effects andMechanism of Constitutive TL1A Expression on Intestinal Mucosal Barrier inDSS-Induced Colitis.[J].Dig Dis Sci,2019,64:1844-1856.
(3)统计学处理
实验结果采用SPSS 20.0统计学软件分析，计量资料以均数±标准差
表示，样本比较采用独立样本t检验分析，p值小于0.05判定为样本间具有显著差异。
实验结果
图2A至图2E的结果显示，IMP处理可引起小鼠结肠炎样症状，包括体重下降、DAI指数增加、结肠通透性增加、以及肠粘膜结构损坏伴中性粒细胞浸润及杯状细胞减少。另外，IMP导致结肠组织中紧密连接蛋白(Occludin、ZO-1)显著减少，并且炎症相关因子NF-κB、iNOS和IL6表达显著增加。动物实验结果表明，IMP可引起结肠炎症及肠屏障损坏，可作为溃疡性结肠炎的一个早期诊断标记物。
以上所述的实施例只是本发明的一种较佳的方案，并非对本发明作任何形式上的限制，在不超过权利要求所记载的技术方案的前提下，还可以有其他的变形。可以理解的是，以上实施例仅仅是为了说明本公开的原理而采用的示例性实施例，然而本公开并不局限于此。对于本领域普通技术人员而言，在不脱离本公开的精神和实质的情况下，可以做出各种变型和改进，这些变型和改进也视为本公开的保护范围。
Claims (10)
1.咪唑丙酸在制备用于预测溃疡性结肠炎的生物标志物中的应用。
2.一种用于预测溃疡性结肠炎的成套用具，包含：
咪唑丙酸；
用于提取样品的提取溶剂、
用于高效液相色谱-串联质谱检测的流动相、以及
用于高效液相色谱-串联质谱检测的色谱柱。
3.根据权利要求2所述的成套用具，其特征在于，所述成套用具包括诊断试剂盒。
4.根据权利要求2或3所述的成套用具，其特征在于，所述提取溶剂包括乙腈。
5.根据权利要求2或3所述的成套用具，其特征在于，所述流动相包含体积比为5-95:5-95的水和乙腈。
6.根据权利要求2或3所述的成套用具，其特征在于，所述色谱柱选自氨基色谱柱和Biphenyl色谱柱中的至少一种。
7.咪唑丙酸作为生物标志物在制备用于预测溃疡性结肠炎的试剂盒中的应用。
8.根据权利要求7所述的应用，其特征在于，所述试剂盒还包含用于提取样品的提取溶剂、用于高效液相色谱-串联质谱检测的流动相和用于高效液相色谱-串联质谱检测的色谱柱。
9.根据权利要求8所述的应用，其特征在于，所述提取溶剂包括乙腈。
10.根据权利要求8所述的应用，其特征在于，所述流动相包含体积比为5-95:40-50的水和乙腈，
所述色谱柱选自氨基色谱柱和Biphenyl色谱柱中的至少一种。
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| CN112689755A (zh) * | 2018-09-11 | 2021-04-20 | 梅塔博隆股份有限公司 | 用于检测和定量微生物区系相关代谢物的质谱测定法 |
| WO2021170848A1 (en) * | 2020-02-28 | 2021-09-02 | Academisch Medisch Centrum | Intervention strategy for prevention or treatment of diabetes mellitus, autoimmune disease, inflammatory disease or cardiovascular disease |
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| CN104768560A (zh) * | 2012-08-29 | 2015-07-08 | 加州理工学院 | 孤独症谱系障碍的诊断和治疗 |
| CN112689755A (zh) * | 2018-09-11 | 2021-04-20 | 梅塔博隆股份有限公司 | 用于检测和定量微生物区系相关代谢物的质谱测定法 |
| WO2021170848A1 (en) * | 2020-02-28 | 2021-09-02 | Academisch Medisch Centrum | Intervention strategy for prevention or treatment of diabetes mellitus, autoimmune disease, inflammatory disease or cardiovascular disease |
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