CN113684260A - Tim-3作为生物标志物在制备脓毒症免疫抑制预测的试剂或试剂盒中的应用 - Google Patents
Tim-3作为生物标志物在制备脓毒症免疫抑制预测的试剂或试剂盒中的应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，具体涉及Tim‑3作为生物标志物在制备脓毒症免疫抑制预测的试剂或试剂盒中的应用。本发明降低了脓毒症免疫抑制的发生率，对提高脓毒症患者预后具有重要临床应用价值。
Description
技术领域
本发明属于生物技术领域，具体涉及Tim-3作为生物标志物在制备脓毒症免疫抑制预测的试剂或试剂盒中的应用。
背景技术
脓毒症是由宿主对感染反应失调所致危及生命的器官功能障碍综合征，进一步发展可导致脓毒症休克、多器官功能障碍综合征(MODS)，其致死率高(王圆圆，张琪，长链非编码RNA在脓毒症多器官功能障碍中的研究进展[J].中华危重症医学杂志:电子版，14(2):4)。
现阶段，创伤脓毒症预测方法大致可分为创伤严重度评分系统、危重病评分系统、临床体征、血液生化指标、病原学指标、炎症介质和代谢变化等几个方面。
临床上，应用较多的是生物标志物，这些生物标志物主要包括C反应蛋白(CRP)、降钙素原(PCT)等。然而，这些指标仅能反映患者检测当时的全身炎症反应程度，且随着检测时间窗的不同，结果变化很大，特异性不高。
发明内容
有鉴于此，本发明的目的在于提供Tim-3作为生物标志物在制备脓毒症免疫抑制预测的试剂或试剂盒中的应用。
为实现上述目的，本发明的技术方案为：
细胞免疫球蛋白粘蛋白-3(Tim-3)作为生物标志物在制备脓毒症免疫抑制预测的试剂或试剂盒中的应用。
进一步，所述试剂或试剂盒是指检测细胞免疫球蛋白粘蛋白-3(Tim-3)阳性CD4 T细胞在CD4 T细胞中所占比例的试剂或试剂盒。
进一步，所述CD4 T细胞是外周血CD4 T细胞。
进一步，所述试剂盒是指检测细胞免疫球蛋白粘蛋白-3(Tim-3)转录水平的试剂或试剂盒。
进一步，所述转录水平是指在在外周血CD4 T细胞中的转录水平。
进一步，检测转录水平所采用的PCR扩增的正向扩增引物的核苷酸序列如SEQ IDNO.1(GACTCTAGCAGACAGTGGGATC)所示，反向扩增引物的核苷酸序列如SEQ ID NO.2(GGTGGTAAGCATCCTTGGAAAGG)所示。
本发明的目的还在于保护细胞免疫球蛋白粘蛋白-3(Tim-3)的抗体在制备脓毒症免疫抑制预测的试剂或试剂盒中的应用。
进一步，所述抗体是指检测细胞免疫球蛋白粘蛋白-3(Tim-3)阳性CD4 T细胞在CD4 T细胞中所占比例的抗体。
本发明的有益效果在于：
本发明将外周血CD4 T细胞中Tim-3阳性CD4 T细胞比例作为生物标志物，制备脓毒症免疫抑制预测的试剂或试剂盒，通过检测外周血CD4 T细胞中Tim-3阳性CD4 T细胞比例或CD4 T细胞中Tim-3的转录水平，从而能够给予个体化干预治疗，降低了脓毒症免疫抑制的发生率，对提高脓毒症患者预后具有重要临床应用价值。
附图说明
图1为实施例1中脓毒症免疫抑制患者和健康志愿者外周血CD4 T细胞中Tim-3+CD4 T细胞比例检测结果图，其中，“+”表示阳性，Ctrl为脓毒症健康志愿者组，Sepsis为免疫抑制患者组，cells为细胞。
图2为实施例2中受试者工作曲线(ROC)分析外周血CD4 T细胞中Tim-3+CD4 T细胞比例在脓毒症免疫抑制发生风险中的早期预测效果示意图。
图3显示为实施例3中脓毒症免疫抑制患者外周血CD4 T细胞中Tim-3转录水平图，其中，“+”表示阳性，Ctrl为脓毒症健康志愿者组，Sepsis为免疫抑制患者组，cells为细胞。
具体实施方式
所举实施例是为了更好地对本发明的内容进行说明，但并不是本发明的内容仅限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
实施例1
研究人群：收集2017年8月至2019年2月在中国人民解放军陆军特色医学中心诊治的脓毒症免疫抑制患者(36例)血液，根据国际脓毒症协会对该病的诊断标准Sepsis 3.0评判患者是否发生了脓毒症，发生脓毒症后采集第14天外周血，其中6例于采集血液之后不同天数后死亡故纳入脓毒症免疫抑制死亡组(6例)，30例采血后存活故纳入脓毒症免疫抑制存活组(30例)。脓毒症免疫抑制死亡组男女占比分别是80.0％(24例)、20.0％(6例)，平均年龄是39.23±9.44岁；脓毒症免疫抑制存活组男女占比分别是83.3％(5例)、16.7％(1例)，平均年龄是45.8±15.0岁。血液采集后2小时内在4℃条件下500g转速下离心10分钟，吸取血液中白细胞所在的白膜层，使用前恢复至室温通过流式细胞分析技术检测CD4 T细胞中Tim-3阳性的CD4 T细胞比例。所有参加研究的患者均经中国人民解放军陆军特色医学中心伦理委员会批准后实施。
收集36例脓毒症患者诊断脓毒症3.0之后14天时的外周血样本(如表1所示)，根据患者采血后的住院时间内是否死亡分为脓毒症免疫抑制死亡组(6例)和脓毒症免疫抑制存活组(30例)，进行队列试验，通过Tim-3和CD4的流式细胞分析技术抗体(Alexa Fluor 488标记的Tim-3抗体购自Abcam公司，货号ab233060；Alexa Fluor 647标记的CD4抗体购自Abcam公司，货号ab196147)检测CD4 T细胞中Tim-3阳性CD4 T细胞比例，该试剂盒采用流式细胞分析法；将裂解红细胞以后的全血白细胞以106个/管分装至1.5ml管内，置于冰上；300g 4℃离心5min，小心吸弃上清；每管加入200ul PBS和1ul Alexa Fluor 488标记的Tim-3抗体，1ulAlexa Fluor 647标记的CD4抗体后重悬，4℃孵育30min；每管直接加入600ulPBS，300g 4℃离心5min，小心吸弃上清；每管加入200ulPBS重悬细胞；置于冰盒内，送检。流式细胞分析技术检测CD4 T细胞中Tim-3阳性CD4 T细胞比例；检测结果采用SPSS22.0软件进行统计学分析，并采用GraphPad Prism 5.0软件绘图，结果如表2和图1所示。
表1脓毒症免疫患者临床数据统计结果
表2脓毒症免疫抑制存活组和死亡组的Tim-3阳性CD4 T细胞/CD4 T细胞比例
由图1和表2可知，与健康志愿者相比，外周血CD4 T细胞中Tim-3阳性CD4 T细胞比例在发生脓毒症免疫抑制患者体内明显升高，其差异具有显著统计学差异(P<0.01)。由此表面，外周血CD4 T细胞中Tim-3阳性CD4 T细胞比例可以作为临床脓毒症免疫抑制预测的指标。
实施例2
根据实施例1外周血CD4 T细胞中Tim-3阳性CD4 T细胞比例值，使用SPSS22.0软件绘制ROC曲线，结果如图2所示，其中，AUC反映了预测效能(AUC＝0.5，没有预测效能：0.5<AUC<0.7，很小的预测价值：0.7<AUC<0.9相当准确的预测价值：0.9<AUC<1，很准确的预测价值)。
由图2可知，外周血CD4 T细胞中Tim-3阳性CD4 T细胞比例的曲线下面积(AUC)为0.85。由此表明，外周血CD4 T细胞中Tim-3阳性CD4 T细胞比例具有很好的脓毒症免疫抑制死亡的预测价值。
实施例3
外周血CD4 T细胞Tim-3转录水平检测：
选取根据实施例1的外周血CD4 T细胞进行Tim-3的定量PCR检测，使用Qiagen公司RNeasy Plus试剂盒提取总RNA(货号74134)；使用1μg RNA，通过Bio-Rad公司的iScriptcDNA合成试剂盒(货号1708890)合成cDNA。使用合成的cDNA、引物和Bio-Rad公司的SsoFastEvaGreen Supermix(货号172-5204)，进行实时定量PCR检测，结果如图3所示。
其中，Tim-3的qPCR检测引物如下：
正向引物：GACTCTAGCAGACAGTGGGATC；
反向引物：GGTGGTAAGCATCCTTGGAAAGG。
PCR反应体系如表3所示。
表3 PCR扩增体系
PCR反应循环体系如下：先在95.0℃下处理10分钟，然后以先在95.0℃下处理15秒、再在72.0℃下处理1分钟为一个循环，循环35次；熔解曲线65℃-95℃，递增0.5℃/5秒。
由图3可知，脓毒症免疫抑制患者CD4 T细胞的Tim-3转录水平显著高于健康对照者(P<0.01)。该结果结果充分表明，CD4 T细胞上的Tim-3参与了脓毒症免疫抑制发生发展的调控。
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。
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<110> 中国人民解放军陆军特色医学中心
<120> Tim-3作为生物标志物在制备脓毒症免疫抑制预测的试剂或试剂盒中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> 人工序列(artificial sequence)
<400> 1
gactctagca gacagtggga tc 22
<210> 2
<211> 23
<212> DNA
<213> 人工序列(artificial sequence)
<400> 2
ggtggtaagc atccttggaa agg 23
Claims (8)
1.细胞免疫球蛋白粘蛋白-3作为生物标志物在制备脓毒症免疫抑制预测的试剂或试剂盒中的应用。
2.如权利要求1所述的应用，其特征在于，所述试剂或试剂盒是指检测细胞免疫球蛋白粘蛋白-3阳性CD4 T细胞在CD4 T细胞中所占比例的试剂或试剂盒。
3.如权利要求2所述的应用，其特征在于，所述CD4 T细胞是外周血CD4 T细胞。
4.如权利要求1所述的应用，其特征在于，所述试剂盒是指检测细胞免疫球蛋白粘蛋白-3转录水平的试剂或试剂盒。
5.如权利要求4所述的应用，其特征在于，所述转录水平是指在在外周血CD4 T细胞中的转录水平。
6.如权利要求4或5所述的应用，其特征在于，检测转录水平所采用的PCR扩增的正向扩增引物的核苷酸序列如SEQ ID NO.1所示，反向扩增引物的核苷酸序列如SEQ ID NO.2所示。
7.细胞免疫球蛋白粘蛋白-3的抗体在制备脓毒症免疫抑制预测的试剂或试剂盒中的应用。
8.如权利要求7所述的应用，其特征在于，所述抗体是指检测细胞免疫球蛋白粘蛋白-3阳性CD4 T细胞在CD4 T细胞中所占比例的抗体。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017122203A1 (en) * | 2016-01-11 | 2017-07-20 | Technion Research & Development Foundation Limited | Methods of determining prognosis of sepsis and treating same |
| CN112011603A (zh) * | 2020-09-04 | 2020-12-01 | 中国人民解放军陆军特色医学中心 | Ier3作为生物标志物在脓毒症早期预测中的应用 |
| WO2021077794A1 (zh) * | 2018-10-25 | 2021-04-29 | 浙江大学 | 可溶性Tim-3重组蛋白及其突变型蛋白的制备和应用 |
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2021
- 2021-08-24 CN CN202110976184.5A patent/CN113684260A/zh active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017122203A1 (en) * | 2016-01-11 | 2017-07-20 | Technion Research & Development Foundation Limited | Methods of determining prognosis of sepsis and treating same |
| WO2021077794A1 (zh) * | 2018-10-25 | 2021-04-29 | 浙江大学 | 可溶性Tim-3重组蛋白及其突变型蛋白的制备和应用 |
| CN112011603A (zh) * | 2020-09-04 | 2020-12-01 | 中国人民解放军陆军特色医学中心 | Ier3作为生物标志物在脓毒症早期预测中的应用 |
Non-Patent Citations (2)
| Title |
|---|
| KARI OTTERDAL等: "Soluble markers of neutrophil, T-cell and monocyte activation are associated with disease severity and parasitemia in falciparum malaria", BMC INFECT DIS * |
| 黄祖华等: "Tim-3和Galectin-9介导在脓毒症中的免疫调节及其机制研究", 临床急诊杂志 * |
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