CN116837084A - 尿液外泌体miRNA标志物在制备急性移植物抗宿主病诊断试剂盒中的应用 - Google Patents
尿液外泌体miRNA标志物在制备急性移植物抗宿主病诊断试剂盒中的应用 Download PDFInfo
- Publication number
- CN116837084A CN116837084A CN202310406141.2A CN202310406141A CN116837084A CN 116837084 A CN116837084 A CN 116837084A CN 202310406141 A CN202310406141 A CN 202310406141A CN 116837084 A CN116837084 A CN 116837084A
- Authority
- CN
- China
- Prior art keywords
- mir
- hsa
- urine
- seq
- diagnosis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000001808 exosome Anatomy 0.000 title claims abstract description 61
- 210000002700 urine Anatomy 0.000 title claims abstract description 57
- 208000024340 acute graft versus host disease Diseases 0.000 title claims abstract description 40
- 238000003745 diagnosis Methods 0.000 title claims abstract description 31
- 239000003550 marker Substances 0.000 title claims abstract description 14
- 239000002679 microRNA Substances 0.000 title claims abstract description 10
- 108091070501 miRNA Proteins 0.000 title claims abstract description 9
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 230000014509 gene expression Effects 0.000 claims abstract description 27
- 108091061676 Homo sapiens miR-411 stem-loop Proteins 0.000 claims abstract description 24
- 108091068992 Homo sapiens miR-143 stem-loop Proteins 0.000 claims abstract description 23
- 108091067471 Homo sapiens miR-211 stem-loop Proteins 0.000 claims abstract description 23
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 238000001514 detection method Methods 0.000 claims description 11
- 238000003753 real-time PCR Methods 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 7
- 238000005199 ultracentrifugation Methods 0.000 claims description 7
- 210000004027 cell Anatomy 0.000 claims description 5
- 238000000464 low-speed centrifugation Methods 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 238000000034 method Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 102100025222 CD63 antigen Human genes 0.000 description 4
- 102000034342 Calnexin Human genes 0.000 description 4
- 108010056891 Calnexin Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 description 4
- 101000613251 Homo sapiens Tumor susceptibility gene 101 protein Proteins 0.000 description 4
- 102100040879 Tumor susceptibility gene 101 protein Human genes 0.000 description 4
- 238000010322 bone marrow transplantation Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- 238000002054 transplantation Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 210000001124 body fluid Anatomy 0.000 description 3
- 239000010839 body fluid Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000004627 transmission electron microscopy Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- 208000009329 Graft vs Host Disease Diseases 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000007667 floating Methods 0.000 description 2
- 208000024908 graft versus host disease Diseases 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 101100532034 Drosophila melanogaster RTase gene Proteins 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 102000015623 Polynucleotide Adenylyltransferase Human genes 0.000 description 1
- 108010024055 Polynucleotide adenylyltransferase Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 238000012339 Real-time fluorescence quantitative polymerase chain reaction Methods 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 208000032023 Signs and Symptoms Diseases 0.000 description 1
- 238000012352 Spearman correlation analysis Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 208000014951 hematologic disease Diseases 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011528 liquid biopsy Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000003253 miRNA assay Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000003921 particle size analysis Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开尿液外泌体miRNA标志物在制备急性移植物抗宿主病诊断试剂盒中的应用,所述尿液外泌体miRNA标志物是hsa‑miR‑411‑5p,hsa‑miR‑143‑3p和hsa‑miR‑211‑5p中至少两种。本发明成功分离和鉴定人尿液外泌体,并检测尿液外泌体miRNA中hsa‑miR‑411‑5p、hsa‑miR‑143‑3p和hsa‑miR‑211‑5p的表达,若hsa‑miR‑411‑5p、hsa‑miR‑143‑3p显著降低,hsa‑miR‑211‑5p显著升高,三者满足其二则可说明患者发生aGVHD。本发明提供的诊断标志物解决了现有aGVHD的诊断局限性,具有早期、准确、无创的诊断优点。
Description
技术领域
本发明属于生物医药领域,具体涉及尿液外泌体miRNA标志物在制备急性移植物抗宿主病诊断试剂盒中的应用
背景技术
虽然异基因骨髓移植(al lo-bmt)是迄今为止治疗白血病等恶性血液病最成功的肿瘤免疫疗法,但会产生与之相关的副作用移植物抗宿主病(GVHD),尤其是急性移植物抗宿主病(aGVHD),仍然是移植后发病率和死亡率较高的主要原因,其累及器官肠道、皮肤或肝脏,影响超过50%的接受移植的患者。鉴于aGVHD的危害性,准确的早期诊断对于指导治疗决策和防止发生长期的严重并发症是至关重要。然而在aGVHD症状出现后,鲜有非侵入性的诊断方法选择。目前,对aGVHD的诊断主要基于临床症状,并需要辅以侵入性检查,但aGVHD的症状和体征随时间变化而变化,这使得aGVHD的诊断极具挑战性。
Mi croRNA(miRNA)是小的非编码RNA,通过抑制翻译或诱导信使RNAs的降解来负向调节基因的表达。其以高度稳定的形式存在于各种体液中。外泌体是一种直径在30-150nm且由细胞持续分泌的杯装囊泡结构,其携带蛋白核酸等多种生物活性分子,在包括尿液在内的各种体液中浓度很高,其内分子受到RNase含量升高和细胞外环境酸性pH的保护而不被降解。
由于尿液相比于血液收集更方便,处理更简单,且更加无创。因此尿液外泌体miRNA可以作为aGVHD液体活检的理想生物标志物,以帮助患者获得更好的治疗及预后。
发明内容
针对现有技术中用于aGVHD诊断方法的特异性低、灵敏度低,以及诊断价值低且具有一定的伤害性的问题,本发明公开尿液外泌体中hsa-miR-411-5p,hsa-miR-143-3p和hsa-miR-211-5p作为aGVHD的诊断标志物。
本发明的技术方案如下:
尿液外泌体miRNA标志物在制备急性移植物抗宿主病诊断试剂盒中的应用,所述尿液外泌体miRNA标志物是hsa-miR-411-5p,hsa-miR-143-3p和hsa-miR-211-5p中至少两种。
进一步方案,所述hsa-miR-411-5p的诊断标记序列如SEQ no.1所示,hsa-miR-143-3p诊断序列如SEQ no.2所示,hsa-miR-211-5p诊断标记序列如SEQ no.3所示;
SEQ no.1:UAGUAGACCGUAUAGCGUACG
SEQ no.2:UGAGAUGAAGCACUGUAGCUC
SEQ no.3:UUCCCUUUGUCAUCCUUCGCCU。
进一步方案,所述诊断试剂盒的判断步骤为:
(1)先根据尿液外泌体miRNA标志物hsa-miR-411-5p、hsa-miR-143-3p和hsa-miR-211-5p的基因和启动子序列设计三对定量引物和内参基因用于荧光定量PCR检测;
(2)提取人的尿液外泌体RNA;
(3)检测尿液外泌体中hsa-miR-411-5p、hsa-miR-143-3p和hsa-miR-211-5p的丰度水平;
(4)根据hsa-miR-411-5p、hsa-miR-143-3p和hsa-miR-211-5p中至少两种在移植后患者中的表达是否有显著性差异来评估是否发生aGVHD。
更进一步方案,步骤(4)中显著性差异是指has-miR-411-5p和/或has-miR-143-3p的表达显著降低,has-miR-211-5p的表达显著增加。
其中,所述定量引物的序列如下:
hsa-miR-411-5p:
F-primer CCCTAGTAGACCGTATAGCGTAC(SEQ no.4),
R-primer AACGCTTCACGAATTTGCGT(SEQ no.5);
hsa-miR-143-3p:
F-primer CCTGAGATGAAGCACTGTAGCTC(SEQ no.6),
R-primer AACGCTTCACGAATTTGCGT(SEQ no.7);
hsa-miR-211-5p:
F-primer TTCCCTTTGTCATCCTTCGCCT(SEQ no.8),
R-primer AACGCTTCACGAATTTGCGT(SEQ no.9)。
所述内参基因的引物序列为:
F-Primer CTCGCTTCGGCAGCACA(SEQ no.10),
R-Primer AACGCTTCACGAATTTGCGT(SEQ no.11)。
进一步方案,步骤(2)中提取人的尿液外泌体RNA,是先将收集的新鲜晨尿进行分梯度离心,然后提取尿液外泌体蛋白。
其中,提取尿液外泌体RNA是用TRIzol法提取外泌体RNA,用GeneCopoe ia成熟体miRNA定量检测试剂盒进行cDNA合成以及miRNA定量检测不同患者尿液样本间hsa-miR-411-5p、hsa-miR-143-3p和hsa-miR-211-5p丰度水平。
具体的,所述分梯度离心包括:先低速离心后高超速梯度离心,其中低速离心为:2000g尿液样本,室温离心20分钟去除细胞和碎片,收取上清液以0.22μm滤膜过滤;高超速梯度离心为:13500g,室温离心20分钟收取上清;在4℃下,200000g,离心120分钟保留沉淀;20mlPBS清洗后,在4℃下,200000g,离心120分钟,最后使用50ulPBS重悬外泌体。
进一步方案,所述诊断试剂盒为miRNA定量检测试剂盒。
本发明公开的诊断标志物为:hsa-miR-411-5p、hsa-miR-143-3p和hsa-miR-211-5p中至少两种在aGVHD患者和移植后病人尿液外泌体中的表达,并探讨其作为aGVHD诊断标志物的可能性。根据hsa-miR-411-5p、hsa-miR-143-3p和hsa-miR-211-5p中至少两种在移植后患者中的表达是否有显著性差异来评估是否发生aGVHD,即当has-miR-411-5p、has-miR-143-3p的表达显著降低、has-miR-211-5p的表达显著增加中发生两项的,则判断移植后患者发生aGVHD。
目前aGVHD的诊断主要基于临床症状并辅以侵入性检查,但aGVHD患者的体征和症状随着时间推移而有所不同,使得aGVHD诊断极具挑战性,同时以往针对aGVHD诊断分子标志物的研究多集中于血液上。本发明从尿液这一收取更方便无创的体液入手,通过高通量测序发现这些分子标志物在移植后病人和aGVHD患者尿液外泌体中呈现差异表达,通过qPCR进行验证后进一步证明其与aGVHD的进程相关。本发明提供的诊断标志物解决了现有aGVHD的诊断局限性,具有早期、准确、无创的诊断优点。
即本发明采用超速离心法分离提取人尿液外泌体,实时荧光定量聚合酶链式反应(qRT-PCR)技术分析三种miRNA标志物在骨髓移植后发生aGVHD的患者和未发病的患者的尿液外泌体中表达和差异。该方法优于现有的aGVHD诊断技术。
本发明的积极效果在于:
本发明成功分离鉴定了人尿液外泌体;并公开尿液外泌体标志物has-miR-411-5p/143-3p/211-5p作为aGVHD的诊断标志物;其中has-miR-411-5p/143-3p在aGVHD患者中的表达较未发病患者低(P<0.05),而has-miR-211-5p在aGVHD患者中的表达则显著增加(P<0.05)。
附图说明
图1为人尿液外泌体透射电镜下形态结构以及标志性蛋白CD63、TSG101和阴性标记Calnexin鉴定;
图2为移植后病人以及aGVHD患者尿液外泌体has-miR-411-5p/143-3p/211-5p表达水平。
具体实施方式
以下是本发明的具体实施例并结合附图,对本发明的技术方案作进一步的描述,但本发明并不限于这些实施例。
实施例1
一、尿液样本收集:
1.尿液样本均收集于中国科学技术大学附属第一医院(后简称为科大附院)。
2021年6月至2023年2月期间对科大附院白血病移植患者进行晨尿留取,将晨尿样本分成训练组(患者入院当天、移植前一天、移植后第五天、第七天和第十天,若发生aGVHD加取一天),验证组(经临床诊断确诊为aGVHD的患者)。
2.样本收集与保存:使用50ml离心管编号后收集晨尿样本,2000g,室温离心20分钟去除细胞和碎片,收取上清液后以0.22μm滤膜过滤后保存于-80℃超低温冰箱。
二、尿液外泌体分离与鉴定:
1.解冻前期收集的晨尿样本,完全融化后13500g,室温离心20分钟收取上清;4℃条件下,200000g,离心120分钟保留沉淀;20mlPBS清洗后4℃条件下,200000g,离心120分钟,最后使用50ulPBS重悬外泌体分装到1.5mlEP管里。
2.外泌体粒径分析:使用NanoFCM仪器(福流生物Flow NanoAnalyzer)分析颗粒粒径分布。将超离得到的外泌体取出5ul稀释30倍检测。
3.透射电镜观察外泌体形态:将外泌体样品稀释两倍后滴加于200目铜网上沉淀1分钟,滤纸吸去浮液;2%磷钨酸10ul滴加于铜网上沉淀1分钟,滤纸吸去浮液;常温干燥数分钟;80kv进行电镜检测成像。透射电镜(HITACHI,HT-7800)观察外泌体形态和大小。
4.Westernblot检测外泌体标志蛋白:采用NP-40裂解液(P0013F,Beyot ime)提取外泌体蛋白,BCA(P0010,Beyot ime)测定蛋白浓度。12%SDS-PAGE分离蛋白,每道上样量为40ug总蛋白。湿转法将蛋白转移到PVDF膜上,5%脱脂奶粉室温封闭1小时,剪膜后分别加入CD63、TSG101和Calnexin抗体(1:1000),4℃摇床封闭过夜,TBST洗5次每次7分钟,加入HRP标记的二抗(1:8000),室温孵育1小时,孵育结束后TBST清洗5次每次7分钟,再按1:1比例配制显影液(P0018FS,Beyot ime),将显影液滴加到膜上,吸去多余显影液,曝光,拍照保存。
尿液外泌体透射电镜下形态结构如图1所示,其中A图为不同样本使用WesternBlot检测外泌体特异性蛋白CD63和TSG101以及阴性蛋白Calnexin的表达,结果显示符合外泌体的表征;B、C图分别为1.0um和200nm尺寸下所得的投射电镜扫描图,形态学符合外泌体的表征,说明提取到的囊泡为尿液外泌体。
尿液外泌体较血液外泌体提取难度高,本发明在参考以往文献和实验室实际操作的基础上,优化了超速离心法的条件,使用低速离心和过滤去除尿液里的细胞和碎片,再使用高超速离心提取尿液外泌体。以此方法得到的尿液外泌体呈杯状、部分有凹陷,粒径在80nm左右,表达CD63和TSG101等外泌体标志分子,不表达阴性蛋白Calnexin,且训练组和验证组无明显差异。
hsa-miR-411-5p的诊断标记序列如SEQ no.1所示,hsa-miR-143-3p诊断序列如SEQ no.2所示,hsa-miR-211-5p诊断标记序列如SEQ no.3所示;
SEQ no.1:UAGUAGACCGUAUAGCGUACG
SEQ no.2:UGAGAUGAAGCACUGUAGCUC
SEQ no.3:UUCCCUUUGUCAUCCUUCGCCU。
三、人尿液外泌体miRNA测定
外泌体RNA抽提和实时荧光定量PCR:50ul外泌体悬液中加入1mlTRIzol,反复吹打混匀后加入200ml氯仿,剧烈震荡2分钟,室温静置5-10分钟。4℃离心15000g,10分钟,离心后分三层分别是最下层红色酚-氯仿相,中间层,最上层无色水相。RNA仅存在于水相中,取上清400ul至新EP管,加入等体积预冷的异丙醇,轻轻混匀后冰上静置10分钟,再4℃离心15000g,10分钟,弃上清得到RNA沉淀。将新配制的75%乙醇1ml加入离心管混匀15000g,10分钟,弃上清重复洗涤一边再弃上清,吸尽管壁水珠后4℃离心15000g,30秒,等乙醇完全挥发,加入20ulDEPC水,用振荡器混匀后测浓度并保存于-80℃冰箱。
用GeneCopoe ia成熟体miRNA定量检测试剂盒进行cDNA合成以及miRNA定量检测。
逆转录:(1)反应液配制:100ng总RNA,2U/ul Poly A Polymerase 1ul,SureScirpt RTase Mix(20×)1ul,5×PAP/RT Buffer I I 4ul,dd H2O补足至20ul,冰上操作。(2)反应条件:37℃孵育1小时,85℃温育5分钟。逆转录产物保存于-20℃。
qPCR定量检测miRNA:(1)反应液配制:2×Al l-in-One qPCR Mix 10ul,相应miRNA qPCR Primer 2ul,Universal Adaptor PCR Primer 2ul,cDNA 2ul,加ddH2O补足至20ul,重复3次;(2)反应条件:95℃预变性10分钟;95℃变性10秒,Tm-2℃退火20秒,72℃延伸10秒(40个循环);95℃15秒;30℃30秒。反应完成后应用GraphPad Pri sm分析数据。
其中三种miRNA的定量引物序列分别为:
hsa-miR-411-5p:
F-primer CCCTAGTAGACCGTATAGCGTAC,
R-primer AACGCTTCACGAATTTGCGT;
hsa-miR-143-3p:
F-primer CCTGAGATGAAGCACTGTAGCTC,
R-primer AACGCTTCACGAATTTGCGT;
hsa-miR-211-5p:
F-primer TTCCCTTTGTCATCCTTCGCCT,
R-primer AACGCTTCACGAATTTGCGT。
内参基因U6的引物序列为:
F-Primer CTCGCTTCGGCAGCACA,
R-Primer AACGCTTCACGAATTTGCGT。
荧光定量PCR定量检测mi croRNA的相对表达变化量时,相对表达量的计算用公式RQ=2-ΔCt,ΔCt=CtmiR-CtmiR-16。统计学分析采用SPSS22.0统计分析软件,P<0.05时,认为结果在统计学上有显著性差异。分析内容为mi croRNA在尿液外泌体中表达的个体差异性分析、ROC诊断分析以及Spearman相关性分析。该结果说明mi croRNA是否可作为急性移植物抗宿主病的生物标志物。
试验例
验证组尿液外泌体miRNA表达:
采用qPCR检测前期测序得到的差异表达基因hsa-miR-411-5p、hsa-miR-143-3p和hsa-miR-211-5p(如图2所示),has-miR-411-5p/143-3p在aGVHD患者中的表达较未发病患者低(P<0.05),而has-miR-211-5p在aGVHD患者中的表达则显著增加(P<0.05)。因此它们可以作为骨髓移植后aGVHD的诊断标志物,且本发明公开的诊断方法创新性和准确性优于现有活检技术。
应用例:
在实际诊断中,收取骨髓移植后患者尿液提取尿液外泌体RNA后进行实时荧光定量PCR,检测其中hsa-miR-411-5p、hsa-miR-143-3p和hsa-miR-211-5p的表达,若hsa-miR-411-5p、hsa-miR-143-3p显著降低,hsa-miR-211-5p显著升高,三者满足其二则可说明患者发生aGVHD。
本文中所描述的具体实施例仅仅是对本发明精神作举例说明。本发明所属技术领域的技术人员可以对所描述的具体实施例做各种各样的修改或补充或采用类似的方式替代,但并不会偏离本发明的精神或者超越所附权利要求书所定义的范围。
Claims (7)
1.尿液外泌体miRNA标志物在制备急性移植物抗宿主病诊断试剂盒中的应用,其特征在于:所述尿液外泌体miRNA标志物是hsa-miR-411-5p,hsa-miR-143-3p和hsa-miR-211-5p中至少两种。
2.根据权利要求1所述的应用,其特征在于:所述hsa-miR-411-5p的诊断标记序列如SEQ no.1所示,hsa-miR-143-3p诊断序列如SEQ no.2所示,hsa-miR-211-5p诊断标记序列如SEQ no.3所示;
SEQ no.1:UAGUAGACCGUAUAGCGUACG
SEQ no.2:UGAGAUGAAGCACUGUAGCUC
SEQ no.3:UUCCCUUUGUCAUCCUUCGCCU。
3.根据权利要求1所述的应用,其特征在于:所述诊断试剂盒的判断步骤为:
(1)先根据尿液外泌体miRNA标志物hsa-miR-411-5p、hsa-miR-143-3p和hsa-miR-211-5p的基因和启动子序列设计三对定量引物和内参基因用于荧光定量PCR检测;
(2)提取人的尿液外泌体RNA;
(3)检测尿液外泌体中hsa-miR-411-5p、hsa-miR-143-3p和hsa-miR-211-5p的表达变化量;
(4)根据hsa-miR-411-5p、hsa-miR-143-3p和hsa-miR-211-5p中至少两种在移植后患者中的表达是否有显著性差异来评估是否发生aGVHD。
4.根据权利要求3所述的应用,其特征在于:步骤(4)中显著性差异是指has-miR-411-5p和/或has-miR-143-3p的表达显著降低,has-miR-211-5p的表达显著增加。
5.根据权利要求3所述的应用,其特征在于:步骤(2)中提取人的尿液外泌体RNA,是先将收集的新鲜晨尿进行分梯度离心,然后提取尿液外泌体蛋白。
6.根据权利要求5所述的应用,其特征在于:所述分梯度离心包括:先低速离心后高超速梯度离心,其中低速离心为:2000g尿液样本,室温离心20分钟去除细胞和碎片,收取上清液以0.22μm滤膜过滤;高超速梯度离心为:13500g,室温离心20分钟收取上清;在4℃下,200000g,离心120分钟保留沉淀;20mlPBS清洗后,在4℃下,200000g,离心120分钟,最后使用50ulPBS重悬外泌体。
7.根据权利要求1所述的应用,其特征在于:所述诊断试剂盒为miRNA定量检测试剂盒。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310406141.2A CN116837084A (zh) | 2023-04-11 | 2023-04-11 | 尿液外泌体miRNA标志物在制备急性移植物抗宿主病诊断试剂盒中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310406141.2A CN116837084A (zh) | 2023-04-11 | 2023-04-11 | 尿液外泌体miRNA标志物在制备急性移植物抗宿主病诊断试剂盒中的应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116837084A true CN116837084A (zh) | 2023-10-03 |
Family
ID=88158739
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310406141.2A Pending CN116837084A (zh) | 2023-04-11 | 2023-04-11 | 尿液外泌体miRNA标志物在制备急性移植物抗宿主病诊断试剂盒中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116837084A (zh) |
-
2023
- 2023-04-11 CN CN202310406141.2A patent/CN116837084A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN113249481B (zh) | 外泌体基因的应用、前列腺癌检测物及其检测试剂盒和检测装置 | |
CN109609630B (zh) | 用于早期胃癌诊断的分子标志物及其应用 | |
CN113151472A (zh) | 胃癌诊断标志物及其应用 | |
CN112575079B (zh) | 外泌体miRNA作为诊断2型糖尿病合并冠心病的分子标志物及其应用 | |
CN111893176B (zh) | 预示荷斯坦奶牛乳房炎乳源外泌体miRNA诊断标志物 | |
CN111471759B (zh) | 一种孤独症血清神经元来源外泌体标志物ostc的应用 | |
CN106987634B (zh) | 血浆外泌体源性miRNAs在制备早期诊断原发性肝癌的试剂盒中的应用 | |
CN110878350B (zh) | 一种用于脓毒症诊断及预后评估的试剂盒 | |
CN116837084A (zh) | 尿液外泌体miRNA标志物在制备急性移植物抗宿主病诊断试剂盒中的应用 | |
CN107583052B (zh) | miR-6734-5p在制备Luminal型乳腺癌诊断工具中的应用 | |
CN110592207A (zh) | 一种外泌体microRNA的应用及制备的试剂盒 | |
RU2708074C1 (ru) | Способ диагностики злокачественных опухолей головного мозга | |
KR102505618B1 (ko) | 신장이식 후 항체 매개성 거부반응의 진단을 위한 소변 엑소좀 유래 miRNA 유전자 바이오마커 및 이의 용도 | |
KR102505617B1 (ko) | 신장이식 후 T 세포 매개성 거부반응의 진단을 위한 소변 엑소좀 유래 miRNA 유전자 바이오마커 및 이의 용도 | |
CN113637753A (zh) | 基于尿液外泌体的膀胱癌标志物lncRNA TERC及其应用 | |
CN108424960B (zh) | 一种LncRNA作为深静脉血栓形成诊断标志物的应用 | |
CN111690746A (zh) | 与肺癌相关的血小板rna标志物及其应用 | |
CN112961912A (zh) | 外泌体miRNA作为诊断冠心病的分子标志物及其应用 | |
CN110042162B (zh) | 一种用于肺癌早期诊断及转移预警的标志物及其制备的试剂盒 | |
CN110042163B (zh) | 一种用于肺癌诊断及转移预警的试剂盒 | |
CN113403390B (zh) | lncRNA在儿童心肌炎诊治中的应用 | |
CN110144352B (zh) | 用于早期诊断骨科疾病的分子标志物 | |
KR102550113B1 (ko) | 전립선암 진단용 바이오마커 및 이의 용도 | |
CN111662985B (zh) | 一种microRNA联合CEA用于制备宫颈癌早期诊断试剂盒的用途 | |
CN113373229B (zh) | 胃癌相关生物标志物及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |