CN113930496A - Application of marrow vascular endothelial cells in myelodysplastic syndrome - Google Patents
Application of marrow vascular endothelial cells in myelodysplastic syndrome Download PDFInfo
- Publication number
- CN113930496A CN113930496A CN202111273168.6A CN202111273168A CN113930496A CN 113930496 A CN113930496 A CN 113930496A CN 202111273168 A CN202111273168 A CN 202111273168A CN 113930496 A CN113930496 A CN 113930496A
- Authority
- CN
- China
- Prior art keywords
- vascular endothelial
- bone marrow
- endothelial cells
- cells
- value
- 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.)
- Granted
Links
- 210000003556 vascular endothelial cell Anatomy 0.000 title claims abstract description 275
- 201000003793 Myelodysplastic syndrome Diseases 0.000 title claims abstract description 107
- 210000001185 bone marrow Anatomy 0.000 claims abstract description 246
- 208000031261 Acute myeloid leukaemia Diseases 0.000 claims abstract description 64
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims abstract description 39
- 230000004069 differentiation Effects 0.000 claims abstract description 32
- 230000004064 dysfunction Effects 0.000 claims abstract description 29
- 238000003745 diagnosis Methods 0.000 claims abstract description 9
- 210000004027 cell Anatomy 0.000 claims description 104
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 claims description 58
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 46
- 208000032839 leukemia Diseases 0.000 claims description 45
- 230000006907 apoptotic process Effects 0.000 claims description 37
- 230000006058 immune tolerance Effects 0.000 claims description 28
- 230000006378 damage Effects 0.000 claims description 26
- 239000000126 substance Substances 0.000 claims description 21
- 108090000623 proteins and genes Proteins 0.000 claims description 19
- 230000014509 gene expression Effects 0.000 claims description 16
- 206010061818 Disease progression Diseases 0.000 claims description 12
- 230000005779 cell damage Effects 0.000 claims description 12
- 208000037887 cell injury Diseases 0.000 claims description 12
- 230000005750 disease progression Effects 0.000 claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims description 10
- 238000012360 testing method Methods 0.000 claims description 10
- 238000013508 migration Methods 0.000 claims description 7
- 230000005012 migration Effects 0.000 claims description 7
- 206010058314 Dysplasia Diseases 0.000 claims description 6
- NNJPGOLRFBJNIW-HNNXBMFYSA-N (-)-demecolcine Chemical compound C1=C(OC)C(=O)C=C2[C@@H](NC)CCC3=CC(OC)=C(OC)C(OC)=C3C2=C1 NNJPGOLRFBJNIW-HNNXBMFYSA-N 0.000 claims description 4
- OMOVVBIIQSXZSZ-UHFFFAOYSA-N [6-(4-acetyloxy-5,9a-dimethyl-2,7-dioxo-4,5a,6,9-tetrahydro-3h-pyrano[3,4-b]oxepin-5-yl)-5-formyloxy-3-(furan-3-yl)-3a-methyl-7-methylidene-1a,2,3,4,5,6-hexahydroindeno[1,7a-b]oxiren-4-yl] 2-hydroxy-3-methylpentanoate Chemical compound CC12C(OC(=O)C(O)C(C)CC)C(OC=O)C(C3(C)C(CC(=O)OC4(C)COC(=O)CC43)OC(C)=O)C(=C)C32OC3CC1C=1C=COC=1 OMOVVBIIQSXZSZ-UHFFFAOYSA-N 0.000 claims description 4
- 208000008601 Polycythemia Diseases 0.000 claims description 2
- 208000011580 syndromic disease Diseases 0.000 claims 1
- 208000016586 myelodysplastic syndrome with excess blasts Diseases 0.000 abstract description 51
- 201000010099 disease Diseases 0.000 abstract description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 11
- 238000001514 detection method Methods 0.000 abstract description 9
- 238000000338 in vitro Methods 0.000 abstract description 4
- 238000011161 development Methods 0.000 abstract description 2
- 230000003211 malignant effect Effects 0.000 abstract description 2
- 239000003550 marker Substances 0.000 abstract description 2
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 206010067959 refractory cytopenia with multilineage dysplasia Diseases 0.000 abstract 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 23
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 20
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 20
- 238000003501 co-culture Methods 0.000 description 17
- 210000002889 endothelial cell Anatomy 0.000 description 17
- 230000003834 intracellular effect Effects 0.000 description 14
- 210000005087 mononuclear cell Anatomy 0.000 description 12
- 238000007619 statistical method Methods 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 11
- 102100032616 Caspase-2 Human genes 0.000 description 9
- 101000867612 Homo sapiens Caspase-2 Proteins 0.000 description 9
- 102100027308 Apoptosis regulator BAX Human genes 0.000 description 8
- 108050006685 Apoptosis regulator BAX Proteins 0.000 description 8
- 108090000397 Caspase 3 Proteins 0.000 description 8
- 102100029855 Caspase-3 Human genes 0.000 description 8
- 238000011529 RT qPCR Methods 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- YXHLJMWYDTXDHS-IRFLANFNSA-N 7-aminoactinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=C(N)C=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 YXHLJMWYDTXDHS-IRFLANFNSA-N 0.000 description 5
- 108700012813 7-aminoactinomycin D Proteins 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 230000022131 cell cycle Effects 0.000 description 5
- 230000001332 colony forming effect Effects 0.000 description 5
- 210000003013 erythroid precursor cell Anatomy 0.000 description 5
- 210000002360 granulocyte-macrophage progenitor cell Anatomy 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 108010016788 Cyclin-Dependent Kinase Inhibitor p21 Proteins 0.000 description 4
- 102100033270 Cyclin-dependent kinase inhibitor 1 Human genes 0.000 description 4
- 101001056180 Homo sapiens Induced myeloid leukemia cell differentiation protein Mcl-1 Proteins 0.000 description 4
- 101000716729 Homo sapiens Kit ligand Proteins 0.000 description 4
- 101000617130 Homo sapiens Stromal cell-derived factor 1 Proteins 0.000 description 4
- 101000851007 Homo sapiens Vascular endothelial growth factor receptor 2 Proteins 0.000 description 4
- 102100026539 Induced myeloid leukemia cell differentiation protein Mcl-1 Human genes 0.000 description 4
- 102100020880 Kit ligand Human genes 0.000 description 4
- 238000003559 RNA-seq method Methods 0.000 description 4
- 108010078814 Tumor Suppressor Protein p53 Proteins 0.000 description 4
- 102000015098 Tumor Suppressor Protein p53 Human genes 0.000 description 4
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 4
- 230000001464 adherent effect Effects 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 210000002960 bfu-e Anatomy 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 230000003394 haemopoietic effect Effects 0.000 description 4
- 230000011132 hemopoiesis Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 230000008506 pathogenesis Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 102000004121 Annexin A5 Human genes 0.000 description 3
- 108090000672 Annexin A5 Proteins 0.000 description 3
- 102000016359 Fibronectins Human genes 0.000 description 3
- 108010067306 Fibronectins Proteins 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 102100037858 G1/S-specific cyclin-E1 Human genes 0.000 description 3
- 101000738568 Homo sapiens G1/S-specific cyclin-E1 Proteins 0.000 description 3
- 101000979342 Homo sapiens Nuclear factor NF-kappa-B p105 subunit Proteins 0.000 description 3
- 102100023050 Nuclear factor NF-kappa-B p105 subunit Human genes 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- 102100021669 Stromal cell-derived factor 1 Human genes 0.000 description 3
- 210000004241 Th2 cell Anatomy 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000000750 progressive effect Effects 0.000 description 3
- 210000003289 regulatory T cell Anatomy 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 102100031351 Galectin-9 Human genes 0.000 description 2
- 108010007707 Hepatitis A Virus Cellular Receptor 2 Proteins 0.000 description 2
- 102100034458 Hepatitis A virus cellular receptor 2 Human genes 0.000 description 2
- 101100382122 Homo sapiens CIITA gene Proteins 0.000 description 2
- 101001130151 Homo sapiens Galectin-9 Proteins 0.000 description 2
- 101000610551 Homo sapiens Prominin-1 Proteins 0.000 description 2
- 102100026371 MHC class II transactivator Human genes 0.000 description 2
- 108700002010 MHC class II transactivator Proteins 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 102100040120 Prominin-1 Human genes 0.000 description 2
- 238000009012 ROS assay kit Methods 0.000 description 2
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 2
- 210000000447 Th1 cell Anatomy 0.000 description 2
- 210000000068 Th17 cell Anatomy 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 230000000735 allogeneic effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 238000012137 double-staining Methods 0.000 description 2
- 238000011134 hematopoietic stem cell transplantation Methods 0.000 description 2
- 238000007417 hierarchical cluster analysis Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 238000000513 principal component analysis Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 108010006464 Hemolysin Proteins Proteins 0.000 description 1
- 101100220044 Homo sapiens CD34 gene Proteins 0.000 description 1
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 description 1
- 101000932478 Homo sapiens Receptor-type tyrosine-protein kinase FLT3 Proteins 0.000 description 1
- 101000799461 Homo sapiens Thrombopoietin Proteins 0.000 description 1
- 101000694103 Homo sapiens Thyroid peroxidase Proteins 0.000 description 1
- 101000891649 Homo sapiens Transcription elongation factor A protein-like 1 Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 101710177504 Kit ligand Proteins 0.000 description 1
- 108010007622 LDL Lipoproteins Proteins 0.000 description 1
- 102000007330 LDL Lipoproteins Human genes 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 101000596402 Mus musculus Neuronal vesicle trafficking-associated protein 1 Proteins 0.000 description 1
- 101000800539 Mus musculus Translationally-controlled tumor protein Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 101000781972 Schizosaccharomyces pombe (strain 972 / ATCC 24843) Protein wos2 Proteins 0.000 description 1
- 101001009610 Toxoplasma gondii Dense granule protein 5 Proteins 0.000 description 1
- 102100040250 Transcription elongation factor A protein-like 1 Human genes 0.000 description 1
- 240000003864 Ulex europaeus Species 0.000 description 1
- 235000010730 Ulex europaeus Nutrition 0.000 description 1
- 244000063464 Vitex agnus-castus Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 235000009347 chasteberry Nutrition 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000009274 differential gene expression Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000008482 dysregulation Effects 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 230000000925 erythroid effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 239000003228 hemolysin Substances 0.000 description 1
- 102000055151 human KITLG Human genes 0.000 description 1
- 102000053400 human TPO Human genes 0.000 description 1
- 210000005104 human peripheral blood lymphocyte Anatomy 0.000 description 1
- 230000008938 immune dysregulation Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000000207 lymphocyte subset Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- 208000025113 myeloid leukemia Diseases 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000035778 pathophysiological process Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
Images
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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
-
- 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
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B40/10—Signal processing, e.g. from mass spectrometry [MS] or from PCR
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B40/30—Unsupervised data analysis
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
- G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
- G16H50/30—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for calculating health indices; for individual health risk assessment
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Medical Informatics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Public Health (AREA)
- Data Mining & Analysis (AREA)
- Urology & Nephrology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Evolutionary Biology (AREA)
- Toxicology (AREA)
- Epidemiology (AREA)
- Databases & Information Systems (AREA)
- Theoretical Computer Science (AREA)
- Bioinformatics & Computational Biology (AREA)
- General Engineering & Computer Science (AREA)
- Evolutionary Computation (AREA)
- Medicinal Chemistry (AREA)
- Artificial Intelligence (AREA)
- Bioethics (AREA)
Abstract
The invention discloses an application of marrow vascular endothelial cells in myelodysplastic syndrome. The invention provides an application of a marrow vascular endothelial cell as a marker in any one of the following: for the preparation of a product for use in the detection or co-detection of the progression of the course of MDS, for the preparation of a product for use in the diagnosis or co-diagnosis of MDS, or for the differentiation or co-differentiation of MDS from non-MDS. The research of the invention finds that the number of the marrow vascular endothelial cells is gradually increased from MDS-MLD and MDS-EB to AML patients, but the dysfunction is gradually increased. Furthermore, as the disease progresses, the bone marrow vascular endothelial cells of MDS patients have a reduced ability to support normal hematopoietic cells in vitro and an increased ability to support malignant hematopoietic cells. The invention has important significance for detecting the occurrence and development of MDS, particularly monitoring the disease course progress of MDS.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to application of a marrow vascular endothelial cell in myelodysplastic syndrome.
Background
Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal diseases of the myeloid lineage characterized by dysplasia of myeloid cells and a predisposition to transformation into Acute Myeloid Leukemia (AML). The pathogenesis of MDS is complex and diverse, the current strategy for treating MDS depends on allogeneic hematopoietic stem cell transplantation, hypomethylation drugs, immunosuppressants and chemotherapy, but the clinical curative effect of the treatments is limited, and only 40-50% of patients can survive for 5 years by taking the allogeneic hematopoietic stem cell transplantation as an example. Therefore, further elucidation of the pathogenesis of MDS and establishment of novel therapeutic strategies thereof are important clinical scientific issues to be urgently solved.
Ineffective hematopoiesis is the major pathophysiological process of MDS. MDS with multiple lineage dysplasia (MDS-MLD), MDS with primitive cytosis (MDS-EB) to Acute Myeloid Leukemia (AML) are typical processes for the progression of MDS. Meanwhile, immune dysregulation, another important pathogenesis of MDS, shows that different T cell subsets are associated with the progression of MDS.
The simultaneous multiple effects of bone marrow vascular endothelial cells as important components of the bone marrow microenvironment on normal, abnormal, and immunoregulatory processes in MDS patients in different clinical stages are not well defined. Furthermore, the relationship between these effects of bone marrow vascular endothelial cells and the progression of MDS is still unknown. Therefore, providing a correlation between bone marrow microenvironment cells and MDS progression is of great importance for the clinical treatment of MDS.
Disclosure of Invention
The invention aims to provide application of marrow vascular endothelial cells in myelodysplastic syndrome.
In a first aspect, the invention claims the use of bone marrow vascular endothelial cells as markers in any one of:
(A1) preparing a product for detecting or assisting in detecting the course of disease progression of myelodysplastic syndrome, or detecting or assisting in detecting the course of disease progression of myelodysplastic syndrome;
(A2) preparing a product for use in, or for aiding in, the diagnosis of myelodysplastic syndrome;
(A3) preparing a product for, or to aid in, distinguishing between myelodysplastic syndrome patients and non-myelodysplastic syndrome patients, or distinguishing between myelodysplastic syndrome patients and non-myelodysplastic syndrome patients.
In a second aspect, the invention claims the use of substance a and/or substance B and/or substance C and/or substance D and/or substance E and/or substance F in any one of the following:
(A1) preparing a product for detecting or assisting in detecting the course of disease progression of myelodysplastic syndrome, or detecting or assisting in detecting the course of disease progression of myelodysplastic syndrome;
(A2) preparing a product for use in, or for aiding in, the diagnosis of myelodysplastic syndrome;
(A3) preparing a product for, or to aid in, distinguishing between myelodysplastic syndrome patients and non-myelodysplastic syndrome patients, or distinguishing between myelodysplastic syndrome patients and non-myelodysplastic syndrome patients.
The substance A is used for detecting the number of vascular endothelial cells in bone marrow.
The substance B is used for detecting the damage condition of vascular endothelial cells in bone marrow.
The substance C is used for detecting the functional state of vascular endothelial cells in bone marrow.
The substance D is used for detecting the supporting capacity of vascular endothelial cells in bone marrow to hematopoietic stem cells.
The substance E is a substance for detecting whether or not vascular endothelial cells in bone marrow cause T cells to differentiate toward an immune tolerance.
The substance F is used for detecting the leukemia cell supporting capacity of vascular endothelial cells in bone marrow.
Further, the vascular endothelial cell damage condition in the bone marrow can be embodied as all or part of the following (the same below): the apoptosis rate of the marrow vascular endothelial cells, the ROS level in the marrow vascular endothelial cells and the expression quantity of apoptosis-related genes in the marrow vascular endothelial cells. The higher the apoptosis rate of the marrow vascular endothelial cells, the higher the intracellular ROS level and the higher the expression level of apoptosis-related genes (such as CASP2, CASP3 and BAX), the more serious the damage condition of the marrow vascular endothelial cells is.
Further, the functional status of vascular endothelial cells in bone marrow may be expressed in whole or in part as follows (the same applies below): the tube forming ability of the marrow vascular endothelial cells and the migration ability of the marrow vascular endothelial cells. The stronger the tube forming ability and the migration ability of the marrow vascular endothelial cells are, the more serious the dysfunction of the marrow vascular endothelial cells is.
Further, the supporting ability of the vascular endothelial cells in the bone marrow to the hematopoietic stem cells can be expressed in whole or in part (the same below): bone marrow CD34 derived from healthy people+Bone marrow CD34 after co-culturing the cells with the bone marrow vascular endothelial cells to be tested+Apoptosis rate, bone marrow CD34+ROS levels in cells, bone marrow CD34+CFU-E, BFU-E, CFU-GM and/or CFU-GEMM forming ability of the cells. Marrow CD34+The higher the rate of apoptosis, the higher the intracellular ROS level, the lower the ability of CFU-E, BFU-E, CFU-GM and CFU-GEMM to form, indicating the lower the ability of vascular endothelial cells in the bone marrow to support hematopoietic stem cells.
Further, whether the vascular endothelial cells in the bone marrow cause differentiation of T cells toward immune tolerance may be embodied as all or part of the following (same below): bone marrow CD3 derived from healthy people+Bone marrow CD3 after co-culture of T cells with test bone marrow vascular endothelial cells+Differentiation of T cells, including Th1/CD4+T cells, Th17/CD4+T cells, Th2/CD4+T cell, Treg/CD4+T cell, Th1/Th2 ratio. Marrow CD3+T cell to Th1/CD4+T cells and Th17/CD4+Less differentiation of T cells to Th2/CD4+T cells and Treg/CD4+The more differentiated the T cells and the lower the Th1/Th2 ratio, the more the bone marrow vascular endothelial cells will cause T cells to become immune-tolerantDirectional differentiation.
Further, the capability of the vascular endothelial cells in the bone marrow to support leukemia cells can be expressed by all or part of the following (the same applies below): proliferation, apoptosis, intracellular ROS levels, leukemia cell colony unit (CFU-Leukemia, CFU-L) formation efficiency, intracellular apoptosis, and/or cell cycle associated gene expression of leukemia cells following co-culture of said leukemia cells with bone marrow vascular endothelial cells of a test subject. The stronger the proliferation of leukemia cells, the lower the apoptosis rate, the lower the intracellular ROS level, the higher the formation efficiency of leukemia cell colony units (CFU-Leukemia, CFU-L), the higher the expression level of intracellular apoptosis and/or cell cycle related genes (e.g., CCNE1, MCL1), and the lower the expression level of intracellular apoptosis and/or cell cycle related genes (e.g., CASP2, CASP3, BAX, TP53, CDKN1A), indicating the stronger the ability of vascular endothelial cells in bone marrow to support leukemia cells.
In a particular embodiment of the invention, the leukemia cells are in particular HL-60 cells.
In each of the above aspects, the non-myelodysplastic syndrome patient is required to satisfy the following conditions: compared with myelodysplastic syndrome patients, the number of vascular endothelial cells in bone marrow is reduced, the damage condition of the vascular endothelial cells in the bone marrow is reduced or no damage occurs, the dysfunction of the vascular endothelial cells in the bone marrow is reduced or no dysfunction occurs, the supporting capacity of the vascular endothelial cells in the bone marrow to hematopoietic stem cells is enhanced, the differentiation of T cells to an immune tolerance direction is not caused by the vascular endothelial cells in the bone marrow, the supporting capacity of the vascular endothelial cells in the bone marrow to leukemia cells is reduced or the leukemia cells have no supporting capacity.
In a particular embodiment of the invention, the non-myelodysplastic syndrome patient is a healthy human.
In a specific embodiment of the present invention, the progression of myelodysplastic syndrome can be embodied in the following three stages (the same applies below): MDS with multiple lineage blood cell dysplasia (MDS-MLD), MDS with primitive cytosis (MDS-EB) to Acute Myeloid Leukemia (AML).
In (a3) of the above aspects, the myelodysplastic syndrome patient may be a patient with MDS with multiple lineage dysplasia (MDS-MLD), a patient with MDS with polycythemia primitive (MDS-EB), or an Acute Myeloid Leukemia (AML) patient.
In both aspects, the product may be specifically a kit.
In the present invention, each of the substances (substance A, B, C, D, E, F) described above may be a substance capable of specifically binding to a specific detection substance or an instrument and kit for detecting each marker.
In a third aspect, the invention claims a system for detecting the progression of the course of myelodysplastic syndrome, comprising:
(B1) reagents and/or instruments;
the reagent and/or apparatus has all or part of the following functions: detecting the quantity of vascular endothelial cells in bone marrow, detecting the damage condition of the vascular endothelial cells in the bone marrow, detecting the functional state of the vascular endothelial cells in the bone marrow, detecting the supporting capacity of the vascular endothelial cells in the bone marrow to hematopoietic stem cells, detecting whether the vascular endothelial cells in the bone marrow can cause the differentiation of T cells towards the immune tolerance direction, and detecting the supporting capacity of the vascular endothelial cells in the bone marrow to leukemia cells.
(B2) A device;
the device comprises a data input module, a threshold storage module, a data comparison module and a judgment module.
The data input module is configured to input (B1) all or part of the detected values: a vascular endothelial cell count value in a test bone marrow from a myelodysplastic syndrome patient; the damage condition value of the vascular endothelial cells in the bone marrow to be detected; the vascular endothelial cell dysfunction value in the bone marrow to be detected; the supporting capacity value of the vascular endothelial cells in the bone marrow to be tested to the hematopoietic stem cells; the vascular endothelial cells in the bone marrow to be detected cause the differentiation value of T cells to the immune tolerance direction; the supporting capacity value of the vascular endothelial cells in the bone marrow to be tested to leukemia cells.
The threshold storage module is configured to store all or part of the following thresholds: a threshold A, a threshold B, a threshold C, a threshold D, a threshold E and a threshold F; the threshold value A is a vascular endothelial cell quantity value in the bone marrow of a healthy person; the threshold B is a vascular endothelial cell damage condition value in the bone marrow of a healthy person; the threshold C is a vascular endothelial cell dysfunction value in the bone marrow of a healthy person; the threshold value D is the supporting capacity value of the vascular endothelial cells in the healthy human bone marrow to the hematopoietic stem cells; the threshold value E is a value of differentiation of T cells to an immune tolerance direction caused by vascular endothelial cells in the bone marrow of a healthy person; the threshold value F is the supporting capacity value of the vascular endothelial cells in the bone marrow of the healthy human to leukemia cells.
The data comparison module is configured to receive all or part of the values obtained by the detection (B1) sent by the data input module (namely, the number value of the vascular endothelial cells in the bone marrow to be detected, the damage condition value of the vascular endothelial cells in the bone marrow to be detected, the function state value of the vascular endothelial cells in the bone marrow to be detected, the support capability value of the vascular endothelial cells in the bone marrow to be detected on hematopoietic stem cells, the differentiation value of the vascular endothelial cells in the bone marrow to be detected in the direction of immune tolerance, the support capability value of the vascular endothelial cells in the bone marrow to be detected on leukemia cells, call the thresholds in the threshold storage module, compare the number value of the vascular endothelial cells in the bone marrow to be detected with the threshold A, and compare the damage condition value of the vascular endothelial cells in the bone marrow to be detected with the threshold B, comparing the dysfunction value of the vascular endothelial cells in the bone marrow to be detected with the threshold value C, comparing the support capacity value of the vascular endothelial cells in the bone marrow to hematopoietic stem cells with the threshold value D, comparing the differentiation value of the vascular endothelial cells in the bone marrow to be detected, which lead T cells to be subjected to immunological tolerance, with the threshold value E, and/or comparing the support capacity value of the vascular endothelial cells in the bone marrow to be detected to leukemia cells with the threshold value F.
The judging module is configured to receive the comparison result sent by the data comparing module and then make a result judgment as follows: the number value of the vascular endothelial cells in the bone marrow to be detected is greater than the threshold a, the value of the vascular endothelial cell injury condition in the bone marrow to be detected is greater than the threshold B (i.e., the vascular endothelial cell injury condition of the bone marrow to be detected is more severe), the value of the vascular endothelial cell dysfunction in the bone marrow to be detected is greater than the threshold C (i.e., the vascular endothelial cell dysfunction of the bone marrow to be detected is more severe), the value of the support ability of the vascular endothelial cells in the bone marrow to hematopoietic stem cells is less than the threshold D (i.e., the support ability of the vascular endothelial cells of the bone marrow to hematopoietic stem cells is less), the value of the differentiation of the vascular endothelial cells in the bone marrow to be detected to the direction of immune tolerance is greater than the threshold E (i.e., the vascular endothelial cells of the bone marrow to be detected cause the differentiation of the T cells to the direction of immune tolerance), and/or the value of the support ability of the vascular endothelial cells in the bone marrow to the leukemia cells is greater than the threshold E The threshold F (i.e., the vascular endothelial cells of the bone marrow to be tested have stronger support ability for leukemia cells), and the larger the absolute value of the difference between each value and the corresponding threshold, the more serious the disease condition of the myelodysplastic syndrome patient.
In a fourth aspect, the invention claims a system for diagnosing or aiding in the diagnosis of myelodysplastic syndrome, comprising:
(C1) reagents and/or instruments;
the reagent and/or apparatus has all or part of the following functions: detecting the quantity of vascular endothelial cells in bone marrow, detecting the damage condition of the vascular endothelial cells in the bone marrow, detecting the functional state of the vascular endothelial cells in the bone marrow, detecting the supporting capacity of the vascular endothelial cells in the bone marrow to hematopoietic stem cells, detecting whether the vascular endothelial cells in the bone marrow can cause the differentiation of T cells towards the immune tolerance direction, and detecting the supporting capacity of the vascular endothelial cells in the bone marrow to leukemia cells.
(C2) A device;
the device comprises a data input module, a threshold storage module, a data comparison module and a judgment module.
The data input module is configured to input (B1) all or part of the detected values: a vascular endothelial cell count value in a test bone marrow from a test subject; the damage condition value of the vascular endothelial cells in the bone marrow to be detected; the vascular endothelial cell dysfunction value in the bone marrow to be detected; the supporting capacity value of the vascular endothelial cells in the bone marrow to be tested to the hematopoietic stem cells; the vascular endothelial cells in the bone marrow to be detected cause the differentiation value of T cells to the immune tolerance direction; the supporting capacity value of the vascular endothelial cells in the bone marrow to be tested to leukemia cells.
The threshold storage module is configured to store all or part of the following thresholds: a threshold A, a threshold B, a threshold C, a threshold D, a threshold E and a threshold F; the threshold value A is a vascular endothelial cell quantity value in the bone marrow of a healthy person; the threshold B is a vascular endothelial cell damage condition value in the bone marrow of a healthy person; the threshold C is a vascular endothelial cell dysfunction value in the bone marrow of a healthy person; the threshold value D is the supporting capacity value of the vascular endothelial cells in the healthy human bone marrow to the hematopoietic stem cells; the threshold value E is a value of differentiation of T cells to an immune tolerance direction caused by vascular endothelial cells in the bone marrow of a healthy person; the threshold value F is the supporting capacity value of the vascular endothelial cells in the bone marrow of the healthy human to leukemia cells.
The data comparison module is configured to receive all or part of the values obtained by the detection (C1) sent by the data input module (namely, the number value of the vascular endothelial cells in the bone marrow to be detected, the damage condition value of the vascular endothelial cells in the bone marrow to be detected, the function state value of the vascular endothelial cells in the bone marrow to be detected, the support capability value of the vascular endothelial cells in the bone marrow to be detected on hematopoietic stem cells, the differentiation value of the vascular endothelial cells in the bone marrow to be detected on the direction of immune tolerance, the support capability value of the vascular endothelial cells in the bone marrow to be detected on leukemia cells), call the thresholds in the threshold storage module, compare the number value of the vascular endothelial cells in the bone marrow to be detected with the threshold A, and compare the damage condition value of the vascular endothelial cells in the bone marrow to be detected with the threshold B, comparing the dysfunction value of the vascular endothelial cells in the bone marrow to be detected with the threshold value C, comparing the support capacity value of the vascular endothelial cells in the bone marrow to hematopoietic stem cells with the threshold value D, comparing the differentiation value of the vascular endothelial cells in the bone marrow to be detected, which lead T cells to be subjected to immunological tolerance, with the threshold value E, and/or comparing the support capacity value of the vascular endothelial cells in the bone marrow to be detected to leukemia cells with the threshold value F.
The judging module is configured to receive the comparison result sent by the data comparing module and then make a result judgment as follows: if the number of the vascular endothelial cells in the bone marrow to be detected is greater than the threshold a, the value of the damage condition of the vascular endothelial cells in the bone marrow to be detected is greater than the threshold B (i.e., the damage condition of the vascular endothelial cells in the bone marrow to be detected is more severe), the value of the dysfunction of the vascular endothelial cells in the bone marrow to be detected is greater than the threshold C (i.e., the dysfunction of the vascular endothelial cells in the bone marrow to be detected is more severe), the value of the support ability of the vascular endothelial cells in the bone marrow to hematopoietic stem cells is less than the threshold D (i.e., the support ability of the vascular endothelial cells in the bone marrow to be detected to hematopoietic stem cells is less strong), the value of the differentiation of the vascular endothelial cells in the direction of immune tolerance in the bone marrow to be detected is greater than the threshold E (i.e., the differentiation of the vascular endothelial cells in the direction of immune tolerance in the bone marrow to be detected causes the T cells), and/or the value of the support ability of the vascular endothelial cells in the bone marrow to leukemia cells is greater than the threshold E And if the threshold value F is higher (namely the vascular endothelial cells of the bone marrow to be detected have stronger supporting capacity on leukemia cells), the patient to be detected is the myelodysplastic syndrome patient.
The research of the invention finds that the number of the marrow vascular endothelial cells (BM EC) is gradually increased from MDS-MLD and MDS-EB to AML patients, but the dysfunction is gradually increased. Furthermore, as the disease progresses, BM ECs in MDS patients have a reduced ability to support normal hematopoietic cells in vitro and an increased ability to support malignant hematopoietic cells. After the culture medium is co-cultured with T cells, the proportions of Th2 and Treg are increased, and the proportions of Th1 and Th17 are reduced. Transcriptome sequencing shows that the expression profiles of the bone marrow vascular endothelial cells of the MDS-MLD patients are more similar to those of Healthy Donors (HD), and MDS-EB is more similar to AML. Mechanistically, the level of CXCL12, SCF and NFKB related genes related to hematopoietic regulation of bone marrow vascular endothelial cells is increased along with the progress of MDS. The invention has important significance for detecting the occurrence and development of MDS, particularly monitoring the disease course progress of MDS. The present invention provides a diagnostic service for MDS.
Drawings
FIG. 1 shows the ratio of the bone marrow vascular endothelial cells in the mononuclear cells of HD, MDS-MLD, MDS-EB, AML patients. Flow cytometry examined the proportion of CD34, CD309, and CD133 positive endothelial cells in HD, MDS with multiple lineage dysplasia (MDS-MLD), MDS with primitive cytosis (MDS-EB), and Acute Myeloid Leukemia (AML) bone marrow mononuclear cells. P <0.05 was considered statistically different. Represents P < 0.05; represents P < 0.005; represents P < 0.001.
FIG. 2 shows the experiment of double-staining of the endothelial cells of the bone marrow of HD, MDS-MLD, MDS-EB and AML patients. A is a typical picture; b is a statistical analysis chart of the double-staining quantity of the endothelial cells of the bone marrow of the patients with HD, MDS-MLD, MDS-EB and AML.
FIG. 3 is a graph of bone marrow vascular endothelial cell damage in MDS patients. A is a statistical analysis chart of the apoptosis of the marrow vascular endothelial cells of patients with HD, MDS-MLD, MDS-EB and AML; b is a graph for statistical analysis of ROS levels in the bone marrow vascular endothelial cells of patients with HD, MDS-MLD, MDS-EB and AML; c is the relative mRNA levels of CASP2, CASP3, and BAX in the bone marrow vascular endothelial cells of HD, MDS-MLD, MDS-EB, and AML patients.
FIG. 4 is a graph of impaired function of myeloid vascular endothelial cells in a subject with MDS. A is the tube forming ability (original magnification, 10 times) of the marrow vascular endothelial cells of the patients with HD, MDS-MLD, MDS-EB and AML; b is the migration capacity (original magnification, 10 times) of the marrow vascular endothelial cells of the patients with HD, MDS-MLD, MDS-EB and AML; c is a tube forming length statistical analysis chart; d is a statistical analysis of the number of cells migrating across the membrane (tube pixels per field). Three fields were counted randomly and averaged for each sample. The scale bar represents 200 μm.
FIG. 5 shows the hematopoietic stem cell supporting ability of bone marrow vascular endothelial cells of MDS and AML patients. A is CD34 after 5 days of co-culture of vascular endothelial cells derived from HD, MDS-MLD, MDS-EB and AML patients+The rate of apoptosis of the cell; b is CD34 after co-culture+Intracellular ROS levels; c is CD34 after co-culture+CFU forming ability of the cells.
FIG. 6 shows that the bone marrow vascular endothelial cells of MDS-MLD, MDS-EB and AML patients caused abnormal differentiation of CD3+ cells. A is the ratio of Th1 cells after co-culture; b is the ratio of Th2 cells after co-culture; c is the ratio of Th17 cells after co-culture; d is the proportion of Treg cells after co-culture; e is the ratio of Th1/Th2 after co-cultivation.
FIG. 7 shows the proliferation, apoptosis, ROS levels of HL-60 cells after co-culture of MDS-MLD, MDS-EB, and AML patients with bone marrow vascular endothelial cells. A is the proliferation of HL-60 cells; b is the apoptosis of HL-60 cells; c is the statistical analysis of the proportion of HL-60 cell EdU positive cells; d is statistical analysis of the apoptosis ratio of HL-60 cells; e is a statistical analysis of the ROS levels (mean fluorescence intensity) of HL-60 cells; f is the CFU-L formation efficiency of HL-60 cells after the co-culture of MDS-MLD, MDS-EB and AML patients' marrow vascular endothelial cells; g is HL-60 cell apoptosis and cell cycle related gene expression level after co-culture.
FIG. 8 shows the results of transcriptome sequencing analysis of the bone marrow vascular endothelial cells of HD, MDS and AML patients. A is a principal component analysis diagram of 12 transcriptome sequencing libraries; b is a gene expression heat map, and hierarchical clustering is carried out according to Euclidean distance; c is differential gene counts between groups; d is the differential expression of the genes related to apoptosis, hematopoiesis and immunity in the sequencing result of the transcriptome; e, verifying the mRNA level of the hematopoietic related gene by adopting qRT-PCR; f, verifying the mRNA level of the immune related gene by adopting qRT-PCR.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 use of bone marrow vascular endothelial cells in MDS
First, experimental material
EGM-2-MV-SingleQuots liquid medium: lonza corporation, usa.
RPMI 1640 liquid medium: gibco, USA.
MethoCult H4434 Classic: STEMCELL Technologies, Canada.
StemBan SFEM: STEMCELL Technologies, Canada.
5. Human peripheral blood lymphocyte isolate (Ficoll): GE Healthcare, USA.
6. Monoclonal antibodies: CD3-APC/H7(Becton Dickinson (BD)), CD8-BV510 (Biolegged), CD25-PE/Cy7(Invitrogen), IFN-. gamma. -PerCP/Cy5.5 (Biolegged), IL-4-PE (BD), IL17A-FITC (BD), FOXP3-APC (Invitrogen), CD 34-perc/Cy 5.5 (Biolegged), CD34-FITC (Biolegged), CD133-APC (Miltenyi), CD45-V500(BD), vascalendecott growth factor 2(VEGFR2, CD309) -PE (BD), BD-NEXIn-V and 7-amino-actinomycin D (7-AAD), mouse-anti-CD 34(BD), and CD133 (Abcari-Biocaman).
DAPI solution: beijing Solaibao Co.
2 ', 7' -dichlorfluoro escein diacetate (DCFH-DA): china bi yun tian company.
9. Reactive Oxygen Species Assay Kit (Reactive Oxygen Species Assay Kit): china Biyuntian Co Ltd
10. Fibronectin (Fibronectin): sigma Co USA
11.5-Ethyl-20-deoxyuridine (EdU): china Ruibo Bio Inc
12. Recombinant human FLT-3: PEPROTECH, USA
13. Recombinant human TPO: PEPROTECH, USA
14. Recombinant human SCF: PEPROTECH, USA
15. Human CD34 sorting magnetic beads: miltenyi, Germany
16. Human CD3 sorting magnetic beads: miltenyi, Germany
17. Hemolysin: BD FACSM dissolving Solution, BD corporation, USA
18. Fetal Bovine Serum (FBS): gibco Inc. of USA
19. Vitex bean lectin I-FITC labeling: FITC-latex led free from Ulex europaeus (FITC-UEA-I): Sigma-Aldrich, USA
20. Diacetylated low density lipoprotein: DiI-AcLDL, Life Technologies, USA
21. Substrate glue: matrigel matrix, Corning USA Inc
Transwell cell: transwell chamber, Corning Inc. of USA
RNA extraction kit: RNeasy Mini kit, QIAGEN, Germany
24. Reverse transcriptase: RT reagent Kit with gDNA Eraser, TaKaRa, Japan
25. Fluorescence quantitative heat-resistant reverse transcription kit: SYBR-Green qRT-PCR kit, Thermo Fisher Scientific, USA
An HL-60 cell line: human myelogenous leukemia cell line, cell bank/stem cell bank of Chinese academy of sciences
27. The cohort study included bone marrow samples from 15 and 15 HD cases each of first-visit MDS-MLD, MDS-EB, or AML patients as healthy controls. Wherein the diagnosis of MDS is according to the 2016 version of the WHO typing standard; the primary AML patients were primary AML and were diagnosed with subtype M2, M4 or M5. The basic characteristics of each group of patients are similar, such as sex, age, etc.
Second, Experimental methods
1. Culture of bone marrow vascular endothelial cells
Separating bone marrow mononuclear cells by adopting a density gradient centrifugation method: bone marrow 1: lymphocyte separation (GE Healthcare, USA) is added and centrifuged at 1800rpm for 18 minutes at room temperature with a gradient. The white and misty mixed solution in the middle layer, i.e., the mononuclear cells, was gently aspirated and washed twice with PBS. Placing bone marrow mononuclear cell in 6-well plate coated with fibronectin, adding endothelial cell induction culture medium EGM-2-MV-SingleQuots and 10% fetal calf serum, at 37 deg.C and 5% CO2The culture was carried out in the incubator for 7 days, and the solution was changed once on the fourth day.
2. Bone marrow vascular endothelial cell number detection
The human bone marrow mononuclear cell is marked with anti-human-CD 45, anti-human-CD 34, anti-human-CD 133 and anti-human-CD 309 at room temperature in dark for 15 min. Washed twice with PBS, centrifuged at 1500rpm for 5 minutes, and assayed. Data were analyzed using BD LSRFortessa software.
② human bone marrow mononuclear cells (BMCs) are cultured for 7 days by Endothelial Cell (EC) induction medium (EGM-2-MV-SingleQuots), then the adherent cells are washed for three times by PBS, and 10 mug/ml DiI-Ac-LDL is added to incubate for 4 hours at 37 ℃. Cells were washed three more times with PBS and fixed with 4% paraformaldehyde for 10 min. PBS was washed twice for 5 minutes each. The fixed cells were incubated with FITC-UEA-110. mu.g/ml for 1 hour at room temperature in the absence of light. The stained cells were observed under a fluorescent microscope (Olympus, Tokyo, Japan). Dil-Ac-LDL stained red, FITC-UEA-1 stained green, and double positive cells were yellow, EC, after coincidence in color. Three fields were counted randomly and averaged.
3. Intracellular Reactive Oxygen Species (ROS) level and apoptosis assays
ROS: the cells were incubated with 10. mu.M 2 ', 7' -dichlorofluoro sequence diacetate (DCFH-DA) for 15min at 37 ℃ and washed twice with PBS followed by flow-testing of the mean fluorescence intensity of intracellular DCFH-DA using BD LSRFortessa software (DCFH-DA is a ROS probe).
Apoptosis: Annexin-V&7-amino-actinomycin D (7-AAD) was incubated for 15 minutes and directly detected by BD LSR Fortessa flow meter. Annexin-V+7-AAD-For early apoptosis, Annexin-V+7-AAD+Late apoptosis.
Data were analyzed using BD LSRFortessa software.
4. Tube formation experiment and migration experiment
Firstly, a tube forming experiment: in a pre-cooled 24-well plate, 200. mu.L of matrigel solution was added per well, and then placed in an incubator at 37 ℃ for 30min to form a gel. Endothelial cell suspension (500. mu.L/well) after 7 days of induction culture was added to the gel surface, and the number of cells per well was 5X 105Adding EC induction medium (EGM-2-MV-SingleQuots), and photographing under microscope after 48 hr. Total 4X 104The individual vascular endothelial cells were transferred to a substrate-coated plate at 37 ℃ with 5% CO2Stored for 48 hours. The relative tube length per field was measured with an inverted optical microscope.
Migration experiment: transwell chamber for cell migration experiments. Cells under digestion were at 5X 10 per well4Cell seed in the upper chamber, the lower chamber is added 500. mu.l EC induction medium (EGM-2-MV-SingleQuots). After culturing the cells at 37 ℃ for 24 hours, the cells on the membrane were fixed with formaldehyde for 30 minutes, the cells on the upper layer were wiped off with a cotton swab, and the cells on the bottom of the membrane were stained with crystal violet for 20 minutes. The stained cells were photographed under a microscope (Olympus, Tokyo, Japan).
5、CD34+Co-culture of cells with bone marrow vascular endothelial cells
Separation of CD34 from bone marrow mononuclear cells of HD using CD34 magnetic beads+Cells, as non-adherent cells, were cultured in contact with HD, MDS-MLD, MDS-EB and AML bone marrow vascular endothelial cells at day 7 of culture in StemBantm SFEM at 37 ℃ with 5% CO2The culture box is cultured for 5 days, and then the subsequent detection is carried out.
6、CD3+Co-culture of cells with bone marrow vascular endothelial cells
Separation of CD3 from bone marrow mononuclear cells (HD) using CD3 magnetic beads+The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin as non-adherent cells and HD, MDS-MLD, MDS-EB and AML bone marrow vascular endothelial cells cultured on day 7, and cultured in contact with 5% CO at 37 deg.C2The culture box is cultured for 3 days, and then the subsequent detection is carried out.
7. Co-culture of HL-60 cells and bone marrow vascular endothelial cells
HL-60 cells as non-adherent cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in contact with HD, MDS-MLD, MDS-EB and AML bone marrow vascular endothelial cells cultured on day 7, and cultured at 37 deg.C and 5% CO2The culture box is cultured for 5 days, and then the subsequent detection is carried out.
8. Detection of Colony Forming Unit (CFU) and leukemia colony Forming Unit (CFU-L)
Harvesting of suspended CD34+Cells or HL-60 cells, 2X 103An individual CD34+Cell/103HL-60 cells in 500 mu LMethoCultTMH4434 Classic Medium was mixed well at 37 ℃ with 5% CO2The culture was carried out in the incubator for 14 days. CD34 after 14 days+The number of erythroid colony forming units (CFU-E), erythroid burst-type colony forming units (BFU-E), granulocyte-macrophage colony forming units (CFU-GM), and granulocyte-erythrocyte-macrophage-megakaryocyte colony forming units (CFU-GEMM) of cell differentiation and CFU-L formation by HL-60 cells were counted.
9. T cell subpopulation analysis
T cells were labeled with the following mouse anti-human monoclonal antibodies: anti-human-CD 3, anti-human-CD 4, anti-human-CD 8, anti-human-CD 25, anti-human-IFN-gamma, anti-human-IL-4, anti-human-Foxp 3, anti-human-IL-17A, lymphocyte subpopulations were quantified by flow cytometry. Th1, Th2, Th17 and Treg cells are CD3+CD8-IFN-γ+、CD3+CD8-IL-4+、CD3+CD8-IL-17A+And CD3+CD8-CD25+Foxp3+. Data were analyzed using BD LSRFortessa software.
10. EdU test
The CO-cultured HL-60 cells were harvested and treated with 50. mu.M EdU at 37 ℃ with 5% CO2Incubate for 1 hour, and the remaining steps were performed according to product instructions. The cell fluorescence intensity was finally detected and analyzed on BD lsrortessa.
11. RNA sequencing (RNA-seq) and real-time quantitative PCR (qRT-PCR)
RNA-seq analysis was performed on bone marrow-derived vascular endothelial cells cultured on day 7 in HD, MDS-MLD, MDS-EB, and AML patients. Hierarchical clustering analysis and differential gene expression analysis were performed using the heatmap and DESeq2 packages in R (1.16.1). To verify the RNA-seq results, the mRNA levels of CASP2, CASP3, BAX, CCNE1, MCL1, p53, p21, CXCL12, KITLG, and NFKB1 were tested using SYBR Green-based qRT-PCR (ViiA7Real-Time PCR System, Thermo Fisher Scientific, USA) and the genes were normalized to 18S mRNA levels.
12. Statistical analysis
Statistical analysis was performed using GraphPad Prism 6.0 (USA). The two component comparisons were tested using Mann-Whitney U. Results are expressed as mean ± SEM, P <0.05 is considered statistically significant.
Third, results and analysis
1. The number of bone marrow vascular endothelial cells of MDS patients gradually increases with disease progression
HD. On the day of extraction of bone marrow mononuclear cells of MDS-MLD, MDS-EB and AML patients (day 0), vascular endothelial cells were delineated by flow antibodies CD34, CD309 and CD 133. The proportion of bone marrow vascular endothelial cells in mononuclear cells is shown in fig. 1, with the MDS-EB group being significantly higher than the MDS-MLD group, and the AML group being significantly higher than the MDS-EB group (fig. 1).
Identification of human bone marrow endothelial cells cultured for 7 days the double-label experiment (A in FIG. 2) was a classical experiment for identifying vascular endothelial cells by using a Dil-ac-LDL/FITC-UEA-1 double-label experiment, labeled with Dil-ac-LDL (red fluorescence) and FITC-UEA-1 (green fluorescence), and cells with double positive staining were vascular endothelial cells (yellow fluorescence). Statistical analysis was performed by counting double positive cells under a fluorescent microscope field. The number of double positive myeloid vascular endothelial cells was significantly greater in AML patients than in MDS-EB patients (figure 2).
In conclusion, the number of myeloid vascular endothelial cells in MDS and AML patients increases, and gradually increases from MDS-MLD, MDS-EB and AML as the disease progresses.
2. Bone marrow vascular endothelial cell injury of MDS patients gradually worsens with disease progression
In order to study the damage condition of the marrow vascular endothelial cells, the expression levels of the apoptosis, ROS and apoptosis-related genes of the marrow vascular endothelial cells of patients with HD, MDS-MLD, MDS-EB and AML are tested. Apoptosis, ROS, was measured by flow assay on day 0 bone marrow vascular endothelial cells. The expression level of the apoptosis-related gene was determined by culturing human bone marrow vascular endothelial cells for 7 days.
The apoptosis rate of the marrow vascular endothelial cells in the MDS-EB group is significantly higher than that in the MDS-MLD group (A in FIG. 3). ROS levels were significantly higher in AML patients than in MDS-EB patients (B in FIG. 3). Bone marrow vascular endothelial cells CASP2, CASP3 and BAX apoptosis related gene expression levels are further analyzed by adopting qRT-PCR. The mRNA level of CASP2 was significantly increased in the bone marrow vascular endothelial cells of AML patients compared to MDS-EB patients (C in FIG. 3), and the expression levels of CASP3 and BAX in the bone marrow vascular endothelial cells of MDS-EB patients were significantly increased compared to MDS-MLD (C in FIG. 3). Taken together, these data suggest MDS, AML, and progressive progression of MDS, with progressive, and progressive, vascular endothelial cell damage to the bone marrow.
3. The dysfunction of marrow vascular endothelial cells of MDS patients gradually worsens along with the disease progression
HD. The function of the MDS-MLD, MDS-EB and AML patients bone marrow-derived vascular endothelial cells will be assessed from their angiogenic capacity. Bone marrow-derived vascular endothelial cells were analyzed for tube-forming ability (a in fig. 4) and migration ability (B in fig. 4) on day 7 of culture. Three fields were counted randomly and averaged for each sample. Statistical analysis of tube length showed that the bone marrow vascular endothelial cells of MDS-EB patients had significantly improved tube forming ability compared to MDS-MLD patients (C in FIG. 4). Human bone marrow endothelial cells migrated transmembrane-wise in the chamber migration assay, and bone marrow vascular endothelial cells from AML patients migrated more strongly than from MDS-MLD patients (D in FIG. 4). These results indicate that there is dysfunction of the myeloid vascular endothelial cells of the MDS and AML patients, and that the dysfunction of the myeloid vascular endothelial cells is increasingly severe as MDS progresses.
4. The supporting capacity of the marrow vascular endothelial cells on the hematopoietic stem cells is weakened along with the progress of MDS
To investigate the effect of bone marrow vascular endothelial cells on hematopoietic stem cells, we sorted donor-derived bone marrow mononuclear cells from CD34 of HD+Co-culturing the cells with the vascular endothelial cells from the bone marrow of patients with HD, MDS-MLD, MDS-EB and AML on the 7 th day of culture, and detecting CD34 after 5 days+Apoptosis of the cells, intracellular ROS levels and hematopoietic stem cell-Colony Forming Unit (CFU) forming capacity. Apoptosis, intracellular ROS levels were detected by flow cytometry. Evaluation of CFU Forming ability 2X 103CD3 after 5 days of co-culture4+Cells were plated evenly on special semi-solid medium for 14 days and colonies of different types were counted. Bone marrow CD34 in coculture with MDS-EB bone marrow vascular endothelial cells, compared to MDS-MLD group+The level of apoptosis was significantly increased (a in fig. 5). AML group CD34+Cellular ROS levels were elevated compared to the MDS-MLD group (B in FIG. 5). CFU-E, BFU-E, CFU-GM and CFU-GEMM-forming ability were decreased in both MDS-EB and AML groups compared to MDS-MLD group (C in FIG. 5). These data indicate that myeloid vascular endothelial cells in patients with MDS, AML, cause hematopoietic stem cell dysfunction, and as MDS progresses, vascular endothelial cells become less and less able to support normal hematopoietic stem cells.
5. As MDS progresses, the myeloid vascular endothelial cells cause T cells to gradually differentiate towards immune tolerance
Considering MDS and immune-related pathogenesis, we further study the in vitro immunoregulation effect of marrow vascular endothelial cells, and we take HD-derived marrow CD3+The T cells were co-cultured with bone marrow vascular endothelial cells of patients with MDS-MLD, MDS-EB and AML, and the difference of T cell subsets was analyzed 3 days later. Th1 in the AML group was CD4 compared to the MDS-EB group+The proportion in T cells decreased significantly (a in fig. 6). Th2 in AML group at CD4+The proportion in T cells (B in FIG. 6) was higher than in the MDS-MLD group. Th17 was in CD4 for MDS-EB group compared to MDS-MLD group+The proportion in T cells decreased significantly (C in fig. 6). Tregs of MDS-EB group are in CD4+The proportion in T cells (D in FIG. 6) was higher than in the MDS-MLD group. The ratio Th1/Th2 was lower in the AML group than in the MDS-EB group, and the ratio Th1/Th2 was significantly lower in the MDS-MLD group (E in FIG. 6). These data suggest that the myeloid vascular endothelial cells of MDS, AML patients are the cause of dysregulation of MDS, and that as the disease progresses, the myeloid vascular endothelial cells may be more inclined to induce T cell differentiation into immune-tolerant cells.
6. The supporting capacity of the marrow vascular endothelial cells of MDS patients on leukemia cells is enhanced along with the disease progression
To investigate the effect of myeloid vascular endothelial cells on AML cells in vitro, we examined the proliferation, apoptosis, intracellular ROS levels and leukemic cell colony-forming monomers of HL-60 cells after 5 days of co-culture with myeloid vascular endothelial cellsThe formation efficiency of the locus (CFU-leukamia, CFU-L) and the relative expression level of the apoptosis and cell cycle related gene mRNA in HL-60 cells after co-culture. Proliferation of HL-60 cells was detected using the EdU (5-ethyl-20-deoxyuridine) assay. HL-60 apoptosis and intracellular ROS level were detected by flow cytometry. CFU-L Forming ability by combining 103And uniformly planting the HL-60 cells which are cultured for 5 days in a special semisolid culture medium for 14 days, and counting the number of leukemia cell colonies. The positive rate of HL-60 cells EdU in the MDS-EB group is obviously higher than that in the MDS-MLD group (A and C in figure 7). Apoptosis rates (B and D in fig. 7) were significantly lower in AML group than in MDS-MLD group, and ROS levels (fold relative to HD) were significantly lower in MDS-EB group than in MDS-MLD group (E in fig. 7). In addition, the CFU-L formation efficiency was significantly higher in the AML group than in the MDS-EB group (F in FIG. 7). Compared with MDS-MLD, the MDS-EB group TP53 and CDKN1A are remarkably reduced, the MCL1 is remarkably increased, and the AML group CASP2, CASP3, BAX, TP53 and CDKN1A are remarkably reduced. Compared with MDS-EB, the AML group CASP2, TP53 and CDKN1A are significantly reduced, and CCNE1 and MCL1 are significantly increased (G in figure 7). These data suggest enhanced leukemia cell support by myeloid vascular endothelial cells in MDS and AML patients. More importantly, the capacity to support leukemia cells is gradually increased as the disease progresses.
7. Heterogeneity and potentially impaired mechanisms of myeloid vascular endothelial cells in MDS progression
In order to confirm the heterogeneity of the myeloid-vascular endothelial cells of the MDS and AML patients at the transcriptome level and further explore the mechanism of the myeloid-vascular endothelial cell damage of the MDS and AML patients, we performed transcriptome sequencing on the bone marrow-derived endothelial cells cultured at day 7 and performed principal component analysis (a in fig. 8), hierarchical cluster analysis (B in fig. 8) and differential gene analysis (C in fig. 8). The results showed that 12 samples were clearly divided into two distinct subgroups, HD and disease groups (a, B in figure 8). More importantly, the heatmap (B in fig. 8) shows the progression of the RNA expression profile of myeloid vascular endothelial cells from HD to AML. The differential gene counts between HD and patients (C in FIG. 8) (e.g., HD and MDS-MLD (3448)) are much higher than between patients (e.g., MDS-MLD vs. MDS-EB (722)). These results reveal the heterogeneity of bone marrow-derived endothelial cells in HD and MDS-MLD, MDS-EB, AML from the transcriptome level. Further analysis of the transcriptome sequencing results revealed that genes associated with apoptosis (CASP2, CASP3, BAX), hematopoiesis (CXCL12, KITLG, NFKB1) and immunity (HAVCR2, LGALS9, CIITA) were elevated in bone marrow-derived endothelial cells of MDS and AML patients, as shown (D in figure 8). And qRT-PCR was used to verify the increase of hematopoietic related genes CXCL12, KITLG, NFKB1 (E in FIG. 8) and immune related genes (HAVCR2, LGALS9, CIITA) as disease progresses (F in FIG. 8). These data further reveal the role of myeloid vascular endothelial cells in regulating hematopoiesis and immunity in MDS, AML, and suggest that the relevant genes may be responsible for the impairment of MDS, AML myeloid vascular endothelial cells.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (8)
1. The application of the marrow vascular endothelial cells as the markers in any one of the following:
(A1) preparing a product for detecting or assisting in detecting the course of disease progression of myelodysplastic syndrome, or detecting or assisting in detecting the course of disease progression of myelodysplastic syndrome;
(A2) preparing a product for use in, or for aiding in, the diagnosis of myelodysplastic syndrome;
(A3) preparing a product for, or to aid in, distinguishing between myelodysplastic syndrome patients and non-myelodysplastic syndrome patients, or distinguishing between myelodysplastic syndrome patients and non-myelodysplastic syndrome patients.
2. Use of substance a and/or substance B and/or substance C and/or substance D and/or substance E and/or substance F in any of:
(A1) preparing a product for detecting or assisting in detecting the course of disease progression of myelodysplastic syndrome, or detecting or assisting in detecting the course of disease progression of myelodysplastic syndrome;
(A2) preparing a product for use in, or for aiding in, the diagnosis of myelodysplastic syndrome;
(A3) preparing a product for, or to aid in, distinguishing between myelodysplastic syndrome patients and non-myelodysplastic syndrome patients, or distinguishing between myelodysplastic syndrome patients and non-myelodysplastic syndrome patients;
the substance A is used for detecting the number of vascular endothelial cells in bone marrow;
the substance B is used for detecting the damage condition of vascular endothelial cells in bone marrow;
the substance C is used for detecting the functional state of vascular endothelial cells in bone marrow;
the substance D is used for detecting the supporting capacity of vascular endothelial cells in bone marrow to hematopoietic stem cells;
the substance E is used for detecting whether the vascular endothelial cells in the bone marrow can cause the T cells to differentiate towards the immune tolerance direction;
the substance F is used for detecting the leukemia cell supporting capacity of vascular endothelial cells in bone marrow.
3. Use according to claim 2, characterized in that: the vascular endothelial cell damage condition in the bone marrow is embodied in whole or part of the following conditions: the apoptosis rate of the marrow vascular endothelial cells, the ROS level in the marrow vascular endothelial cells and the expression quantity of apoptosis-related genes in the marrow vascular endothelial cells; and/or
The functional state of vascular endothelial cells in the bone marrow is represented by all or part of the following: the tube forming ability of the marrow vascular endothelial cells and the migration ability of the marrow vascular endothelial cells.
4. Use according to any one of claims 1 to 3, characterized in that: the non-myelodysplastic syndrome patient satisfies the following conditions: compared with myelodysplastic syndrome patients, the number of vascular endothelial cells in bone marrow is reduced, the damage condition of the vascular endothelial cells in the bone marrow is reduced or no damage occurs, the dysfunction of the vascular endothelial cells in the bone marrow is reduced or no dysfunction occurs, the supporting capacity of the vascular endothelial cells in the bone marrow to hematopoietic stem cells is enhanced, the differentiation of T cells to an immune tolerance direction is not caused by the vascular endothelial cells in the bone marrow, the supporting capacity of the vascular endothelial cells in the bone marrow to leukemia cells is reduced or the leukemia cells have no supporting capacity.
5. Use according to claim 4, characterized in that: the non-myelodysplastic syndrome patient is a healthy person.
6. Use according to any one of claims 1 to 5, characterized in that: in (A3), the myelodysplastic syndrome patient is a patient with MDS with multilineage dysplasia, a patient with MDS with polycythemia, or a patient with acute myeloid leukemia.
7. A system for detecting the progression of the course of myelodysplastic syndrome, comprising:
(B1) reagents and/or instruments;
the reagent and/or apparatus has all or part of the following functions: detecting the number of vascular endothelial cells in bone marrow, detecting the damage condition of the vascular endothelial cells in the bone marrow, detecting the functional state of the vascular endothelial cells in the bone marrow, detecting the supporting capacity of the vascular endothelial cells in the bone marrow to hematopoietic stem cells, detecting whether the vascular endothelial cells in the bone marrow can cause the differentiation of T cells towards the immune tolerance direction, and detecting the supporting capacity of the vascular endothelial cells in the bone marrow to leukemia cells;
(B2) a device;
the device comprises a data input module, a threshold storage module, a data comparison module and a judgment module;
the data input module is configured to input (B1) all or part of the detected values: a vascular endothelial cell count value in a test bone marrow from a myelodysplastic syndrome patient; the damage condition value of the vascular endothelial cells in the bone marrow to be detected; the vascular endothelial cell dysfunction value in the bone marrow to be detected; the supporting capacity value of the vascular endothelial cells in the bone marrow to be tested to the hematopoietic stem cells; the vascular endothelial cells in the bone marrow to be detected cause the differentiation value of T cells to the immune tolerance direction; the supporting capacity value of the vascular endothelial cells in the bone marrow to be detected to leukemia cells;
the threshold storage module is configured to store all or part of the following thresholds: a threshold A, a threshold B, a threshold C, a threshold D, a threshold E and a threshold F; the threshold value A is a vascular endothelial cell quantity value in the bone marrow of a healthy person; the threshold B is a vascular endothelial cell damage condition value in the bone marrow of a healthy person; the threshold C is a vascular endothelial cell dysfunction value in the bone marrow of a healthy person; the threshold value D is the supporting capacity value of the vascular endothelial cells in the healthy human bone marrow to the hematopoietic stem cells; the threshold value E is a value of differentiation of T cells to an immune tolerance direction caused by vascular endothelial cells in the bone marrow of a healthy person; the threshold value F is the supporting capacity value of the vascular endothelial cells in the bone marrow of the healthy human to leukemia cells;
the data comparison module is configured to receive all or part of the detected values (B1) sent by the data input module, calling each threshold in the threshold storage module, comparing the quantity value of the vascular endothelial cells in the bone marrow to be detected with the threshold A, comparing the damage condition value of the vascular endothelial cells in the bone marrow to be detected with the threshold B, comparing the dysfunction value of the vascular endothelial cells in the bone marrow to be detected with the threshold C, comparing the supporting capacity value of the vascular endothelial cells in the bone marrow to be detected on hematopoietic stem cells with the threshold D, comparing the differentiation value of T cells towards the immune tolerance direction caused by the vascular endothelial cells in the bone marrow to be detected with the threshold E, and/or comparing the leukemia cell supporting capacity value of the vascular endothelial cells in the bone marrow to be detected with the threshold value F;
the judging module is configured to receive the comparison result sent by the data comparing module and then make a result judgment as follows: the number value of the vascular endothelial cells in the bone marrow to be detected is greater than the threshold value A, the damage condition value of the vascular endothelial cells in the bone marrow to be detected is greater than the threshold value B, the dysfunction value of the vascular endothelial cells in the bone marrow to be detected is greater than the threshold value C, the supporting capacity value of the vascular endothelial cells in the bone marrow to be detected to hematopoietic stem cells is less than the threshold value D, the differentiation value of T cells to the immune tolerance direction caused by the vascular endothelial cells in the bone marrow to be detected is greater than the threshold value E, and/or the supporting capacity value of the vascular endothelial cells in the bone marrow to be detected to leukemia cells is greater than the threshold value F, and the larger the absolute value of the difference between each value and the corresponding threshold value is, the more serious the illness state of the myelodysplastic syndrome patient is.
8. A system for diagnosing or aiding in the diagnosis of myelodysplastic syndrome, comprising:
(C1) reagents and/or instruments;
the reagent and/or apparatus has all or part of the following functions: detecting the number of vascular endothelial cells in bone marrow, detecting the damage condition of the vascular endothelial cells in the bone marrow, detecting the functional state of the vascular endothelial cells in the bone marrow, detecting the supporting capacity of the vascular endothelial cells in the bone marrow to hematopoietic stem cells, detecting whether the vascular endothelial cells in the bone marrow can cause the differentiation of T cells towards the immune tolerance direction, and detecting the supporting capacity of the vascular endothelial cells in the bone marrow to leukemia cells;
(C2) a device;
the device comprises a data input module, a threshold storage module, a data comparison module and a judgment module;
the data input module is configured to input (B1) all or part of the detected values: a vascular endothelial cell count value in a test bone marrow from a test subject; the damage condition value of the vascular endothelial cells in the bone marrow to be detected; the vascular endothelial cell dysfunction value in the bone marrow to be detected; the supporting capacity value of the vascular endothelial cells in the bone marrow to be tested to the hematopoietic stem cells; the vascular endothelial cells in the bone marrow to be detected cause the differentiation value of T cells to the immune tolerance direction; the supporting capacity value of the vascular endothelial cells in the bone marrow to be detected to leukemia cells;
the threshold storage module is configured to store all or part of the following thresholds: a threshold A, a threshold B, a threshold C, a threshold D, a threshold E and a threshold F; the threshold value A is a vascular endothelial cell quantity value in the bone marrow of a healthy person; the threshold B is a vascular endothelial cell damage condition value in the bone marrow of a healthy person; the threshold C is a vascular endothelial cell dysfunction value in the bone marrow of a healthy person; the threshold value D is the supporting capacity value of the vascular endothelial cells in the healthy human bone marrow to the hematopoietic stem cells; the threshold value E is a value of differentiation of T cells to an immune tolerance direction caused by vascular endothelial cells in the bone marrow of a healthy person; the threshold value F is the supporting capacity value of the vascular endothelial cells in the bone marrow of the healthy human to leukemia cells;
the data comparison module is configured to receive all or part of the detected values (C1) sent by the data input module, calling each threshold in the threshold storage module, comparing the quantity value of the vascular endothelial cells in the bone marrow to be detected with the threshold A, comparing the damage condition value of the vascular endothelial cells in the bone marrow to be detected with the threshold B, comparing the dysfunction value of the vascular endothelial cells in the bone marrow to be detected with the threshold C, comparing the supporting capacity value of the vascular endothelial cells in the bone marrow to be detected on hematopoietic stem cells with the threshold D, comparing the differentiation value of T cells towards the immune tolerance direction caused by the vascular endothelial cells in the bone marrow to be detected with the threshold E, and/or comparing the leukemia cell supporting capacity value of the vascular endothelial cells in the bone marrow to be detected with the threshold value F;
the judging module is configured to receive the comparison result sent by the data comparing module and then make a result judgment as follows: if the number value of the vascular endothelial cells in the bone marrow to be detected is greater than the threshold value A, the value of the damage condition of the vascular endothelial cells in the bone marrow to be detected is greater than the threshold value B, the dysfunction value of the vascular endothelial cells in the bone marrow to be detected is greater than the threshold value C, the value of the supporting capacity of the vascular endothelial cells in the bone marrow to be detected to hematopoietic stem cells is less than the threshold value D, the differentiation value of the vascular endothelial cells in the bone marrow to be detected to the immune tolerance direction is greater than the threshold value E, and/or the value of the supporting capacity of the vascular endothelial cells in the bone marrow to be detected to leukemia cells is greater than the threshold value F, the patient to be detected is a myelodysplasia syndrome patient.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111273168.6A CN113930496B (en) | 2021-10-29 | 2021-10-29 | Application of marrow vascular endothelial cells in myelodysplastic syndrome |
| CN202210451953.4A CN114807349A (en) | 2021-10-29 | 2021-10-29 | Application of bone marrow vascular endothelial cells in myelodysplastic syndrome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111273168.6A CN113930496B (en) | 2021-10-29 | 2021-10-29 | Application of marrow vascular endothelial cells in myelodysplastic syndrome |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210451953.4A Division CN114807349A (en) | 2021-10-29 | 2021-10-29 | Application of bone marrow vascular endothelial cells in myelodysplastic syndrome |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113930496A true CN113930496A (en) | 2022-01-14 |
| CN113930496B CN113930496B (en) | 2022-06-14 |
Family
ID=79284858
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111273168.6A Active CN113930496B (en) | 2021-10-29 | 2021-10-29 | Application of marrow vascular endothelial cells in myelodysplastic syndrome |
| CN202210451953.4A Pending CN114807349A (en) | 2021-10-29 | 2021-10-29 | Application of bone marrow vascular endothelial cells in myelodysplastic syndrome |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210451953.4A Pending CN114807349A (en) | 2021-10-29 | 2021-10-29 | Application of bone marrow vascular endothelial cells in myelodysplastic syndrome |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN113930496B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005221323A (en) * | 2004-02-04 | 2005-08-18 | Nippon Medical School | Method for detecting osteomyelodysplasia syndrome |
| CN102768283A (en) * | 2012-07-23 | 2012-11-07 | 北京大学人民医院 | Kit for detecting or supplementarily detecting medullary leukemia cell differentiation stages |
| WO2020200295A1 (en) * | 2019-04-03 | 2020-10-08 | Nanjing Iaso Biotherapeutics Co., Ltd. | Ex-vivo cultured macrophage population and use thereof |
-
2021
- 2021-10-29 CN CN202111273168.6A patent/CN113930496B/en active Active
- 2021-10-29 CN CN202210451953.4A patent/CN114807349A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005221323A (en) * | 2004-02-04 | 2005-08-18 | Nippon Medical School | Method for detecting osteomyelodysplasia syndrome |
| CN102768283A (en) * | 2012-07-23 | 2012-11-07 | 北京大学人民医院 | Kit for detecting or supplementarily detecting medullary leukemia cell differentiation stages |
| WO2020200295A1 (en) * | 2019-04-03 | 2020-10-08 | Nanjing Iaso Biotherapeutics Co., Ltd. | Ex-vivo cultured macrophage population and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| SYED A. MIAN等: "Nature or Nurture? Role of the Bone Marrow Microenvironment in the Genesis and Maintenance of Myelodysplastic Syndromes", 《CANCER》 * |
| 吕扬扬等: "骨髓微环境与骨髓增生异常综合征", 《天津医药》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113930496B (en) | 2022-06-14 |
| CN114807349A (en) | 2022-07-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Russo et al. | Modeling the interplay between neurons and astrocytes in autism using human induced pluripotent stem cells | |
| Yu et al. | IL15 promotes growth and invasion of endometrial stromal cells and inhibits killing activity of NK cells in endometriosis. | |
| Bergmann et al. | T regulatory type 1 cells in squamous cell carcinoma of the head and neck: mechanisms of suppression and expansion in advanced disease | |
| Yang et al. | The characteristics of endothelial progenitor cells derived from mononuclear cells of rat bone marrow in different culture conditions | |
| Essid et al. | Induction of epithelial-mesenchymal transition (EMT) and Gli1 expression in head and neck squamous cell carcinoma (HNSCC) spheroid cultures | |
| CN107064085B (en) | Method for measuring natural killer cell activity | |
| Horzum et al. | CD66b+ monocytes represent a proinflammatory myeloid subpopulation in cancer | |
| JPWO2020045368A1 (en) | Evaluation method for anti-infective drugs, vaccines, etc. using immortalized monocyte cells and inducible cells | |
| Nalin et al. | Notch regulates innate lymphoid cell plasticity during human NK cell development | |
| Hu et al. | Differentiation of monocytes into phenotypically distinct macrophages after treatment with human cord blood stem cell (CB-SC)-derived exosomes | |
| CN115094034A (en) | Human NKT cell line and application thereof | |
| Slone et al. | Modeling chemotherapy resistant leukemia in vitro | |
| Wang et al. | Impact of cell fusion in myeloma marrow microenvironment on tumor progression | |
| CN113930496B (en) | Application of marrow vascular endothelial cells in myelodysplastic syndrome | |
| WO2015169781A1 (en) | Predictive markers for successful cancer immunotherapy | |
| Liu et al. | Immunoregulatory functions of mature CD10+ and immature CD10–neutrophils in sepsis patients | |
| Yu et al. | Purification, visualization, and molecular signature of neural stem cells | |
| Lubin et al. | Human Dendritic Cell Enrichment and Their Activation of T Cells | |
| WO2022148015A1 (en) | Signaling pathway inhibitor and drug for up-regulating mllt3 gene expression in hematopoietic stem cells, and use of signaling pathway inhibitor | |
| Yao et al. | The effect of regulating the Wnt signaling pathway on the proliferation and differentiation of spermatogonial stem cells | |
| Tang et al. | Improved function and balance in T cell modulation by endothelial cells in young people | |
| WO2020232512A1 (en) | Microglial cells and methods of use thereof | |
| Jang et al. | Induction of a memory‐like CD4+ T‐cell phenotype by airway smooth muscle cells | |
| CN115896015B (en) | In-vitro culture method of myeloid-derived suppressor cells | |
| CN115198017B (en) | Application of macrophage subgroup based on high-expression PLIN2 in diagnosis and treatment of ovarian cancer ascites |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230731 Address after: 100020 101, 201, 301 and 401, 1 / F, 2 / F, building 26, yard 6, Jingshun East Street, Chaoyang District, Beijing Patentee after: BEIJING JINYU MEDICAL EXAMINATION LABORATORY Co.,Ltd. Address before: 100044 No. 11 South Main Street, Xicheng District, Beijing, Xizhimen Patentee before: PEKING UNIVERSITY PEOPLE'S Hospital |