CN107287273B - Application of peripheral blood NK (natural killer) cells expressing Tim-3 in preparation of natural abortion biomarker - Google Patents
Application of peripheral blood NK (natural killer) cells expressing Tim-3 in preparation of natural abortion biomarker Download PDFInfo
- Publication number
- CN107287273B CN107287273B CN201610202196.1A CN201610202196A CN107287273B CN 107287273 B CN107287273 B CN 107287273B CN 201610202196 A CN201610202196 A CN 201610202196A CN 107287273 B CN107287273 B CN 107287273B
- Authority
- CN
- China
- Prior art keywords
- cells
- tim
- pregnancy
- peripheral blood
- cell
- 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.)
- Expired - Fee Related
Links
- 101001068133 Homo sapiens Hepatitis A virus cellular receptor 2 Proteins 0.000 title claims abstract description 156
- 102100034458 Hepatitis A virus cellular receptor 2 Human genes 0.000 title claims abstract description 155
- 210000005259 peripheral blood Anatomy 0.000 title claims abstract description 62
- 239000011886 peripheral blood Substances 0.000 title claims abstract description 62
- 239000000090 biomarker Substances 0.000 title claims abstract description 18
- 206010000210 abortion Diseases 0.000 title abstract description 28
- 231100000176 abortion Toxicity 0.000 title abstract description 28
- 210000000822 natural killer cell Anatomy 0.000 claims abstract description 183
- 230000035935 pregnancy Effects 0.000 claims abstract description 121
- 206010000234 Abortion spontaneous Diseases 0.000 claims abstract description 52
- 208000000995 spontaneous abortion Diseases 0.000 claims abstract description 52
- 230000002829 reductive effect Effects 0.000 claims abstract description 30
- 230000022534 cell killing Effects 0.000 claims abstract description 6
- 230000014509 gene expression Effects 0.000 claims description 55
- 102000004388 Interleukin-4 Human genes 0.000 claims description 28
- 108090000978 Interleukin-4 Proteins 0.000 claims description 28
- 230000001965 increasing effect Effects 0.000 claims description 18
- 230000000694 effects Effects 0.000 claims description 15
- 102000004887 Transforming Growth Factor beta Human genes 0.000 claims description 13
- 108090001012 Transforming Growth Factor beta Proteins 0.000 claims description 13
- 230000006058 immune tolerance Effects 0.000 claims description 13
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 claims description 13
- 102000003814 Interleukin-10 Human genes 0.000 claims description 10
- 108090000174 Interleukin-10 Proteins 0.000 claims description 10
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 claims description 9
- 229930192851 perforin Natural products 0.000 claims description 9
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 8
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims description 8
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 claims description 7
- 238000010172 mouse model Methods 0.000 claims description 3
- 102000004503 Perforin Human genes 0.000 claims 2
- 108010056995 Perforin Proteins 0.000 claims 2
- 210000004027 cell Anatomy 0.000 abstract description 50
- 238000012546 transfer Methods 0.000 abstract description 11
- 230000004064 dysfunction Effects 0.000 abstract description 7
- 230000002265 prevention Effects 0.000 abstract description 6
- 238000012360 testing method Methods 0.000 abstract description 5
- 230000003042 antagnostic effect Effects 0.000 abstract description 2
- 231100000419 toxicity Toxicity 0.000 abstract description 2
- 230000001988 toxicity Effects 0.000 abstract description 2
- 102100031351 Galectin-9 Human genes 0.000 description 51
- 101710121810 Galectin-9 Proteins 0.000 description 51
- 230000002093 peripheral effect Effects 0.000 description 43
- 241000699670 Mus sp. Species 0.000 description 31
- 210000001161 mammalian embryo Anatomy 0.000 description 28
- 108090000695 Cytokines Proteins 0.000 description 25
- 102000004127 Cytokines Human genes 0.000 description 24
- 230000002147 killing effect Effects 0.000 description 20
- 238000002474 experimental method Methods 0.000 description 18
- 210000000952 spleen Anatomy 0.000 description 18
- 230000006870 function Effects 0.000 description 17
- 230000001105 regulatory effect Effects 0.000 description 16
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 14
- 238000010521 absorption reaction Methods 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 12
- 230000019491 signal transduction Effects 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 11
- 238000011161 development Methods 0.000 description 10
- 230000018109 developmental process Effects 0.000 description 10
- 238000000684 flow cytometry Methods 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 10
- 108010077544 Chromatin Proteins 0.000 description 9
- 210000003483 chromatin Anatomy 0.000 description 9
- 102000013968 STAT6 Transcription Factor Human genes 0.000 description 8
- 108010011005 STAT6 Transcription Factor Proteins 0.000 description 8
- 230000003213 activating effect Effects 0.000 description 8
- 230000033228 biological regulation Effects 0.000 description 8
- 239000005556 hormone Substances 0.000 description 8
- 229940088597 hormone Drugs 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 102000001398 Granzyme Human genes 0.000 description 7
- 108060005986 Granzyme Proteins 0.000 description 7
- 239000000186 progesterone Substances 0.000 description 7
- 229960003387 progesterone Drugs 0.000 description 7
- 108010074328 Interferon-gamma Proteins 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 102000040945 Transcription factor Human genes 0.000 description 6
- 108091023040 Transcription factor Proteins 0.000 description 6
- 230000003915 cell function Effects 0.000 description 6
- 230000004069 differentiation Effects 0.000 description 6
- 210000002257 embryonic structure Anatomy 0.000 description 6
- 230000003827 upregulation Effects 0.000 description 6
- 102100037850 Interferon gamma Human genes 0.000 description 5
- 210000003785 decidua Anatomy 0.000 description 5
- 230000002950 deficient Effects 0.000 description 5
- 230000001404 mediated effect Effects 0.000 description 5
- 230000003472 neutralizing effect Effects 0.000 description 5
- 230000026731 phosphorylation Effects 0.000 description 5
- 238000006366 phosphorylation reaction Methods 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 230000000638 stimulation Effects 0.000 description 5
- 230000005856 abnormality Effects 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 101100013967 Mus musculus Gata3 gene Proteins 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 210000001616 monocyte Anatomy 0.000 description 3
- 230000001072 progestational effect Effects 0.000 description 3
- 230000000306 recurrent effect Effects 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 102000015872 Human beta Subunit Chorionic Gonadotropin Human genes 0.000 description 2
- 108010010590 Human beta Subunit Chorionic Gonadotropin Proteins 0.000 description 2
- 101150111463 ID2 gene Proteins 0.000 description 2
- 101000687343 Mus musculus PR domain zinc finger protein 1 Proteins 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000002981 blocking agent Substances 0.000 description 2
- 230000016396 cytokine production Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000031261 interleukin-10 production Effects 0.000 description 2
- 230000017307 interleukin-4 production Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 101150023701 nfil3 gene Proteins 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 210000002993 trophoblast Anatomy 0.000 description 2
- 230000006433 tumor necrosis factor production Effects 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 238000001353 Chip-sequencing Methods 0.000 description 1
- 101710112752 Cytotoxin Proteins 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 101150046249 Havcr2 gene Proteins 0.000 description 1
- 101710083479 Hepatitis A virus cellular receptor 2 homolog Proteins 0.000 description 1
- 101001002709 Homo sapiens Interleukin-4 Proteins 0.000 description 1
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 1
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 1
- 102100022297 Integrin alpha-X Human genes 0.000 description 1
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 1
- 101100445364 Mus musculus Eomes gene Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 229940126547 T-cell immunoglobulin mucin-3 Drugs 0.000 description 1
- 210000000447 Th1 cell Anatomy 0.000 description 1
- 210000004241 Th2 cell Anatomy 0.000 description 1
- 101100445365 Xenopus laevis eomes gene Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000007621 cluster analysis Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000009274 differential gene expression Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000032692 embryo implantation Effects 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 102000055229 human IL4 Human genes 0.000 description 1
- 102000049018 human NCAM1 Human genes 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 description 1
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000036244 malformation Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 208000015994 miscarriage Diseases 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 210000000581 natural killer T-cell Anatomy 0.000 description 1
- 230000008775 paternal effect Effects 0.000 description 1
- 230000033064 perforin production Effects 0.000 description 1
- 239000000583 progesterone congener Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000009933 reproductive health Effects 0.000 description 1
- 210000005000 reproductive tract Anatomy 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 229940125712 tocolytic agent Drugs 0.000 description 1
- 239000003675 tocolytic agent Substances 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Images
Classifications
-
- 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
- G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
Abstract
The invention belongs to the technical field of biology, and relates to application of peripheral blood NK (natural killer) cells expressing Tim-3 in preparation of a natural abortion biomarker. The NK cell has tolerant phenotype and low cell killing toxicity, and tests prove that the peripheral blood NK cell expressing Tim-3 is highly expressed in the peripheral blood of normal pregnancy, and the number of the cells of patients with spontaneous abortion is reduced and the cells have dysfunction; antagonizing Tim-3 of NK cells to cause pregnancy failure, and the adoptive transfer of the NK cell subgroup can save the pregnancy failure caused by an immune spontaneous abortion model and NK knockout; the peripheral blood NK cell expressing Tim-3 can be used for preparing a natural abortion biomarker and further used as a novel clinical unknown natural abortion prediction and prevention target.
Description
Technical Field
The invention belongs to the field of biotechnology. Relates to a biomarker for predicting and preventing spontaneous abortion, in particular to application of peripheral blood NK (natural killer) cells expressing Tim-3 in preparation of a biomarker for spontaneous abortion. The biomarker of the spontaneous abortion can be used as a target for predicting and preventing early pregnancy failure.
Background
The prior art discloses a prediction method related to pregnancy outcome, which mainly bases on the peripheral blood HCG-beta and progesterone level of pregnant women, but a great deal of evidence shows that the relationship between the P level of the peripheral blood of pregnant women and the pregnancy outcome is not completely consistent in practice; although HCG- β can be relatively true in response to pregnancy prognosis in pregnant women, increasing clinical data suggest: once the peripheral blood HCG of pregnant women has decreased (except for the physiological decrease of HCG- β), the embryo has failed to rescue; in addition, some pregnant women stop developing embryos (or empty blastocysts) despite continuing to secrete large amounts of HCG- β and P; obviously, HCG-beta and P have a plurality of defects as indicators for detecting pregnancy outcome, and clinical practice urgently needs a biological indicator for reflecting early prediction and prevention of pregnancy state in early pregnancy.
Studies have disclosed that embryos express paternal antigens and can therefore be considered as a semi-allograft, where embryos that are semi-allograft are able to survive within the mother until delivery actually reflects the mother's immune tolerance to the embryo. Once this maternal-fetal immune tolerance is breached, pregnancy failure or spontaneous abortion and complications of pregnancy will result. Spontaneous abortion is one of the common diseases seriously harming human reproductive health, and the incidence rate of spontaneous abortion tends to rise year by year. According to statistical data, the occurrence rate of spontaneous abortion reaches up to 15-40%, and even in the current situation that the assisted reproduction technology is rapidly developed, pregnancy failure caused by immune regulation disorder of the mother-fetus cannot be overcome, and unfortunately, an effective early diagnosis and prevention strategy is still lacked in clinical practice. The spontaneous abortion is mostly caused by heredity, infection, endocrine, genital tract malformation and the like, but the clinical causes of more than 50 percent of abortion, particularly recurrent spontaneous abortion are still unknown, wherein the imbalance of maternal-fetal immunoregulation is considered to be one of the important causes; studies have shown that immune cells are the performers and effectors of maternal-fetal immune tolerance, where NK cells play a crucial regulatory role in maternal-fetal tolerance; earlier stage research of the application finds that decidua NK cells are important for establishment and maintenance of maternal-fetal tolerance, and decidua NK cells in gestation maintain progression of maternal-fetal tolerance and normal pregnancy through the action of cell surface molecules and various intracellular factors, but decidua tissues in gestation are not easy to obtain, so that the decidua tissues in gestation are unlikely to be used for clinical diagnosis and prevention targets. Therefore, in clinical practice, a simple and reliable method for objectively evaluating the immunological state of early spontaneous abortion and intervening the immunological state of early spontaneous abortion is urgently needed so as to achieve the purposes of preventing and treating spontaneous abortion and saving pregnant embryos.
Based on the current situation of the prior art, the inventor of the application intends to provide a biomarker for predicting and preventing spontaneous abortion, in particular to application of peripheral blood NK (Natural killer) cells expressing Tim-3 in preparation of a biomarker for spontaneous abortion. The biomarker of the spontaneous abortion can be used as a target for predicting and preventing early pregnancy failure.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides a biomarker for predicting and preventing spontaneous abortion, and particularly relates to application of peripheral blood NK (natural killer) cells expressing Tim-3 in preparation of a biomarker for spontaneous abortion. The biomarker of the spontaneous abortion can be used as a target for predicting and preventing early pregnancy failure.
In the present invention, peripheral blood Tim-3 of different pregnancy states was performed+NK cell percentage comparison experiments and differential analysis are carried out, and expression characteristics and regulation conditions of peripheral blood NK cells of Tim-3 in different pregnancy states are analyzed; the result shows that the expression level of Tim-3 in NK cells is obviously different in different gestational periods, the expression of the NK cells Tim-3 in peripheral blood at the early stage of pregnancy is obviously increased, and the expression level of the NK cells in the middle stage of pregnancy is reduced to the level of the non-pregnancy; in the early stage of pregnancy, pregnancy-associated hormones (mainly progesterone) and Th2 type cytokine (IL-4) are involved in the upregulation of Tim-3 expression of peripheral NK cells;
in the invention, a Galectin-9/Tim-3 signal mediated early pregnancy peripheral NK cell immune tolerance test is carried out, and the result shows that: and Tim3-NK cell comparison, Tim-3+NK cells produce higher level of Th2 type cytokines and lower level of Th1 type cytokines and present weaker killing activity, and the phenotype characteristics are more favorable for maintaining the tolerance state of pregnancy peripheral immunity; the Tim-3 receptor on the NK cell interacts with Galectin-9, and promotes the expression of transcription factors Id-2, Blimp1 and Gata-3 by activating Akt and JNK signal paths, so as to induce the differentiation of the peripheral NK cell to an immune tolerance phenotype; the Tim-3/Gal-9 signal is involved in the dominant generation of Th2 type cytokines by NK cells;
in the invention, the test of NK dysfunction and pregnancy failure caused by early pregnancy peripheral NK cell Galectin-9/Tim-3 signal abnormality is carried out, and the result shows that the reduction of the expression of the peripheral NK cell Tim-3 and the reduction of the generation level of the peripheral Gal-9 destroy the immune tolerance of the peripheral NK cell by secreting cell factors and maintaining lower killing activity, thereby causing natural abortion;
furthermore, the invention carries out the test of the influence of the interference of a Gal-9/Tim-3 signal channel on the pregnancy outcome and the adoptive transfer of Tim-3+The NK cell rescue pregnancy failure experiment shows that Tim-3 plays a role in protecting by regulating the function of peripheral NK cells in the normal pregnancy process, if a signal path mediated by Tim-3 is damaged, the function of the peripheral NK cells and even differentiation and development can be influenced, so that the normal progress of pregnancy and the development of embryos are influenced, and particularly, the dysfunction of early pregnancy peripheral blood NK cells to cause poor pregnancy fatality can be caused by the abnormal Galectin-9/Tim-3 signal path; IL-4 and progesterone stimulated Tim-3+The NK cells effectively relieve the high embryo absorptivity of the abortion model; adoptive transfer of Tim-3+NK cells reduce embryo absorption rate of NK cell-deficient mice, and Ti m-3 is adoptively transferred-NK cells do not affect pregnancy outcome.
The experiment proves that the tolerance NK subgroup is highly expressed in the peripheral blood of normal pregnancy, and the number of the cells of a patient suffering from spontaneous abortion is reduced, and the cells are disordered; antagonizing Tim-3 of NK cells to cause pregnancy failure, and the adoptive transfer of the NK cell subgroup can save the pregnancy failure caused by an immune spontaneous abortion model and NK knockout; the peripheral blood NK cell expressing Tim-3 can be used for preparing a natural abortion biomarker and further used as a novel clinical unknown natural abortion prediction and prevention target.
The invention has the advantages that:
peripheral blood NK cells expressing Tim-3 can be used for preparing natural abortion biomarkers and further used as novel clinical prediction and prevention targets for unexplained natural abortion;
the Tim-3+The NK cells exist in the peripheral blood of women, the phenotype and the function of the NK cells are highly consistent with those of the decidua part at the periphery of the early pregnancy, and the immune tolerance state of the uterus part and the whole body of the pregnant woman can be monitored by only extracting a little peripheral blood and analyzing by flow cytometry; furthermore, the cells of the population are targeted by in vitro needlingAdoptive transfer of this subset of cells after stimulation (e.g., IL-4, P) can be used to directly prevent recurrent pregnancy failure in patients with recurrent spontaneous abortion.
Drawings
FIG. 1: shows the expression condition of different immune cell subsets Tim-3 in human early pregnancy peripheral blood and Tim-3+The proportion of NK cells in different gestational periods, wherein Tim-3+The proportion of NK cells in the early pregnancy period is obviously higher than that in the non-pregnancy and middle pregnancy periods.
FIG. 2, shows that IL-4/STAT6 signaling correlates with pregnancy-associated progestogen upregulation of peripheral blood Tim3+The proportion of NK cells is determined,
wherein, Th2 type cell factor IL-4 up-regulates Tim-3 expression by activating STAT6 signal, STAT6 inhibitor and Th1 type cell factor inhibit the regulation of IL-4, and classical tocolytic agent progesterone also promotes pNK cell to express Tim-3.
FIG. 3, shows human early pregnancy peripheral blood Tim-3+The NK cells exhibit an immune-tolerant phenotype,
wherein, the chip analysis result shows that the Tim-3+ NK cells and the Tim-3-NK cells are two groups of NK cell subsets with different gene expression, Tim-3+865 genes are up-regulated and 100 genes are down-regulated in NK cells, and the up-regulated expression of the genes is mostly related to Th2 type immune response and immune tolerance; FCM analysis of human early pregnancy peripheral blood Tim-3+And Tim-3-NK cells of (1), found to be related to Tim-3-NK cell comparison, Tim-3+NK cells produce higher levels of IL-4, IL-10 and TGF-beta, lower levels of TNF-alpha and Perforin; the killing activity to the trophoblast is also obviously reduced.
FIG. 4: Galectin-9/Tim-3 signals regulate and control peripheral NK cells to present a tolerant phenotype through activating JNK and AKT signals, wherein a natural ligand Gal-9 of Tim-3 stimulates pNK cells, and qPCR and western blot show that after Gal-9 stimulation, the levels of transcription factors related to development and differentiation of NK cells and cytotoxicity are activated, IL-4/IL-10 is up-regulated, and TNF-1/Perforin and cytotoxicity are down-regulated.
FIG. 5: the expression of peripheral blood NK cells Tim-3/Gal-9 of patients with spontaneous abortion is abnormal and is accompanied with NK cell dysfunction, wherein, FIG. 5.1 shows the table of peripheral blood NK cells Tim-3 of patients with spontaneous abortionDecrease in peripheral blood Gal-9 level accompanied by Tim-3+The chromatin accessibility of NK cell anti-inflammatory cytokine gene promoter and enhancer regions is obviously reduced, the production of proinflammatory cytokines is reduced, and Th2/Th1 type cytokine disorder and enhanced cell killing toxicity are caused; FIG. 5.2 shows that peripheral blood and spleen NK cells Gal-9/Tim-3 expression of early pregnancy mice is higher than during mid-pregnancy; the group of mice with spontaneous abortion was lower than the normal pregnant mice; natural abortion mouse peripheral blood and spleen Tim-3+NK cells produce Th2/Th1 type cytokine disorder.
FIG. 6: disruption of NK cell function by TIM-3 signaling leads to pregnancy failure, where,
the embryo absorption and development and the NK cell function of a normal pregnancy and spontaneous abortion model mouse are shown, a Tim-3 signal is blocked, the litter number of the normal pregnancy and spontaneous abortion mouse is reduced, the embryo absorption is increased, and the embryo growth is inhibited; the effect is more remarkable in the spontaneous abortion group, and exogenous Gal-9 administration does not affect the litter number, embryo size and absorptivity of normal pregnancy and NK cell function, but remarkably inhibits embryo absorption of mice with spontaneous abortion and improves the function of NK cells.
FIG. 7: adoptive transfer of normal spleen Tim-3 in pregnancy+The NK cells relieve the loss of the embryo,
wherein A is an experimental flow chart, B, C, D and E show that the spleen Tim-3 is normally pregnant by adoptive transfer+NK cells relieve embryo absorption of spontaneous abortion model, and adoptive transfer of mice with spontaneous abortion Tim-3-NK has no improving effect; treating NK cells of mice with spontaneous abortion by IL-4 and P to obtain Tim-3+After NK is adoptively transferred to mice with spontaneous abortion, the embryo absorption rate is obviously reduced; the embryo absorption rate of the NK cell deficient mouse pregnancy model is obviously improved, and the normal Tim-3 is transmitted by adoptive transfer+NK other than Tim-3-NK cells, significantly reducing embryo absorption rate of NK deficient mice.
FIG. 8 is a general diagram of the experimental flow of target spots for predicting and preventing early pregnancy failure by using peripheral blood NK cells expressing Tim-3 as a biomarker for spontaneous abortion.
Detailed Description
Example 1:different pregnancy state peripheral blood Tim-3+NK cell percentage comparison and differential analysis/analysis of expression characteristics and regulation of Tim-3 in peripheral blood NK cells of different pregnancy states
1) Analysis of expression characteristics of Tim-3 in peripheral blood NK cells of different pregnancy states
First, the peripheral blood of normal non-pregnant, early pregnant and middle pregnant women is separated, the lymphocyte is separated by Ficoll, and the Tim-3 is analyzed by FCM in CD4+T cell, CD8+The expression of T cells, NK cells, NKT cells, DC cells and monocytes (shown in figure 1A), results show that the expression levels of the NK cells, the DC cells and the monocytes are high-expression Tim-3 (respectively 80%, 80% and 90%), the expression levels of the Tim-3 in the NK cells are obviously different in different pregnancy periods, and the expression levels of the peripheral blood NK cells Tim-3 in the early pregnancy period are obviously up-regulated and are reduced to the level of the non-pregnancy period in the middle pregnancy period as shown in figures 1B and C.
2) Expression regulation of Tim-3 in human early pregnancy peripheral blood NK cells
As the peripheral blood and maternal-fetal interface of early pregnancy show the advantage of Th2 and the pregnancy related hormone such as E, P, hCG is also increased, the invention further discusses the possible factor of the expression up-regulation of NK cell Tim-3, after the non-pregnant woman peripheral NK cells are separated and purified by magnetic beads, recombinant human IL-4(100ng/ml) and IFN-gamma (35ng/ml) are added singly or jointly for culturing for 48h, the cells are collected, and the expression condition of the NK cell Tim-3 is detected by flow cytometry; FIG. 2 shows that Th2 type cytokine IL-4 significantly upregulates Tim-3 expression after NK cell treatment, this effect being attenuated by IFN-. gamma.; in order to further discuss the action mechanism of IL-4/IFN-gamma for regulating the expression of NK cell Tim-3, the experiment firstly uses flow cytometry to detect the phosphorylation level of STAT6 (downstream signal molecule of IL-4 action) in NK cells treated by the method, and the result shows that the phosphorylation level of STAT6 of the NK cell group treated by IL-4 is really and obviously improved, and the combination of IFN-gamma and IL-4 inhibits the phosphorylation level of STAT6 induced by IL-4 (figures 2C and D). The Western Blot detection results show that consistent results are shown in figure 2E), a specific blocker A77-1726 of STAT6 can inhibit the up-regulation effect of IL-4 on Tim-3 (shown in figure 2F), the Th2 type cytokine IL-4 further promotes the expression of NK cell Tim-3 by activating downstream molecules STAT6, and the Th1 type cytokine IFN-gamma can antagonize the effect;
in the early stage of pregnancy, not only the Th2/Th1 cytokine pattern in peripheral blood is changed, but also the hormone level of a parent body is obviously changed, therefore, the experiment further observes whether pregnancy related hormone can also influence the expression of the peripheral NK cells Tim-3, female, progestational hormone and hCG-beta with different concentration gradients are used for stimulating the purified peripheral NK cells of non-pregnant women, and the expression condition of the NK cells Tim-3 is detected by flow cytometry, as shown in figure 2G, the progestational hormone regulates and controls the expression of the NK cells Tim-3 in a concentration-dependent manner, the progestational hormone with lower concentration promotes the expression of the NK cells Tim-3, the up-regulation effect disappears and even inhibits the expression of the NK cells Tim-3 at higher concentration, and the estrogen and the hCG-beta have no obvious influence on the expression of the NK cells Tim-3;
the results show that pregnancy related hormone (mainly progesterone) and Th2 type cytokine (IL-4) are involved in the upregulation of Tim-3 expression of peripheral NK cells in the early stage of pregnancy.
Example 2: Galectin-9/Tim-3 signal mediated early pregnancy peripheral NK cell immune tolerance experiment
1) Phenotypic comparison of peripheral blood Tim-3+ NK and Tim3-NK cells
In order to investigate whether the expression of Tim-3 on NK cells is related to NK cell function, the separated peripheral mononuclear cells are further separated into Tim-3 by magnetic beads+NK and Tim3-NK cells, the identification and sorting purity is 92%, the experimental requirements are met, and the whole genome chip analysis is carried out after total RNA is extracted; as shown in FIG. 3A, B, Tim-3+ and Tim-3-NK cells are two subsets of NK cells with differential gene expression, Tim-3+865 genes are up-regulated and 100 genes are down-regulated in NK cells, and the up-regulated expression of the genes is mostly related to Th2 type immune response and immune tolerance; the protein expression levels of Th2 and Th1 cytokines were further detected by flow-testing after 4-6 hours of stimulation with PMA, ionomycin and BFA in this experiment, as shown in FIGS. 3C and D, the Th2 cytokines IL-4, IL-10 and TGF-beta were in Tim-3+The expression level of pNK cells is obviously higher than Tim-3-pNK cells and Th1 type cytokine TNF-alpha are expressed at opposite levels in the two populations of cells; IFN-gamma in Tim-3+NK and Tim3-There was little difference in NK cell expression (as shown in fig. 3E, F);
further comparison of Tim-3+NK and Tim3-NK cell killing activity of the two purified populations of cells are cultured in contact with target cells HTR-8 (extravillous cytotrophoblast line) separately
Detection of cell killing Activity with Non-Radioactive cytotoxin Assay kit, Tim-3, FIG. 3I+NK and Tim3-The killing activity of NK cells is reduced along with the reduction of the effective target ratio, but Tim-3+The killing activity of NK cells is always lower than Tim3-An NK cell; flow cytometry analysis of Tim-3+NK and Tim3-NK cells produced perforin levels, and the results showed perforin at Tim-3+The expression level of NK cells is obviously lower than that of Tim-3-NK cells (as shown in FIG. 3G, H); experiments the results show that: and Tim3-NK cell comparison, Tim-3+NK cells produce higher levels of Th2 type cytokines and lower levels of Th1 type cytokines and exhibit weaker killing activity, and these phenotypic characteristics will be more favorable for maintaining the tolerance state of pregnancy peripheral immunity.
2) Expression regulation of peripheral blood NK cell differentiation development and function related transcription factor by Tim-3 natural ligand Gal-9
The expression of transcription factors Id-2, Blimp1 and Gata-3 related to the differentiation development and functions of NK cells can be obviously promoted by treating peripheral NK cells with natural ligand Galectin-9 of Tim-3 at the mRNA level, while the expression of transcription factors Eomes for regulating the killing activity of the NK cells is obviously reduced, and further analysis shows that the Gal-9 promotes the NK cells to generate Th2 cell factors, inhibits the generation of Th1 cell factors and effectively reduces the killing activity of the NK cells by activating two signal paths of Akt and JNK; therefore, the Tim-3 receptor on the NK cell interacts with Galectin-9, and promotes the expression of transcription factors Id-2, Blimpl and Gata-3 by activating Akt and JNK signal pathways, thereby inducing the differentiation of peripheral NK cells to an immune tolerance phenotype.
3) Influence of Gal-9/Tim-3 Signal on cytokine secretion Pattern and killing Activity of peripheral blood NK cells
Research suggests that Tim-3 may participate in the phenotype and function regulation of NK cells by combining with ligand Gal-9, therefore, rhGal-9 or anti-Tim-3 neutralizing antibody is further added into the culture system of peripheral NK cells, after 48 hours of treatment, the IL-4 and IL-10 production levels of the rhGal-9 treatment group are obviously increased, and the TNF-alpha production level is obviously reduced; the cytokine production level of the group treated with the anti-Tim-3 neutralizing antibody alone did not change significantly before and after the treatment, but the IL-4 and IL-10 production levels of the group treated with rhGal-9 and anti-Tim-3 were significantly lower than those of the group treated with rhGal-9, and the TNF- α production levels were opposite; the experimental results suggest that Tim-3/Gal-9 signals are involved in the dominant production of Th2 type cytokines by NK cells (as shown in FIG. 4C);
in addition, the killing activity of peripheral blood NK cells is obviously reduced after rhGal-9 treatment, the effect is partially recovered by the anti-Tim-3 neutralizing antibody, and the effect is not obvious when the anti-Tim-3 neutralizing antibody is singly used; similarly, flow cytometry was used to detect the level of NK cell perforin production following rhGal-9 or anti-Tim-3 neutralizing antibody treatment (as shown in FIG. 4D, E).
4) Experiment for regulating peripheral blood NK cell function through activation of JNK and Akt signals by Gal-9/Tim-3 interaction
In order to further analyze the signal transduction mechanism of Tim-3/Gal-9 regulation peripheral blood NK cells, rhGal-9 is used for stimulating purified peripheral blood NK cells for 10 minutes, 30 minutes and 60 minutes respectively in the experiment, Western Blot is used for detecting the expression condition of molecules related to signal pathways of peripheral blood NK cells, as shown in figure 4F, compared with the single culture of NK cells, after the stimulation of rhGal-9, the phosphorylation levels of Akt, JNK and Erk are obviously increased, and the phosphorylation level of NK-kB is not obviously changed before and after the stimulation, which indicates that Tim-3/Gal-9 is likely to regulate and control the functions of peripheral NK cells by activating three signal pathways of Akt, JNK and Erk;
in order to clarify the possible signal pathways, specific inhibitors of three signal pathways of Akt, JNK and Erk were added to a culture system of NK cells treated with Gal-9, and then the production levels of NK cell cytokines and perforin were detected by flow cytometry, as shown in FIG. 4G, a specific blocking agent of JNK or AKT could inhibit the regulatory effect of Gal-9 on NK cells, while a specific blocking agent of Erk had no significant effect; the results show that Gal-9 further changes the biological functions of NK cells by activating two signal pathways of JNK and Akt, but not Erk.
Example 3: experiment on NK dysfunction and pregnancy failure caused by early pregnancy peripheral NK cell Galectin-9/Tim-3 signal abnormality
1) The expression level of peripheral NK cells Tim-3 of the patient with spontaneous abortion is reduced, the generation level of Gal-9 is reduced, and NK cells are thin
Disturbance of cytokine secretion level and enhancement of killing activity
To investigate that the abnormality of the number and function of peripheral NK cells is closely related to the occurrence of spontaneous abortion, it was further confirmed whether there is a difference in expression of NK cell Tim-3 in peripheral blood of patients with normal pregnancy and spontaneous abortion, there is a change in Gal-9 content in blood, and there is a corresponding change in NK cell function? The experiment collects the peripheral blood of patients with early normal pregnancy and spontaneous abortion of unknown reason, the flow cytometry is used for detecting and analyzing the problems, the result shows that although the proportion of Tim-3+ NK cells in peripheral immunocompetence cells is not obviously increased or decreased, the fluorescence density of Tim-3 expressed in NK cells is obviously reduced in the peripheral blood of the patients with abortion (as shown in figure 5.1A), the difference of the peripheral Tim-3+ NK cells of the patients with normal pregnancy and repeated spontaneous abortion is further systematically compared, the ATAC-seq technology is used for detecting and analyzing the chromatin accessibility of the peripheral Tim-3+ NK cells and the Tim-3-NK cells of the patients with normal pregnancy and repeated spontaneous abortion, and the analysis result of main components shows that the Tim-3+ NK cells and the Tim-3-NK cells of the patients can be well distinguished from the normal NK cells (as shown in figure 5.1D), the result of the cluster analysis is consistent with the result of the main component analysis, which indicates that the Tim-3+ NK cells are functional cells closely related to pregnancy outcome; further comparing and analyzing the peripheral Tim-3+ NK cells of the abortion patients and the normal pregnant women, finding that the chromatin accessibility of 260 gene sites in the Tim-3+ NK cells of the abortion patients is reduced, the chromatin accessibility of 360 gene sites is increased (as shown in figure 5.1E), combining with the known ChIP-seq result analysis of human CD56+ NK cells, the gene sites with reduced chromatin accessibility are mainly enhancers, the gene sites with increased chromatin accessibility are mainly promoters, wherein, the chromatin accessibility of the 3 'end of TGF-beta 1 is most obviously reduced, meanwhile, the chromatin accessibility of the promoter region of TGF-beta 1 is also obviously reduced, which indicates that the TGF-beta 13' end region can play the role of enhancers and coordinate with the TGF-beta 1 promoter to regulate the expression of TGF-beta 1 (as shown in figure 5.1F), the same changes in chromatin accessibility of IL-10 (as shown in FIG. 5.1G);
further examination of NK cell production of Th2, Th1 type cytokines and perforin in response to its killing activity was found by Tim-3+The production of Th2 cytokines (L-4, IL-10 and TGF-beta 1) by pNK cells is obviously reduced in peripheral blood of a patient with the production, and on the contrary, Th1 cytokines (TNF-alpha and IFN-gamma) are obviously increased, and Tim-3 is obviously improved-The levels of cytokines produced by pNK cells did not differ significantly between the two samples (as shown in FIG. 5.1H, I); similarly, Tim-3+pNK cells instead of Tim-3-pNK cell production perforin was significantly upregulated in the peripheral blood of patients with abortion (as shown in FIG. 5.1J), suggesting that the killing activity of peripheral NK cells was significantly enhanced in patients with abortion, mainly due to Tim-3+An increase in pNK cytotoxicity;
as shown in FIG. 5.1B, the secretion level of Gal-9 in the peripheral blood of the aborted patient was reduced from 1.84ng/ml in the normal case to 0.965ng/ml, and in addition, the respective peripheral immunocompetent cells including NK cells themselves (CD 4)+、CD8+T cells，CD14+Monocytes and CD11c+Dendritic cells) also had a significant down-regulation of the level of Gal-9 production (as shown in FIG. 5.1C); the above results indicate that the reduction of expression of Tim-3 and the down-regulation of the level of Gal-9 production in peripheral NK cells may destroy the immune tolerance of peripheral NK cells by secreting cytokines and maintaining low killing activity, thereby causing spontaneous abortion.
Example 4: experiment for influence of interference of Gal-9/Tim-3 signal path on pregnancy outcome
Based on the fact that the abnormal expression of Gal-9/Tim-3 in the peripheral blood of early pregnancy is possibly related to spontaneous abortion, in order to further analyze the influence of the pair of molecular-mediated signal paths on pregnancy outcome and molecular mechanisms, the experiment carries out human intervention on Gal-9/Tim-3 signals of normal and aborted mouse models; firstly, establishing normal and abortive mouse models, and comparing the expression of NK cells Tim-3 of peripheral (including peripheral blood and spleen) NK cells of mice in early pregnancy and middle pregnancy, wherein the result shows that the expression of the peripheral NK cells Tim-3 of the mice in the early pregnancy of the normal pregnancy is higher than that of the mice in the middle pregnancy and is consistent with that of people, namely Tim-3+ NK cells are enriched in the early pregnancy; FIG. 5.1A shows that differential expression of peripheral NK cells Gal-9/Tim-3 in aborted patients is accompanied by NK cell dysfunction, and this experimental analysis compares the expression of Tim-3 and Gal-9 on peripheral NK cells in early pregnancy in normal pregnancy and spontaneous abortion models, as shown in FIGS. 5.2A, B, and compared with normal pregnant mice, NK cells in spontaneous abortion mice express Tim-3 and Gal-9 in peripheral blood and spleen at significantly lower levels, not only that the level of Th2 type cytokines (IL-4, IL-10 and TGF-beta) produced by Tim-3+ pNK in spontaneous abortion model, but also that the level of Th1 type cytokines (TNF-alpha) produced is increased; analyzing Granzyme A (Granzyme A which reflects killing activity of NK cells) by flow cytometry, and finding that the expression of the Granzyme A in peripheral Tim-3+ NK cells of a natural abortion model is obviously higher than that of the peripheral Tim-3+ NK cells of a normal pregnancy model; analyzing the above index in the spleen, consistent with the results of peripheral blood; (FIG. 5.2C, D) shows that the function of Tim-3-NK secretion of cytokines and killing activity were almost not different in the normal pregnancy and the model of spontaneous abortion regardless of peripheral blood or spleen, and that Gal-9/Tim-3 was closely related to the development of normal function of pregnant peripheral NK cells;
in order to clarify the role of Tim-3/Gal-9 in pregnancy, recombinant exogenous Gal-9 and RM3-23 (blockers of Tim-3 signal pathway) are used in the experiment to interfere with the Gal-9/Tim-3 signal pathway in pregnant mice, and after the Tim-3 signal pathway is blocked by RMT3-23 in a normal pregnancy model and a natural abortion model, the embryo absorptivity of the two groups is obviously increased, the embryo absorptivity of the normal pregnancy model is increased to 17% from about 5%, while the embryo absorptivity of the natural abortion model which is originally higher is increased to 46% from about 27% (FIG. 6A, D); correspondingly, the number of born survived in the normal pregnancy model is reduced from 10 to 6, and the number of born survived in the natural abortion model is reduced from 7 to 4 before intervention (as shown in figures 6A and C); although the embryo absorption rate of the normal pregnancy model and the spontaneous abortion model is obviously different, the size of the surviving embryo is not different; however, after the mice were injected with RMT3-23, the embryo volume of the mice of both models was significantly smaller than that of the non-injected group, and it is speculated that the blockage of Tim-3 signal not only affects the embryo absorption rate, but also has significant inhibitory effect on the growth and development of the embryo (as shown in FIGS. 6B and E); in order to prove whether the blockage of Tim-3 signals can influence the functions of NK cells, the experiment analyzes the peripheral blood and spleen NK cells of the mice of the treated group by flow cytometry, and the result shows that the number of the NK cells in the peripheral blood of the mice injected with RMT3-23 is reduced sharply from 5-10% before treatment to 1-2% and even reaches a deletion level, but the number of the NK cells in the spleen is not changed; further analyzing the functions of spleen NK cells, and finding that the production levels of spleen NK cells IL-4, IL-10 and TGF-beta of normal pregnant mice are obviously reduced after Tim-3 signals are blocked, TNF-alpha is obviously increased, and INF-has no obvious difference before and after blocking; the effect of Tim-3 signal blocking on spleen NK cells in normal pregnancy was more pronounced in the spontaneous abortion model (as shown in FIG. 6F); the detection of spleen NK cell Granzyme A (Granzyme A) shows that after Tim-3 is blocked, a abortion model generates a higher level of Granzyme A, which prompts that the killing activity of NK cells is enhanced, and Tim-3 blocking does not enhance the killing activity of NK cells in normal pregnancy groups, but shows a certain level of reduction, and experimental data prompts that Tim-3 plays a role in protecting by regulating the function of peripheral NK cells in the normal pregnancy process, if a signal path mediated by Tim-3 is damaged, the function, even differentiation and development of the peripheral NK cells can be influenced, so that the normal progress of pregnancy and the development of embryos can be influenced;
the experiment injects exogenous recombinant Gal-9 into pregnant mice to improve the pregnancy outcome, and the result shows that the exogenous Gal-9 can improve the pregnancy outcome to a certain extent: for a natural pregnancy model, the embryo absorption rate can be reduced, the survival litter size can be increased, but the pregnancy outcome of a Tim-3 blocked natural abortion model and a normal pregnancy model is not obviously improved; the function of spleen NK cells is detected, and the generation levels of NK cells IL-4 and TGF-beta of a natural abortion model injected with Gal-9 are increased, INF-gamma is reduced, granzyme A is also reduced, and IL-10 and TNF-alpha are not obviously changed; the normal pregnancy model and the natural abortion model of Tim-3 are blocked by RMT3-23 in advance, and the cytokine production level and the killing activity of NK cells are not obviously changed after Gal-9 is given; the above experimental data show that Gal-9 regulates the function of peripheral NK cells by binding and interacting with its receptor Tim-3 to maintain normal pregnancy, and if the signal pathway of Tim-3 is blocked, Gal-9 cannot normally bind to Tim-3 to exert regulation (as shown in FIG. 6);
therefore, abnormalities in the Galectin-9/Tim-3 signaling pathway can cause dysfunction of early pregnancy peripheral blood NK cells leading to poor pregnancy outcome.
Example 5: adoptive transfer of Tim-3+NK cell rescue pregnancy failure experiment
Flow-sorting Tim-3 from spleen of Normal pregnant mice+NK，Tim-3-NK and total NK cells, the three cells are respectively resuspended in 200uLPBS, and are adoptively transfused to miscarriage mice at 4.5 days of pregnancy by tail vein injection; tim-3 was obtained from the spleen of aborted mice in the same manner+NK，Tim-3-NK and Total NK cells, Tim-3+NK，Tim-3-NK and a part of NK cells were adoptively transferred to aborted mice by the same method, and the other part of NK cells were stimulated with IL-4 and progesterone in vitro, and after 48 hours, Tim-3 was again flow sorted+NK，Tim-3-NK cells were adoptively transferred to aborted mice by the above method, the aborted mice were sacrificed at 14.5 days of pregnancy, embryo absorption was observed (as shown in FIG. 7A), and the result showed that Tim-3 was observed in mice with normal pregnancy+NK reduces embryo absorptivity of abortion model, and aborted mice Tim-3+NK has no significant effect, but Tim-3 stimulated by IL-4 and progesterone+NK cells efficiently alleviated abortion model high embryo uptake (shown in fig. 7D);
establishment of NK cell deficient mice (Nfil 3)-/-) The early pregnancy model of (1), the normal pregnancy Nfil3 was adoptively transfused in 4.5 days of pregnancy by the above method+/+Tim-3 of mouse spleen+NK and Tim-3-NK cells, killing of Nfil3 at 14.5 days gestation-/-Pregnant mouse, observing embryo implantation number and embryo absorption rate, and displaying the result of adoptive transfer Tim-3+NK cells reduce embryo absorption rate of NK cell-deficient mice, and Ti m-3 is adoptively transferred-NK cells did not affect pregnancy outcome (as shown in fig. 7E, F).
Claims (1)
1. Use of peripheral blood NK cells expressing Tim-3 in preparation of biomarkers for spontaneous abortion, characterized in that the expression of the T-3 is related to Tim-3-Compared with NK cells, the NK cells have immune tolerance phenotype and low cell killing activity in the early pregnancy period of human, the NK cells highly express IL-4, IL-10 and TGF-beta, and lowly express TNF-alpha and Perforin; compared with normal pregnancy, the NK cell expresses IL-4, IL-10 and TGF-beta in a spontaneous abortion patient and a spontaneous abortion mouse model to be reduced, expresses TNF-alpha to be increased, has high cell killing activity and has high Perforin expression.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610202196.1A CN107287273B (en) | 2016-03-31 | 2016-03-31 | Application of peripheral blood NK (natural killer) cells expressing Tim-3 in preparation of natural abortion biomarker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610202196.1A CN107287273B (en) | 2016-03-31 | 2016-03-31 | Application of peripheral blood NK (natural killer) cells expressing Tim-3 in preparation of natural abortion biomarker |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107287273A CN107287273A (en) | 2017-10-24 |
| CN107287273B true CN107287273B (en) | 2021-03-09 |
Family
ID=60087449
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610202196.1A Expired - Fee Related CN107287273B (en) | 2016-03-31 | 2016-03-31 | Application of peripheral blood NK (natural killer) cells expressing Tim-3 in preparation of natural abortion biomarker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107287273B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112414925A (en) * | 2020-11-27 | 2021-02-26 | 中国医科大学附属盛京医院 | Application of peripheral blood NK (Natural killer) cells expressing NKG2C as natural abortion biomarker |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015082499A2 (en) * | 2013-12-03 | 2015-06-11 | Iomet Pharma Ltd | Pharmaceutical compound |
| CN105143253A (en) * | 2013-03-14 | 2015-12-09 | 美国安进公司 | Interleukin-2 muteins for the expansion of t-regulatory cells |
-
2016
- 2016-03-31 CN CN201610202196.1A patent/CN107287273B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105143253A (en) * | 2013-03-14 | 2015-12-09 | 美国安进公司 | Interleukin-2 muteins for the expansion of t-regulatory cells |
| WO2015082499A2 (en) * | 2013-12-03 | 2015-06-11 | Iomet Pharma Ltd | Pharmaceutical compound |
Non-Patent Citations (4)
| Title |
|---|
| Jintang Sun等.Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway.《PLOS ONE》.2016,第11卷(第1期),摘要、第9页第2段和第10页第1段、图6. * |
| The Galectin-9/Tim-3 pathway is involved in the regulation of NK cell function at the maternal–fetal interface in early pregnancy;Yan-Hong Li等;《Cellular & Molecular Immunology》;20160131;第13卷(第1期);图5 * |
| Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway;Jintang Sun等;《PLOS ONE》;20160120;第11卷(第1期);摘要、第2页第1段、第5页第3段、第9页第2段、第10页第1-2段、图6 * |
| Tim-3分子表达异常与原因不明复发性流产的关系;张铭等;《中国优生与遗传杂志》;20121231;第20卷(第2期);第31-34页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107287273A (en) | 2017-10-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kalagiri et al. | Inflammation in complicated pregnancy and its outcome | |
| Belderbos et al. | Skewed pattern of Toll-like receptor 4-mediated cytokine production in human neonatal blood: low LPS-induced IL-12p70 and high IL-10 persist throughout the first month of life | |
| Hou et al. | IL-37 ameliorating allergic inflammation in atopic dermatitis through regulating microbiota and AMPK-mTOR signaling pathway-modulated autophagy mechanism | |
| Cappelletti et al. | Inflammation and preterm birth | |
| Shynlova et al. | Myometrial immune cells contribute to term parturition, preterm labour and post‐partum involution in mice | |
| Tian et al. | The PD-1/PD-L1 inhibitory pathway is altered in pre-eclampsia and regulates T cell responses in pre-eclamptic rats | |
| Hsiao et al. | Activation of the maternal immune system induces endocrine changes in the placenta via IL-6 | |
| Sisino et al. | Diabetes during pregnancy influences Hofbauer cells, a subtype of placental macrophages, to acquire a pro-inflammatory phenotype | |
| Gomez-Lopez et al. | Macrophages exert homeostatic actions in pregnancy to protect against preterm birth and fetal inflammatory injury | |
| Tang et al. | Progress in the understanding of the etiology and predictability of fetal growth restriction | |
| Edey et al. | The local and systemic immune response to intrauterine LPS in the prepartum mouse | |
| Sha et al. | Alteration of Th17 and Foxp3+ regulatory T cells in patients with unexplained recurrent spontaneous abortion before and after the therapy of hCG combined with immunoglobulin | |
| Xie et al. | Toll‐like receptors 2 and 4 and the cryopyrin inflammasome in normal pregnancy and pre‐eclampsia | |
| Jonsson et al. | Comparison of metabolic, hematological, and peripheral blood leukocyte cytokine profiles of dairy cows and heifers during the periparturient period | |
| Gomez‐Lopez et al. | Interleukin‐6 controls uterine Th9 cells and CD8+ T regulatory cells to accelerate parturition in mice | |
| Chen et al. | Contribution of regulatory T cells to immune tolerance and association of microRNA‑210 and Foxp3 in preeclampsia | |
| Yin et al. | IL-27 activates human trophoblasts to express IP-10 and IL-6: implications in the immunopathophysiology of preeclampsia | |
| Holmes et al. | Plasma levels of the immunomodulatory cytokine interleukin-10 during normal human pregnancy: a longitudinal study | |
| Berraïes et al. | Increased expression of thymic stromal lymphopoietin in induced sputum from asthmatic children | |
| Yanai et al. | Diabetic pregnancy activates the innate immune response through TLR5 or TLR1/2 on neonatal monocyte | |
| Huggard et al. | Altered toll-like receptor signalling in children with Down syndrome | |
| Rogovskii | Immune tolerance as the physiologic counterpart of chronic inflammation | |
| Busse et al. | Regulatory B cells are decreased and impaired in their function in peripheral maternal blood in pre-term birth | |
| Li et al. | The role of Tim-3 on dNK cells dysfunction during abnormal pregnancy with Toxoplasma gondii infection | |
| Wu et al. | Regulation of activin A release from murine bone marrow-derived neutrophil precursors by tumour necrosis factor-α and insulin |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210309 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |