CN107287273A - Express purposes of the Tim-3 peripheral blood NK cell in spontaneous abortion biomarker is prepared - Google Patents
Express purposes of the Tim-3 peripheral blood NK cell in spontaneous abortion biomarker is prepared Download PDFInfo
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Abstract
The invention belongs to biological technical field, it is related to purposes of expression Tim 3 peripheral blood NK cell in spontaneous abortion biomarker is prepared.NK cells of the present invention have tolerance phenotype and low cell killing toxicity, tests prove that height expression of described expression Tim 3 peripheral blood NK cell in normal pregnancy peripheral blood, and this cell quantity of spontaneous abortion declines, dysfunction；The Tim 3 of antagonism NK cells causes Pregnancy failure, adopts and transfers the NK cell subsets and can save the Pregnancy failure caused by immunity abortion-prone modelses and NK are knocked out；Described expression Tim 3 peripheral blood NK cell can be used for preparing spontaneous abortion biomarker, be further used as new prediction and the preventing and treating target spot of clinical Unexplained spontaneous abortion.
Description
Technical field
The invention belongs to biological technical field.It is related to the biomarker of prediction and preventing and treating spontaneous abortion, and in particular to expression
Purposes of the Tim-3 peripheral blood NK cell in spontaneous abortion biomarker is prepared.The biomarker of the spontaneous abortion
It is used as prediction and the preventing and treating target spot of early pregnancy failure.
Background technology
It is mainly the peripheral blood HCG- β and progesterone according to pregnant woman prior art discloses the Forecasting Methodology about pregnancy outcome
Substantial amounts of evidence shows that maternal blood P levels are not fully consistent with pregnancy outcome relation in level, but practice；Although
HCG- β can more really react the pregnant prognosis of pregnant woman, but increasing clinical data shows：Once pregnant woman periphery
Blood-prostatae barrier declines (except HCG- β physiologicals decline), and embryo can not save；In addition, Pregnant Women is although persistently divide
Substantial amounts of HCG- β and P are secreted, but embryo still stops development (or empty blastular)；Obvious HCG- β and P is pregnant as detecting
There is many defects in the index for final result of being pregnent, clinical practice is in the urgent need to the early prediction of a reflection trimester pregnant state
With the Biological indicators of preventing and treating.
Research is disclosed, the paternal antigen of embryo's expression, thus can be considered a kind of half alloplast, of the same race as this half
The embryo of graft can survive in parent until childbirth has really reacted immune tolerance of the parent to embryo.Once this
Plant mother-tire immune tolerance to be broken, it will cause Pregnancy failure or spontaneous abortion and complications of pregnancy.Spontaneous abortion is tight
One of common diseases of human reproduction's health are endangered again, and the trend risen year by year is presented in its incidence.Data shows according to statistics,
At present, spontaneous abortion incidence is up to 15~40%, the today even developed rapidly in Assisted Reproductive Technology ART, still can not
Overcome the Pregnancy failure caused by mother-tire immunoregulatory disorder, regrettably still lack in clinical practice to effective morning
Phase diagnosis and treatment strategy.Heredity, infection, endocrine, reproductive malformation etc. are attributed to the generation of spontaneous abortion more,
But clinic still has more than 50% miscarriage especially recurrent spontaneous abortion etiology unknown, wherein, mother-tire immunological regulation is unbalance quilt
It is considered one of major reason；There are executor and effect person that studies have shown that immunocyte is mother-tire immune tolerance, its
Middle NK cells play vital adjustment effect in mother-tire tolerance；The early-stage Study discovery of the application, decidua NK
Foundation and maintenance of the cell to mother-tire tolerance are most important, and gestational period decidua NK cells pass through cell surface molecule and cell
The effect of the interior various factors, maintains mother-tire tolerance and the progress of normal pregnancy, is difficult yet with gestational period decidua tissue
Obtain, it is several without possible with preventing and treating target spot for clinical diagnosis.Therefore, in the urgent need to one simple and reliable in clinical practice
Method carries out objective assessment to the amynologic state of Early-stage cervical cancer, and can to progress intervene to reach preventing and treating nature
The purpose of pregnant embryo is saved in miscarriage.
Present situation based on prior art, present inventor intends providing a kind of biomarker predicted with preventing and treating spontaneous abortion
Thing, especially expresses Tim-3 peripheral blood NK cell for preparing the purposes in spontaneous abortion biomarker.Should be certainly
The biomarker so miscarried is used as prediction and the preventing and treating target spot of early pregnancy failure.
The content of the invention
It is that the defect for overcoming prior art to exist is marked there is provided a kind of predict with the biological of preventing and treating spontaneous abortion the mesh of the present invention
Remember thing, and in particular to expression Tim-3 peripheral blood NK cell is for preparing the purposes in spontaneous abortion biomarker.
The biomarker of the spontaneous abortion is used as prediction and the preventing and treating target spot of early pregnancy failure.
In the present invention, different pregnant state peripheral blood Tim-3 have been carried out+The comparative experiments of NK cell percentages and variance analysis,
Tim-3 is analyzed in the expression characteristic of different pregnant state peripheral blood NK cells and its regulation situation；As a result show, Tim-3 is in NK
Cellular expression levels have notable difference in different pregnancy phases, and First Trimester peripheral blood NK cell Tim-3 expression is obvious to raise,
Second trimester of pregnancy is down to non-pregnancy level；First Trimester, pregnancy associated hormone (mainly progesterone) and Th2 cytokines
(IL-4) the Tim-3 up-regulated expressions of periphery NK cells are participated in；
In the present invention, Galectin-9/Tim-3 signals mediation early pregnancy periphery NK cellular immunity resistance tests, knot have been carried out
Fruit shows：With Tim3-NK cells are compared, Tim-3+NK cells produce the Th2 cytokines and relatively low water of higher level
Flat Th1 cytokines, and weaker killing activity is showed, these phenotypic characteristics would be even more beneficial to maintain gestation outer
The tolerance status that week is immunized；, the Tim-3 acceptors on NK cells interact with Galectin-9, by activate Akt,
JNK signal paths, promote transcription factor Id-2, Blimp1, Gata-3 expression, so inducing peripheral NK cells to
Immune tolerance phenotypic differentiation；Tim-3/Gal-9 signals participate in NK cells advantage and produce Th2 cytokines；
In the present invention, carried out early pregnancy periphery NK cell Galectin-9/Tim-3 abnormal signals cause NK dysfunctions with
The experiment of Pregnancy failure, as a result shows, the reduction of periphery NK cells Tim-3 expression and periphery Gal-9 produce level
The immune tolerance effect that destruction periphery NK cells are played by secrete cytokines and the relatively low killing activity of maintenance is lowered,
And then cause spontaneous abortion；
Further, the present invention has carried out the influence experiment for disturbing Gal-9/Tim-3 signal paths to pregnancy outcome and adopted
Transfer Tim-3+NK cell rescues Pregnancy failure is tested, and is as a result shown, Tim-3 passes through regulation in serum of women during normal pregnancy
The function of periphery NK cells shields, if the signal path that Tim-3 is mediated is destroyed, can influence periphery
The function of NK cells even differentiation and development, so as to influence the development being normally carried out with embryo of gestation, especially Galectin-9/
Tim-3 signal paths can cause the dysfunction of early pregnancy peripheral blood NK cell to cause Averse pregnancy outcomes extremely；By IL-4 and
The Tim-3 of Progesterone stimulated+NK cells effectively alleviate the high embryo-resorption rate of abortion model；Adopt and transfer Tim-3+NK cells
The embryo-resorption rate of NK Cell Deficient Mice Infecteds is reduced, adopts and transfers Tim-3-NK cells do not influence pregnancy outcome.
The present invention is tests prove that a kind of tolerance NK subgroups are in the height expression of normal pregnancy peripheral blood, and spontaneous abortion
This cell quantity of patient declines, dysfunction；The Tim-3 of antagonism NK cells causes Pregnancy failure, adopts and transfers this
NK cell subsets can save immunity abortion-prone modelses and NK knocks out caused Pregnancy failure；Described expression Tim-3
Peripheral blood NK cell can be used for prepare spontaneous abortion biomarker, be further used as clinical Unexplained spontaneous abortion
New prediction with preventing and treating target spot.
The advantage of this invention is：
Peripheral blood NK cell there is provided expression Tim-3 can be used for preparing spontaneous abortion biomarker, be further used as
The new prediction of clinical Unexplained spontaneous abortion and preventing and treating target spot；
Described Tim-3+NK cells exist in women's peripheral blood, and its phenotype is with function in early pregnancy periphery and decidua local height
Unanimously, a little peripheral blood only need to be extracted, the uterus that can monitor pregnant woman by flow cytometry is local immune with whole body
Tolerance status；Moreover, for this group of cells, the subgroup is transferred by adopting after stimulated in vitro (such as IL-4, P)
Cell can be used for the Pregnancy failure again for directly preventing and treating recurrent spontaneous abortion.
Brief description of the drawings
Fig. 1：Show the different immunocyte subgroup Tim-3 of people's early pregnancy peripheral blood expression and Tim-3+NK cells are in difference
The ratio in pregnancy period, wherein Tim-3+NK cells are in trimester ratio apparently higher than non-pregnant with the middle pregnancy period.
Fig. 2, the display IL-4/STAT6 signals progestational hormone up-regulation peripheral blood Tim3 related to gestation+NK cell proportions,
Wherein, Th2 cytokines IL-4 by activate STAT6 signals raise Tim-3 expression, STAT6 inhibitor with
Th1 cytokines suppress IL-4 this adjustment effect, and classical agent progesterone of preventing miscarriage also promotes pNK cells to express
Tim-3。
Fig. 3, display people's early pregnancy peripheral blood Tim-3+Immune tolerance phenotype is presented in NK cells,
Wherein, Microarray results show that Tim-3+NK cells and Tim-3-NK cells are two groups of variant gene expression
NK cell subsets, Tim-3+865 gene upregulation expression in NK cells, 100 gene deregulation expression, and on
The gene that mileometer adjustment reaches is how relevant with Th2 types immune response and immune tolerance；FCM analysis people's early pregnancy peripheral bloods Tim-3+With
Tim-3-NK cells, find and Tim-3-NK cells are compared, Tim-3+NK cells produce higher level IL-4, IL-10
With TGF-β, lower level TNF-α and Perforin；Its killing activity to trophocyte is also decreased obviously.
Fig. 4：Tolerance phenotype is presented by activating JNK and AKT signals-modulatings periphery NK cells in Galectin-9/Tim-3 signals,
Wherein show, Tim-3 native ligand Gal-9 stimulates pNK cells, and qPCR and western blot show Gal-9 thorns
Swash the transcription factor level that post activation and NK cell developments break up and cytotoxicity is related, raise IL-4/IL-10, under
Adjust TNF-1/Perforin and cytotoxicity.
Fig. 5：Spontaneous abortion peripheral blood NK cell Tim-3/Gal-9 abnormal expressions, with NK cell functional disorders,
Wherein, Fig. 5 .1 show that spontaneous abortion peripheral blood NK cell Tim-3 expression decline, peripheral blood Gal-9 levels are low
Under, with Tim-3+NK cell anti-inflammatory cytokine gene promoters and the chromatin accessibility in enhancer region are notable
Reduction, proinflammatory cytokine generation reduction causes the disorder of Th2/Th1 cytokines, the enhancing of cell killing toxicity；Figure
5.2 display early pregnancy mouse peripheral bloods are higher than the middle pregnancy period with spleen NK cells Gal-9/Tim-3 expression；Spontaneous abortion mouse group
Less than normal pregnancy mouse；Spontaneous abortion mouse peripheral blood and splenic T im-3+NK cells produce Th2/Th1 types cell because
It is sub disorderly.
Fig. 6：TIM-3 signal impairment NK cell functions are blocked to cause Pregnancy failure, wherein,
Embryo absorption and development and the function of NK cell of the normal pregnancy with abortion-prone modelses mouse are illustrated, is blocked
Tim-3 signals, which reduce the nest son number of normal pregnancy and spontaneous abortion mouse, increase its embryo absorbs, suppresses embryo growth；
It is this to act on that spontaneous abortion group is more notable, it is exogenous to give nest son number, the embryo's size that Gal-9 does not influence normal pregnancy
With absorptivity and NK cell functions, but significantly inhibit the embryo of spontaneous abortion mouse and absorb, and improve NK cells
Function.
Fig. 7：Adopt and transfer normal pregnancy splenic T im-3+NK cells alleviate embryo's loss,
Wherein, A be experiment flow figure, B, C, D, E, which shows to adopt, transfers normal pregnancy splenic T im-3+NK cells alleviate nature
Abortion model embryo absorbs, and adopts and transfer spontaneous abortion mouse Tim-3-NK is then without improvement result；Spontaneous abortion is small
Tim-3 of the mouse NK cells after IL-4, P processing+NK, which adopts, to be transferred to after spontaneous abortion mouse, and embryo-resorption rate shows
Write and decline；NK Cell Deficient Mice Infected Pregnancy Models, its embryo-resorption rate is significantly raised, and is adopted and is transferred normal Tim-3+NK
Rather than Tim-3-NK cells, significantly reduce the embryo-resorption rate of NK defect mouse.
Fig. 8 is used for early pregnancy failure for expression Tim-3 peripheral blood NK cell as spontaneous abortion biomarker
Prediction and preventing and treating target spot experiment flow total figure.
Embodiment
Embodiment 1：Different pregnant state peripheral blood Tim-3+NK cell percentages compare and variance analysis/analysis Tim-3
Expression characteristic and its regulation in different pregnant state peripheral blood NK cells
1) expression characteristics of the analysis Tim-3 in different pregnant state peripheral blood NK cells
Normal non-pregnant, early pregnancy, middle pregnant women peripheral blood, Ficoll separation lymphocytes, FCM analyses are separated first
Tim-3 is in CD4+T cell, CD8+T cell, NK cells, NKT cells, DC cells and monocyte expression (such as
Shown in Figure 1A), as a result show, NK cells, DC cells and monocyte height expression Tim-3 (be respectively 80%, 80%,
90%), Tim-3 has notable difference, Figure 1B, shown in C, outside First Trimester in NK cellular expression levels in different pregnancy phases
All obvious up-regulations of blood NK cells Tim-3 expression, second trimester of pregnancy is down to non-pregnancy level.
2) Expression modulations of the Tim-3 in people's early pregnancy peripheral blood NK cell
Because Th2 advantages are presented in trimester peripheral blood and Materno-fetal interface, pregnancy associated hormone such as E, P, hCG are also raised,
The of the invention possible factor for further inquiring into NK cell Tim-3 up-regulated expressions, with magnetic bead by unpregnancy women periphery NK cells
After isolating and purifying, recombined human IL-4 (100ng/ml) and IFN-γ (35ng/ml) culture 48h are added alone or in combination,
Cell is collected, with Flow cytometry NK cells Tim-3 expression；Fig. 2 shows Th2 cytokines
Significantly up-regulation Tim-3 expression after IL-4 processing NK cells, this effect is weakened by IFN-γ；In order to further inquire into
The above-mentioned side of Flow cytometry is used in the mechanism of action of the gamma regulated NK cells Tim-3 expression of IL-4/IFN-, this experiment first
After method processing in NK cells STAT6 (downstream signaling molecule of IL-4 effects) phosphorylation level, as a result show, IL-4
The NK groups of cells of processing, its STAT6 phosphorylation level is significantly improved really, and use in conjunction IFN-γ and IL-4
Suppress the STAT6 phosphorylation levels (Fig. 2 C, D) of IL-4 inductions.Western Blot detections obtain consistent result such as
Shown in Fig. 2 E), STAT6 specific inhibition agent A77-1726 can suppress up-regulations of the IL-4 to Tim-3 and act on (such as
Shown in Fig. 2 F), by activation, molecule STAT6 further promotes NK cells Tim-3 to Th2 cytokines IL-4 downstream
Expression, and Th1 cytokines IFN-γ can be with this effect of antagonism；
First Trimester, not only the Th2/Th1 cytokines general layouts in peripheral blood change, the hormonal readiness of parent
Occurs obvious change, therefore, this experiment further looks at whether pregnancy associated hormone can also influence periphery NK cells
Tim-3 expression, the unpregnancy women periphery NK cells of purifying are stimulated with the Estrogen and progestin of various concentrations gradient, hCG- β,
Flow cytometry NK cells Tim-3 expression, as shown in Figure 2 G, progestational hormone is in the way of concentration dependant pair
NK cells Tim-3 expression is regulated and controled, and the progestational hormone of low concentration promotes NK cells Tim-3 expression, more highly concentrated
When spending, this up-regulation, which acts on just disappearing, even suppresses NK cells Tim-3 expression, and estrogen and hCG- β are to NK
Cell Tim-3 expression does not make significant difference；
As a result show, First Trimester, pregnancy associated hormone (mainly progesterone) is joined with Th2 cytokines (IL-4)
With the Tim-3 up-regulated expressions of periphery NK cells.
Embodiment 2：Galectin-9/Tim-3 signals mediation early pregnancy periphery NK cellular immunity tolerance tests
1) peripheral blood Tim-3+NK is compared with the phenotype of Tim3-NK cells
In order to inquire into, whether expression of the Tim-3 on NK cells be related to NK cell functions, by the isolated single core in periphery
Cell further goes out Tim-3 with magnetic bead sorting+NK and Tim3-NK cells, it is 92% to identify separating purity, meets requirement of experiment,
Extract and full-length genome chip analysis is carried out after total serum IgE；As shown in Fig. 3 A, B, Tim-3+ and Tim-3-NK cells are variant
Two groups of NK cell subsets of gene expression, Tim-3+865 gene upregulation expression, 100 gene deregulations in NK cells
Expression, and the gene of up-regulated expression is how relevant with Th2 types immune response and immune tolerance；This experiment PMA, ion are mould
Element and BFA stimulate 4 to 6 hours after on further use flow cytometer detection Th2 types, the protein expression level of Th1 cytokines,
Such as Fig. 3 C, shown in D, Th2 cytokines IL-4, IL-10, TGF-β are in Tim-3+The expression of pNK cells is substantially high
In Tim-3-PNK cells and Th1 cytokines TNF-α is then opposite in the expression of two groups of cells；IFN-γ exists
Tim-3+NK and Tim3-Several indifferences of expression (such as Fig. 3 E, shown in F) of NK cells；
Further compare Tim-3+NK and Tim3-The killing activity of NK cells, by this two groups of cells of purifying respectively with target cell
HTR-8 (the outer langhans cell system of fine hair) contact culture, is used Non-Radioactive
Cytotoxicity Assay kit detection cell killing activities, as shown in fig. 31, Tim-3+NK and Tim3-NK cells
Killing activity with effect target than decline and reduce, but Tim-3+The killing activity of NK cells is consistently lower than Tim3-NK
Cell；Flow cytometry Tim-3+NK and Tim3-NK cells produce perforin perforin level, as a result show
Perforin is in Tim-3+The expression of NK cells is significantly lower than Tim-3-NK cells (such as Fig. 3 G, H)；A little results are tested to show
Show：With Tim3-NK cells are compared, Tim-3+NK cells produce the Th2 cytokines of higher level and the Th1 of reduced levels
Cytokines, and weaker killing activity is showed, these phenotypic characteristics would be even more beneficial to maintain pregnant periphery immune
Tolerance status.
2) Expression modulation of Tim-3 native ligands Gal-9 human peripheral blood NK development and cell differentiations and function associated transcription factor
It can be obviously promoted and NK development and cell differentiations with Tim-3 native ligand Galectin-9 processing periphery NK cells
And function related transcription factor Id-2, Blimp1, Gata-3 are in the expression of mRNA level in-site, and regulate and control NK cells and kill
The transcription factor Eomes expression for hindering activity is then substantially lowered, and further analysis is found, Gal-9 by activate Akt,
The signal paths of JNK two, promote NK cells to produce Th2 cytokines, suppress the generation of its Th1 cytokines, and
And effectively reduce NK cell killing activities；Therefore, the Tim-3 acceptors on NK cells interact with Galectin-9,
By activating Akt, JNK signal path, promote transcription factor Id-2, Blimpl, Gata-3 expression, and then induce
Periphery NK cells are to immune tolerance phenotypic differentiation.
3), the influence of the cytokine secretion general layout of Gal-9/Tim-3 signals human peripheral blood NK cells and killing activity
Research prompting Tim-3 may participate in the phenotype and function point analysis of NK cells by being combined with its part Gal-9, because
This, further adds rhGal-9 or anti-Tim-3 neutrality antibodies in the cultivating system of periphery NK cells, place
Reason is after 48 hours, and rhGal-9 treatment groups IL-4, IL-10 generation level is significantly raised, and TNF-α produces level then
Substantially reduction；Individually with the generation levels of anti-Tim-3 neutrality antibody treatment group cell factors before treatment after without bright
Aobvious change, but rhGal-9 and anti-Tim-3 Combined Treatments group IL-4, IL-10 produces level and is significantly lower than rhGal-9
Treatment group, it is then opposite that TNF-α produces level；Experimental result prompting Tim-3/Gal-9 signals participate in the production of NK cells advantage
Raw Th2 cytokines (as shown in Figure 4 C)；
In addition, the peripheral blood NK cell killing activity after rhGal-9 processing is remarkably decreased, this effect is by anti-Tim-3
Neutrality antibody part is recovered, and individually with anti-Tim-3 neutrality antibodies then without obvious effect；Equally, streaming is used
The production of NK perforins perforin after cell art detection rhGal-9 or anti-Tim-3 neutrality antibody processing
Unboiled water is flat also to there occurs above-mentioned change (such as Fig. 4 D, shown in E).
4) Gal-9/Tim-3 interactions are by activating JNK, Akt Signal Regulation peripheral blood NK cell functional experiment
In order to further parse the signal transduction mechanism that Tim-3/Gal-9 adjusts peripheral blood NK cell, this experiment rhGal-9
The peripheral blood NK cell of purifying is stimulated respectively 10 minutes, 30 minutes, 60 minutes, Western Blot detection peripheral bloods
The expression of NK cell-signaling pathways correlation molecules, as illustrated in figure 4f, compared with NK cells individually culture, rhGal-9
After stimulation, Akt, JNK and Erk phosphorylation level are significantly raised and NK- κ B phosphorylation level is stimulating front and rear
There is no a significant change, point out Tim-3/Gal-9 very possible by activating Akt, JNK and Erk this three signal paths
To regulate and control the function of periphery NK cells；
For clearly above-mentioned possible signal path, by the specific inhibitor of the signal paths of Akt, JNK and Erk tri-
In the cultivating system for adding Gal-9 processing NK cells, then with Flow cytometry NK cell cytokines and wearing
Kong Su generation level, as shown in Figure 4 G, Gal-9 can be suppressed to NK with JNK or AKT specific inhibition agent
The adjustment effect of cell, and Erk specific inhibition agent is then without obvious effect；As a result show, Gal-9 is by work
Change the biological function that the signal paths of JNK, Akt two further change NK cells, rather than Erk.
Embodiment 3：Early pregnancy periphery NK cell Galectin-9/Tim-3 abnormal signals cause NK dysfunctions real with Pregnancy failure
Test
1) spontaneous abortion periphery NK cell Tim-3 expressions decline Gal-9 generation level reductions, and NK cells are thin
Intracellular cytokine secretion level is disorderly, killing activity enhancing
It is that research periphery NK cell quantities and the exception of function and the generation of spontaneous abortion are closely related, further confirms just
Often whether the peripheral blood NK cell Tim-3 of gestation and spontaneous abortion expression is variant, and Gal-9's contains in blood
Whether also whether amount has a change, the functions of NK cells respective changeThis experiment acquires early stage normal pregnancy and not clear original
Because of the peripheral blood of spontaneous abortion, detected and analyzed for problem above with flow cytometry, as a result found,
Although obvious lifting, Tim-3 do not occur for Tim-3+NK cells ratio shared in the immunocompetent cell of periphery
The fluorescence intensity expressed in NK cells significantly reduces (as shown in Fig. 5 .1A), further system in spontaneous abortion peripheral blood
Compare the difference of normal pregnancy and recurrent spontaneous abortion peripheral t im-3+NK cells, examined using ATAC-seq technologies
Survey and analyze the dyeing of normal pregnancy and recurrent spontaneous abortion peripheral t im-3+NK cells and Tim-3-NK cells
Matter accessibility, Principle components analysis result shows, patient Tim-3+NK cells and Tim-3-NK cells with it is normal
NK cells of human beings can be very good to distinguish (as shown in Fig. 5 .1D), result and the Principle components analysis result of clustering
It is consistent, it is the functioning cell closely related with pregnancy outcome to point out Tim-3+NK cells；Further to spontaneous abortion
It is compared and analyzes with women with normal peripheral t im-3+NK cells, finds spontaneous abortion Tim-3+NK cells
In have 260 gene locis chromatin accessibility decline, the chromatin accessibility rise of 360 gene locis is (such as
Shown in Fig. 5 .1E), with reference to the ChIP-seq interpretations of result of known people CD56+NK cells, chromatin accessibility drop
Low gene loci is mainly enhancer, and the elevated gene loci of chromatin accessibility importantly promoter, wherein,
The chromatin accessibility reduction at 3 ' ends of TGF-β 1 is most notable, at the same time, the dyeing of the promoter region of TGF-β 1
Matter accessibility is significantly reduced, and points out the end regions of TGF-β 13 ' to play the effect of enhancer, with TGF-β 1
The expression (as shown in Fig. 5 .1F) of promoter coordinated regulation TGF-β 1, IL-10 chromatin accessibility also has same
Change (as shown in Fig. 5 .1G)；
Further detection NK cells produce Th2, Th1 cytokines and react the perforin perforin of its killing activity
It was found that, by Tim-3+The Th2 cytokines (L-4, IL-10 and TGF-β 1) that pNK cells are produced are suffered from miscarriage
Person's peripheral blood is decreased obviously, on the contrary, Th1 cytokines (TNF-α, IFN-γ) are then significantly raised, Tim-3-pNK
The cytokine levels that cell is produced have no obvious difference (such as Fig. 5 .1H, shown in I) in two kinds of samples；Similarly,
Tim-3+PNK cells rather than Tim-3-The perforin perforin that pNK cells are produced is bright in spontaneous abortion peripheral blood
Aobvious up-regulation points out spontaneous abortion periphery NK cell killing activities to be remarkably reinforced (as shown in Fig. 5 .1J), and this be mainly because
For Tim-3+The rise of pNK cytotoxicities；
As shown in Fig. 5 .1B, spontaneous abortion peripheral blood Gal-9 secretion levels are dropped to by 1.84ng/ml under normal circumstances
0.965ng/ml, in addition, each periphery immunocompetent cell (CD4 including NK cells itself+、CD8+T cells,
CD14+Monocyte and CD11c+BMDC) level that produces Gal-9 also has and significantly lowers (such as Fig. 5 .1C institutes
Show)；The above results illustrate that the reduction of periphery NK cells Tim-3 expression and the downward of periphery Gal-9 generation levels may be broken
The immune tolerance effect that periphery NK cells are played by secrete cytokines and the relatively low killing activity of maintenance is broken, is entered
And cause spontaneous abortion.
Embodiment 4：Disturb influence experiment of the Gal-9/Tim-3 signal paths to pregnancy outcome
Based on Gal-9/Tim-3 is had been verified that, unconventionality expression may be relevant with spontaneous abortion in First Trimester peripheral blood,
In order to further parse influence and molecular mechanism of this pair numerator mediated signal paths to pregnancy outcome, this experiment is aligned
The Gal-9/Tim-3 signals of normal and abortion mouse model carry out human intervention；Normal and abortion mouse model is initially set up,
Compare early pregnancy and middle pregnancy period mouse periphery (including peripheral blood and spleen) NK cells Tim-3 expression, as a result show,
The trimester periphery NK cells Tim-3 expression of normal pregnancy mouse is higher than the middle pregnancy period, is consistent with people, that is to say, that
Tim-3+NK cells are enriched with First Trimester；Fig. 5 .1A result shows spontaneous abortion periphery NK cells Gal-9/Tim-3
Differential expression with NK cell functional disorders, this experimental analysis compare Tim-3 and Gal-9 in normal pregnancy model and
Expression on the NK cells of abortion-prone modelses trimester periphery, such as Fig. 5 .2A, shown in B, relative to normal pregnancy mouse,
The level that spontaneous abortion NK cells in mice expresses Tim-3 and Gal-9 in peripheral blood and spleen is decreased obviously, not only
In this way, abortion-prone modelses Tim-3+pNK produces the level drop of Th2 cytokines (IL-4, IL-10 and TGF-β)
It is low, and the level (TNF-α) for producing Th1 cytokines is then raised；Flow cytometry granzyme A (Granzyme
A, reflects the killing activity of NK cells) it has also been found that the expression in abortion-prone modelses peripheral t im-3+NK cells is bright
It is aobvious to be higher than normal pregnancy model Tim-3+NK；The These parameters in spleen are analyzed, the result with peripheral blood is consistent；
(Fig. 5 .2C, D) is shown either in peripheral blood still, in spleen, the function of Tim-3-NK secrete cytokines and is killed
Wound activity, almost without difference, as a result shows Gal-9/Tim-3 and pregnant periphery in normal pregnancy and self-heating abortion model
The performance of NK cell normal functions is closely related；
For effects of the clear and definite Tim-3/Gal-9 in gestation, this experiment with the exogenous Gal-9 of restructuring and
RM3-23 (blocking agent of Tim-3 signal paths) intervenes Gal-9/Tim-3 signal paths, normal pregnancy in pregnant mouse body
Model and abortion-prone modelses with RMT3-23 after Tim-3 signal paths are blocked, and two groups of embryo-resorption rate is obvious
Rise, normal pregnancy model increases to 17% by 5% or so embryo-resorption rate, and abortion-prone modelses are originally just higher
Embryo-resorption rate increase to about 46% (Fig. 6 A, D) by about 27%；Accordingly, the fetus that normal pregnancy model is survived
Number drops to 6 by 10, and 7 then before intervening of abortion-prone modelses drop to 4 (such as Fig. 6 A, shown in C)；Although
The embryo-resorption rate of normal pregnancy model and abortion-prone modelses has obvious difference, but the embryo's size and indifference of survival
It is different；However, being injected with RMT3-23 after mouse, the mice embryonic volume of two kinds of models, which is significantly less than, does not inject group,
Embryo-resorption rate can not only be influenceed by speculating the blocking of Tim-3 signals, have significant inhibitory action to growing also for embryo
(such as Fig. 6 B, shown in E)；In order to prove whether blocking for Tim-3 signals can produce influence, this reality to the function of NK cells
The peripheral blood that above-mentioned treatment group mouse is analyzed with Flow cytometry and spleen NK cells are tested, is as a result shown, is injected
NK cell quantities in its peripheral blood of RMT3-23 mouse are drastically reduced, and 1-2% is dropped to by the 5-10% of before processing,
Level is even up to deleted, but the NK cell quantities in spleen do not change；Further analyze spleen NK
The function of cell, it is found that normal pregnancy natural killer cells in mice IL-4, IL-10, the generation level of TGF-β are being blocked
It is remarkably decreased after Tim-3 signals, TNF-α then significantly rises, the upper no significant differences before and after blocking of INF-；Tim-3 believes
Number block to normal pregnancy spleen NK cells act in abortion-prone modelses embody more significantly (such as Fig. 6 F institutes
Show)；Detection to spleen NK cell granulations enzymes A (Granzyme A) finds that abortion model is after Tim-3 blockings
Higher levels of Granzyme A are produced, the killing activity enhancing of NK cells is pointed out, and Tim-3 is blocked without enhancing just
The killing activity of normal gestation group NK cells, shows the reduction of certain level on the contrary, and experimental data prompting, Tim-3 exists
Shielded in serum of women during normal pregnancy by adjusting the function of periphery NK cells, if the signal that Tim-3 is mediated leads to
Road is destroyed, and the function even differentiation and development of periphery NK cells can be influenceed, so as to influence being normally carried out and embryo for gestation
Development；
Whether this is tested recombinates Gal-9 to pregnant mouse injection of exogenous, to be made moderate progress to pregnancy outcome, as a result show,
Pregnancy outcome can be improved to a certain extent really by giving exogenous Gal-9：For natural pregnancy model, embryo can be reduced
Absorptivity, increases the litter size of survival, but the gestation of the Tim-3 abortion-prone modelses blocked and normal pregnancy model is tied
Office is without obvious improvement result；Detect that the function of spleen NK cells is found, inject the NK of Gal-9 abortion-prone modelses
Cell IL-4, the generation level of TGF-β rise, and INF- γ decline, and GranzymeA also declines, and IL-10, TNF-
α does not substantially change；Tim-3 normal pregnancy model and abortion-prone modelses is blocked with RMT3-23 in advance, is given
The cell factor of NK cells produces level and killing activity without significant changes after Gal-9；Above-mentioned experimental data explanation,
Gal-9 is the function of adjusting periphery NK cells by being combined and being interacted with its acceptor Tim-3 so as to remain normal
Gestation is smoothed out, if Tim-3 signal path is blocked, and Gal-9 can not normally be combined performance with Tim-3 and be adjusted
Section acts on (as shown in Figure 6)；
Therefore, Galectin-9/Tim-3 signal paths can cause early pregnancy peripheral blood NK cell dysfunction cause bad extremely
Pregnancy outcome.
Embodiment 5：Adopt and transfer Tim-3+NK cell rescues Pregnancy failure is tested
Airflow classification goes out Tim-3 from the spleen of normal pregnancy mouse+NK, Tim-3-NK and total NK cells, respectively
Above-mentioned three kinds of cells are resuspended in 200uLPBS, adopting through tail vein injection, it is small to the miscarriage of pregnancy the 4.5th day to transfer
Mouse；Tim-3 is obtained from the spleen of abortion mouse with same method+NK, Tim-3-NK and total NK cells,
Tim-3+NK, Tim-3-NK and a portion NK cells are adopted with same method to be transferred to abortion mouse, another
Part NK cells use IL-4 and progesterone stimulated in vitro respectively, after 48 hours, again airflow classification Tim-3+NK, Tim-3-
NK cells, adopt transfer to abortion mouse according to the method described above, put to death within pregnant 14.5 days abortion mouse, and observation embryo absorbs
Situation (as shown in Figure 7 A), as a result shows, normal pregnancy mouse Tim-3+NK reduction abortion model embryos absorb
Rate, and abortion mouse Tim-3+NK passes through IL-4 and the Tim-3 of Progesterone stimulated without remarkable effect+NK cells
Effectively alleviate the high embryo-resorption rate of abortion model (shown in Fig. 7 D)；
Set up NK Cell Deficient Mice Infecteds (Nfil3-/-) Early pregnancy model, adopted as stated above in pregnant 4.5 days and transfer just
Normal gestation Nfil3+/+The Tim-3 of mouse spleen+NK and Tim-3-NK cells, Nfil3 was put to death in pregnant 14.5 days-/-Pregnant mouse,
Embryo nidation number and embryo-resorption rate are observed, as a result display, which is adopted, transfers Tim-3+NK cells reduce NK Cell Deficient Mice Infecteds
Embryo-resorption rate, adopt and transfer Tim-3-NK cells do not influence pregnancy outcome (such as Fig. 7 E, shown in F).
Claims (8)
1. express purposes of the Tim-3 peripheral blood NK cell in spontaneous abortion biomarker is prepared；Institute
The NK cells stated have immune tolerance phenotype and low cell killing activity.
2. the purposes as described in claim 1, it is characterised in that described NK cell predominant expressions IL-4,
IL-10 and TGF-β, low expression TNF-α；Low cell killing activity and Perforin expression rates.
3. the purposes as described in claim 1, it is characterised in that described NK cells are in spontaneous abortion
Patient is significantly lower than normal pregnancy with the cell quantity in mouse model.
4. the purposes as described in claim 1, it is characterised in that described NK cells are in spontaneous abortion
The chromatin proximity of patient's Tim-3+NK cells TGF-β and IL-10 promoters and enhancer is reduced.
5. the purposes as described in claim 1, it is characterised in that described NK cells are in spontaneous abortion
Patient declines with the Tim-3+NK cells expression IL-4 in mouse model with TGF-β, on expression TNF-α
Rise；Cell killing activity increases with Perforin expression.
6. the purposes as described in claim 1, it is characterised in that described NK cell antagonisms Tim-3
Signal, influence NK cell developments and immune tolerance phenotype, normal pregnancy failure.
7. the purposes as described in claim 1, it is characterised in that described NK cells, which are adopted, to be transferred
Tim-3+NK cells, save the embryo being at death's door.
8. the peripheral blood NK cell for expressing Tim-3 is early in preparation prediction as the biomarker of spontaneous abortion
Purposes in phase Pregnancy failure and preventing and treating target spot.
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| 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》 * |
| YAN-HONG LI等: "The Galectin-9/Tim-3 pathway is involved in the regulation of NK cell function at the maternal–fetal interface in early pregnancy", 《CELLULAR & MOLECULAR IMMUNOLOGY》 * |
| 张铭等: "Tim-3分子表达异常与原因不明复发性流产的关系", 《中国优生与遗传杂志》 * |
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