CN102429911A - Application of ZSTK474 to preparation of medicine for treating autoimmune diseases - Google Patents

Abstract

The invention discloses that 2-(2-difluoromethylbenzimidazol-1-yl)-4,6-dimorpholinyl-1,3,5-triazine (ZSTK474 for short) is used for the treatment of autoimmunity, Drug use in inflammatory diseases. The experimental results of the present invention show that ZSTK474 can inhibit the differentiation of human immune dendritic cells and the maturity of human immune dendritic cells activated by LPS; it can inhibit the antigen uptake ability of immature dendritic cells and the Antigen presentation ability can inhibit T cell proliferative response stimulated by dendritic cells. In particular, ZSTK474 can reduce the severity of experimental autoimmune encephalomyelitis in mice. Therefore, the present invention is expected to become a medicine for treating autoimmune inflammatory diseases such as multiple sclerosis and acute disseminated encephalomyelitis clinically.

Description

The application of ZSTK474 aspect preparation treatment self property exempted from disease medicament

Technical field

The invention belongs to the technical field of the new usefulness of old medicine, relate to 2-(2-difluoromethyl benzo imidazoles-1-yl)-4, the application of 6-dimorpholine base-1,3,5-triazines (ZSTK474) aspect preparation treatment self property exempted from, diseases associated with inflammation medicine.

Background technology

(dendritic cells is that (antigen-presenting cells APC), also is the unique APC that can directly activate primary tape T cell that finds at present to the antigen presenting cell that function is the strongest in the known body DC) to BMDC ^[1]When being inflamed; DC contacts and then capture antigen with antigen at first; Antigenic peptides is combined with the MHC molecule and offer other immunocytes surface, cause cytokine secretion, under first, second signal combined effect; Activate the T lymphocyte, produce the antigen-specific immune responses reaction; DC can break up regulatory cell on the other hand, keeps self tolerance, becomes the key factor of mediation immunne response and tolerance ^[2]Therefore, APC is immunoreactive primary link.

It is generally acknowledged that human body DC has different DC subgroups to exist.If the four-stage that mainly experiences according to its differentiation and development is a differentiation degree; DC can be divided into the remote ancestor's cell that originates from the CD34+ pluripotent stem cell in the bone marrow again; Precursor in the peripheral blood, be settled in immature DC in the peripheral tissues (immature DC, iDC); Be settled in ripe DC in the secondary lymphoid tissue (mature DC, mDC) ^[3]Seeing that the DC precursor in the different microenvironments, is being brought into play different biological functions in different differential periods.Banchereau is divided into 4 stages with the growth of DC: the CFU-GM in (1) bone marrow; (2) discern the preceding DC precursor that in blood, lymphatic vessel and lymphoid tissue, goes on patrol each other with pathogen, they can secrete a large amount of cytokines makes inflammation localization; (3) the immature DC of resident tissue, they have powerful phagocytic function and are convenient to antigenic picked-up; (4) ripe DC comes across in the secondary lymphatic organ, and the collaborative stimulation molecule of high expressed carries out antigenic offering ^[4]Therefore, the differentiation of DC and sophisticated state have determined the result that immunoreactive initial sum fine equilibrium is regulated.And the initial or immunologic balance of immunity is regulated is the main cause that causes immunity and autoimmune disease unusually.

The final result of the maturity state decision immunne response of DC.IDC resembles intravital " soldier ", plays a part immune surveillance, and it is distributed in nearly all tissue and organ, has powerful endocytosis, migrates to local secondary lymphoid tissue from peripheral tissues's antigen constantly, but its angtigen presentation hypofunction.Under the normal physiological state that does not have the damage of infection and inorganization, the iDC that is present in peripheral tissues constantly absorbs the protein (like the protein in respiratory tract and the digestive tract) in autoantigen and the non-infection environment, gets into draining lymph node, induces peripheral tolerance ^[5]" toleration DC " possibly induce tolerance through following mechanism: the incapability or the low energy reactions of (1) inducing T cell.Toleration DC low expression level MHC molecule is expressed CD40 hardly, CD80, CD86, IFA3, the necessary secondary signal molecule of activated T cells such as ICAM-1; (2) inducing T cell apoptosis.The bonded while of MHC of the DC of T cells with antigenic specificity TCR and expression FasL, its Fas combines with DC surface FasL, mediates these t cell proliferations ^[6](3) induce regulatory T cells to produce.Toleration DC can promote that T cells is converted into T-reg, and can to induce the DC precursor conversion again be toleration DC to T-reg conversely ^[7]This mechanism makes " danger signal " (the danger signal) when inflammation and tissue injury discerned and defendd to the immune system of body can better, and autoantigen and harmless exogenous antigen are kept tolerance.

DC is a double-edged sword, but both inducing immune tolerances also can stimulate intensive immunne response, and its maturity state plays a decisive role in immunologic balance.Immature DC has the ability of very strong capture antigen, but stimulates the ability of T cell very low.Only (like IL-1 β, during TNF) or by bacterial stimulations, DC turns to ripe DC by the immature stage, and the surperficial costimulatory molecules of high expressing cell is moved to the propagation of lymph node inducing antigen-specific T cell simultaneously irritating signal occurring.Activation the T cell be signal through CD 40 conversely again, discharge cytokine stimulation DC, induce the DC activation.In multiple disease, find that the DC of incidence tissue often is in a kind of jejune state, can not move to lymph node ^[8]In autoimmune disease, DC differentiation, activation, antigen recognition or dysfunction or too activation such as offer, the perhaps unusual or excessive activation of T cell function that stimulates of DC.Therefore cause immune system recognition autoantigen and then attack autogenous cell or tissue and produce autoimmune disease.

Chemical name 2-(2-difluoromethyl benzo imidazoles-1-yl)-4,6-dimorpholine base-1,3,5-triazines, (being called for short ZSTK474), it is a kind of novel S-pyrrolotriazine derivatives, is a strong PI3K inhibitor, has high selectivity.Reported once that ZSTK474 suppressed the growth of tumor cell consumingly, had antitumor curative effect in vitro and in vivo, blocked and anti-angiogenesis activity with 1 phase of G 0/G.ZSTK47 has powerful antitumor activity when being used for transplanting and give mice oral to human cancer.Furthermore, ZSTK474 did not show significant toxicity at the doses tested. in addition, ZSTK474 does not show the dosage of apparent toxotest.ZSTK474 is that a kind of novel special PI3K targeting suppresses medicine, and its specificity suppresses the PI3K signal path, can be issued to the effect (concentration of 1 μ M can suppress fully) that suppresses fully at extremely low concentration; Specificity is good, and to the protein kinase unrestraint effect of more than 130 kinds of different approaches, pair cell toxicity is low during 30 μ M.ZSTK474 come to light at first can the strong inhibition tumor cell growth, and be fit to oral; When testing anti-tumor activity in vivo, injection human tumor cell's the major organs of mice is not had toxicity [1-5], got into the clinical II phase at present to test, be expected to become new type antineoplastic medicine.

The PI3K inhibitor works to the panimmunity cell through suppressing the PI3K/Akt path.Research to the PI3K signal transduction pathway at present focuses mostly on aspect the PI3K/Akt path.Akt is silk/Soviet Union's kinase amino acid that proto-oncogene c-akt expresses coding, has and the homologous PH domain of multiple protein kinases, and be the direct target protein of PI3K.PI3K (phosphatidylinositol 3-kinase) is the lipid kinase family of 3-oh group on the inositol ring of a phosphorylation phosphoinositide; It is the important molecule in the somatomedin superfamily signal conductive process; Can be activated by the various kinds of cell factor and chemical factors; Regulate the various kinds of cell function,, and in the pathogenesis of inflammation, tumor, metabolism and cardiovascular disease, play an important role like apoptosis, propagation, metabolism, growth conversion, film transhipment, secretion and chemotactic etc.PI3K is by regulating the heterodimer that subunit p85 and catalytic subunit p110 constitute.The adjusting subunit p85 that finds now has 5 kinds of isomers at least: p85 α, p85 β, p55 γ, p50 α, p55 α (wherein p50 α, p55 α are the coded products that p85 α gene is sheared in turn); Its sequence has high homology; Aminoterminal has a SH3 district, and c-terminus has two SH2 districts, has high-affinity with receptor pYXXM motif, and the intermediate code district combines with the p110 of catalytic subunit.Catalytic subunit p110 has 4 kinds of isomers: p110 α, p110 β, p110 γ and p110 δ by the different genes coding, all comprise catalytic domain, PIK domain, C2 domain and Ras binding structural domain respectively.Regulate subunit and catalytic subunit and have multiple isomer, having tissue specificity also can be in different receptors bind, and this multiple function with its participation various kinds of cell is relevant.Construction features and the bonded specificity of substrate according to them are divided into three types. Main phosphorylation phosphoinositide 4,5 diphosphonic acid of I class PI3K (being called for short PIP2) generate phosphatidylinositols 3,4,5 InsP3s (PIP3), and PIP3 raises with phosphorylation Akt's.Therefore, in cell effect (like propagation), brought into play important effect.Usually PI3K refers to I class PI3K, and the function of other two types of PI3K is seldom known.I class PI3K has four isomers, is respectively α, β, δ and γ.I class PI3K α plays an important role in tumor takes place, because the altofrequency of PIK3CA gene mutation, the p110 α of this gene code finds in human cancer.Have recently and report that PI3K β plays a significant role in the tumor of PTEN Tumor Suppressor Gene (phosphatase and tensin omology deleted on chromosome ten) takes place.And PI3K δ and PI3K γ also are considered to participate in various inflammatory reactions and immune system ^[9]The invention reside in and find that ZSTK474 suppresses the application on autoimmune and inflammatory reaction and the relevant disease thereof.

DCs plays important effect congenital the reaction in the initiation with adaptive immunity.Though the generally effect of DCs is the lymphocytic activation of angtigen presentation and T, DCs has different surface mark, migration model and cytokine production in the different phase of differentiation.These differences have determined their interactional destiny of T cell.Initial t cell activation depends on traction and the CD28 and the CD80/86 costimulatory signal through on APCs (for example DCs) surface, expressing of TXi Baoshouti.Therefore, we pass through to detect DC cytokine production and surface marker CD80, CD83, and whether ZSTK474 is investigated in the expression of CD86 and HLA-DR influences DC differentiation and activation.We also study ZSTK474 to the antigen uptake ability of immature DC and the T cell proliferation stimulating activity of ripe DC.This research provides the physiological function of ZSTK474 and the further understanding of medical application.

Summary of the invention

The objective of the invention is to disclose a kind of 2-(2-difluoromethyl benzo imidazoles-1-yl)-4, the application of 6-dimorpholine base-1,3,5-triazines aspect preparation conduct treatment LADA, diseases associated with inflammation medicine.

Autoimmune disease of the present invention refers to: the autoimmune disease that immune BMDC or T cell are relevant.The autoimmune disease that wherein immune BMDC or T cell are relevant refers to: multiple sclerosis, LADA retinitis, optic neuromyelitis, systemic lupus erythematosus (sle), diabetes, thyroid, hemolytic anemia, atrophic orchitis, AT, myasthenia gravis, primary biliary cirrhosis, aggressive chronic hepatitis, scorching, routed colitis, dermatitis, myositis, the systemic sclerosis of hindering of chronic glomerulus.

The nervous system inflammation property disease that diseases associated with inflammation of the present invention refers to.Particularly multiple sclerosis and acute disseminated encephalomyelitis.

Immunologically competent cell such as BMDC etc. have the identification exotic antigen or think mechanism to the deleterious autoantigen of organism.Immunocyte is discerned autoantigen and is caused that autoimmune response is the pathogenesis basis of panimmunity disease.Multiple sclerosis (Multiple Sclerosis for example; MS), optic neuromyelitis (neuromyelitis optica), LADA retinitis, systemic lupus erythematosus (sle) (Systemic Lupus Erythematosus, SLE), diabetes, thyroid, hemolytic anemia, atrophic orchitis, goodpasture's disease (Goodpasturee ' s disease), AT, myasthenia gravis, primary biliary cirrhosis (primary biliary cirrhosis), aggressive chronic hepatitis, chronic glomerulus inflammation, Si Yegelun syndrome (Sjogren syndrome), burst and hinder colitis, dermatitis, Reiter syndrome (Reiter syndrome), myositis (miositis), systemic sclerosis etc.And the inhibition immunocyte, the material that especially suppresses the BMDC with professional antigen presentation ability in immune system forward position will have the purposes of potential treatment autoimmune disease.

Application of the present invention; Wherein autoimmune disease refers to the autoimmune disease of above-mentioned indication; Especially inflammatory disease of the central nervous system, for example people's multiple sclerosis and acute disseminated encephalomyelitis or the like are typically people's multiple sclerosis.

Multiple sclerosis is an autoimmune inflammation property disease; Characteristic is the variation of the sings and symptoms of central nervous system dysfunction; Cooperating repeatedly alleviation and recurrence. modal disease symptom is a paraesthesia, appears at one or several limbs, the face of a trunk or a side; Lower limb or hands unable or clumsy; Or visual disorder, pain, blurred vision or dim spot when for example simple eye part property is lost one's sight with ocular movemeut. other common early symptoms comprise the diplopia that ophthalmoplegia causes; One or more limbs of short duration unable; Slight gait disorder, limbs slight stiff and unusual easy to be tired, bladder control difficulty; Dizzy, and slight dysthymic disorder.

Autoimmune inflammation refers specifically to people's multiple sclerosis, optic neuromyelitis and acute disseminated encephalomyelitis, and cytologic experiment such as differentiation, maturation, antigen presentation and T cell proliferation through suppressing immune BMDC and mouse disease animal model experiment prove: can treat LADA inflammatory disease of the central nervous system such as multiple sclerosis and acute disseminated encephalomyelitis.Optic neuromyelitis is thought a kind of of multiple sclerosis the earliest, along with the development of clinical diagnosis and pathological study, just is subdivided into the LADA optic neuromyelitis in recent years.Acute disseminated encephalomyelitis; Claim encephalomyelitis after postinfectious encephalomyelitis, the prophylactic immunization again; Be meant acute eruption property diseases such as being secondary to fiber crops are examined, rubella, chickenpox, variola, or after the prophylactic immunization, because of immunodeficiency disease causes the demyelination in the central nervous system.

Experimental autoimmune encephalomyelitis (Experimental autoimmune encephalomyelitis; EAE) be people's multiple sclerosis (Multiple Sclerosis; MS) and acute disseminated encephalomyelitis (Acute disseminated encephalomyelitis; ADEM) etc. the animal model of marrowbrain inflammation property disease also is the typical animal model of research LADA and diseases associated with inflammation.It is the struvite demyelination of a kind of central nervous system's autoimmune.Its pathogeny is the autoimmune inflammation reaction by immunocyte (helper T lymphocyte TH1 and TH17 cell) mediation.The present invention is an example with the typical animal model-mouse experiment systemic autoimmune encephalomyelitis of research LADA and diseases associated with inflammation, finds generation and development that ZSTK474 can effectively alleviate mouse experiment systemic autoimmune encephalomyelitis disease.Therefore, the present invention is expected to become the medicine of autoimmune inflammation property diseases such as treatment multiple sclerosis and acute disseminated encephalomyelitis clinically.

The application of ZSTK474 chemical compound disclosed by the invention aspect treatment autoimmune, diseases associated with inflammation medicine; Normally take with the form of pharmaceutical composition; Orally-ingestible or non-oral administration are perhaps with the oral or non-oral administration of chemical compound (like tablet, slow releasing preparation, capsule, injection, the solution) safety that forms with pharmaceutically acceptable carrier, excipient and other additive.When oral administration, compositions can be mixed with tablet, sugar-coat agent or capsule.For the preparation combination of oral medication can adopt lactose or starch to do carrier, gelatin, sodium carboxymethyl cellulose, methylcellulose, polyvinylpyrrolidone etc. are suitable bonding agent or become an agent.Can select starch or microcrystalline Cellulose for use as disintegrating agent, often with Pulvis Talci, santocedl, tristerin, calcium stearate or magnesium etc. are as suitable antiadhesives and lubricant.For example, can prepare tablet through the compacting wet granular.Active component and carrier and optionally with a disintegrate additive composition mixture; The aqueous solution of this mixture and binding agent; Alcohol property or aqueous alcohol property solution carry out granulating in suitable device; Dried particles adds other disintegrating agent subsequently, and lubricant and antiplastering aid are with this mixture tabletting.Series compound of the present invention can the injection form administration, though dosage changes according to treatment target, administering mode, symptom and other factor.The dosage of the actual ZSTK474 chemical compound of taking should be decided according to relevant situation by the doctor; These situation comprise by the condition of therapist; The person's of choosing route of administration, age, body weight, patient are to the individual reaction of medicine, order of severity of patient's symptom or the like.

Description of drawings:

Fig. 1 suppresses the differentiation figure (flow cytometer datagram) of people's dendritic cell for ZSTK474;

Fig. 2 suppresses the differentiation figure (block diagrams of corresponding flow cytometer data) of people's dendritic cell for ZSTK474;

Fig. 3 suppresses the maturation figure (flow cytometer datagram) of the activated people's dendritic cell of LPS for ZSTK474; It can suppress immune activation, therefore can treat autoimmune disease;

Fig. 4 suppresses the maturation figure (block diagrams of corresponding flow cytometer data) of the activated people's dendritic cell of LPS for ZSTK474; It can suppress immune activation, therefore can treat autoimmune disease;

Fig. 5 can try hard to for the antigen uptake that ZSTK474 suppresses the immaturity dendritic cell; It can suppress immune activation, therefore can treat autoimmune disease;

Fig. 6 suppresses allosome T cell proliferative response (CFSE method) figure that dendritic cell stimulate for ZSTK474; It can treat the cell-mediated autoimmune diseasees of T such as multiple sclerosis;

Fig. 7 is the secretion figure of the cytokine of the complementary cell differentiation of promotion T of ZSTK474 inhibition dendritic cell mediation; The secretion of its main inflammation-inhibiting sexual cell factor possibly be used to treat the cell-mediated diseases associated with inflammation of T;

Fig. 8 alleviates the occurring degree figure of mouse experiment systemic autoimmune encephalomyelitis for ZSTK474;

Fig. 9 alleviates the gross score figure of the morbidity clinical score of mouse experiment systemic autoimmune encephalomyelitis for ZSTK474;

Figure 10 alleviates the highest score figure of the morbidity clinical score of mouse experiment systemic autoimmune encephalomyelitis for ZSTK474.

  

The specific embodiment:

Below in conjunction with embodiment the present invention is described, the scheme of embodiment described here does not limit the present invention; One of skill in the art can make improvements and change according to spirit of the present invention, and described these improvement and variation all should be regarded as within the scope of the invention, and scope of the present invention and essence are limited claim; 2-(2-difluoromethyl benzo imidazoles-1-yl)-4 wherein; 6-dimorpholine base-1,3,5-triazines (ZSTK474) has commercially available; The reagent that other is used all has commercially available except that special mark.Wherein blood need pass through the approval of administration of health department.

Embodiment 1

1. 1 human peripheral blood mononuclear cell's separation

(1) fresh blood (through administration of health department approval) is got at the station of dehematizing.Be approximately 40 mL.

(2) PBS with 3 times of volumes dilutes (blood sample of dilution is many more, and monocytic purification is good more).The totally 160 mL Ficoll Paque that gets 15 mL adds the centrifuge tube of 50 mL, and centrifuge tube inclination 45o draws the blood of 35 mL dilution, is superimposed upon on the separating medium along tube wall separating liquid level 1 cm place, does not destroy separating interface.(divide 4 pipes, in the Ficoll Paque of every pipe 15 mL, add 35 mL blood, the blood of average residual more earlier)

(3) centrifugal 30 min of room temperature 400g slowly slow down, and the brake shelves can not be set.

(4) steadily take out centrifuge tube, orlop is erythrocyte and granulocyte, and the intermediate layer is a lymphocyte separation medium, and the superiors are blood plasma and diluent etc.Muddy the sucking-off upper strata stays mononuclear cell cellular layer (one deck tunica albuginea) to greyish white layer (tunica albuginea) in order to be prone to for plasma layer and separating medium intersection, be distributed in practise physiognomy on.

(5) carefully shift tunica albuginea (lymphocyte, mononuclear cell, platelet) to new 50mL centrifuge tube.Be transferred to two pipes.

(6) PBS of 3 times of volumes of adding in centrifuge tube mixes centrifugal 10 min of room temperature 300g.Carefully remove supernatant fully.

(7) in order to remove platelet, re-suspended cell precipitates in the buffer of 50 mL, and centrifugal 15 min of 200g carefully remove supernatant fully under the room temperature.When 200g was centrifugal, the platelet major part rested in the supernatant.

(8) count behind the PBS mixing of 3 times of volumes of adding.

(9) repeat again to wash 1 time.Annotate: PMBC possibly be stored in 5% BSA or serum and the anticoagulant (heparin, EDTA or citric acid phosphoric acid glucose) and spend the night.Cell is stored in refrigerator and is no more than one day.

1.2 human CD14 ⁺Monocytic purification

(1) gets the cell suspending liquid of 80 μ L and the CD14 of 20 μ L ⁺Hatch 15 min for 4 ℃ behind the microballon mixing, (cell and microballon are pressed 4:1 and added) will be mixed once in the centre.(all hemocytees add 300 μ L magnetic beads, add the PBS that 1200 μ L contain 0.5%BSA again)

(2) centrifugal 5 min of PBS (containing 0.5%BSA) 200g with 20 times of volumes (2mL) pre-cooling wash once, abandon supernatant, add the PBS of 500 μ L.

(3) during centrifugal, the MACS discrete magnets be installed on the MACS multipurpose bracket, and MS sorting post is placed in the magnet, the sorting post is transferred a clean aseptic centrifuge tube.With meeting cold MACS buffer flushing sorting post 3 times, each 0.5mL.Abandon eluent, again at clean aseptic centrifuge tube of sorting post underlying, with on the cell suspension appearance to the sorting post.

(4) with MACS buffer flushing detached dowel, each 0.5mL washes 3 times (first 0.5mL adds slowly in order to avoid the cell in the disturbance sorting post).

(5) treat that the cell suspension all-pass is crossed post after, take off the sorting post from magnet, be placed on the aseptic 15mL core barrel.In the sorting post, add the full RPMI1640 culture medium of 1mL, piston is filled in detached dowel and eluting CD14 ⁺Mononuclear cell, counting.With CD14 ^-Cell cryopreservation is used for the T cell proliferation experiment.

(6) flow cell sorter is confirmed purity (95%)

(7) with containing 1000U/mL GM-CSF, the complete RPMI1640 culture medium of 1000U/mL IL-4 (Sodium Pyruvate that contains 10mM, the glutamine of 10 mM, 100 those mycins of μ g/mL) adjustment cell concentration is 1X10 ⁶Individual/mL also adds medicine.Cell suspending liquid 2 mL/ holes are inoculated in 12 orifice plates.

1.3. ZSTK474 processing time and concentration are confirmed

The DC that obtains is divided into three groups, and every group has been carried out various concentrations over control treatment:

First group (differentiation): dosing in first day, carried out Flow cytometry on the 5th day.ZSTK474 handles different concentration: the normal control group;

0.008 μ M/L ZSTK474 handles five days groups;

0.04 μ M/L ZSTK474 handles five days groups;

0.2 μ M/L ZSTK474 handles five days groups.

Second group (differentiation): dosing in the 5th day, carried out Flow cytometry on the 7th day.ZSTK474 handles different concentration:

the normal control group;

0.02 μ M/L ZSTK474 handles two days groups; 0.04 μ M/L ZSTK474 handles two days groups;

0.2 μ M/L ZSTK474 handles two days groups.

The 3rd group (maturation): dosing in the 5th day and LPS (1 μ g/ml), carried out Flow cytometry on the 7th day.ZSTK474 handles different concentration:

negative control group (ZSTK474 and LPS do not add); matched group (only adding LPS);

0.02 μ M/L ZSYK474+LPS handles two days groups;

0.04 μ M/L ZSTK474+LPS handles two days groups;

0.2 μ M/L ZSTK474+LPS handles two days groups.

At the 3rd day that cultivates, draw 50% supernatant, add the GM-CSF that contains 1000 U/ml of equivalent, the RPMI1640 culture medium of the IL-4 of 1000 U/ml also adds be used as medicine (adding by 2 ml).Annotate: at the 5th day, in the hole of not dosing, add LPS and medicine respectively, and, continue in the RPMI1640 culture medium of the IL-4 of 1000 U/mL to cultivate two days at the GM-CSF of 1000 U/mL that contain equivalent.

1.4 flow cytometer showed

(1) at sophisticated the 5th day, the 7th day last collecting cell of cell differentiation to the 1.5mL centrifuge tube, two pipes, the centrifugal 10min of 2000 r shifts supernatant again to the 1.5mL centrifuge tube, keeps supernatant;

(2) with containing 2 mLPBS washed cells, according to per 10 ⁵The amount of cell/2 μ L adds DC surface markers antibody CD80, CD86, and CD83, HLA-DR, 4 ℃ of following lucifuges are hatched 30min;

(3) discard dyestuff, once with 2 mL PBS washed cells;

(4) with 2mL PBS re-suspended cell, the paraformaldehyde that adds 1mL 2% is again fixed, and 300 mesh filter screens filter, and flow cytometer showed is subsequent use.

(5) detect with flow cytometer.The FACS data are used the CELLQuest software analysis.

1.5 FITC-dextran absorbs detection

The differentiation immature cell of collecting the 5th day is by 10 ⁵Cells/ml is suspended from the RPMI1640 complete medium, adds FITC-dextran (0.1mg/mL) again, and 37 ℃ of incubators are hatched 30 min; Centrifugal 10 min of 1000 r/min, collecting cell cleans 2 times with PBS; Be resuspended in the streaming pipe at last; Detecting the fluorescence intensity of cell respectively, is benchmark with 4 ℃ of conditions, judges the antigen uptake ability of cell.

1.6 ZSTK474 suppresses the allosome T cell proliferation that ripe DC causes

Utilize CD4+ magnetic bead sorting allosome CD4+ cell, PBS washes twice, 37 ℃ of labelling CFSE (hydroxyl fluorescein diacetate butanimide fat) dyestuff 10 min, 2 * 10 ⁵Cell/200 μ l plants in round bottom 96 orifice plates; The ripe DC that handled by 1: 10,1: 20,1: 40 (DC/CD4+) adding ametycin and ZSTK474 again; After the co-cultivation 4 days, collecting cell is done flow cytometer showed; Decay number of times showed cell propagation with the CFSE fluorescence intensity changes, and sees Fig. 4.

1.7 suppressing ripe DC, ZSTK474 produces the cytokine that promotes Th1 and Th17 differentiation

Ripe DC is produced the influence of cytokine for the ZSTK474 that studies variable concentrations; When we break up the 6th day at DC, add variable concentrations (0.02 μ M/L, 0.04 μ M/L; 0.2 ZSTK474 pretreatment 3h μ M/L); LPS with 100ng/ml stimulates its maturation then, and respectively at 0h, 1h, 2h, 4h, 8h, 16h collecting cell that LPS adds, fluorescence quantitative PCR detection IL-12p35, IL-12/23p40, IL-23p19, IL-6, TNF-α, IFN-γ expression of gene are seen Fig. 5.

1.8 the construction method of EAE mouse model

Experimental technique: the 8-10 week big identical female C57BL/6 wild-type mice of sex, age and body weight of inducing of EAE in mice is used for dividing into groups to induce EAE, every group of n=12 mice.The mycobacterium (Mycobacterium tuberculosis) of 100 μ g MOG35-55 polypeptide (MEVGWYRSPFSRVVHLYRNGK) and 500 μ g deactivations mixes with 100 μ l normal saline and 100 μ l Freund ' s adjuvants fully; Emulsifying, four some injections of the subcutaneous branch of mice back (50 μ l/ point).While is every mice difference lumbar injection 200ng pertussis toxin, PT (pertussis toxin) after the same day and immune 48 hours.The administration group is since the ZSTK474 of per kilogram of body weight filling the 1st day every day stomach 50mg.Mice begins to take place EAE after 9-14 days.The classification of the clinical function of nervous system of inspection record disease every day.

The EAE in mice animal model function of nervous system concrete standard of marking is following:

0 minute, Non Apparent Abnormality; 0.5 divide, the part tail is unable lax; 1 minute, tail paralysis fully; 2 minutes, back myasthenia of limbs was slow in action, mild ataxia; 2.5 divide acroparalysis after one; 3 minutes, acroparalysis after two; 3.5 divide back acroparalysis, preceding myasthenia of limbs; 4 minutes, preceding acroparalysis; 5 minutes, be at death's door.EAE took place in mice in the time of 9-14 days, inspection every day and the classification of the clinical function of nervous system of record disease.

The experimental result explanation of above-mentioned 1.1-1.8:

(1) differentiation, maturation and the antigen presentation through suppressing immune BMDC and then suppress immune initial sum and activate.

(2) the T cell proliferation that mediates through the immune BMDC of inhibition, and then the relevant disease of suppressor T cell mediation.

(3) secretion through suppressing the struvite cytokine of immune BMDC and promoting the cytokine of TH1 and TH17 cell differentiation, and then inflammation-inhibiting relevant disease.

(4) the score explanation of disease clinical score, overall score and the disease through suppressing mouse experiment systemic autoimmune encephalomyelitis is expected to treat diseases such as multiple sclerosis and acute disseminated encephalomyelitis.

(5) comprehensive above result can administer autoimmune inflammation property diseases such as multiple sclerosis and acute disseminated encephalomyelitis.

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Claims (5)

1. 2-(2-difluoromethylbenzimidazol-1-yl)-4,6-dimorpholinyl-1,3,5-triazine in the preparation of drugs for the treatment of autoimmune and inflammatory diseases Applications. 2. The application according to claim 1, wherein said autoimmune disease refers to autoimmune disease related to immune dendritic cells or T cells. 3. The application according to claim 2, wherein the autoimmune diseases related to immune dendritic cells or T cells refer to: multiple sclerosis, autoimmune retinitis, neuromyelitis optica, systemic lupus erythematosus, diabetes, Thyroid, hemolytic anemia, atrophic orchitis, autoimmune thrombocytopenia, myasthenia gravis, primary biliary cirrhosis, aggressive chronic hepatitis, chronic glomerulitis, ulcerative colitis, dermatitis, Myositis, systemic sclerosis. 4. The application of claim 1, wherein the inflammatory disease refers to an inflammatory disease of the nervous system. 5. The application according to claim 4, wherein the inflammatory diseases of the nervous system refer to multiple sclerosis and acute disseminated encephalomyelitis.

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