CN103977401A - Medicinal composition for treating multiple sclerosis - Google Patents
Medicinal composition for treating multiple sclerosis Download PDFInfo
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- CN103977401A CN103977401A CN201310120331.4A CN201310120331A CN103977401A CN 103977401 A CN103977401 A CN 103977401A CN 201310120331 A CN201310120331 A CN 201310120331A CN 103977401 A CN103977401 A CN 103977401A
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Abstract
The invention discloses a medicinal composition for treating multiple sclerosis, and the medicinal composition comprises BAFF (B cell activating factor belonging to TNF family) inhibitor (TACI-IgG) and IL-15 inhibitor (anti IL-15 antibody). The medicinal composition is used for treating a murine model EAE (experimental allergic encephalomyelitis) mice subjected to the multiple sclerosis, and the results showed that the medicinal composition can significantly reduce the number of B cells, helper T cells and memory B / T cells in spleen and lymph nodes, inhibits the production of activation and autoimmune antibodies of the B cells, and relieves clinical autoimmune symptoms. The medicinal composition is effective in the treatment of the multiple sclerosis.
Description
Technical field
The invention belongs to biomedical sector, relate to a kind of pharmaceutical composition for the treatment of autoimmune disease multiple sclerosis (MS).Particularly, compositions of the present invention comprises BAFF inhibitor and IL-15 inhibitor, and zoopery shows that described pharmaceutical composition can effectively suppress autoimmune generation and development.
Background technology
Along with the development of social times, the sickness rate of autoimmune disease in all kinds of crowds of China constantly rises.In view of this after being ill the phase can to life in the future of people, work even self health cause serious harm, further strengthen to the basic research of autoimmune disease will help the mankind understand this type of disease generation, develop and the mechanism of action such as lapse to, thereby improve its prevention, diagnosis and treatment level.This is healthy to ensureing human lives, promotes socioeconomic development all to have important theoretical and practical significance.
Autoimmune disease can be divided into two large classes according to immunologic injury mechanism: a class is that the autoimmune disease that causes of autoantibody is as systemic lupus erythematosus (sle) (SLE), myasthenia gravis (MG) and idiopathic thrombocytopenic purpura (ITP) etc., another kind of is that the autoimmune disease that causes of autoreactive T cell is as multiple sclerosis [MS, its animal model is autoimmunity encephalomyelitis (EAE)] and insulin-dependent diabetes (IDDM).
Many experimental evidences show that T cell participates in autoimmune disease directly, and multiple organ specific autoimmune disease is all cell-mediated by T, as type i diabetes, autoimmune thyroiditis, multiple sclerosis (MS) etc.In antibody-mediated autoimmune disease, the activation of B cell also depends on the auxiliary of Th cell simultaneously.Recently, many evidences are presented in the autoimmune disease of T cellular driven, and autoreactivity B cell is also being brought into play very important effect.Therefore, numerous autoimmune diseasees normally coordinates and mediates generation jointly by autoreactivity T, B cell.
The treatment of autoimmune disease still lacks desirable method.Conventionally for the pathological change of disease and the consequence due to tissue injury, treat, also can be by regulating the links blocking-up disease process of immunne response to reach the object for the treatment of.At present some common Therapeutic Method comprise: use heavy dose of 17-hydroxy-11-dehydrocorticosterone, salicylic acid or various synthetic prostaglandin inhibitor, suppress the inflammatory reaction due to some serious symptom autoimmune diseases; Or come Immunosuppression system to play a role by immunosuppressant.But these two kinds of methods all can only slow down the development speed of the state of an illness and not effect a radical cure, and also can produce serious side effect simultaneously.In recent years along with the pathogenesis of autoimmune disease is progressively illustrated, for this sick Therapeutic Method also among continuous research.
BAFF claims again B-cell stimulating factor (B lymphocyte stimulator, BLyS), belongs to tumor necrosis factor superfamily (tumor necrosis factor superfamily, TNSF) member.BAFF plays a significant role in the morbidity of B cell development, function adjusting and autoimmune disease, and its defect or overexpression all can cause that immunity of organism is unbalance, relevant with the generation of some autoimmune disease.In the autoimmune disease patients serums such as SLE, SS and RA, find that BAFF level obviously raises, BAFF cross expression can cause autoreactivity B cell to escape normal immunity thereby to screen out mechanism ripe be antibody secreted B cell or plasma cell, further confirmed that BAFF has participated in generation and the development [Editorial, et al.The B cell:a new therapeutic target in rheumatoid arthritis and other autoimmune diseases.Joint Bone Spine2004.71:357-360] of these diseases closely.
T cell is considered to main pathogenesis [the Behi ME of MS, et al.New insights into cell responses involved in experimental autoimmune encephalomyelitis and multiple sclerosis.Immunology Letters2005.96 (1): 11-26], closely related with the cellular immunization of Th1 mediation.The molecular simulation theory of main flow is thought, between MS patient infection's virus and central nervous system's myelin protein composition or oligodendrocyte, exists cross-reacting antigen.After viral infection, the T cell being activated in body, when removing is viral, due to the composition generation cross reaction with myelin, thereby causes maincenter demyelination.Because MS damage zone contains a large amount of T cells, the T cell that therefore uses myelin basic protein (MBP) polypeptide fragment to activate is transferred to the animal model that mice can cause MS---tentative autoimmunity encephalomyelitis (EAE).But the continuous intensification to B cell function understanding in recent years, the effect of B cell in the generating process of MS is also taken seriously gradually, and we early-stage Study show, and in autoimmune disease MS, the BAFF level in serum raises.
The expression of BAFF is only adenoid 1/10th in normal human's brain, and being on close level in the expression of MS patient damage zone BAFF and lymphoid tissue.And because the BAFF of the astrocyte secretion of activation is far above mononuclear cell and macrophage, so MS patient's damage zone becomes the main source of BAFF in brain.The research that comes from relapsing remitting EAE and chronic recurrence type EAE also shows, laboratory animal is in morbidity or recurrence phase, and central nervous system BAFF level increases, and catabasis BAFF horizontal down-regulation.In view of BAFF plays a significant role in the growth of B cell, differentiation, again the pathogenic molecule of key in various autoimmune disease simultaneously, so BAFF has been used as the treatment target spot of inhibition B cytological effect important in various autoimmune disease, in March, 2011, anti-BAFF antibody belimumab is named as the listing of the Benlysta Bing U.S..But also there is certain defect in the method for blocking-up BAFF treatment, in MS patient's brain, exist lymphoid follicle structure, B cell can constantly increase and break up in these structures becomes plasma cell or autoreactivity memory B cell, by discharging autoantibody or as antigen-presenting cell, the state of an illness of MS constantly also being worsened repeatedly.Although independent anti-BAFF antibody can significantly reduce the number of peripheral blood mature B cell, but it can not reduce the quantity [Vincent FB, et al.BAFF and innate immunity:new therapeutic targets for systemic lupus erythematossu.Immunol Cell Biol.2012.90:293-303] of plasma cell and autoreactivity memory B cell.Therefore as long as autoreactivity memory B cell is present in body, humoral immune reaction just can be activated again.So delete autoreactivity memory B cell by finding the method for therapeutic alliance, the patient who suffers from autoimmune disease will likely obtain treating completely and rehabilitation.
IL-15 is a kind of important multi-functional cell regulating factor, not only can stimulate CD4
+and CD8
+the propagation of T cell and the generation that can induce CTL, also mediate by IgM specific antibody or CD40L simultaneously and stimulate the synthetic of the B cell proliferation that causes and immunoglobulin.In the generation, propagation and the activation process that stimulate NK cell, IL-15 also plays a significant role.So IL-15 is the key factor of Memorability T/B cell survival and propagation, BAFF may carry out by suppressing IL-15 the quantity of negative regulator Memorability T/B cell.In addition, IL-15 or a kind of pro-inflammatory cytokine of many biological effects, can be by suppressing the AICD of IL-2 induction and promoting CD8
+the existence of memory t cell and cause the generation of autoimmune disease.IL-15 is at CD4
+in T cell, can promote by activation STAT5 expression [Pandiyan P, the et al.The role of IL-15in activating STAT5and fine-tuning IL-17A production in CD4 of IL-17A
+t lymphocytes.J Immunol.2012.189:4237-46], and then propagation, maturation and the chemotactic of induction neutrophilic granulocyte; Can also promote various kinds of cell subgroup to produce the proinflammatory cytokines such as TNF, IL-1, IL-6, IL-8 and GM-CSF; Stimulating expression of macrophage produces IL-1 β, tumor necrosis factor-alpha and PGE2 etc. and produces synergism to amplify inflammatory reaction simultaneously.Experiment shows, the IL-15 in various autoimmune Disease body expresses imbalance, and generation and development that the activity of blocking-up IL-15 can control inflammation realize treatment and take the various diseases that immunomediated inflammatory is main mechanism.
Summary of the invention
For solving the defect of existing medicine in treatment autoimmune disease, and the deficiency of using separately BAFF inhibitor for treating autoimmune disease, the invention discloses a kind of pharmaceutical composition, described pharmaceutical composition comprises BAFF inhibitor and IL-15 inhibitor.
The preferred TACI-IgG of BAFF inhibitor in aforementioned pharmaceutical compositions, the preferred Anti-IL-15 antibody of IL-15 inhibitor, described antibody is monoclonal antibody or polyclonal antibody.
Pharmaceutical composition of the present invention, wherein the mass ratio of BAFF inhibitor and IL-15 inhibitor is 0.5～10: 1, wherein preferably 4: 1.
Pharmaceutical composition of the present invention also comprises adjuvant conventional in pharmaceutical preparation, as: excipient, emulsifying agent, solubilizing agent, antioxidant, controlled release agent, as long as it is suitable for corresponding drug delivery system and keeps rightly BAFF inhibitor and the activity of the molecule of IL-15 inhibitor.
The present invention has evaluated the curative effect of aforementioned pharmaceutical compositions in treatment autoimmune disease by zoopery, specifically comprises the following steps:
1) foundation of animal model: the present invention chooses the mouse model of multiple sclerosis, i.e. tentative autoimmunity encephalomyelitis (EAE) mouse model.The present invention adopts MOG35-55 and complete Freund's adjuvant to prepare antigen Emulsion, after mouse subcutaneous injection antigen Emulsion, gives lumbar injection pertussis toxin, PT.
2) treatment grouping and Therapeutic Method: zoopery was provided with normal mouse group, PBS matched group, single inhibitor group and compositions treatment group, by the administration course for the treatment of 2 weeks to 24 weeks.Wherein preferably administering mode is to be administered once for 3 days, intravenously administrable pneumoretroperitoneum administration in 2 weeks 2 weeks.
3) therapeutic evaluation:
The present invention effectively reduces the number of B cell and the activation of B cell in Mice Body with TACI-IgG and Anti-IL-15 medicine composite for curing EAE mice.
The present invention has effectively reduced the number of helper T lymphocyte Th1 and Th17 with TACI-IgG and Anti-IL-15 medicine composite for curing EAE mice.
The present invention has effectively reduced the number of the interior Memorability T of Mice Body and B cell with TACI-IgG and Anti-IL-15 medicine composite for curing EAE mice.
The clinical symptoms of mice has effectively been alleviated in the present invention with TACI-IgG and Anti-IL-15 medicine composite for curing EAE mice.
The present invention effectively reduces the level of autoreactivity antibody in Mice Body with TACI-IgG and Anti-IL-15 medicine composite for curing EAE mice, reduced the autoimmune of EAE mice.
The present invention has detected the therapeutic effect of aforementioned pharmaceutical compositions by experiment, experimental result shows, pharmaceutical composition of the present invention significantly reduces EAE clinical symptoms, significantly reduced the reaction of mice to autoantigen, pharmaceutical composition therapeutic effect of the present invention is significantly better than TACI-IgG or anti-IL-15 antibody is treated separately.
Pharmaceutical composition of the present invention can effectively be treated the cell-mediated autoimmune disease of T, comprises autoimmunity encephalomyelitis, multiple sclerosis, insulin-dependent diabetes.
Accompanying drawing explanation
The number of B cell in the antibody combined treatment of Fig. 1 TACI-IgG and anti-IL-15 EAE mice;
The activation of B cell in the antibody combined treatment of Fig. 2 TACI-IgG and anti-IL-15 EAE mice;
The number of Th1 and Th17 cell in the antibody combined treatment of Fig. 3 TACI-IgG and anti-IL-15 EAE mice;
The number of Memorability T and B cell in Fig. 4 TACI-IgG and anti-IL-15 antibody combined treatment EAE mice spleen and lymph node;
The clinical symptoms of the antibody combined treatment of Fig. 5 TACI-IgG and anti-IL-15 EAE mice;
Fig. 6 separate groups of mice is reactive to autoantigen.
Embodiment 1TACI-IgG and the impact of the antibody combined treatment of anti-IL-15 on B cell
One materials and methods
1, material
BALB/c mouse is bought from Military Medical Science Institute's animal center; Mice myelin oligodendrocyte glycoprotein 33-55 (MOG33-55), complete Freund's adjuvant (CFA), pertussis toxin, PT are purchased from Sigma company; The anti-Mus B220 of rabbit monoclonal antibody, the anti-Mus CD3 of rabbit monoclonal antibody, the anti-Mus CD21 of rabbit monoclonal antibody, the anti-Mus CD23 of rabbit monoclonal antibody are purchased from Abcam; BAFF inhibitor TACI-IgG, anti-mice IL-15 neutrality antibody is purchased from R & D company.
2, method
2.1, EAE mouse model builds
With the PBS of 0.01mol/L, by MOG35-55 dilution, be 2mg/mL, then diluent fully mixed with the complete freund adjuvant of equivalent, by tee T, Emulsion is lashed as Water-In-Oil state, be the antigen Emulsion of induction EAE.EAE group mice is in both sides, back subcutaneous injection antigen Emulsion (only including MOG antigen 200～250 μ g/).Matched group will not be processed by antigen.After immunity, within the 0th, the 48th hour, give whole mices (comprising matched group) lumbar injection pertussis toxin, PT, every animal 500ng/0.2mL.
2.2, experiment grouping
Experiment mice is divided into 5 groups: control mice, PBS treatment EAE mice, TACI-IgG treatment EAE mice, Anti-IL-15 treatment EAE mice, TACI-IgG and Anti-IL-15 therapeutic alliance EAE mice.Every group of 12 animals.By tail vein injection TACI-IgG and Anti-IL-15, arrive in the EAE Mice Body of morbidity, injection in every 3 days 1 time, treats after 2 weeks continuously, uses abdominal cavity continual cure instead 2 weeks.TACI-IgG consumption: each 2mg/kg; Anti-IL-15 antibody consumption: each 0.5mg/kg.
2.4, adenoid pretreatment
A) piece of tissue is put into plate, added a small amount of normal saline;
B) with shears by tissue shear to homogenate shape;
C) add 10ml normal saline;
D) with suction pipe, draw tissue homogenate, first with 100 order nylon net filters in test tube;
E) centrifugation 1000rpm, 3-5min, then wash 3 times with normal saline, with low speed (500-800rpm) centrifugation in short-term, remove cell debris at every turn;
F) with 300 order nylon wire elimination cell masses;
G) cell saves backup.
2.3, fluidic cell sample treatment and detection
A) get 2 * 10
6individual cell, with cold PBS2ml washed cell once, the centrifugal 5min of 800rpm;
B) with 2ml4% paraformaldehyde room temperature fixed cell 40min, the centrifugal 5min of 800rpm; 2ml PBS re-suspended cell, the centrifugal 5min of 800rpm;
C) the PBS re-suspended cell containing 0.2%Triton-X100 and 5% serum with 1ml, places 10min on ice, the centrifugal 5min of 800rpm;
D) add the unmarked antibody of Sa (the anti-Mus B220 of rabbit, CD3, CD21, CD23 antibody), place 40min on ice, the centrifugal 5min of 800rpm, with the cold PBS re-suspended cell of 2ml, the centrifugal 5min of 800rpm, washes away not in conjunction with primary antibodie, repeats once;
E) add fluorescently-labeled goat anti-rabbit igg two anti-, lucifuge is placed after 40min on ice, and the centrifugal 5min of 800rpm, removes supernatant, PBS washed twice;
F) add 0.5ml1% paraformaldehyde re-suspended cell, fixing to be measured;
G) flow cytometer detects fluorescent value, 10000 cells of every pipe counting.
Two results
Data analysis: adopt SPSS16.0 software data to be carried out to statistical analysis, the relatively employing t method of inspection between two groups.
Fig. 1 demonstration is compared with PBS treatment group, and TACI-IgG treatment causes B cell in spleen and lymph node significantly to reduce (P < 0.01).Compare with PBS matched group, therapeutic alliance causes spleen and enlargement of lymph node B cell significantly to reduce (P < 0.01).Compare with the independent treatment group of TACI-IgG, therapeutic alliance group does not significantly reduce B cell number.This demonstration, TACI-IgG suppresses B cell specifically, and anti-IL-15 antibody does not have the function of this respect, but TACI-IgG is similar with the independent treatment group of the antibody combined therapeutic combination TACI-IgG of anti-IL-15, can significantly reduce B cell number in EAE mice spleen and lymph node.
Fig. 2 demonstration is compared with PBS treatment group, TACI-IgG treatment causes B cell activation sign CD21 in spleen and lymph node significantly to reduce (P < 0.01), and therapeutic alliance group also causes B cell activation in spleen and lymph node significantly to reduce (P < 0.01).Compare with the independent treatment group of TACI-IgG, therapeutic alliance group does not significantly reduce B cell activation sign.This demonstration, TACI-IgG suppresses B cell activation specifically, and in the EAE mice spleen of TACI-IgG and the antibody combined treatment of anti-IL-15 and lymph node, B cell activation sign CD21 reduces significantly.
Embodiment 2TACI-IgG and the impact of the antibody combined treatment of anti-IL-15 on helper T lymphocyte (Th1, Th17)
One materials and methods
1, material
BALB/c mouse is bought from Military Medical Science Institute's animal center; Mice myelin oligodendrocyte glycoprotein 33-55 (MOG33-55), complete Freund's adjuvant (CFA), pertussis toxin, PT are purchased from Sigma company; The anti-Mus CD4 of rabbit monoclonal antibody, the anti-Mus IL-17 of rabbit monoclonal antibody, the anti-Mus IFN of rabbit γ monoclonal antibody are purchased from Abcam; BAFF inhibitor TACI-IgG, anti-mice IL-15 neutrality antibody is purchased from R & D company.
2, method
2.1, EAE mouse model builds
With the PBS of 0.01mol/L, by MOG35-55 dilution, be 2mg/mL, then diluent fully mixed with the complete freund adjuvant of equivalent, by tee T, Emulsion is lashed as Water-In-Oil state, be the antigen Emulsion of induction EAE.EAE group mice is in both sides, back subcutaneous injection antigen Emulsion (only including MOG antigen 200～250 μ g/).Matched group will not be processed by antigen.After immunity, within the 0th, the 48th hour, give whole mices (comprising matched group) lumbar injection pertussis toxin, PT, every animal 500ng/0.2mL.
2.2, experiment grouping
Experiment mice is divided into 5 groups: control mice, PBS treatment EAE mice, TACI-IgG treatment EAE mice, Anti-IL-15 treatment EAE mice, TACI-IgG and Anti-IL-15 therapeutic alliance EAE mice.Every group of 12 animals.By tail vein injection TACI-IgG and Anti-IL-15, arrive in the EAE Mice Body of morbidity, injection in every 3 days 1 time, treats after 2 weeks continuously, uses abdominal cavity continual cure instead 2 weeks.TACI-IgG consumption: each 2mg/kg; Anti-IL-15 antibody consumption: each 0.5mg/kg.
2.3, adenoid pretreatment
A) piece of tissue is put into plate, added a small amount of normal saline;
B) with shears by tissue shear to homogenate shape;
C) add 10ml normal saline;
D) with suction pipe, draw tissue homogenate, first with 100 order nylon net filters in test tube;
E) centrifugation 1000rpm, 3-5min, then wash 3 times with normal saline, with low speed (500-800rpm) centrifugation in short-term, remove cell debris at every turn;
F) with 300 order nylon wire elimination cell masses;
G) cell saves backup.
2.4, fluidic cell sample treatment and detection
A) get 2 * 10
6individual cell, with cold PBS2ml washed cell once, the centrifugal 5min of 800rpm;
B) with 2ml4% paraformaldehyde room temperature fixed cell 40min, the centrifugal 5min of 800rpm; 2ml PBS re-suspended cell, the centrifugal 5min of 800rpm;
C) the PBS re-suspended cell containing 0.2%Triton-X100 and 5% serum with 1ml, places 10min on ice, the centrifugal 5min of 800rpm;
D) add the unmarked antibody of Sa (the anti-Mus CD4 of rabbit, IL-17, IFN gamma antibodies), place 40min on ice, the centrifugal 5min of 800rpm, with the cold PBS re-suspended cell of 2ml, the centrifugal 5min of 800rpm, washes away not in conjunction with primary antibodie, repeats once;
E) add fluorescently-labeled goat anti-rabbit igg two anti-, lucifuge is placed after 40min on ice, and the centrifugal 5min of 800rpm, removes supernatant, PBS washed twice;
F) add 0.5ml1% paraformaldehyde re-suspended cell, fixing to be measured;
G) flow cytometer detects fluorescent value, 10000 cells of every pipe counting.
Two results
Data analysis: adopt SPSS16.0 software data to be carried out to statistical analysis, the relatively employing t method of inspection between two groups.
Fig. 3 shows in the EAE mice spleen of TACI-IgG and the antibody combined treatment of anti-IL-15 and lymph node that Th1 and Th17 cell significantly reduce.Autoimmune disease as EAE in, Th1 and Th17 cell significantly raise.Compare with PBS treatment group, TACI-IgG treatment group can not reduce Th1 and Th17 cell.Compare with the independent treatment group of TACI-IgG, therapeutic alliance group significantly reduces Th1 and Th17 cell.Compare with PBS treatment group, anti-IL-15 Antybody therapy group Th1 and Th17 cell reduce significantly.This explanation, aspect inhibition Th1 and Th17 cell, anti-IL-15 antibody is being brought into play Main Function.
Embodiment 3TACI-IgG and the impact of the antibody combined treatment of anti-IL-15 on Memorability T/B cell
One materials and methods
1, material
BALB/c mouse is bought from Military Medical Science Institute's animal center; Mice myelin oligodendrocyte glycoprotein 33-55 (MOG33-55), complete Freund's adjuvant (CFA), pertussis toxin, PT are purchased from Sigma company; The anti-Mus B220 of rabbit monoclonal antibody, the anti-Mus CD44 of rabbit monoclonal antibody, the anti-Mus CD27 of rabbit monoclonal antibody, the anti-Mus CD3 of rabbit monoclonal antibody are purchased from Abcam; BAFF inhibitor TACI-IgG, anti-mice IL-15 neutrality antibody is purchased from R & D company.
2, method
2.1, EAE mouse model builds
With the PBS of 0.01mol/L, by MOG35-55 dilution, be 2mg/mL, then diluent fully mixed with the complete freund adjuvant of equivalent, by tee T, Emulsion is lashed as Water-In-Oil state, be the antigen Emulsion of induction EAE.EAE group mice is in both sides, back subcutaneous injection antigen Emulsion (only including MOG antigen 200～250 μ g/).Matched group will not be processed by antigen.After immunity, within the 0th, the 48th hour, give whole mices (comprising matched group) lumbar injection pertussis toxin, PT, every animal 500ng/0.2mL.
2.2, experiment grouping
Experiment mice is divided into 5 groups: control mice, PBS treatment EAE mice, TACI-IgG treatment EAE mice, Anti-IL-15 treatment EAE mice, TACI-IgG and Anti-IL-15 therapeutic alliance EAE mice.Every group of 12 animals.By tail vein injection TACI-IgG and Anti-IL-15, arrive in the EAE Mice Body of morbidity, injection in every 3 days 1 time, treats after 2 weeks continuously, uses abdominal cavity continual cure instead 2 weeks.TACI-IgG consumption: each 2mg/kg; Anti-IL-15 antibody consumption: each 0.5mg/kg.
2.2, adenoid pretreatment
A) piece of tissue is put into plate, added a small amount of normal saline;
B) with shears by tissue shear to homogenate shape;
C) add 10ml normal saline;
D) with suction pipe, draw tissue homogenate, first with 100 order nylon net filters in test tube;
E) centrifugation 1000rpm, 3-5min, then wash 3 times with normal saline, with low speed (500-800rpm) centrifugation in short-term, remove cell debris at every turn;
F) with 300 order nylon wire elimination cell masses;
G) cell saves backup.
2.3, fluidic cell sample treatment and detection
A) get 2 * 10
6individual cell, with cold PBS2ml washed cell once, the centrifugal 5min of 800rpm;
B) with 2ml4% paraformaldehyde room temperature fixed cell 40min, the centrifugal 5min of 800rpm; 2ml PBS re-suspended cell, the centrifugal 5min of 800rpm;
C) the PBS re-suspended cell containing 0.2%Triton-X100 and 5% serum with 1ml, places 10min on ice, the centrifugal 5min of 800rpm;
D) add the unmarked antibody of Sa (the anti-Mus primary antibodie of rabbit), place 40min on ice, the centrifugal 5min of 800rpm, with the cold PBS re-suspended cell of 2ml, the centrifugal 5min of 800rpm, washes away not in conjunction with primary antibodie, repeats once;
E) add fluorescently-labeled goat anti-rabbit igg two anti-, lucifuge is placed after 40min on ice, and the centrifugal 5min of 800rpm, removes supernatant, PBS washed twice;
F) add 0.5ml1% paraformaldehyde re-suspended cell, fixing to be measured;
G) flow cytometer detects fluorescent value, 10000 cells of every pipe counting.
Two results
Data analysis: adopt SPSS16.0 software data to be carried out to statistical analysis, the relatively employing t method of inspection between two groups.
Fig. 4 demonstration is compared with healthy mice, and in autoimmune disease EAE, Memorability T and B cell significantly raise.Compare TACI-IgG treatment group Memorability T and B cell rising (P < 0.05) with PBS treatment group.Compare with the independent treatment group of TACI-IgG, therapeutic alliance group significantly reduces Memorability T and B cell (P < 0.01).Compare with PBS treatment group, anti-IL-15 Antybody therapy group Memorability T and B cell reduce (P < 0.01) significantly.This demonstration, TACI-IgG suppresses B cell activation specifically, and can not suppress the formation of Memorability T/B cell, and anti-IL-15 antibody specificity ground suppresses the function of Memorability T and B cell.Therefore, in the EAE mice spleen of TACI-IgG and the antibody combined treatment of anti-IL-15 and lymph node, Memorability T and B cell significantly reduce.
The impact of the antibody combined treatment of embodiment 4TACI-IgG and anti-IL-15 EAE on the clinical state of an illness and autoantigen reaction
One materials and methods
1, material
BALB/c mouse is bought from Military Medical Science Institute's animal center; Mice myelin oligodendrocyte glycoprotein 33-55 (MOG33-55), complete Freund's adjuvant (CFA), pertussis toxin, PT are purchased from Sigma company; BAFF inhibitor TACI-IgG, anti-mice IL-15 neutrality antibody is purchased from R & D company.
2, method
2.1, EAE mouse model builds
With the PBS of 0.01mol/L, by MOG35-55 dilution, be 2mg/mL, then diluent fully mixed with the complete freund adjuvant of equivalent, by tee T, Emulsion is lashed as Water-In-Oil state, be the antigen Emulsion of induction EAE.EAE group mice is in both sides, back subcutaneous injection antigen Emulsion (only including MOG antigen 200～250 μ g/).Matched group will not be processed by antigen.After immunity, within the 0th, the 48th hour, give whole mices (comprising matched group) lumbar injection pertussis toxin, PT, every animal 500ng/0.2mL.
2.2, experiment grouping
Experiment mice is divided into 5 groups: control mice, PBS treatment EAE mice, TACI-IgG treatment EAE mice, Anti-IL-15 treatment EAE mice, TACI-IgG and Anti-IL-15 therapeutic alliance EAE mice.Every group of 12 animals.By tail vein injection TACI-IgG and Anti-IL-15, arrive in the EAE Mice Body of morbidity, injection in every 3 days 1 time, treats after 2 weeks continuously, uses abdominal cavity continual cure instead 2 weeks.TACI-IgG consumption: each 2mg/kg; Anti-IL-15 antibody consumption: each 0.5mg/kg.
2.3 symptom scores
5 point-score standards of grading classifications are as follows: 0 is divided into and does not fall ill; 1 to be divided into tail unable; 2 are divided into slight rear myasthenia of limbs; 3 are divided into serious rear acroparalysis; 4 are divided into quadriplegia; 5 be divided into dying or dead.Played every day mice is weighed the immune same day, and adopt double-blind method to carry out function of nervous system's scoring.By two non-experimenters, observe and evaluate, with reference to score by rules, mark.First by personnel, carry out 5 point-scores and 7 point-scores are marked, both can carry out simultaneously.After above-mentioned scoring 30min, then carry out 15 point-score scorings by an other observer.Because mice diel movement is widely different, all evaluated at 8 o'clock in the morning every day.
2.4, lymph node is to the reactive detection of autoantigen MOG33-55
A) lymph node is put into plate, added a small amount of normal saline;
B) with shears and tweezers, lymph node is cut and pinched to homogenate shape;
C) add 10ml normal saline;
D) with the homogenate of suction pipe absorptive tissue, first with 100 order nylon net filters in test tube;
E) centrifugation 1000rpm, 3-5min, is washing with normal saline 3 times, removes cell debris with low speed (500-800rpm) centrifugation in short-term at every turn;
F) with 300 order nylon wire elimination cell masses;
G) with the separated lymphocyte of lymphocyte separation medium;
H) with the autoantigen MOG33-55 of a series of concentration (0,5,10,20 μ g/ml), stimulate lymphocyte 3 days;
I) at the 3rd day, add 0.5 μ Ci3H-TdR;
J), after 16 hours, with cell scintiloscope reading out data, result is expressed as CPM ± S.E.
Two results
Data analysis: adopt SPSS16.0 software data to be carried out to statistical analysis, the relatively employing t method of inspection between two groups.
Fig. 5 shows that the EAE mice clinical symptoms of TACI-IgG and the antibody combined treatment of anti-IL-15 significantly reduces.Autoimmune disease as EAE in, clinical symptoms significantly raises.Compare with PBS treatment group, TACI-IgG treatment group clinical symptoms significantly reduces (p < 0.05).Compare with PBS treatment group, anti-IL-15 Antybody therapy group clinical symptoms reduces (p < 0.05) significantly.Compare with TACI-IgG or the independent treatment group of anti-IL-15 antibody, therapeutic alliance group significantly reduces clinical symptoms.Therefore, therapeutic alliance effect is significantly better than TACI-IgG or anti-IL-15 antibody is treated separately.
Fig. 6 shows that the EAE mouse lymphocyte of TACI-IgG and the antibody combined treatment of anti-IL-15 significantly reduces autoantigen MOG33-55 reactive polypeptide.Autoimmune disease as EAE in, to autoantigen is reactive, significantly raise.Compare with PBS treatment group, the independent treatment group autoantigen of TACI-IgG or anti-IL-15 antibody is reactive significantly to be reduced.Compare with TACI-IgG or the independent treatment group of anti-IL-15 antibody, therapeutic alliance group significantly reduces the reaction to autoantigen.Therefore, therapeutic alliance effect is significantly better than TACI-IgG or anti-IL-15 antibody is treated separately.
Claims (8)
1. a pharmaceutical composition, is characterized in that, described pharmaceutical composition comprises TACI-IgG and anti-IL-15 antibody.
2. pharmaceutical composition claimed in claim 1, is characterized in that, the mass ratio of TACI-IgG and anti-IL-15 antibody inhibition is 0.5～10: 1.
3. pharmaceutical composition claimed in claim 1, is characterized in that, the mass ratio of TACI-IgG and anti-IL-15 antibody suppression is 4: 1.
4. pharmaceutical composition claimed in claim 1, is characterized in that, described anti-IL-15 antibody is monoclonal antibody.
5. the pharmaceutical composition described in any one in claim 1-4, treats the application in autoimmune disease medicine in preparation.
6. autoimmune disease claimed in claim 5, is characterized in that, described autoimmune disease is the cell-mediated autoimmune disease of T, comprises multiple sclerosis, insulin-dependent diabetes.
7. the application of the pharmaceutical composition described in any one in preparation treatment autoimmunity encephalomyelitis medicine in claim 1-4.
8. the pharmaceutical composition described in any one in claim 1-4, is characterized in that, described pharmaceutical composition also comprises one or more in excipient, emulsifying agent, solubilizing agent, antioxidant, controlled release agent.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310120331.4A CN103977401B (en) | 2013-04-09 | 2013-04-09 | A kind of pharmaceutical composition treating multiple sclerosis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310120331.4A CN103977401B (en) | 2013-04-09 | 2013-04-09 | A kind of pharmaceutical composition treating multiple sclerosis |
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| CN103977401A true CN103977401A (en) | 2014-08-13 |
| CN103977401B CN103977401B (en) | 2016-08-03 |
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| CN201310120331.4A Expired - Fee Related CN103977401B (en) | 2013-04-09 | 2013-04-09 | A kind of pharmaceutical composition treating multiple sclerosis |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6036956A (en) * | 1987-08-18 | 2000-03-14 | The Leland Stanford Junior University | Method and dosage form using an antagonist to gamma interferon to control MHC-associated autoimmune disease |
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2013
- 2013-04-09 CN CN201310120331.4A patent/CN103977401B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6036956A (en) * | 1987-08-18 | 2000-03-14 | The Leland Stanford Junior University | Method and dosage form using an antagonist to gamma interferon to control MHC-associated autoimmune disease |
Non-Patent Citations (5)
| Title |
|---|
| FABIEN B VINCENT ET AL.: "BAFF and innate immunity: new therapeutic targets for systemic lupus erythematosus", 《IMMUNOLOGY AND CELL BIOLOGY》 * |
| FUMI MIYAGAWA: "IL-15 serves as a co-stimulator in determining the activity of autoreactive CD8 Tcells in an experimental mouse model of graft vs. host like disease", 《J IMMUNOL》 * |
| GEORGE D KALLIOLIAS1 AND LIONEL B IVASHKIV: "Targeting cytokines in inflammatory diseases: focus on interleukin-1-mediated autoinflammation", 《F1000 BIOLOGY REPORTS》 * |
| JENNIFER H. ANOLIK ET.AL.: "New treatments for SLE: cell-depleting and anti-cytokine therapies", 《BEST PRACTICE & RESEARCH CLINICAL RHEUMATOLOGY》 * |
| MARTA BENITO-MIGUEL: "IL-15 expression on RA synovial fibroblasts promotes B cell survival", 《PLOS ONE》 * |
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| Publication number | Publication date |
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| CN103977401B (en) | 2016-08-03 |
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