CN103977401B - A kind of pharmaceutical composition treating multiple sclerosis - Google Patents
A kind of pharmaceutical composition treating multiple sclerosis Download PDFInfo
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- CN103977401B CN103977401B CN201310120331.4A CN201310120331A CN103977401B CN 103977401 B CN103977401 B CN 103977401B CN 201310120331 A CN201310120331 A CN 201310120331A CN 103977401 B CN103977401 B CN 103977401B
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Abstract
The invention discloses a kind of pharmaceutical composition treating multiple sclerosis, pharmaceutical composition of the present invention includes BAFF inhibitor (TACI IgG) and IL 15 inhibitor (anti-IL 15 antibody).Utilize the mouse model EAE mice of medicine composite for curing multiple sclerosis of the present invention, result shows that pharmaceutical composition of the present invention can significantly reduce B cell in spleen and lymph node, helper T lymphocyte, the quantity of memory B/T cell, inhibit the activation of B cell and the generation of autoimmune antibody, alleviate autoimmune clinical symptoms.Pharmaceutical composition of the present invention has effectively treated multiple sclerosis.
Description
Technical field
The invention belongs to biomedical sector, relate to one and treat the medicine of autoimmune disease multiple sclerosis (MS)
Compositions.Specifically, compositions of the present invention includes BAFF inhibitor and IL-15 inhibitor, and zoopery shows described
Pharmaceutical composition can effectively inhibit autoimmune generation and development.
Background technology
Along with the development of social times, autoimmune disease sickness rate in China various people constantly rises.In view of this
People's life in the future, the even own health that works can be caused serious harm by the phase after being ill, strengthen autoimmune further
The basic research of property disease will help the mankind to understand the generation of this type of disease, develop and lapse to etc. the mechanism of action, thus carries
High prevention, diagnosis and treatment level to it.This, to ensureing that human lives is healthy, promotes that socioeconomic development all has important
Theoretical and practical significance.
Autoimmune disease can be divided into two big classes according to immunologic injury mechanism: a class be autoantibody cause self
Immune disease such as systemic lupus erythematosus (sle) (SLE), myasthenia gravis (MG) and idiopathic thrombocytopenic purpura (ITP)
Deng, another kind of is that [MS, its animal model is self to the autoimmune disease such as multiple sclerosis that causes of autoreactive T cell
Allergic encephalomyelitis (EAE)] and insulin-dependent diabetes (IDDM).
Many experimental evidences show that T cell directly participates in autoimmune disease, multiple organ specific autoimmune disease
All mediated by T cell, such as type i diabetes, autoimmune thyroiditis, multiple sclerosis (MS) etc..Simultaneously at antibody
In the autoimmune disease of mediation, the activation of B cell also relies on the auxiliary of Th cell.Recently, many evidences show at T thin
In the autoimmune disease that born of the same parents drive, autoreactivity B cell also plays considerable effect.Therefore, numerous self exempt from
Epidemic disease disease is typically jointly coordinated by autoreactivity T, B cell and mediate generation.
The treatment of autoimmune disease still lacks preferable method.It is generally directed to caused by pathological change and the tissue injury of disease
Consequence treat, it is possible to by regulation immunne response links block disease process reach treatment purpose.Mesh
More front common Therapeutic Method include: use heavy dose of 17-hydroxy-11-dehydrocorticosterone, salicylic acid or the prostaglandin inhibitor of various synthesis,
Suppress some inflammatory reactions caused by serious symptom autoimmune disease；Or use immunosuppressant to suppress immune system to play to make
With.But both approaches all can only slow down the development speed of the state of an illness and not effect a radical cure, and the most also can produce serious side effect.
Pathogenesis recently as autoimmune disease is progressively illustrated, and the Therapeutic Method for this disease is also constantly being studied
Among.
BAFF, also known as B-cell stimulating factor (B lymphocyte stimulator, BLyS), belongs to tumor necrosis factor
Superfamily (tumor necrosis factor superfamily, TNSF) member.BAFF B cell grow, function point analysis and
Playing a significant role in the morbidity of autoimmune disease, its defect or overexpression all can cause immunity of organism unbalance, with certain
The generation of a little autoimmune diseasees is relevant.BAFF level is found in autoimmune disease patient's serum such as SLE, SS and RA
Significantly raised, BAFF process LAN can cause autoreactivity B cell to escape normal immunity screening out mechanism thus ripe for antibody
Secretion B cell or plasma cell, further demonstrate that BAFF closely take part in these diseases generation and development [Editorial,
Et al.The B cell:a new therapeutic target in rheumatoid arthritis and other
Autoimmune diseases.Joint Bone Spine2004.71:357-360].
T cell is considered as primary pathogenic event [Behi ME, the et al.New insights into cell of MS
responses involved in experimental autoimmune encephalomyelitis and multiple
, closely related with the cellular immunization of Th1 mediation sclerosis.Immunology Letters2005.96 (1): 11-26].Main
The molecular simulation theory of stream is thought, the virus that MS patient infects and central nervous system's myelin protein composition or oligodendrocyte
Between there is cross-reacting antigen.After virus infects, the internal T cell being activated is being removed while virus, due to the becoming of myelin
The raw cross reaction of distribution, thus cause maincenter demyelination.Owing to MS damage zone is contained within substantial amounts of T cell, therefore use myelin
The T cell that basic protein (MBP) polypeptide fragment activates is transferred to mice and can cause the animal model of MS---tentative self exempts from
Epidemic disease encephalomyelitis (EAE).But the continuous intensification to B cell function understanding in recent years, B cell is in the generating process of MS
Effect is the most gradually taken seriously, and we show early-stage Study, and in autoimmune disease MS, the BAFF level in serum raises.
In normal human's brain, the expression of BAFF is only adenoid 1/10th, and the table of MS patient damage zone BAFF
Reach being on close level in level and lymphoid tissue.And owing to the BAFF of the astrocyte secretion of activation is thin far above monokaryon
Born of the same parents and macrophage, the main source of BAFF in therefore the damage zone of MS patient becomes brain.Come from relapsing remitting EAE with slow
Property relapsing EAE research also indicate that, laboratory animal morbidity or recurrence the phase, central nervous system's BAFF level increases, and delays
Solution phase BAFF horizontal down-regulation.In view of BAFF plays a significant role in the growth, differentiation of B cell, it is again multiple self exempting from simultaneously
Key pathogenetic molecule in epidemic disease disease, so BAFF is imitated as suppression B cell important in various autoimmune disease
The therapy target answered, in March, 2011, anti-BAFF antibody belimumab is named as Benlysta and lists in the U.S..But resistance
The method of disconnected BAFF treatment there is also certain defect, in the brain of MS patient, there is lymphoid follicle structure, and B cell is permissible
Constantly amplification break up and become plasma cell or autoreactivity memory B cell in these structures, by release autoantibody or
As antigen-presenting cell so that the state of an illness of MS the most repeatedly and deteriorates.Outside although single anti-BAFF antibody can significantly reduce
The number of all blood mature B cells, but it can not reduce the quantity [Vincent of plasma cell and autoreactivity memory B cell
FB, et al.BAFF and innate immunity:new therapeutic targets for systemic lupus
Erythematossu.Immunol Cell Biol.2012.90:293-303].If therefore autoreactivity memory B cell
Being present in internal, humoral immune reaction just can be activated again.So deleting self by the method finding therapeutic alliance
Reactive memory B cell, suffers from the patient of autoimmune disease it would be possible to obtain fully treating and rehabilitation.
IL-15 is a kind of important multi-functional cell regulating factor, is possible not only to stimulate CD4+And CD8+The propagation of T cell
And the generation of CTL can be induced, the most also mediate by IgM specific antibody or CD40L stimulate the B cell proliferation caused and
The synthesis of immunoglobulin.In stimulating the generation of NK cell, propagation and activation process, IL-15 also plays a significant role.So
IL-15 is the key factor of Memorability T/B cell survival and propagation, and BAFF then may carry out negative regulator by suppression IL-15 and remember
The quantity of the property recalled T/B cell.It addition, IL-15 or the pro-inflammatory cytokine of a kind of many biological effects, it is possible to by suppression
The AICD and promotion CD8 of IL-2 induction+The existence of memory t cell and cause the generation of autoimmune disease.IL-15 exists
CD4+T cell can be promoted expression [Pandiyan P, the et al.The role of IL-of IL-17A by activation STAT5
15in activating STAT5and fine-tuning IL-17A production in CD4+T lymphocytes.J
Immunol.2012.189:4237-46], and then the induction propagation of neutrophilic granulocyte, maturation and chemotactic；Can also promote multiple
Cell subsets produces the proinflammatory cytokines such as TNF, IL-1, IL-6, IL-8 and GM-CSF；Stimulating expression of macrophage produces IL-1 simultaneously
β, tumor necrosis factor-alpha and PGE2 etc. produce synergism to amplify inflammatory reaction.Experiment shows, various autoimmune disease
IL-15 in patient's body expresses imbalance, blocks generation and development that the activity of IL-15 can control inflammation, it is achieved treat with
Immunomediated inflammatory is the various diseases of main mechanism.
Summary of the invention
For solving existing medicine defect in treatment autoimmune disease, and it is used alone BAFF inhibitor for treating
The deficiency of autoimmune disease, the invention discloses a kind of pharmaceutical composition, and described pharmaceutical composition includes BAFF inhibitor and IL-
15 inhibitor.
The preferred TACI-IgG of BAFF inhibitor in aforementioned pharmaceutical compositions, the preferred Anti-IL-15 of IL-15 inhibitor are anti-
Body, 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, the most preferably 4: 1.
Pharmaceutical composition of the present invention also includes in pharmaceutical preparation conventional adjuvant, such as: excipient, emulsifying agent,
Solubilizing agent, antioxidant, controlled release agent, if its be suitable for corresponding drug delivery system and be properly maintained BAFF inhibitor and
The activity of the molecule of IL-15 inhibitor.
The present invention have rated aforementioned pharmaceutical compositions curative effect in treatment autoimmune disease by zoopery, specifically wraps
Include following steps:
1) foundation of animal model: the present invention chooses the mouse model of multiple sclerosis, the most tentative autoimmunity brain
Myelitis (EAE) mouse model.The present invention uses MOG35-55 and complete Freund's adjuvant to prepare antigen Emulsion, mouse subcutaneous injection
Lumbar injection pertussis toxin, PT is given after antigen Emulsion.
2) treatment packet and Therapeutic Method: zoopery be provided with normal mouse group, PBS control group, single inhibitor group and
Compositions treatment group, by being administered 2 weeks to 24 weeks the course for the treatment of.The most preferably administering mode is to be administered once for 3 days, intravenously administrable 2 weeks
Pneumoretroperitoneum is administered 2 weeks.
3) therapeutic evaluation:
Present invention TACI-IgG and Anti-IL-15 medicine composite for curing EAE mice effectively reduce B in Mice Body
The number of cell and the activation of B cell.
It is thin that present invention TACI-IgG and Anti-IL-15 medicine composite for curing EAE mice effectively reduce complementary T
The number of born of the same parents Th1 and Th17.
Present invention TACI-IgG and Anti-IL-15 medicine composite for curing EAE mice are remembered in effectively reducing Mice Body
The property recalled T and the number of B cell.
Present invention TACI-IgG and Anti-IL-15 medicine composite for curing EAE mice effectively alleviate the clinic of mice
Symptom.
Present invention TACI-IgG and Anti-IL-15 medicine composite for curing EAE mice effectively reduce in Mice Body from
The level of body reactive antibody, reduces the autoimmune of EAE mice.
The present invention have detected the therapeutic effect of aforementioned pharmaceutical compositions by experiment, and experimental result shows, the medicine of the present invention
Compositions significantly reduces EAE clinical symptoms, significantly reduces the mice reaction to autoantigen, the pharmaceutical composition of the present invention
Therapeutic effect is considerably better than TACI-IgG or anti-IL-15 antibody monotherapy.
The pharmaceutical composition of the present invention can effectively treat the autoimmune disease of T cell mediation, including autoimmunity
Encephalomyelitis, multiple sclerosis, insulin-dependent diabetes.
Accompanying drawing explanation
The number of B cell in Fig. 1 TACI-IgG and anti-IL-15 antibody combined treatment EAE mice；
The activation of B cell in Fig. 2 TACI-IgG and anti-IL-15 antibody combined treatment EAE mice；
The number of Th1 and Th17 cell in Fig. 3 TACI-IgG and anti-IL-15 antibody combined treatment EAE mice；
Memorability T and B cell in Fig. 4 TACI-IgG and anti-IL-15 antibody combined treatment EAE mice spleen and lymph node
Number；
The clinical symptoms of Fig. 5 TACI-IgG and anti-IL-15 antibody combined treatment EAE mice；
Fig. 6 separate groups of mice is reactive to autoantigen.
Embodiment 1TACI-IgG and the anti-IL-15 antibody combined treatment impact on B cell
One material and method
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；Rabbit anti-Mus B220 monoclonal antibody, the anti-Mus of rabbit
CD3 monoclonal antibody, rabbit anti-Mus CD21 monoclonal antibody, rabbit anti-Mus CD23 monoclonal antibody are purchased from Abcam；BAFF inhibitor TACI-IgG, anti-mouse 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, MOG35-55 is diluted to 2mg/mL, then by the complete Fu Shi assistant of diluent with equivalent
Agent is sufficiently mixed, and is lashed by Emulsion as Water-In-Oil state by tee T, is the antigen Emulsion of induction EAE.EAE group mice in
Both sides, back subcutaneous injection of antigens Emulsion (only includes MOG antigen 200～250 μ g/).Matched group is refused antigen and is processed.After immunity
Within 0th, the 48th hour, give whole mice (including matched group) lumbar injection pertussis toxin, PT, every animal 500ng/0.2mL.
2.2, experiment packet
Experiment mice being divided into 5 groups: control mice, PBS treats EAE mice, and TACI-IgG treats EAE mice, Anti-
IL-15 treats EAE mice, TACI-IgG and Anti-IL-15 therapeutic alliance EAE mice.Often 12 animals of group.By tail vein
Injection TACI-IgG and Anti-IL-15, in the EAE Mice Body of morbidity, injects 1 time, after treating 2 weeks continuously, uses abdomen instead for every 3 days
Chamber continual cure 2 weeks.TACI-IgG consumption: 2mg/kg every time；Anti-IL-15 antibody consumption: 0.5mg/kg every time.
2.4, adenoid pretreatment
A) piece of tissue is put in plate, add a small amount of normal saline；
B) with shears by tissue shear to being homogenized shape；
C) 10ml normal saline is added；
D) it is homogenized with suction pipe aspirates tissue, first with in 100 mesh nylon net filters to test tube；
E) centrifugation 1000rpm, 3-5min, then wash 3 times with normal saline, every time with low speed (500-800rpm) in short-term
Cell debris is removed in centrifugation；
F) cell mass is filtered off with 300 mesh nylon wires；
G) cell saves backup.
2.3, fluidic cell sample treatment and detection
A) 2 × 10 are taken6Individual cell, with cold PBS2ml washed cell once, 800rpm is centrifuged 5min；
B) fixing cell 40min by 2ml4% paraformaldehyde room temperature, 800rpm is centrifuged 5min；2ml PBS re-suspended cell,
800rpm is centrifuged 5min；
C) with 1ml containing 0.2%Triton-X100 and the PBS re-suspended cell of 5% serum, 10min, 800rpm are placed on ice
Centrifugal 5min；
D) add the unmarked antibody of Sa (rabbit anti-Mus B220, CD3, CD21, CD23 antibody), place 40min on ice,
800rpm is centrifuged 5min, and with PBS re-suspended cell cold for 2ml, 800rpm is centrifuged 5min, washes away uncombined one and resists, is repeated once；
E) adding fluorescently-labeled goat anti-rabbit igg two to resist, after lucifuge places 40min on ice, 800rpm is centrifuged 5min, goes
Clearly, PBS washes twice；
F) 0.5ml1% paraformaldehyde re-suspended cell is added, fixing to be measured；
G) flow cytomery fluorescent value, often 10000 cells of pipe counting.
Two results
Data analysis: using SPSS16.0 software that data carry out statistical analysis, the comparison between two groups uses t method of inspection.
Fig. 1 shows compared with PBS treatment group, and TACI-IgG treatment causes B cell in spleen and lymph node to substantially reduce (P ＜
0.01).Compared with PBS control group, therapeutic alliance causes spleen and enlargement of lymph node B cell to substantially reduce (P ＜ 0.01).With TACI-
IgG monotherapy group is compared, and therapeutic alliance group does not significantly reduce B cell number.This display, TACI-IgG specifically presses down
B cell processed, and anti-IL-15 antibody does not has a function of this respect, but TACI-IgG and the antibody combined therapeutic combination of anti-IL-15
TACI-IgG monotherapy group is similar, can significantly reduce B cell number in EAE mice spleen and lymph node.
Fig. 2 shows compared with PBS treatment group, and TACI-IgG treatment causes B cell Activation markers CD21 in spleen and lymph node
Substantially reducing (P ＜ 0.01), therapeutic alliance group also causes B cell activation in spleen and lymph node to substantially reduce (P ＜ 0.01).With
TACI-IgG monotherapy group is compared, and therapeutic alliance group does not significantly reduce B cell Activation markers.This display, TACI-IgG
Specifically B cell in the activation of suppression B cell, TACI-IgG and the EAE mice spleen of the antibody combined treatment of anti-IL-15 and lymph node
Activation markers CD21 significantly decreases.
Embodiment 2TACI-IgG and the anti-IL-15 antibody combined treatment impact on helper T lymphocyte (Th1, Th17)
One material and method
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；Rabbit anti-Mus CD4 monoclonal antibody, the anti-Mus of rabbit
IL-17 monoclonal antibody, rabbit anti-Mus IFN γ monoclonal antibody are purchased from Abcam；BAFF inhibitor TACI-IgG, anti-mouse 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, MOG35-55 is diluted to 2mg/mL, then by the complete Fu Shi assistant of diluent with equivalent
Agent is sufficiently mixed, and is lashed by Emulsion as Water-In-Oil state by tee T, is the antigen Emulsion of induction EAE.EAE group mice in
Both sides, back subcutaneous injection of antigens Emulsion (only includes MOG antigen 200～250 μ g/).Matched group is refused antigen and is processed.After immunity
Within 0th, the 48th hour, give whole mice (including matched group) lumbar injection pertussis toxin, PT, every animal 500ng/0.2mL.
2.2, experiment packet
Experiment mice being divided into 5 groups: control mice, PBS treats EAE mice, and TACI-IgG treats EAE mice, Anti-
IL-15 treats EAE mice, TACI-IgG and Anti-IL-15 therapeutic alliance EAE mice.Often 12 animals of group.By tail vein
Injection TACI-IgG and Anti-IL-15, in the EAE Mice Body of morbidity, injects 1 time, after treating 2 weeks continuously, uses abdomen instead for every 3 days
Chamber continual cure 2 weeks.TACI-IgG consumption: 2mg/kg every time；Anti-IL-15 antibody consumption: 0.5mg/kg every time.
2.3, adenoid pretreatment
A) piece of tissue is put in plate, add a small amount of normal saline；
B) with shears by tissue shear to being homogenized shape；
C) 10ml normal saline is added；
D) it is homogenized with suction pipe aspirates tissue, first with in 100 mesh nylon net filters to test tube；
E) centrifugation 1000rpm, 3-5min, then wash 3 times with normal saline, every time with low speed (500-800rpm) in short-term
Cell debris is removed in centrifugation；
F) cell mass is filtered off with 300 mesh nylon wires；
G) cell saves backup.
2.4, fluidic cell sample treatment and detection
A) 2 × 10 are taken6Individual cell, with cold PBS2ml washed cell once, 800rpm is centrifuged 5min；
B) fixing cell 40min by 2ml4% paraformaldehyde room temperature, 800rpm is centrifuged 5min；2ml PBS re-suspended cell,
800rpm is centrifuged 5min；
C) with 1ml containing 0.2%Triton-X100 and the PBS re-suspended cell of 5% serum, 10min, 800rpm are placed on ice
Centrifugal 5min；
D) add the unmarked antibody of Sa (rabbit anti-Mus CD4, IL-17, IFN γ antibody), place 40min on ice,
800rpm is centrifuged 5min, and with PBS re-suspended cell cold for 2ml, 800rpm is centrifuged 5min, washes away uncombined one and resists, is repeated once；
E) adding fluorescently-labeled goat anti-rabbit igg two to resist, after lucifuge places 40min on ice, 800rpm is centrifuged 5min, goes
Clearly, PBS washes twice；
F) 0.5ml1% paraformaldehyde re-suspended cell is added, fixing to be measured；
G) flow cytomery fluorescent value, often 10000 cells of pipe counting.
Two results
Data analysis: using SPSS16.0 software that data carry out statistical analysis, the comparison between two groups uses t method of inspection.
Fig. 3 shows that in TACI-IgG and the EAE mice spleen of the antibody combined treatment of anti-IL-15 and lymph node, Th1 and Th17 is thin
Born of the same parents significantly reduce.In autoimmune disease is such as EAE, Th1 and Th17 cell significantly raises.Compared with PBS treatment group,
TACI-IgG treatment group can not reduce Th1 and Th17 cell.Compared with TACI-IgG monotherapy group, therapeutic alliance group significantly drops
Low Th1 and Th17 cell.Compared with PBS treatment group, anti-IL-15 Antybody therapy group Th1 and Th17 cell significantly decrease.This says
Bright, in terms of suppression Th1 and Th17 cell, anti-IL-15 antibody plays Main Function.
Embodiment 3TACI-IgG and the anti-IL-15 antibody combined treatment impact on Memorability T/B cell
One material and method
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；Rabbit anti-Mus B220 monoclonal antibody, the anti-Mus of rabbit
CD44 monoclonal antibody, rabbit anti-Mus CD27 monoclonal antibody, rabbit anti-Mus CD3 monoclonal antibody are purchased from Abcam；BAFF inhibitor TACI-IgG, anti-mouse 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, MOG35-55 is diluted to 2mg/mL, then by the complete Fu Shi assistant of diluent with equivalent
Agent is sufficiently mixed, and is lashed by Emulsion as Water-In-Oil state by tee T, is the antigen Emulsion of induction EAE.EAE group mice in
Both sides, back subcutaneous injection of antigens Emulsion (only includes MOG antigen 200～250 μ g/).Matched group is refused antigen and is processed.After immunity
Within 0th, the 48th hour, give whole mice (including matched group) lumbar injection pertussis toxin, PT, every animal 500ng/0.2mL.
2.2, experiment packet
Experiment mice being divided into 5 groups: control mice, PBS treats EAE mice, and TACI-IgG treats EAE mice, Anti-
IL-15 treats EAE mice, TACI-IgG and Anti-IL-15 therapeutic alliance EAE mice.Often 12 animals of group.By tail vein
Injection TACI-IgG and Anti-IL-15, in the EAE Mice Body of morbidity, injects 1 time, after treating 2 weeks continuously, uses abdomen instead for every 3 days
Chamber continual cure 2 weeks.TACI-IgG consumption: 2mg/kg every time；Anti-IL-15 antibody consumption: 0.5mg/kg every time.
2.2, adenoid pretreatment
A) piece of tissue is put in plate, add a small amount of normal saline；
B) with shears by tissue shear to being homogenized shape；
C) 10ml normal saline is added；
D) it is homogenized with suction pipe aspirates tissue, first with in 100 mesh nylon net filters to test tube；
E) centrifugation 1000rpm, 3-5min, then wash 3 times with normal saline, every time with low speed (500-800rpm) in short-term
Cell debris is removed in centrifugation；
F) cell mass is filtered off with 300 mesh nylon wires；
G) cell saves backup.
2.3, fluidic cell sample treatment and detection
A) 2 × 10 are taken6Individual cell, with cold PBS2ml washed cell once, 800rpm is centrifuged 5min；
B) fixing cell 40min by 2ml4% paraformaldehyde room temperature, 800rpm is centrifuged 5min；2ml PBS re-suspended cell,
800rpm is centrifuged 5min；
C) with 1ml containing 0.2%Triton-X100 and the PBS re-suspended cell of 5% serum, 10min, 800rpm are placed on ice
Centrifugal 5min；
D) add the unmarked antibody of Sa (the anti-Mus of rabbit one resists), place 40min, 800rpm on ice and be centrifuged 5min, use
PBS re-suspended cell cold for 2ml, 800rpm is centrifuged 5min, washes away uncombined one and resists, is repeated once；
E) adding fluorescently-labeled goat anti-rabbit igg two to resist, after lucifuge places 40min on ice, 800rpm is centrifuged 5min, goes
Clearly, PBS washes twice；
F) 0.5ml1% paraformaldehyde re-suspended cell is added, fixing to be measured；
G) flow cytomery fluorescent value, often 10000 cells of pipe counting.
Two results
Data analysis: using SPSS16.0 software that data carry out statistical analysis, the comparison between two groups uses t method of inspection.
Fig. 4 shows compared with healthy mice, and in autoimmune disease EAE, Memorability T and B cell significantly raise.With
PBS treatment group is compared, and TACI-IgG treatment group Memorability T and B cell raise (P ＜ 0.05).With TACI-IgG monotherapy group
Comparing, therapeutic alliance group significantly reduces Memorability T and B cell (P ＜ 0.01).Compared with PBS treatment group, anti-IL-15 antibody is controlled
Treatment group Memorability T and B cell significantly decrease (P ＜ 0.01).This display, TACI-IgG specifically suppresses B cell to activate, and
The formation of Memorability T/B cell can not be suppressed, and anti-IL-15 antibody specificity ground suppresses Memorability T and the function of B cell.Cause
This, in TACI-IgG and the EAE mice spleen of the antibody combined treatment of anti-IL-15 and lymph node, Memorability T and B cell significantly reduce.
Embodiment 4TACI-IgG and anti-IL-15 antibody combined treatment EAE are on the clinical state of an illness and the impact of autoantigen reaction
One material and method
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, MOG35-55 is diluted to 2mg/mL, then by the complete Fu Shi assistant of diluent with equivalent
Agent is sufficiently mixed, and is lashed by Emulsion as Water-In-Oil state by tee T, is the antigen Emulsion of induction EAE.EAE group mice in
Both sides, back subcutaneous injection of antigens Emulsion (only includes MOG antigen 200～250 μ g/).Matched group is refused antigen and is processed.After immunity
Within 0th, the 48th hour, give whole mice (including matched group) lumbar injection pertussis toxin, PT, every animal 500ng/0.2mL.
2.2, experiment packet
Experiment mice being divided into 5 groups: control mice, PBS treats EAE mice, and TACI-IgG treats EAE mice, Anti-
IL-15 treats EAE mice, TACI-IgG and Anti-IL-15 therapeutic alliance EAE mice.Often 12 animals of group.By tail vein
Injection TACI-IgG and Anti-IL-15, in the EAE Mice Body of morbidity, injects 1 time, after treating 2 weeks continuously, uses abdomen instead for every 3 days
Chamber continual cure 2 weeks.TACI-IgG consumption: 2mg/kg every time；Anti-IL-15 antibody consumption: 0.5mg/kg every time.
2.3 symptom scores
5 point-score standards of grading classifications are as follows: 0 is divided into and not falling ill；1 is divided into flaccid tail；2 are divided into slight hind limb weakness；3 points
For serious hindlimb paralysis；4 are divided into quadriplegia；5 be divided into dying or dead.Immunity played every day to mouse weights the same day, and used
Double-blind method carries out Neuroscore.By two non-experiment personal observations evaluating, mark with reference to score by rules.First by
One personnel carries out 5 point-scores and the scoring of 7 point-scores, and both can be carried out simultaneously.After above-mentioned scoring 30min, then by an other sight
The person of examining carries out 15 point-score scorings.Owing to mice diel movement is widely different, every day is all evaluated when the morning 8.
2.4, the detection that lymph node is reactive to autoantigen MOG33-55
A) lymph node is put in plate, add a small amount of normal saline；
B) with shears and tweezers lymph node cut and pinch to homogenate shape；
C) 10ml normal saline is added；
D) it is homogenized by suction pipe absorptive tissue, first with in 100 mesh nylon net filters to test tube；
E) centrifugation 1000rpm, 3-5min, is washing 3 times with normal saline, every time with low speed (500-800rpm) in short-term
Cell debris is removed in centrifugation；
F) cell mass is filtered off with 300 mesh nylon wires；
G) with lymphocyte separation medium separation lymphocyte；
H) lymphocyte is stimulated 3 days with the autoantigen MOG33-55 of a series of concentration (0,5,10,20 μ g/ml)；
I) 0.5 μ Ci3H-TdR was added at the 3rd day；
J) after 16 hours, reading data with Cytoscint scintillation instrument, result is expressed as CPM ± S.E.
Two results
Data analysis: using SPSS16.0 software that data carry out statistical analysis, the comparison between two groups uses t method of inspection.
Fig. 5 shows that the EAE mice clinical symptoms of TACI-IgG and the antibody combined treatment of anti-IL-15 significantly reduces.At self
In immune disease such as EAE, clinical symptoms significantly raises.Compared with PBS treatment group, TACI-IgG treatment group clinical symptoms is notable
Reduce (p ＜ 0.05).Compared with PBS treatment group, anti-IL-15 Antybody therapy group clinical symptoms significantly decreases (p ＜ 0.05).With
TACI-IgG or anti-IL-15 antibody monotherapy group is compared, and therapeutic alliance group significantly reduces clinical symptoms.Therefore, therapeutic alliance
Effect is considerably better than TACI-IgG or anti-IL-15 antibody monotherapy.
Fig. 6 shows that the EAE mouse lymphocyte of TACI-IgG and the antibody combined treatment of anti-IL-15 is to autoantigen MOG33-
55 reactive polypeptides significantly reduce.In autoimmune disease is such as EAE, notable rising reactive to autoantigen.With PBS treatment group
Comparing, TACI-IgG or anti-IL-15 antibody monotherapy group autoantigen reactivity significantly reduces.With TACI-IgG or anti-IL-
15 antibody monotherapy groups are compared, and therapeutic alliance group significantly reduces the reaction to autoantigen.Therefore, therapeutic alliance effect is notable
It is better than TACI-IgG or anti-IL-15 antibody monotherapy.
Claims (5)
1. a pharmaceutical composition is preparing the application treated in multiple sclerosis medicine, it is characterised in that described drug regimen
Thing includes TACI-IgG and anti-IL-15 antibody.
2. the application described in claim 1, it is characterised in that the mass ratio of TACI-IgG and anti-IL-15 antibody is 0.5～10:
1。
3. the application described in claim 1, it is characterised in that the mass ratio of TACI-IgG and anti-IL-15 antibody is 4: 1.
4. the application described in claim 1, it is characterised in that described anti-IL-15 antibody is monoclonal antibody.
5. application described in any one in claim 1-4, it is characterised in that described pharmaceutical composition also include excipient,
One or more in emulsifying agent, solubilizing agent, antioxidant, controlled release agent.
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| CN201310120331.4A CN103977401B (en) | 2013-04-09 | 2013-04-09 | A kind of pharmaceutical composition treating multiple sclerosis |
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| CN201310120331.4A 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 (4)
| Title |
|---|
| BAFF and innate immunity: new therapeutic targets for systemic lupus erythematosus;Fabien B Vincent et al.;《Immunology and Cell Biology》;20120110;第90卷;第298页左栏第2段 * |
| IL-15 expression on RA synovial fibroblasts promotes B cell survival;Marta Benito-miguel;《Plos One》;20120709;第7卷(第7期);全文 * |
| New treatments for SLE: cell-depleting and anti-cytokine therapies;Jennifer H. Anolik et.al.;《Best Practice & Research Clinical Rheumatology》;20051231;第19卷(第5期);第866页第2段、第866页图2、第867页第2段、第870页第7段和第871页第2段 * |
| Targeting cytokines in inflammatory diseases: focus on interleukin-1-mediated autoinflammation;George D Kalliolias1 and Lionel B Ivashkiv;《F1000 Biology Reports》;20091231;第1卷(第70期);表2 * |
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