CN104726410A

CN104726410A - Exosome with immunosuppression function and application thereof

Info

Publication number: CN104726410A
Application number: CN201310719722.8A
Authority: CN
Inventors: 王建莉; 蔡志坚; 余磊
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2013-12-23
Filing date: 2013-12-23
Publication date: 2015-06-24
Anticipated expiration: 2033-12-23
Also published as: CN104726410B

Abstract

The invention provides an exosome with immunosuppression function and application thereof. Particularly, the exosome has the following characteristics: (a) the exosome is a vesica structure secreted by immature dendritic cells (imDCs), and the imDCs is modified by an exogenous membrane expression cell factor gene to express the membrane expression cell factor; and (b) the exosome carries the membrane expression cell factor. The exosome can inhibit immunoreaction and induce the reconstruction of immunologic tolerance of the organisms, thereby preventing and/or treating autoimmune diseases.

Description

A kind of efflux body and application thereof with immune suppression function

Technical field

The invention belongs to pharmacy research and development, cytobiology, biology field, relate to the preparation of efflux body in marrow immature dendritic cell source of transmembrane TGF-β 1 genetic modification and the application in prevention and therapy autoimmune disorder thereof.

Background technology

Autoimmune disorder is the disease because body immune system causes self component generation immunne response, has higher sickness rate in crowd.Multiple sclerosis (Multiple Sclerosis, MS) common mainly betides youthful central nervous system demyelination, and think at present, it is that relevant with environment and heredity but the detailed cause of disease is unclear.Clinically, the medicine for the treatment of MS mainly contains IFN-β, glatiramer, mitoxantrone, but effect is unsatisfactory, and side effect is huge.

Dendritic cell (Dendritic cells, DCs) be professional antigen presenting cell (the Antigen presenting cells that body function is the strongest, APCs), it can absorb efficiently, processing treatment and present antigen, immature DC s (immature DCs) has stronger transfer ability, mature DCs (mature DCs) can effectively activate primary tape T cell, is in startup, regulates and controls and maintains the key link of immunne response.

In human body, most of DCs is in non-maturity state, express low-level costimulating factor and adhesion factor, the external ability of mixed lymphocytes proliferative response of the same race that excites is lower, but immature DC s has extremely strong antigen phagocytic activity, be divided into mature DCs in antigen uptaking (comprising external processing) or when being subject to the stimulation of some factor, and the DCs of maturation expresses high-caliber costimulating factor and adhesion factor.

DCs, in the process of maturation, is moved by the peripheral tissues of contact antigen and enters secondary lymphatic organ, contact and excite immunne response with T cell.DCs can high expression level MHC-I class and MHC-class Ⅱmolecule, and the tumour antigen that MHC molecule catches processing with it is combined, and form peptide-MHC molecular complex, and submission is to T cell, thus starts the reaction of MHC-I class Restricted CTL and the restrictive CD4 of MHC-II class ⁺thl reacts.Meanwhile, DCs also provides T cell activation necessary second signal by the costimulatory molecules (CD80/B7-1, CD86/B7-2, CD40 etc.) of its high expression level, starts immunne response.

Act on different in view of ripe in human body with immature DCs, in recent years develop the potential drug that the immunity moderation based on imDCs reacts successively.But owing to cannot overcome the restriction of MHC-II quasi-molecule, these potential drugs based on imDCs all have toxicity in various degree to human body, and effect is poor.

Therefore, this area can effectively suppression of autoimmune responses, regulation and control immunne response in the urgent need to developing one, and safe and effective medicine.

Summary of the invention

The invention provides a kind of there is film expression type TGF-β 1 and not by the efflux body exosomes of MHC-II restriction, the reconstruction of body immunological tolerance can be induced, prevention and therapy autoimmune disorder from immature dendritic cell.

The invention provides a kind of exosomes and mTGF-β 1-EXO of marrow immature dendritic cell source of transmembrane TGF-β 1 genetic modification, it expresses membranous type TGF-β 1.All there is certain immunosuppressive action in vivo and in vitro and the restriction of MHC-II can be broken through, also having therapeutic action to allogeneic mice with experimental autoimmune encephalomyelitis, being expected to the general preparation becoming treatment autoimmune disorder.

First aspect present invention, provide a kind of efflux body (exosomes), described efflux body has following characteristics:

A () described efflux body is the capsule balloon-shaped structure secreted by immature dendritic cell (imDCs cell), described immature dendritic cell through the film expression cytokines gene of external source modification thus express described film expression cytokines; With

B () described efflux body carries described film expression cytokines.

In another preference, described efflux body diameter is 50-100nm.

In another preference, the outside surface of the vesica film of described efflux body has and shows the film expression cytokines described in (at least partially).

In another preference, described efflux body has the activity suppressing MHC-II.

In another preference, in described immature dendritic cell, costimulatory molecules CD80, CD86 and MHC-II (major histocompatibility complex II) low expression.

In another preference, to described MHC-II express suppression, be caused by described film expression cytokines or part cause.

In another preference, described efflux body also has one or more features following:

D () described efflux body has the activity of Immunosuppression or Immunosuppression system;

E () described efflux body expressing protein comprises Hsp70, Tsg101 or CD63;

F () described efflux body does not express ER molecular chaperones Grp94.

In another preference, described film expression cytokines comprises the cytokine of immunosuppression class, preferably comprises TGF-β 1, IL-10, IL-4, FasL or its combination.

In another preference, described efflux body has one or more activity following:

I () cause Treg (regulatory T cells) grows;

(ii) effect of Th1 polarization is suppressed;

(iii) Th17 cytodifferentiation is suppressed.

Second aspect present invention, provides a kind of pharmaceutical composition, and described pharmaceutical composition contains the efflux body described in first aspect present invention of safe and effective amount as activeconstituents; With pharmaceutically acceptable carrier.

In another preference, described pharmaceutical composition is vaccine composition.

Third aspect present invention, provides the purposes of the efflux body described in first aspect present invention, for the preparation of the pharmaceutical composition preventing and/or treating autoimmune disorder.

In another preference, described autoimmune disorder comprises nervus centralis Demyelination, preferably, described nervus centralis Demyelination comprises multiple sclerosis (Multiple Sclerosis MS), experimental autoimmune encephalomyelitis (experimentalautoimmune encephalomyelitis, EAE), acute disseminated encephalomyelitis, diffuse cerebrosclerosis, optic neuromyelitis and Secondary cases demyelinating disease.

In another preference, described Secondary cases demyelinating disease causes primarily of systemic disease, as Periventricular Leukomalacia in Prematures disease etc.

Fourth aspect present invention, provide a kind of immature dendritic cell for generation of the efflux body described in first aspect present invention (Immature Dendritic Cells, imDCs), described immature dendritic cell through the film expression cytokines gene of external source modification thus express described film expression cytokines, and the described efflux body of immature dendritic cell secretion power described in first aspect present invention, and described efflux body carries described film expression cytokines.

In another preference, described film expression cytokines comprises the cytokine of immunosuppression class, preferably comprises TGF-β 1, IL-10, IL-4, FasL etc.

In another preference, the expression of described immature dendritic cell MHC-II is downtrod.

Fifth aspect present invention, provides a kind of method preparing efflux body described in first aspect present invention, comprises step:

A () cultivates the immature dendritic cell described in fourth aspect present invention, thus obtain the mixture of the efflux body of described immature dendritic cell and secretion thereof;

B the efflux body of secretion described in (a) from described mixture separation and purifying, thus is obtained efflux body described in first aspect present invention by ().

In another preference, described separation comprises standard four step centrifuging.

Sixth aspect present invention, provides a kind of expression vector of knowing clearly, and described expression vector contains film expression type TGF-β 1 gene, and described film expression type TGF-β 1 gene comprises TGF-β 1 gene be connected with film expression homing sequence operability.

In another preference, described film expression homing sequence comprises TM protein sequence and GPI anchorin sequence.

In another preference, described TM protein sequence comprises the TM albumen coming from HO8910 clone, and its sequence is as shown in SEQ ID NO.:1; Or come from gastric carcinoma cell lines MKN-7, the nucleotide sequence Genbank accession number of shown stomach cancer cell MKN-7 is X03363.1, GI:31197, CDS is 175-3939, wherein, the nucleotide sequence of encode transmembrane section albumen, as shown in SEQ ID NO.:9, corresponds to the albumen of the 654-678 of gastric carcinoma cell lines MKN-7, and its sequence is as shown in SEQ ID NO.:10.

In another preference, described expression vector is adenovirus carrier.

In another preference, described HO8910 clone cross-film section consecutive nucleotides sequence is as shown in SEQ IDNO.:1; The protein sequence of its coding is as shown in SEQ ID NO.:4.

In another preference, described TGF-β 1 gene order Genbank accession number NM_011577GI:6755774, its sequence is as shown in SEQ ID NO.:2.

Seventh aspect present invention, provides a kind of method preventing and/or treating autoimmune disorder, uses the efflux body described in first aspect present invention of safe and effective amount or the pharmaceutical composition described in second aspect present invention to required object.

In another preference, described object is Mammals, more preferably, is people.

Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.

Accompanying drawing explanation

Fig. 1 shows Ad-mTGF-β 1 and to check order peak figure.

Fig. 2 shows recombination adenovirus construction strategy.

Fig. 3 shows the phenotype that Ad-mTGF-β 1 infects rear BMDCs, namely uses the protein marker on Flow cytometry BMDCs surface after adenovirus infection.

Fig. 4 shows the function that Ad-mTGF-β 1 infects rear BMDCs, and after Fig. 4 A shows adenovirus infection, DCs resists the ability that LPS induces IL-12P70 secretion; Fig. 4 B shows the effect in mixed lymphocyte reacion of DCs after adenovirus infection.

Fig. 5 shows the phenotype of mTGF-β 1-EXO, and Fig. 5 A shows the form of efflux body under Electronic Speculum, scale=200 nanometer; Fig. 5 B shows application fluorescence antibody mark, the phenotype of flow cytometer showed efflux body; Fig. 5 C shows the WB detected result of efflux body characteristic protein; Fig. 5 D shows the expression of TGF-β 1 in flow cytometer detection efflux body; After Fig. 5 E shows the latex particle seizure efflux body adsorbing anti-TGF-beta 1 monoclonal antibody in advance, the expression level of CD9 on flow cytometer detection latex particle.

Fig. 6 shows the severity that mTGF-β 1-EXO treatment can alleviate EAE, and Fig. 6 A shows the impact that preventive administration is fallen ill on EAE; Fig. 6 B shows the impact that therapeutic is fallen ill on EAE; Fig. 6 C shows EAE Mice brain tissues and observes inflammatory cell infiltration situation through H & E dyeing; Demyelinating Condition is observed in LFB dyeing, and magnification is 400.

Fig. 7 show mTGF-β 1-EXO suppress MOG cause T cell propagation and Th1 polarization, Fig. 7 A show MOG peptide stimulated in vitro EAE mouse originate splenocyte or CD4 ⁺after T lymphocyte, AlamarBlue method detects cell proliferative conditions.The ELISA method that shows Fig. 7 B detects spleen or the CD4 of the stimulation of MOG peptide ⁺the level of IFN-γ and IL-10 in T lymphocyte supernatant.

Fig. 8 shows the differentiation that mTGF-β 1-EXO suppresses Th17.Fig. 8 A shows the change of IL-17 in flow cytometer detection different treatment group mice serum; Fig. 8 B shows the change of Th17 cell proportion in Flow cytometry different treatment group mouse spleen; Fig. 8 C shows the change of Th17 cell proportion in Flow cytometry different treatment group mouse inguinal lymph nodes.

Fig. 9 shows mTGF-β 1-EXO and induces Treg to alleviate the occurring degree of EAE.Fig. 9 A shows after AmTGF-β 1-EXO treats, the ratio of Tregs in EAE mouse spleen and inguinal lymph nodes lymphocyte.Fig. 9 B shows the CD4 that normal mouse feeds back each group of EAE mouse source separately ⁺cD25 ⁺carry out EAE induction after Tregs, observe the EAE incidence of mouse every other day.

Figure 10 shows the EAE morbidity that mTGF-β 1-EXO can alleviate allogeneic BALB/c mouse.Figure 10 A shows the propagation of the mTGF-β 1-EXO vitro inhibition BALB/c mouse T cell in C57BL/6 mouse source; Figure 10 B shows the external evoked BALB/c mouse Treg of mTGF-β 1-EXO in C57BL/6 mouse source; After Figure 10 C shows 1 week and 4 weeks that feeds back efflux body, ELISA detects the level of IgE and IgG in BALB/c mouse serum.Figure 10 D shows the effect of the mTGF-β 1-EXO treatment BALB/c mouse EAE in C57BL/6 mouse source.

Embodiment

The present inventor is through extensive and deep research, prepare a kind of secretion first from immature dendritic cell, the efflux body with immunosuppressive action, after efflux body of the present invention adopts film expression type immuno-suppressing cytokine genetic modification, its vesica film has the TGF-β 1 of transmembrane, the character of mTGF-β 1 genetic modification imDCs can be reflected, there is very strong immunosuppressant function.The CD4 that mTGF-β 1-EXO of the present invention can suppress CFSE to mark ⁺t lymphopoiesis, and the generation of a large amount of induction regulatory T cells.In addition, experiment finds, mTGF-β 1-EXO can alleviate EAE symptom by the Th17 cell lowering mouse spleen and lymphoglandula, and research shows that mTGF-β 1-EXO suppresses to produce IL-6 through the DCs of LPS activation further.Sorting treats the CD4 of the mouse spleen after EAE through each group of exosomes ⁺foxp3 ⁺regulatory T-cell, by its adoptive transfer in normal mouse body, and then induce EAE, result shows that the Treg that mTGF-β 1-EXO treatment group spleen is originated significantly suppresses developing of EAE, and the LacZ-EXO in the DCs source of transfection Ad LacZ, the function of the Treg inflammation-inhibiting in the Control-EXO in untreated fish group DCs source, sTGF-β 1-EXO and untreated group source weakens greatly.The more important thing is, the mTGF-β 1-EXO in C57BL/6 mouse source can break through the restricted of MHC, also can have therapeutic action to the EAE of the BALB/c mouse that allogeneic PLP induces, and therefore it is expected to the general preparation becoming treatment autoimmune disorder.On this basis, the present invention is completed.

In addition, present invention also offers the pharmaceutical composition containing efflux body of the present invention and therepic use thereof.

Efflux body

Efflux body is the secretion of various viable cell, and non-plasma membrane source, diameter is between the vesicles with lipid bilayer Rotating fields of 50-100nm.This vesicles indicates as CD9, CD63, CD81 and CD82 jointly except the efflux body containing different cell derived, heat shock protein A subunit, HSP90, Tsg101, the protein moleculars such as Alix, MHC-I, MHC-II molecule also containing reflection imDCs cell characteristics, the protein that CD80, CD86, ICAM-I etc. are special, and express lower than mDCs.

Film expression cytokines

Body based intracellular cvtokine deposits mainly with secretor type form, but also have a few cell factor to be present in cell surface in the mode of transmembrane.Research show the biologic activity of secretor type and transmembrane cytokine and the mode of action not the same.Such as, Transmembrane TNF α is by cell and cell contact, play a role in local, Secretary TNF α then can enter circulation of blood and act on remote histoorgan, and Transmembrane TNF α kills knurl spectrum more extensively than Secretary TNF α, it effectively can kill and wound the tumour cell to Secretary TNF α tolerance.In addition, CD4 is expressed in ⁺the TNF-α of the secretor type of T cell and membrane-bound TNF defend antileishmanial ability also obviously different at activated macrophage.

Film expression cytokines used in the present invention is the film expression cytokines with immune suppression function, and described film expression cytokines can be that natural existence or synthetic are expressed.It can be that the trans-membrane region encoding sequence of the cytogene of inhibitive ability of immunity and specific cells is spliced rear formation that a kind of preferred synthetic expresses film expression cytokines.

A kind of preferred inhibitive ability of immunity cytokine used in the present invention is TGF-β 1.The feature of the exosomes in the powerful immunoloregulation function had in view of TGF-β 1 and imDCs source, therefore the cytokine of secretor type is probably wrapped up by exosomes, then TGF-β 1 function can be caused impaired.

TGF-β 1 adenovirus carrier (the Adenovirus expressing membrane-associated TGF-β 1 containing TM cross-film section that the present invention adopts the method for molecular bypass to build, Ad mTGF-β 1), can see from the amino acid nature in coding TM district, most amino acid is hydrophobic, and this is conducive to its cross-film effect.Extract the exosomes in the imDCs source of Ad mTGF-β 1 genetic modification, find to there is its character that can reflect mTGF-β 1 genetic modification imDCs very strong immunosuppressant function, alleviate EAE symptom further.

In addition, other inhibitive ability of immunity cytokines are connected the exosomes that also can be used for producing the imDCs containing film expression type and originate with transmembrane protein.Such as, can be used for inhibitive ability of immunity cytokine of the present invention and also comprise TGF-β 1, IL-10, IL-4, FasL or its combination.Described TM protein sequence also can comprise the TM albumen coming from HO8910 clone, and its sequence is as shown in SEQ ID NO.:1; Or coming from the TM albumen of gastric carcinoma cell lines MKN-7, its sequence is as shown in SEQ ID NO.:10, and the nucleotide sequence of this albumen of encoding is as shown in SEQ ID NO.:9.

TGF-β 1 and film expression type TGF-β 1 (mTGF-β 1)

Transforming Growth Factor-beta1 (TGF-beta1) is a kind of containing 112 ammonia amino acid, protein dimer (the Genbank accession number AAH13738GI:15489275 of molecular weight 25KD, its protein sequence is as shown in SEQ ID NO.:3, and the nucleotide sequence of this albumen of encoding is as shown in SEQ ID NO.:2.

(between each kind high conservative, such as people and rat, mouse have identical aminoacid sequence for TGF-β 1.Nearly all cell has the acceptor of TGF-β 1, and it is very widely on the growth of cell, the impact of differentiation.

TGF-β 1 plays a significant role in maintenance immunological homeostasis, and the mouse that TGF-β 1 lacks has the inflammatory reaction of serious multiple organ and multiple MHC that organizes raises extremely.Close TGF-β 1 signal of T cell, mouse suffers from the autoimmune disorder for feature with lung and colon inflammatory cell infiltration, and the antibody of autoimmunity detected in blood.

As used herein, term " film expression type TGF-β 1 ", " transmembrane TGF-β 1 " and " film mating type TGF-β 1 " are used interchangeably, all refer to that N that imDCs is connected with cell transmembrane section TM holds be positioned at the outer C of cytolemma and hold and be positioned at cytolemma, TGF-β 1 factor of cell can be directly acted on.The nucleotide sequence of a kind of preferred film expression type TGF-β 1 is as shown in SEQ ID NO.:11.

In the present invention, on the cell that film expression type TGF-β 1 can be positioned at imDC cell or cytolemma.Preferably, the TGF-β 1 of about 20-30%% can localization and expression on imDCs, and its N end is positioned at outside cytolemma.The vesica of Exosomes to be the diameter discharged by viable cell the be lipid bilayer of 50-100nm, a kind of subcellular organelle many vesicas body (Multivesicular Bodies in they and cell, MVBs) after combining, exosomes is formed by internal membrane blastogenesis effect, then by the direct fusion of MVBs and cytolemma, exosomes is released in extracellular matrix.Therefore Exosomes is being carried in the process of exocytosis by endochylema in cell and some albumen on cytolemma and molecule.Exosomes has several functions, and this depends on the character of the cell that they are originated.

Experiment shows, Th1 reaction and the IFN-γ of mTGF-β 1-EXO minimizing antigen-specific produce, but promote the generation of IL-10; MTGF-β 1-EXO reduces EAE mouse Th17 and produces, and P38, Erk, Stat3 and NF-κ B of this DCs that may activate with its downward expresses thus to cause IL-6 to decline relevant; In addition mTGF-β 1-EXO increases the generation of spleen and lymphoglandula Treg, adoptive transfer mTGF-β 1-EXO treatment group CD4 ⁺foxp3 ⁺regulatory T cells obviously reduces the morbidity of EAE in mice.

Immature dendritic cell

Dendritic cell (Dendritic Cells, DCs), as the strongest professional antigen presenting cells of function, plays effect in the generation, progress of EAE.

Ripe DCs expresses high-caliber costimulatory molecules and major histocompatibility complex MHC-II, activation such as proinflammatory cytodifferentiation such as induction Th1, Th17, B cell and scavenger cell etc., the hemato encephalic barrier that the autoreactivity inflammatory cell after activation and the cytokine of generation thereof and demyelination antibody pass through to damage is circulated by body and enters CNS.These cells trigger sequence of events by secretion inflammation cytokines, produce myelin and the microglia infringement of immune response mediation, thus make aixs cylinder exposed, can not conduction action potentials effectively, cause neurologic symptom.

Immature DCs (immature Dendritic Cells, imDCs) expresses low-molecular-weight MHC-II and costimulatory molecules, and inducing T cell anergy in vitro, also can induce CD4 ⁺cD25 ⁺regulate T (regulatory T cells, Tregs) cell, Th3 or Tr1 differentiation, these cells pass through the cytokines such as secretion IL-10, TGF-β 1, soluble HLA-G, or by contacting with T cell or interacting to play the symptom that immunoloregulation function alleviates EAE with DCs.But imDCs is under inflammatory environment effect, may be ripe further, thus form mDCs.

Autoimmune disorder

Autoimmune disorder (autoimmune diseases), refers to that body causes the disease caused by damaged self tissue to autoantigen generation immune response.In the present invention, autoimmune disorder comprises nervus centralis Demyelination or local inflammation pathology.

As used herein, nervus centralis Demyelination refers to that a class cause of disease is not identical, clinical manifestation is different but has the acquired illness of roughly the same feature, and the pathological change of its feature is the myelinoclasis of nerve fiber and neurocyte keeps complete relatively.Because the cause of disease that nervus centralis Demyelination is common is that nervus centralis local inflammation venereal disease becomes, or nervus centralis Demyelination can coexist with local inflammation pathology, therefore, nervus centralis Demyelination of the present invention also comprises the Demyelination that local inflammation pathology causes.

In another preference, described nervus centralis Demyelination comprises multiple sclerosis (MultipleSclerosis, MS), experimental autoimmune encephalomyelitis (Experimental AutoimmuneEncephalomyelitis, EAE), acute disseminated encephalomyelitis, diffuse cerebrosclerosis, optic neuromyelitis etc.; Secondary cases demyelinating disease causes primarily of systemic disease, as Periventricular Leukomalacia in Prematures disease etc.In experimental zoology, being most widely used also the most typical nervus centralis demyelinating disease animal model is experimental autoimmune encephalomyelitis.

Experimental animal model

Experimental autoimmune encephalomyelitis (Experimental AutoimmuneEncephalomyelitis, EAE) be with central nervous system (central nervous system, CNS) local inflammation and demyelination are the autoimmune disorder of principal character, it is the important experimental model of research multiple sclerosis (multiple sclerosis, MS) pathogeny and treatment plan.

Research shows, uses MOG _35-55immature dendritic shape cell TGF-beta receptor signal inactivated mice, find that wherein neural system has a large amount of T cell to infiltrate, there is high-caliber Th1 and Th17 cytokine periphery, and EAE symptom can not be alleviated.After the MOG-TCR mouse hybrid having autoimmunization to be inclined to, show and infiltrate with the myelin specific T-cells of early stage a large amount of activation, microglia, mouse movement is lacked of proper care, and premature death is the idiopathic EAE symptom of feature.

The TGF-β 1 giving doses through nasal feeding can suppress delay-relapsing EAE, and research shows that this suppresses DCs to produce nitrogen protoxide by TGF-β 1 and realizes further, and the latter again can induction of T cell apoptosis.TGF-β 1 can also by Smad4 promote to have the IL-10+IFN γ of immunoregulation effect+Th1 cell produce, the latter stops the inflammatory cell recruitment such as Th1 and Th17 to central nervous system by secretion IL-10, thus alleviation EAE symptom.

Pharmaceutical composition

Present invention also offers a kind of pharmaceutical composition.Described pharmaceutical composition contains the efflux body of the present invention of pharmaceutically acceptable carrier (comprising thinner, vehicle etc.) and significant quantity as activeconstituents.The quantity of efflux body of the present invention is generally 10 microgram-100 milligrams/agent, is preferably 100-1000 microgram/agent.

As used herein, term " significant quantity " refers to therapeutic agent treats, alleviation or prevents the amount of target disease or situation, or shows the amount of detectable treatment or preventive effect.Accurate significant quantity for a certain object depend on the build of this object and healthy state, symptom nature and extent and select the combination of therapeutical agent and/or the therapeutical agent given.

In order to the object of the invention, effective dosage for giving individuality about 0.01 mg/kg to 50 mg/kg, the preferably efflux body of the present invention of 0.05 mg/kg to 10 mg/kg body weight.

Pharmaceutical composition is also containing pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to the carrier being used for the treatment of agent (such as efflux body) administration.This term refers to some medicament carriers like this: they itself are not induced and produce the antibody harmful to the individuality accepting said composition, and do not have undue toxicity after administration.These carriers are well-known to those skilled in the art.

In therapeutic drug composition Chinese materia medica, acceptable carrier can contain liquid, as water, salt solution, glycerine and ethanol.In addition, in these carriers, also may there is complementary material, as wetting agent or emulsifying agent, pH buffer substance etc.In addition, immunological adjuvant can also be contained in immune composition.

Usually, therapeutic composition can be made injectable agent, such as liquor or suspension; Be applicable to allocating in solution or suspension before also can be made into injection, the solid form of liquid vehicle.

Once be made into composition of the present invention, directly object can be given by it.Wait that the object preventing or treat can be animal, especially people.

Treatment containing efflux body of the present invention or prophylactic medicament, can oral administration, subcutaneous, intracutaneous, in chamber, diseased region, intraarticular, Jing arteries and veins Zhu She Alto which apply, therapeutic dose scheme can be single dose regimen also can be multiple doses.

Beneficial effect of the present invention:

1. efflux body of the present invention has good immunosuppressant effect, thus treatment autoimmune disorder.

2. efflux body vesica film of the present invention has or shows film expression type TGF-β 1, can effectively Immunosuppression cell proliferation, differentiation, and cause Treg breaks up, and treats EAE.

3. efflux body of the present invention can suppress the expression of MHC-II, thus does not limit by MHC-II, gives full play to the effect of Immunosuppression, safely, effectively.

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number are weight percent and parts by weight.

The structure of the adenovirus carrier of the recombinant replication-defective of embodiment 1 containing film expression type TGF-β 1 gene, and the packaging of recombinant adenovirus Ad-mTGF-β 1

Method:

By the method for pcr amplification, with the cDNA of 3LL cell (given by the immunity of Shanghai The 2nd Army Medical College) for template, according to people TGF-β 1 full-length gene order meter primer, its total serum IgE is extracted from ovarian cancer cell line HO8910 cell (given by the immunity of Shanghai The 2nd Army Medical College), after reverse transcription, cDNA is template, with two for Cerb-B2 gene order (Genbank accession number: M11730.1; GI:183986) Auele Specific Primer (as Suo Shi SEQ ID NO.:7 and 8) in encode transmembrane (Transmembrane, TM) district fragment (2041-2161 position) in, pcr amplification TM fragment; Montage overlap-extension PCR (Splicing Overlap Extension, SOE) method is adopted to connect TGF-β 1 and TM two gene fragments; Fusion gene TM-TGF-β 1 is connected with cloning vector pDC315 orientation.

After order-checking is correct, by there being the plasmid co-transfection packing cell Hek293 of goal gene mTGF-β 1 recombinant shuttle plasmid and band viral backbone, obtain recombinant adenovirus (see accompanying drawing 1) by homologous recombination.Specific as follows:

1) from 3LL cell, total serum IgE is extracted, with TGF-β 1 primer

Forward：5'-ATTGAATTCATGCCGCCCTCGGGGCTGCGGCTAC-3'(SEQ ID NO.:5)；

Reverse:5'-GCTCTCTGCTCGGCGCTGCACTTGCAGGAGCGCA-3'(SEQ ID NO.:6) carry out reverse transcription;

From HO8910 cell, extract total serum IgE, carry out reverse transcription with TM primer

Forward：5'-TGCGCTCCTGCAAGTGCAGCGCCGAGCAGAGAGC-3'(SEQ ID NO.:7)；

Reverse：5'-CGTGTCGACCGTGTACTTCCGGATCTTCTGCTGC-3'(SEQ ID NO.:8)。

TGF-β 1 fragment PCR reaction conditions is: 95 DEG C of 5min; 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 80s, 35 circulations of increasing; 72 DEG C of 10min.

TM fragment PCR reaction conditions is: 95 DEG C of 5min; 94 DEG C of 30s, 63 DEG C of 30s, 72 DEG C of 11s, 35 circulations of increasing; 72 DEG C of 10min.After PCR, cut respective strap, gel reclaims.

2) survey gel reclaim after TGF-β 1 and the concentration of TM, add the two by equimolar ratio, with the rear chain of the front chain of TGF-β 1 and TM section for primer, carry out bridging PCR, gene fusion construct mTGF-β 1.

PCR reaction conditions is 95 DEG C of 5min; 80 DEG C of 3min; 94 DEG C of 30s, 55 DEG C of 40s, 72 DEG C of 90s, 35 circulations of increasing; 72 DEG C of 10min.

3) gel reclaims object band, and after cutting pDC315 plasmid and TGF-β 1-Tm with restriction endonuclease ECOR I and SaL I enzyme, 16 DEG C of connections are spent the night, and after YC conversion method transforms, random choose 6 bacterium colonies do PCR qualification, and corresponding bacterium colony shakes bacterium.Extract the plasmid of the positive bacterium of PCR qualification after 16h, do enzyme and cut qualification, positive send company to carry out two-way order-checking, the results are shown in Figure 2.

4) before transfection 24h Hek293 with 4 × 105/hole bed board, plasma-free DMEM medium 1ml is changed after cytogamy degree is greater than 80%, by specification adds liposome 10 microlitre simultaneously, 3 μ g pBHGlox Δ E1,3 μ gCre (given by the immunity of Shanghai The 2nd Army Medical College), the pDC315 containing TGF-β 1-Tm gene fragment of 2 μ g, mends complete DMEM substratum 1ml after 6 hours.Observation of cell changes, timely fluid infusion.After 10 days, collecting cell and supernatant, carry out PCR qualification and streaming qualification.

The induction of embodiment 2:BMDCs, amplification and adenovirus infection

Cultivate BMDCs, after infecting 24 hours, cleaned by remaining adenovirus PBS with Ad-mTGF-β 1, BMDCs continues cultivation 48 hours, collects supernatant, for extracting mTGF-β 1-EXO.Concrete operations are as follows:

1) cultivation of BMDCs: Cells Derived from Dendritic was in normal six weeks female C57BL/6 (H-2Kb) mouse, aseptic taking-up mouse femur and shin bone, medullary cell is gone out with serum-free RPMI1640, abolish after red corpuscle through Tris-NH4Cl, in cultivating containing in 10% foetal calf serum RPMI1640, add the GM-CSF of 20ng/ml, the IL-4 of 1ng/ml in 37 DEG C, 5%CO ₂middle cultivation, after 48 hours, washes away non-adherent cell with the substratum of pre-temperature, and attached cell DC substratum continues cultivation 48 hours, blows and beats the results DCs of the 5th day gently and is immature DC s.

2) Ad-mTGF-β 1 infects BMDCs: the DCs accurate counting cultivating the 5th day, then use Ad-sTGF-β 1 (secretion-type T GF-β 1) and Ad-mTGF-β 1 (transmembrane TGF-β 1) or Ad-Lac Z with MOI (multiplicity of infection, MOI)=100 transfectional cells, and establish the blank group not turning virus.

Transfection process is as follows: 1 × 10 ⁷/ ml DCs is resuspended in the eppendorf pipe of 2ml with serum free medium, adds corresponding virus liquid, shakes 2 hours in 37 DEG C of constant-temperature tables with 60 rpms.With 2 × 10 after 2 hours ⁶/ ml supplies perfect medium, puts 37 DEG C of incubators and continues to cultivate centrifugal segregation substratum after 18h, and wash twice to wash away residual virus with PBS.Cell continues to cultivate at DC substratum afterwards, and in substratum, contained foetal calf serum eliminates serum origin efflux body through 100,000g × 1h ultracentrifugation in advance.After 48 hours, collecting cell is used for the analysis of phenotype and function, and preserves the detection of cells and supernatant for cytokine and the preparation of efflux body.

The isolation and purification of embodiment 3:mTGF-β 1-EXO

Standard four step centrifuging the cells and supernatant of aforementioned reservation is adopted to prepare efflux body.Specific as follows:

At 4 DEG C, high speed centrifugation 300g × 10min, 1200g × 20min, 10000g × 20min, remove cell debris and other aggegation material; Then ultracentrifugation 100000g × 60min, institute obtains precipitation with a large amount of PBS resuspended ultracentrifugation again 100000g × 60min to remove residual media, and gained precipitation is purer efflux body.Efflux body is resuspended with a small amount of PBS, adopts BCA method to measure its protein content.The efflux body that separation obtains is through being placed in-80 DEG C of preservations after packing.

Embodiment 4BMDCs infects the qualification of the rear phenotype of Ad-mTGF-β 1

The different adenovirus of transfection by Flow cytometry also continues the expression of the surface molecular of the DCs of cultivation after 24 hours, operate as follows: the DCs of collection different treatment group, 1 × 10 ⁶dCs cell be resuspended in 100 μ lPBS, require the fluorescent-labeled antibody adding corresponding dosage to specifications: MHC-II, CD80, CD86, CD40 and CD11c, hatch 30min on ice, after then washing three times with PBS, detect with flow cytometer, CellQuest software analysis.

Result is as shown in Figure 3: wherein, and Ad-sTGF-β 1 and Ad-LacZ represents the control group with the adenovirus infection containing secretion-type T GF-β 1 and LacZ sequence respectively, and Control is without adenovirus infection group.

Result shows, each group all expresses mouse DCs surface markers CD11c, and its surperficial MHC-class Ⅱmolecule of the Control group DCs of untransfected adenovirus, costimulatory molecules are as all very low in the expression of CD86, CD80, meet the phenotypic characteristic of immature DC s.And after infecting Ad LacZ, not making the phenotypic characteristic of this cell change, this shows that Ad-LacZ can not affect the maturation of DCs.After Ad-mTGF-β 1 transfection, MHC-II, the CD80 on DCs surface expresses and is all suppressed, and wherein the most obvious to the restraining effect of MHC-II, although Ad sTGF-β 1 also can suppress the expression of the MHC-II of DCs, restraining effect is not as good as the former.

Embodiment 5BMDCs infects the qualification of the rear function of Ad-mTGF-β 1

The immature DC s surface costimulatory molecules expression level such as MHC molecule and CD86 is very low, can not provide required activation signals for T cell, and therefore the ability of its activated T cell, induction of immunity reaction is very weak.Immature DC s can be ripe rapidly and secrete IL-12 under the stimulation of LPS.Therefore infect the change of the rear function of Ad-mTGF-β 1 to observe BMDCs, the present embodiment have detected the secretion change of BMDCs IL-12p70 under LPS stimulates and the effect in mixed lymphocyte reacion thereof of the different adenovirus of transfection, specific as follows:

1) ELISA of IL-12p70 detects: collect above-mentioned each group of transfection adenovirus 24 hours and the DCs of untransfected adenovirus, and with the lipopolysaccharides (lipopolysaccharide of 1 μ g/ml, LPS) stimulate, and establish corresponding non-stimulated control group, often organize 3 multiple holes.Continue cultivation 24 hours, collect culture supernatant, detect the concentration of IL-12p70 with double antibody sandwich ELISA.

2) mixed lymphocyte reacion: get BALB/c (H-2Kd) mouse spleen, the splenocyte suspension of aseptic preparation mouse, magnetic bead sorting goes out CD4 ⁺t cell, adjustment CD4 ⁺t cell concentration is 2 × 10 ⁶/ ml, adds 96 orifice plates with every hole 100 μ l volume.Derive from each group of DCs of C57BL/6 (H-2Kb) mouse bone marrow cells after getting above-mentioned transfection adenovirus 24h, add 50 μ g/ml ametycins, after 37 DEG C of water-baths deactivation in 30 minutes, PBS washes twice, and adjusting its concentration is 2 × 10 ⁵/ ml, adds 96 orifice plates with DC:T=1:10,1:20,1:40, supplies substratum and makes its final volume be 200 μ l, often organize and all establish three wells.37 DEG C hatch 4 days afterwards every hole add 20 μ l alamar Blue dyestuffs and dye.Continue to hatch with 535nm excitation wavelength after 24 hours, 595nm emission wavelength, fluorescence intensity.

Result as shown in Figure 4 shown in:

Fig. 4 A shows: comparatively control group is low for the level of transfection Ad-mTGF-β 1 and Ad-sTGF-β 1 rear DCs secretion IL-12p70, after LPS stimulates, and untreated Control DCs and LacZ DCs energy maturation rapidly, and the level of secreting IL-12p70 obviously increases.And the IL-12p70 secretion level lifting amplitude of Ad-mTGF-β 1-DCs and Ad-sTGF-β 1-DCs is less, causing maturation factor to LPS etc. after DCs transfection Ad-m TGF-β 1 and Ad s-TGF-β 1 is described has resistant function, but the former effect is better than the latter.

Fig. 4 B shows: after transfection Ad-mTGF-β 1 and Ad-sTGF-β 1, and DCs stimulates the ability of T cell propagation to be obviously suppressed.Therefore there is through the BMDCs of Ad-mTGF-β 1 transfection the functional character of immature DC s.

The phenotypic evaluation of embodiment 6mTGF-β 1-EXO

Be separated mTGF-β 1-EXO, and by transmission electron microscope, Western Blot, flow cytometry, its phenotype analyzed, specific as follows:

The electronic microscope photos of 6.1 efflux bodies: efflux body suspension PBS washes twice, with stationary liquid (2% paraformaldehyde, 0.25% glutaraldehyde) 4 DEG C fix 1 hour, wash with PBS and once make suspension and drip sheet, 1% osmic acid is fixed, gradient alcohol dehydration, takes the photograph sheet in the electric Microscopic observation of projection after the plumbous uranium dyeing of ultrathin section(ing).

As shown in Figure 5A, the efflux body that we are separated has typical lipid bilayer Rotating fields to result, and is the vesicles that diameter concentrates on 50-100nm.

6.2Western blot analyzes: efflux body measures protein concentration through BCA method, add sample-loading buffer, 5min is boiled in boiling water, in 12%SDS-PAGE electrophoretic separation, every hole adds 20 μ g efflux bodies, electrotransfer is on nitrocellulose filter, after 5% skimmed milk is closed, add mouse anti-mouse Hsp70, anti-mouse Grp94 respectively, sheep anti-Mouse Tsg101, rabbit anti-mouse TGF-β 1 polyclonal antibody, combine at 4 DEG C and to spend the night hour, PBST fully washs, add corresponding two anti-binding 1.5 hours, develop by enhanced chemiluminescence test kit specification sheets.

As shown in fig. 5-c, wherein Lac-Z-EXO and sTGF-β 1-EXO refers to go out efflux body from separation and purification the BMDCs supernatant having infected Ad-LacZ and Ad-sTGF-β 1 respectively result.

Result shows, each group efflux body all expresses efflux body associated protein Hsp70 and Tsg101, CD63, does not express endoplasmic reticulum molecular chaperone Grp94, and only has mTGF-β 1-EXO and sTGF-β 1-EXO to express TGF-β 1.

The FACS qualification of 6.3 efflux body phenotypes: efflux body adds 5 × 10 with saturated amount ⁵/ 100 μ l latex particles, after incubated at room 30min, adding PBS to final volume is 1ml, 37 DEG C of incubator overnight.Then the latex particle of absorption efflux body is divided equally, add fluorescent-labeled antibody respectively: TGF-β 1, MHC-II, CD80, CD86, CD40 and CD11c, final concentration is 5 μ g/ml, 20min is hatched under normal temperature, after washing twice with PBS, detect with flow cytometer, CellQuest software analysis.

Result is as shown in Figure 5 B: each group efflux body all expresses mouse DCs surface characteristic marker molecule CD11c, and MHC-II expresses hardly at mTGF-β 1-EXO, and CD80 and control group are than also lowering to some extent.This also illustrates that efflux body can reflect the characteristic of its DCs originated, and has certain immunoregulation effect.

The expression-form of 6.4 checking TGF-β 1 on efflux body: the expression (Fig. 5 D) TGF-β 1 being detected on mTGF-β 1-EXO instead of sTGF-β 1-EXO.In order to verify the expression-form of TGF-β 1 on efflux body further, the monoclonal antibody of 5 microlitre latex particles and 50 microgram anti-TGF-β 1 is hatched 1h at normal temperatures, then centrifugal, close with FCS, the latex particle of anti-TGF-β 1mAb bag quilt and the efflux body of 20 μ g are in 4 DEG C of incubator overnight.After PBS washing, mark PE-anti-CD9 antibody, FACS detects the expression of CD9.

Result is as shown in fig. 5e: mTGF-β 1-EXO can be caught by the latex particle of anti-TGF-β 1mAb bag quilt, FACS detects CD9 and expresses positive, the sTGF-β 1-EXO of expression-secretion type TGF-β 1 does not then express CD9, and this illustrates that in mTGF-β 1-EXO, TGF-β 1 is film expression more.

Embodiment 7mTGF-β 1-EXO prevention and therapy can alleviate the evaluation of EAE effect

Adopt the EAE model of mouse, and apply mTGF-β 1-EXO prevention and therapy intervention is carried out to it, its effect is evaluated.Specific as follows:

1) EAE induction and disease progression assessment

By MOG35-55 (purchased from the raw work in Shanghai) or PLP _180-199(purchased from the raw work in Shanghai) mixes with equal-volume complete Freund's adjuvant with tuberculin, repeatedly lashes and makes stable water-in-oil-type antigen emulsion.The MOG of every C57BL/6 mouse 3 subcutaneous injection 200 μ g _35-55with 200 μ g tuberculin, the PLP of every Balb/c mouse 3 subcutaneous injection 300 μ g _180-199with 200 μ g tuberculin, and after immunity the 0th and 48h tail vein injection 200ng novain.

Adopt double-blind method to assess mouse, Continuous Observation 36 days, carries out Neuroscore by following standard: 0 point, without exception; 1 point, reduce under tail tension force; 2 points, hind leg paresis, instability of gait and/or wave; 3 points, hind leg is not exclusively paralysed; 4 points, hind leg is paralysed completely; 5 points is moribund condition, hind leg companion fore-limb paralysis.We find that the 10th day mouse starts morbidity, and peak at 14-16 days EAE mouse invasions, severe patient clinical manifestation is hind limb paralysis, even dead.

2) EAE mouse Prevention and Curation

The female C57BL/6 in 6-8 week is equally divided into five groups, often organizes 10, is grouped as follows:

mTGF-β1-EXO

sTGF-β1-EXO

LacZ-EXO

Control-EXO

No treatment

Respectively in first 8 days of modeling, first 5 days and first 2 days through tail vein injection 10 μ g/ each group of efflux body only, at the 0th day induction EAE, record scoring, observed and compares mTGF-β 1-EXO, sTGF-β 1-EXO to the prophylactic effect of EAE.Further detection mTGF-β 1-EXO, sTGF-β 1-EXO, to the therapeutic action of EAE, at each group of efflux body of 14 days, 17 days and 21 days tail vein injections and prevention group same dose being in onset peak period, observe scoring.

Result is as shown in figure 6 a and 6b: the Primary preventive intervention of mTGF-β 1-EXO and sTGF-β 1-EXO obviously can reduce the scoring of EAE, but the former than the latter's effect more obviously (Fig. 6 A).And after EAE modeling, gave mTGF-β 1-EXO treated at the 14th, 17 and 21 day, can significantly improve EAE symptom, clinical score decreases compared with other treatment group efflux body, and some animals even can complete incidence graph (Fig. 6-B).

3) EAE mouse tissue pathology detect

In treatment after two weeks, 4% paraformaldehyde heart perfusion, rapid separation cerebral tissue, 4% neutral formalin stationary liquid is fixed, 6 micrometer thick sections are made after paraffin embedding, routine hematoxylin-Yihong (HE) dyeing and cresyl viollet dyeing (Luxol fast blue, LFB), and in light Microscopic observation inflammatory cell infiltration and disentwining angle velocity situation.

Result is as shown in Figure 6 C: mTGF-β 1-EXO obviously reduces EAE mouse tricorn inflammatory cell infiltration and Demyelinating Condition, through sTGF-β 1-EXO treatment group then without this effect.

Conclusion: above result shows that mTGF-β 1-EXO has strong immunosuppression capability, can the developing of EAE of prevention and prohibition C57BL/6 mouse effectively.

The T cell propagation that embodiment 8mTGF-β 1-EXO suppresses MOG to cause and the evaluation of Th1 polarization effect

The progress of ART and Th1 cell and EAE is closely related, and therefore the present embodiment observes the impact of T cell propagation that mTGF-β 1-EXO causes MOG and Th1 polarization effect.

Method:

After Exosomes treats two weeks, be separated each treatment group Mouse spleen cells and CD4 ⁺t cell, 2 × 10 ⁵/ hole is laid on 96 orifice plates, and adds the MOG of 10 μ g/ml _35-55, at CD4 ⁺t cell group, adds 2 × 10 with the every hole of the ratio of 1:10 ⁴the DCs cell of individual ametycin deactivation, often group establishes three multiple holes, and cultivate after 48 hours and add 20 μ l alamarBlue, DTX880multimode detector (Beckman Coulter, PaloAlto, CA) detects its fluorescence intensity.Under identical culture condition, cultivate 72 h before harvest respectively organize supernatant, ELISA detects the expression amount of IL-10, IL-6, IL-17 and IFN-γ in each group of supernatant.

Result shows: after by 10 μ g/ml MOG peptide stimulated in vitro 3 days, can observe spleen and the CD4 in the EAE mouse source that mTGF-β 1-EXO treats ⁺t lymphopoiesis, compared with other treatment group mouse, obviously suppressed (Fig. 7 A).In addition, mTGF-β 1-EXO treats the spleen and CD4 that the EAE mouse of MOG peptide stimulated in vitro can also be suppressed to originate ⁺the secretion of T lymphocyte IFN-γ, promotes the secretion (Fig. 7 B) of IL-10.

Conclusion: the result for the treatment of to EAE that mTGF-β 1-EXO produces may polarize suppressed relevant with the propagation of autoreactivity Th cell and Th1.

Embodiment 9mTGF-β 1-EXO suppresses the evaluation of the differentiation effect of Th17

The serum application ELSA getting mouse in treatment after two weeks detects IL-17 level, and has been separated the lymphocyte of mouse spleen and inguinal lymph nodes, have detected the distribution of Th17.

Result shows that the mouse IL-17 expression amount that mTGF-β 1-EXO treats is starkly lower than other groups as shown in Figure 8, and spleen and enlargement of lymph node Th17 cell proportion are also remarkable in other groups.This shows that the result for the treatment of to EAE that mTGF-β 1-EXO produces may be relevant to the suppression of Th17 cell with it.

Embodiment 10mTGF-β 1-EXO induces Treg to alleviate the effect assessment of the occurring degree of EAE

Whether the present embodiment have detected mTGF-β 1-EXO and the Tregs in EAE mouse can be induced to produce.

10.1

Method: Tregs is the important attemperator of autoimmune disorder.First we have detected the function whether mTGF-β 1-EXO has induction EAE mouse Tregs, and we have been separated spleen and the lymphoglandula of each treatment group mouse after exosomes treats two weeks, and mark anti-CD4 and anti-Foxp3 streaming antibody, FACS detects CD4 ⁺foxp3 ⁺the ratio of regulatory T cells.

Result is as shown in Figure 9 A: mTGF-β 1-EXO treatment obviously can increase the ratio of Tregs in EAE mouse spleen and inguinal lymph nodes lymphocyte.

10.2 be the Tregs that induces of further clear and definite mTGF-β 1-EXO to the provide protection of EAE mouse, with Foxp3-GFP reporter gene mouse as EAE model, after exosomes treats two weeks, the CD4 of each treatment group spleen of airflow classification and lymphoglandula ⁺foxp3 ⁺the equal 89%-92% of its purity of Treg cell, with 1 × 10 ⁶/ dosage only, by the mode of tail vein injection by its adoptive transfer in normal mouse body, induce EAE two days later, observed and recorded scoring.

Result is as shown in Figure 9 B: come from the CD4 that normal mouse and mTGF-β 1-EXO treat EAE mouse ⁺foxp3 ⁺tregs obviously can alleviate the incidence of new induction EAE mouse.

Result shows, the Tregs that mTGF-β 1-EXO induces has provide protection to EAE mouse.

Embodiment 11mTGF-β 1-EXO alleviates the evaluation of the EAE morbidity effect of allogeneic BALB/c mouse

Whether the 11.1 mTGF-β 1-EXO first detecting C57BL/6 mouse source can suppress propagation and the induction Tregs of BALB/c mouse T cell in vitro.

Method: the CD4 of magnetic bead sorting BALB/c mouse spleen ⁺t cell, 1 × 10 ⁷cD4 ⁺t cell is resuspended in the PBS of 100 μ l, add the CFSE(5 that final concentration is 10mM, 6-carboxyfluoresceindiacetate, succinimidyl ester, CFSE), 37 DEG C hatch 3min after, wash twice immediately with precooling containing 1640 perfect mediums of 10% calf serum, adjustment concentration is 1 × 10 ⁶/ ml, every 1ml cell adds 1 μ l anti-CD3/CD28 magnetic bead, 200 μ l/ holes are laid on 96 orifice plates, then 0.5 μ g/ml is added respectively, 1 μ g/ml, the exosomes of the difference group of 2 μ g/ml, and separately establish and independent do not add any exosomes group in contrast, often group establishes three wells, hatches FACS fluorescence intensity after 3 days for 37 DEG C.

Exosomes induces in the experiment of Treg differentiation, first magnetic bead sorting BALB/c mouse spleen CD4 ⁺t cell, 1 × 10 ⁶the CD4 of/ml ⁺add 1 μ l anti-CD3/CD28 magnetic bead in T cell, get 200 μ l and add 96 orifice plates.Add simultaneously or do not add each group of the 2 μ g/ml exosomes after the HCL acidifying of 1N, separately establish a contrast, namely the anti-TGF-β 1mAbs of mTGF-β 1-EXO and 1 μ g/ml hatches 1h at 37 DEG C, and after three days, FACS detects CD4 ⁺foxp3 ⁺the ratio of Tregs.

Result is as shown in Figure 10 A-B, and the mTGF-β 1-EXO in C57BL/6 mouse source also obviously can suppress the propagation of BALB/c mouse T cell and significantly induce the vitro differentiation of its Tregs.

Secondly 11.2 have rated the security feeding back allogeneic efflux body, and every BALB/c mouse gives the Control-EXO in the C57BL/6 source of 10 μ g, every three days through tail vein injection once, and injection three times altogether.Then surrounding is observed.

In addition, have detected first week of injection Control-EXO respectively and the expression level of 4th week serum IgG and IgE, all in range of normal value.Also have detected first week and the lesion tissue situation of the 4th week mouse major organs heart, liver, spleen, lung, kidney simultaneously, not variant.

As illustrated in figure 10 c, body weight and the Behavioral feature of mouse all do not have noticeable change to result.After feeding back 1 week and 4 weeks of efflux body, in mice serum, the level of IgE and IgG does not have considerable change yet.This illustrates that feeding back allogeneic efflux body does not cause significant acute and chronic rejection.

The 11.3 mTGF-β 1-EXO utilizing C57BL/6 mouse to originate treat the EAE of BALB/c mouse

Method: female C57BL/6 or BALB/C in 6-8 week is equally divided into five groups is mTGF-β 1-EXO, sTGF-β 1-EXO, LacZ-EXO, Control-EXO and No treatment group respectively, often organizes 10.Respectively at first 8 days of modeling, first 5 days and the first 2 days exosomes through tail vein injection 10 μ g/ each group of C57BL/6 source only, at the 0th day induction EAE, record scoring, observed and compares mTGF-β 1-EXO, sTGF-β 1-EXO to the prophylactic effect of EAE.Further detection mTGF-β 1-EXO, sTGF-β 1-EXO, to the therapeutic action of EAE, at each group of exosomes of the 14th day, 17 days and 21 days tail vein injections and prevention group same dose being in onset peak period, observe scoring.

As shown in Figure 10 D, the EAE of mTGF-β 1-EXO to BALB/c mouse in C57BL/6 mouse source has obvious result for the treatment of to result.This shows that the result for the treatment of of mTGF-β 1-EXO to EAE mouse does not limit by MHC.

Discuss:

The ideal vaccine of widespread use should be break through MHC restriction, and the DCs of genetic modification effectively can treat autoimmune disorder, but is subject to the restriction of MHC, and this may make it limited in large-scale application.Experiment proves, the data effect of efflux body of the present invention to Autoimmune Disease Models does not receive MHC restriction,

Compared with imDCs vaccine, the exosomes vaccine in the imDCs of genetic modification source has the following advantages: (1), containing the albumen relevant to own cells specific function, exosomes is by carrying inhibitive ability of immunity albumen and target cell is had an effect; (2) stability is strong, can chilled storage for a long time, eliminates fresh separated and the inconvenience of long-term cultured and difficulty; (3) acellular components, volume is little, easy-clear, and without fecundity, antigenicity is weak, is applied to human body toxic side effect very little, stability and high efficiency.

Therefore, the present invention can have wide application prospect in the vaccine of autoimmune disease and treatment.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims

1. an efflux body (exosomes), it is characterized in that, described efflux body has following characteristics:

B () described efflux body carries described film expression cytokines.

2. efflux body as claimed in claim 1, it is characterized in that, described efflux body also has one or more features following:

E () described efflux body expressing protein comprises Hsp70, Tsg101 or CD63;

F () described efflux body does not express ER molecular chaperones Grp94.

3. efflux body as claimed in claim 1 or 2, it is characterized in that, described film expression cytokines comprises the cytokine of immunosuppression class, preferably comprises TGF-β 1, IL-10, IL-4, FasL or its combination.

4. efflux body as claimed in claim 1 or 2, is characterized in that, described efflux body has one or more activity following:

I () cause Treg (regulatory T cells) grows;

(ii) effect of Th1 polarization is suppressed;

(iii) Th17 cytodifferentiation is suppressed.

5. a pharmaceutical composition, is characterized in that, described pharmaceutical composition contains the efflux body according to claim 1 of safe and effective amount as activeconstituents; With pharmaceutically acceptable carrier.

6. the purposes of efflux body according to claim 1, is characterized in that, for the preparation of the pharmaceutical composition preventing and/or treating autoimmune disorder.

7. purposes as claimed in claim 6, it is characterized in that, described autoimmune disorder comprises nervus centralis Demyelination, preferably, described nervus centralis Demyelination comprises multiple sclerosis (Multiple Sclerosis MS), experimental autoimmune encephalomyelitis (experimentalautoimmuneencephalomyelitis, EAE), acute disseminated encephalomyelitis, diffuse cerebrosclerosis, optic neuromyelitis and Secondary cases demyelinating disease.

8. (the ImmatureDendritic Cells of the immature dendritic cell for generation of efflux body according to claim 1, imDCs), it is characterized in that, described immature dendritic cell through the film expression cytokines gene of external source modification thus express described film expression cytokines, and described immature dendritic cell secretes efflux body according to claim 1, and described efflux body carries described film expression cytokines.

9. prepare the method for efflux body described in claim 1, it is characterized in that, comprise step:

A () cultivates immature dendritic cell according to claim 8, thus obtain the mixture of the efflux body of described immature dendritic cell and secretion thereof;

B the efflux body of secretion described in (a) from described mixture separation and purifying, thus is obtained efflux body according to claim 1 by ().

10. an expression vector, is characterized in that, described expression vector contains film expression type TGF-β 1 gene, and described film expression type TGF-β 1 gene comprises TGF-β 1 gene be connected with film expression homing sequence operability.