CN100434519C - Curing and preventing method - Google Patents

Abstract

The present invention relates generally to a method for treating or preventing or otherwise ameliorating the effects of inflammatory conditions such as but not limited to chronic immune-mediated inflammatory diseases. The present invention further provides pharmaceutical compositions comprising agents which inhibit one or more inflammatory cytokines and/or which down-regulate expression of genes which encode inflammatory cytokines. Such compositions are useful in the treatment and prophylaxis of inflammatory conditions such as inflammatory arthritis amongst other chronic immune-mediated inflammatory diseases. The present invention further provides an animal model for studying the kinetics of and/or screening for agents useful in the treatment or prophylaxis of inflammatory conditions.

Description

The purposes of the inhibitor of granulocyte colony-stimulating factor or granulocyte colony stimulating factor receptor

Invention field

Present invention relates in general to be used for the treatment of or prevent or improve the method for the influence of inflammatory states, for example, but be not limited to chronic immune-mediated inflammatory diseases.The present invention also provides the medicinal compositions of the reagent that contains the genetic expression that can suppress one or more inflammatory cytokines and/or can descend tone coded inflammatory cytokine.Described composition can be used for treatment and prevention inflammatory situation, as inflammatory arthritis, and other chronic immune-mediated inflammatory diseasess.The present invention also provide be used to study be used for the treatment of or prevent the inflammatory situation reagent kinetics and/or screen the animal model of this reagent.

Background of invention

The details of the bibliography of the reference that provides is in this manual listed in the ending of this specification sheets.

In this manual, not, and should not be regarded as admitting or hint that in any form the prior art has constituted the part of the conventional, well-known general knowledge of any country quoting of any prior art.

Granulocyte colony-stimulating factor (G-CSF is by the CSF-3 genes encoding) is a hemopoieticgrowth factor, and it can regulate and control granulocytic production (Nicola etc., Nature 314:625,1985; Metcalf, International Journal of Cancer 25:225,1980; Nicola etc., Journal of Biological Chemistry 258:9017,1983).G-CSF by with the member's of granulocyte colony stimulating factor receptor (G-CSFR is by the CSFR-3 genes encoding)-I cytokines receptor superfamily its effect (Demetri etc., Blood 78:2791-2808,1991) of interaction mediation.G-CSF comprises production and release (Souza etc., Science232:61,1986 of the neutrophilic granulocyte that increases marrow at people and the intravital main biological action of mouse; Lord etc., Proc.Natl.Acad.Sci.USA 86:9499-9503,1989), hemopoietic progenitor cell is transferred to (Bungart etc., BritishJournal of Haematology 22:1156,1990 in the peripheral blood from marrow; De Haan etc., Blood 86:2986-2992,1995; Roberts etc., Blood 89:2736-2744,1997) and regulate differentiation and effector function (Yong etc., BuropeanJournal of Haematology 49:251-259,1992 of sophisticated neutrophilic granulocyte; Colotta etc., Blood 80:2012-2020,1992; Rex etc., Transfusion 35:605-611,1995; Gericke etc., Journal of Leukocyte Biology 57:455-461,1995; Xu etc., British Journal of Haematology 93:558-568,1996; Yong, BritishJournal of Haematology 94:40-46,1996; Jacob etc., Blood 92:353-361,1998).G-CSF is used for the treatment of neutropenia, and is used for mobilizing hematopoietic stem cells (HSC), so that carry out from body and allos stem cell transplantation (Welter etc., Blood 88:1907-1929,1996).

G-CSF is used for HSC shifts increase the weight of (Snowden etc., Bone Marrow Transplantation 22:1035-1041,1998) that may cause rheumatoid arthritis (RA).G-CSF and G CFS GM-CSF and M-CSF are (Leizer etc., Blood 76:1989-1996,1990 that produced by people's synovioblast and chondrocyte in external reaction at IL-1 and TNF; Hamilton etc., Blood 79:1413-1419,1992), and in RA patient's serum and synovia, found G-CSF (Tanabe etc., Rheumatology International 16:67-76,1996 already; Nakamura etc., Clinical and Experimental Rheumatology 18:713-718,2000).Confirmed already that systemic administration G-CSF can increase the weight of the collagen-induced sacroiliitis of muroid (CIA), at DBA/l mouse (Campbell etc., Journal of Leukocyte Biology 68:144-150,2000) (Miyahara etc. and in the passive transfer model of rat CIA, ClinicalImmunology and Immunopathology 69:69-76,1993) seriousness and sickness rate increase.The G-CSF transgenic mice has the bone forming (Takahashi etc., Laboratory Investigation 74:827-834,1996) that the marrow that has strengthened absorbs and weakened, and has shown that G-CSF may play a role in the bone turnover.

G-CSF can enlarge the monocyte/macrophage subgroup, and induces anti-inflammatory cytokines, and this factor can prevent that mouse from suffering from endotoxemia (Gorgen etc., Journal of Immunology149:918,1992).Reported that also G-CSF can weaken allos and mitogenetic t cell responses (Foster etc., Transplantation 59:1557,1995 in people and mouse body; Pan etc., Blood 86:4422,1995), and cause T cell cytokine curve deflection Th2 cytokine production, corresponding weakened Th1 IFN-γ express (Pan etc., 1995, the same; Franzke etc., Blood 102:734-739).In muroid research, already with this deviate from the Th2 cytokine production with to the acute graft versus host disease, the provide protection of experimental autoimmune encephalomyelitis (EAE) and spontaneous systemic lupus erythematous link together (Pan etc., 1995, the same; Zavala etc., Journal of Immunology 163:5125-5132,1999; Zavala etc., Journal of Immunology 168:2011-2019,2000).The glomerulonephritis of neutrophilic granulocyte-mediation can not take place in the G-CSF defective type of mouse, but can not prevent the glomerulonephritis (Kitching etc. of T cell/scavenger cell-mediation, Journal of the American Society of Nephrologh 13:350-358,2000).

Therefore, G-CSF is the pleiotropy molecule with multiple function.Be necessary these functions of more complete sign, and set forth regulation and control to these functions and whether can cause benefit health.

Summary of the invention

In this manual, unless situation has special requirement, word " comprises " or version, be understood as that as " comprising (comprises) " or " comprising (comprising) " hint comprises the group of specified key element or integer or key element or integer, but, do not get rid of the group of any other key element or integer or key element or integer.

In arthritic muroid model, studied the effect of neutrophilic granulocyte.Described muroid model comprises use antibody elimination neutrophilic granulocyte, and the mouse of using G-CSF to knock out.Determined already that described mouse was a high resistance acute arthritis inductive, but as if this effect can not be explained by less neutrophilic granulocyte counting.Find similarly, can directly induce the joint inflammation by topical application G-CSF, and systemic administration G-CSF can replace the IL-1 in the described acute arthritis model.

Collagen-induced sacroiliitis (CIA) is chronic autoimmunization model, and it is widely used in studying rheumatoid arthritis (RA) pathogeny, and is used to assess the treatment of expection.In order to check in the chronic joint disease demand, with being present in chicken II Collagen Type VI (CII) in the Freund's complete adjuvant (CFA) to G-CSF to endogenous G-CSF ^-/-And wild-type (WT) mouse carries out immunity, so that induce CIA.In the G-CSF deficient mice, disease is had significant protective effect, thereby can determine the main effect of G-CSF in CIA.In the mouse that G-CSF knocks out, be normal to the t cell responses of CII.

In a word, this means that endogenous G-CSF plays main effect in inflammatory arthritis.Local or reduce the G-CSF activity capapie, or reduce G-CSF level or inhibition or reduce G-CSF acceptor (G-CSFR), recommended as the mechanism for the treatment of or alleviate the seriousness of inflammatory situation, as chronic inflammatory arthritis and rheumatoid arthritis, or other chronic immune-mediated inflammatory diseases situations.

Therefore, the present invention relates to be used to prevent and/or treat the method for inflammatory situation, comprise using and to suppress described inflammatory cytokine activity to the subject, or reduce its content, this cytokine for example but is not limited to G-CSF or its functionally equivalent or homologue or its acceptor (G-CSFR).

With described acute arthritis model measurement use the neutralizing monoclonal antibody (mAb) of G-CSF treatment render a service.In BM, caused the dose-dependently of marrow pedigree cell to weaken at inhibition G-CSF during the acute arthritis, in peripheral blood, caused neutropenia, and reduced the cellular infiltration of associated joint.

The present invention also provides reagent and has contained the medicinal compositions of described reagent, it can suppress to encode G-CSF and/or other inflammatory cytokines and/or their acceptor gene active or reduce its expression.

The present invention also provides the animal model that is used to study chronic inflammatory diseases, and it is used for screening is used for treating and/or preventing the inflammatory situation, as the purposes of the reagent of inflammatory arthritis.

This paper is provided in table 1 employed shortenings tabulation.

Table 1

Shortenings

Shortenings	Explanation
		Acute arthritis	Bovine serum albumin(BSA)/IL-1--inductive sacroiliitis methylates
Ab	Antibody
		B6	C57BL/6
BM	Marrow
		BSA	Bovine serum albumin(BSA)
CFA	Freund's complete adjuvant
		CIA	Collagen--inductive sacroiliitis
CII	The II Collagen Type VI
		CSF3/Csf3	G CFS	3 genes (people/mouse)
CSF3R	G CFS				3 acceptor genes (people)
		DNP	[dinitrophenyl] ethanoyl
BAE	Experimental autoimmune encephalomyelitis
		FACS	Fluorescence amplifying cell separator
FCS	Foetal calf serum
		FITC	Fluorescein isothiocyanate
G-CSF	Granulocyte colony-stimulating factor

Shortenings	Explanation
		G-CSF -/-	Granulocyte colony-stimulating factor-defective type
G-CSFR	Granulocyte colony stimulating factor receptor
		GM-CSF	Granulocyte-macrophage colony stimutaing factor
H&E	Phenodin and eosin
		HRP	Horseradish peroxidase
HSC	Hemopoietic stem cell
		i.a.	Intraarticular ()
i.d.	Intracutaneous ()
		Ig	Immunoglobulin (Ig)
IL-	Interleukin--
		IFN-γ	Interferon-
i.p.	Intraperitoneal ()
		i.v.	Intravenously
KLH	Keyhole limpet hemocyanin (keyhole limper cyanin)
		LN	Lymphoglandula
mAb	Monoclonal antibody
		mBSA	Bovine serum albumin(BSA) methylates
MBSA/IL-1-inductive sacroiliitis	Bovine serum albumin(BSA)-inductive sacroiliitis methylates
		M-CSF	Macrophage colony stimulating factor
M-CSFR	Macrophage colony-stimulating factor receptor
		NMS	Normal mouse serum
NP-	[4-hydroxyl-3-nitrophenyl] ethanoyl
		PBS	Phosphate-buffered saline
PE	Phycoerythrin
		RA	Rheumatoid arthritis
RHuG-CSF (filgrastim)	The recombinant human g-csf
		s.c.	Subcutaneous ()

Shortenings	Explanation
		TdR	Thymidine
TNF	Tumour necrosis factor
		TNP	Trinitrophenyl
WEHI	The Walter and Eliza Hall Institute of Medical Research
		WT	Wild-type

The accompanying drawing summary

Fig. 1 is the figure of the intraarticular effect of expression G-CSF.A. use filgrastim (G-CSF; 0.1,0.5, or 1 μ g), IL-1 (25ng) or salt solution (vehicle) carry out intraarticular for three days on end to mouse to be handled, and carries out histological examination at the 3rd day.Compare * P＜0.05 with saline control;

B. the joint transudate cell to the joint of G-CSF (0.5,1.5 μ g) or IL-1 injection carries out quantitatively.In the joint of G-CSF-injection, there is a large amount of monocyte/macrophage transudates.N＞12 joint/groups.

Fig. 2 A and B are that expression G-CSF is that the joint inflammation of local I L-1 inductive is necessary, rather than IL-1 inductive proteoglycan reduces necessary figure.At the 0th, 1 and 2 day, with IL-1 (25ng) to B6 and G-CSF-/-mouse carries out intra-articular injection, and carried out Histological assessment at the 3rd day, the seriousness of assessment A. inflammatory feature and destructive characteristics, and the minimizing of B. joint cartilage proteoglycan.The result is expressed as mean value ± SEM (from the score of 5 tests).Data are from 1 time in 2 tests; N=14 joint/group; * p＜0.05.

Fig. 3 A and 3B are illustrated in the acute mBSA/IL-1-inductive model, replace the figure of the systemic effect of IL-1 with filgrastim.With mBSA intra-articular injection B6 mouse, and the 0th, 1, IL-1 (250ng) [grey rod] or filgrastim (15 μ g) [white rod] carried out subcutaneous injection with salt solution [black rod] in 2 days.Show A. at the 2nd day peripheral blood counting and B. in the 7th Tian De lymphonodi poplitei (LN) and spleen weight.Data represented 3 tests, n＞5 mouse/groups.Compare * P＜0.05 with mBSA/ saline control group;

Fig. 4 A and 4B are illustrated in the figure that replaces the effect of IL-1 in the acute arthritis model with general G-CSF.With mBSA mouse is carried out intra-articular injection, and use salt solution vehicle, IL-1, or G-CSF carries out subcutaneous injection, and the knee of injecting is carried out histological examination at the 7th day.A. total average tissue branch ± SEM that learns.The representational histology of B, expression i. uses the joint of mBSA/ brine treatment, in the joint space, has MIN transudate cell (100X), the joint that ii.mBSA/IL-1-handles has moderate to remarkable transudate and synovitis, and the joint that iii.mBSA/G-CSF-handles has moderate inflammatory feature (200X).N 〉=10 joints/group/test; Three representative tests.Compare * p＜0.05 with the mBSA/ saline control.

Fig. 5 A-C G-CSF-/-mouse resists mBSA/IL-1-inductive sacroiliitis mutually.With mBSA to B6 and G-CSF-/-mouse carries out intraarticular handles, and carries out subcutaneous injection with IL-1 or salt solution, and carries out Histological assessment at the 7th day.The histology seriousness score that histogram graph representation A is total, and the B chondroproteoglycan reduces (by safranin O dyeing).C represent from mBSA/IL-1-handle (i, iii) B6 and (ii, iv) G-CSF-/-the H ﹠amp of mouse; The representative slice of E (top picture) and safranin O (bottom picture) section, n＞6 joints/group/experiment; 3 representative tests.

Fig. 6 represents to use by oneself B6mBSA/ salt solution, the representative FACS curve of the leukocyte population of the infiltration synovial tissue of B6mBSA/IL-1 and G-CSF-/-the 3rd day of mouse that mBSA/IL-1 handles and the 7th day.A the 3rd day and B the 7th day, from surpass 6 joint/groups, dissect synovial membrane, in enzyme mixture, dissociate, then special marking is dyeed.In A and B, by determining total infiltration white corpuscle (A, B with CD45.2 dyeing; The top picture).Only CD45.2+ colony is used for analysis subsequently.C will be from B6mBSA/ salt solution, B6mBSA/IL-1 and G-CSF-/-same adherent the spending the night of synovial membrane Digestive system of the mouse that mBSA/IL-1 handles, and non-adherent cell is dyeed, determine that CD4 expresses.Data represented 2 tests.

Fig. 7 A and 7B be WT B6 and G-CSF-/-acute arthritis of mouse in, consume the figure of the effect of neutrophilic granulocyte.Before mBSA/IL-1-inductive sacroiliitis is induced, with the expendable mAb or the isotype control treatment mouse of neutrophilic granulocyte.A is at the 0th, 2 and 7 day, to WT and G-CSF-/-the acute arthritis model of mouse carries out the peripheral blood analysis, described mouse is handled with neutrophilic granulocyte-expendable mAb or isotype contrast mAb.B with the WT of anti--neutrophilic granulocyte mAb or isotype control treatment and G-CSF-/-total histology score of mouse.N＞5 joint/groups compares * P＜0.05 with the mouse neutrophilic granulocyte level that WT isotype contrast mAb-handles.Compare with the control group PTS that anti--neutrophilic granulocyte mAb-handles with the contrast of WT isotype,

Fig. 8 be expression with the B6 mouse relatively G-CSF-/-figure of the CIA that weakened in the mouse.In tail root position mouse is carried out intradermal injection with the CII that is dissolved among the CFA, and strengthened at the 21st day.Carry out the clinical assessment of disease since the 21st day to mouse, for each claw is chosen score from 0 (normally) to 3 (seriously); Maximum score is 12 (relevant details is referring to embodiment 8).A. the CIR of representing disease.B.B6 and G-CSF-/-the clinical severity of CIA in the mouse.Data are collected from 3 tests; N＞30 mouse/groups.Compare * P＜0.001 with G-CSF-1-.

Fig. 9 be illustrated in B6 and G-CSF-/-mouse in the result of Histological assessment of CIA.From four B6 the most serious clinically and G-CSF-/-joint of mouse related organization pathology seriousness must be divided into 0-3.A is normal, and is slight, the figure of the per-cent in medium and serious pathology joint.B represents representational H ﹠amp; E section, described section be from the non-sacroiliitis B6 of i. joint, the B6CIA joint of the serious inflammation of ii., and iii. do not show the general G-CSF-of inflammation/-joint.

Figure 10 be B6 and G-CSF-/-mouse is at the figure of external t cell responses to CII.Independent inguinal region LN suspension is paved plate, and stimulate with the CII of sex change.With [ ³H] the TdR pair cell carries out 8 hours pulse, and the absorption of mensuration radioactivity, so that assessment T cell proliferation.A.B6 and G-CSF-/-the proliferative stimulation index of LN cell.B。The 64th hour by elisa assay from (i) IFN-γ in the supernatant liquor of culture and the (ii) level of IL-2.N＞6 holes/sample.

Figure 11 A and B represent the B6 of CIA-immunity and G-CSF-/-anti--CII Abs of mouse.Serum was gathered at A. the 30th day and B. on the 62nd day, and resisted-the total IgG of CIIAb-, IgM and isotype IgG2b, IgG2c, IgG1 and IgG3 by elisa assay.Data are collected from 3 tests; N＞30 sample/groups.*P＜0.05，

Figure 12 be represent the B6 of not contacted antigenic and CII/CFA-immunity and G-CSF-/-figure of basic Ig level in the mouse and non-specific total IgG level.A. to not contacted antigenic B6 and G-CSF-/-mouse (n=6 mouse/group) carries out bloodletting, and detects the total IgG of round-robin in the serum, the level of IgM and isotype (IgG2b, IgG2c, IgG1, IgG3 and IgA) by ELISA.B. analyzed the 62nd day from the B6 of CIA-immunity and G-CSF-/-non-specific total IgG of the serum of mouse.Comprised mouse from 3 independent experiments; N 〉=16 mouse/groups; * P＜0.05.

Figure 13 represents to compare with the B6 mouse, G-CSF-/-mouse in to the not antigenic Ab reaction of dependent form of T-dependent form and T-.Be used in sedimentary T-dependent form Ag NP-KLH in the alum or be present among the PBS T-not dependent form antigen DNP-dextran to B6 and G-CSF-/-mouse stimulates, and in the date bloodletting of regulation.At the 42nd day the NP-KLH group is carried out reinforced immunological.By elisa assay serum, so that assessment A.T-dependent form NP-KLH reaction, it is to pass through i.NP ₂(total) and ii.NP ₂₀(high-affinity)-specific IgG 1, iii.NP ₂₀The assessment of tiring of Ig2b, and B.T not dependent form DNP-dextran anti--NP IgM.N＞4 mouse/groups.

Figure 14 is illustrated in G-CSF neutral effect in the described acute arthritis model.The 0th, 1,2,3 and 5 days with neutrality anti--G-CSF (50 and 250 μ g) or isotype contrast mAb treatments B 6 mouse.Figure A. counts (n=5) at the peripheral blood neutrophilic granulocyte of the 4th day and the 7th day.B. the average clinical macroscopic score (from 4) in each joint (n=10).C. the average synovial tissue cellularity (n=3) in each mouse joint.D. the neutrophilic granulocyte (GR1hi CD11bhi) that oozes out of expression and the spot figure of monocyte/macrophage (CD11bhi GR1lo).The result of data represented 1 test; * P＜0.01.

Detailed description of preferred embodiments

Part has been inferred the present invention according to the further elaboration of the effect of inflammatory cytokine in inflammatory process.More particularly, suppose that the effect of G-CSF is influential to the inflammatory situation, as the inflammation of chronic immunity-mediation.Therefore, the someone the present invention that gives chapter and verse, local or suppress the activity of inflammatory cytokine capapie, and/or the inflammatory situation can be treated or prevent to the expression of gene of tone coded inflammatory cytokine down.

Therefore, one aspect of the present invention relates to the method that is used in animal or treatment of birds species or prevention inflammatory situation, this method comprises the reagent of using significant quantity to described animal or birds species, and this reagent can suppress the active of inflammatory cytokine or its acceptor and/or reduce the expression of gene level of described inflammatory cytokine of coding or its acceptor.

The present invention is specifically related to G-CSF and its homologue and derivative.Said " G-CSF " or its full name claim homologue and the derivative that " granulocyte colony-stimulating factor " comprises it." homologue " or " derivative " comprises the polymorphism variant of G-CSF.This paper can also be regarded as can be applicable to other inflammatory cytokines to quoting of G-CSF.

Therefore, another aspect of the present invention provides the method that is used for treating and/or preventing at animal or birds species the inflammatory situation, this method comprises to described animal uses reagent, and this reagent can suppress the active of G-CSF and/or can reduce the expression of gene level of coding G-CSF or G-CSFR.

Propose as top, " G-CSF " that mentioned comprises its homologue and its derivative.

Use can be general or partial.Topical application is specially adapted to part or inflammatory states such as arthritic treatment.But G-CSF might be to hematopoietic cell generation effect, so systemic administration can be used for regulating on the whole immunity system." whole body " mentioned comprises intraarticular, and intravenously is used in the intraperitoneal, subcutaneous and sheath, and by the oral cavity, rectum and nasal are used.

Term " inflammatory situation " is to use with its generalized implication, but, and particularly including the inflammatory situation of immune-mediated.In particularly preferred embodiments, the inflammatory situation is an inflammatory arthritis, comprises rheumatoid arthritis (RA).

Therefore, in preferred embodiments, the present invention relates to be used for treat and/or prevent the method for the chronic immune-mediated inflammatory situation of inflammatory arthritis or other at animal or birds species, this method comprises to described animal or birds species uses reagent, and this reagent can suppress the active of G-CSF or G-CSFR and/or can reduce the expression of gene level of coding G-CSF or G-CSFR.

Preferred animal is a Mammals, as people and other primates, domestic animal (for example, sheep, cow, horse, donkey, pig), laboratory animal (for example, rabbit, mouse, hamster, cavy), companion animals (for example, dog, cat), and captive wildlife.The birds species comprise tame bird (for example, chicken, duck, goose, turkey, Bantam), game birds (for example, duck, emu, pheasant) and the birds of raising in cages.The people is the most preferred animal of primate.Horse is particularly preferred domestic animal.

Therefore, in preferred embodiments, the invention provides the method that is used for the treatment of and/or prevents people's inflammatory situation, this method comprises to described people uses reagent, and this reagent can suppress the active of G-CSF or G-CSFR and/or can reduce the expression of gene level of coding G-CSF or G-CSFR.

Described reagent can be protein, non-protein (for example chemical entity) or nucleic acid molecule.

Protein and non-protein molecule comprise peptide, and polypeptide and albumen are small-sized, medium or large-scale chemical molecular, and the molecule of identifying by natural product screening or screen chemical libraries.The natural product screening comprises screening from plant, microorganism, and riverbed soil, coral, the extract of aquatic environment and extraterrestrial environment or sample are to obtain G-CSF activity or influential molecule of G-CSF gene expression dose or group of molecules.Described molecule also may influence G-CSF/G-CSFR and interact.

A kind of example of reagent is the antibody of G-CSF or G-CSFR or its epi-position.This antibody can use or local the use on general ground.

The use of monoclonal antibody is particularly preferred, because have their ability of mass production, and the homogeneity of described product.Preparation by the hybridoma cell line that is used for the manufacture order clonal antibody that merges immortal cell line and the lymphocyte of immunogenic formulation sensitization is obtained, can (for example finish by technology well-known to those skilled in the art, referring to Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium ofImmunology Vol.II, Schwartz edits, and 1981; Kohler and Milstein, Nature 256:495-499,1975; Kohler and Milstein, European Journalof Immunology 6:511-519,1976).

Another example of useful reagent is the G-CSFR of soluble form, the G-CSFR interaction that it is relevant with film with the G-CSF competition.

In addition, can screening reagent and G-CSF or G-CSFR-genetic stocks bonded ability.In one embodiment, G-CSF-or G-CSFR-coding property cDNA or genomic dna or mRNA transcript or its part as EST or SAGE mark, are fixed on the solid support, as nano particle or microsphere.Allow potential reagent contact then with the fixed nucleic acid molecule, and by radiation, emission, atomic excitation, the change of quality and/or density detects combination.

In case after identifying, can be with described reagent wash-out from the nucleic acid molecule, and do more detailed the sign.For example, can suppress to express (transcribe and/or translate) with G-CSF/G-CSFR genetic stocks bonded reagent.

The invention still further relates to the antagonist that the chemical analog of G-CSF or G-CSFR is used as G-CSF or its acceptor.Just as noted above, can also use soluble g-CSF acceptor.

Chemical analog involved in the present invention includes, but are not limited to side chain and modifies, at peptide, polypeptide or albumen are between synthesis phase, the integration of alpha-non-natural amino acid and/or their derivative, and the use of linking agent, and the additive method that protein molecule or their analogue is constituted the conformation constraint.

The example that side chain involved in the present invention is modified comprises amino modification, as carrying out standard reductive alkylation by reacting with aldehyde, uses NaBH then ₄Reduction; (methylacetimidate) carries out amidation with the methyl acetimidate; Use acetic anhydride acylation; With cyanate amino is carried out carbamoylation; With 2,4,6-trinitro-benzene-sulfonic acid (TNBS) carry out trinitrophenylization to amino; With succinyl oxide and tetrahydronaphthalic anhydride amino is carried out acidylate; And Methionin is carried out the pyrrole polyacidization with pyridoxal 5-phosphate, then by using NaBH ₄Reduction.

Can be by using such as 2, the 3-dimethyl diketone, phenylglyoxal and oxalic dialdehyde reagent form the heterocycle condensation product, modify the guanidino group of arginine residues.

Can activate by carbodiimide carboxyl is modified, this activation forms by O-acyl group isourea and carries out derivatize subsequently, for example, forms corresponding amide.

Can modify mercapto groups by the following method, such as with iodoacetic acid or iodo-acid amide carboxymethylation; Performic oxidation is become cysteic acid; Form blended disulphide with other mercaptan compounds; With maleimide, the maleimide that maleic anhydride or other replaced reacts; Use the 4-chloromercuri-benzoate, 4-chlorine mercury phenylbenzimidazole sulfonic acid, phenylmercuric chloride, 2-chlorine mercury-4-nitrophenols and other mercury reagents form mercurous derivative; Under alkaline pH, carry out carbamoylation with cyanate.

For example, can tryptophan residue be modified by indole ring being carried out alkylation with the N-bromosuccinimide oxidation or with 2-hydroxyl-5-nitrobenzyl bromine or sulfenyl halides.On the other hand, can be by using the nitrated change tyrosine residues of tetranitromethane, so that form 3-nitrotyrosine derivative.

Can be by carrying out the modification of N-carbethoxylation realization to the imidazole ring of histidine residues with the alkylation of iodoacetic acid derivative or with diethylpyrocarbonate.

The alpha-non-natural amino acid that mixes between synthesis phase at peptide and the example of derivative comprise, but be not limited to the use nor-leucine, 4-aminobutyric acid, 4-amino-3-hydroxyl-5-phenylpentanoic acid, 6-aminocaprolc acid, tertiary butyl glycine, norvaline, phenylglycocoll, ornithine, sarkosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or amino acid whose D-isomer.The tabulation of the alpha-non-natural amino acid that arrives involved in the present invention has been shown in table 2.

Table 2

Unconventional amino acid	Code	Unconventional amino acid	Code
				Butyrine	Abu	The L-N-methylalanine	Nmala
Alpha-amino group-iophenoxic acid methyl esters	Mgabu	The L-N-methylarginine	Nmarg
				The 1-aminocyclopropane-1-carboxylic acid ester	Cpro	The L-N-methylasparagine	Nmasn
		The L-N-methylaspartic acid	Nmasp
				Aminoisobutyric acid	Aib	L-N-methyl halfcystine	Nmcys
Amino norcamphyl carboxylicesters	Norb	L-N-methyl glutamine	Nmgln
						L-N-methyl L-glutamic acid	Nmglu
Cyclohexylalanine	Chexa	The L-N methylhistidine	Nmhis
				The cyclopentyl L-Ala	Cpen	L-N-methyl Isoleucine	Nmile
The D-L-Ala	Dal	The L-N-methylleucine	Nmleu
				The D-arginine	Darg	The L-N-methyllysine	Nmlys
The D-aspartic acid	Dasp	The L-N-methylmethionine	Nmmet

Unconventional amino acid	Code	Unconventional amino acid	Code
				The D-halfcystine	Dcys	L-N-methyl nor-leucine	Nmnle
The D-glutamine	Dgln	L-N-methyl norvaline	Nmnva
				D-L-glutamic acid	Dglu	L-N-methyl ornithine	Nmorn
The D-Histidine	Dhis	L-N-methylbenzene L-Ala	Nmphe
				The D-Isoleucine	Dile	The L-N-methylproline	Nmpro
The D-leucine	Dleu	L-N-methyl Serine	Nmser
				D-Methionin	Dlys	The L-N-methylthreonine	Nmthr
The D-methionine(Met)	Dmet	The L-N-methyl tryptophan	Nmtrp
				The D-ornithine	Dorn	The L-N-methyltyrosine	Nmtyr
The D-phenylalanine	Dphe	The L-N-methylvaline	Nmval
				The D-proline(Pro)	Dpro	L-N-methylethyl glycine	Nmetg
The D-Serine	Dser	L-N-methyl tertbutyl glycine	Nmtbug
				The D-Threonine	Dthr	The L-nor-leucine	Nle
The D-tryptophane	Dtrp	The L-norvaline	Nva
				D-tyrosine	Dtyr	Alpha-Methyl-aminoisobutyric acid esters	Maib
The D-Xie Ansuan	Dval	Alpha-Methyl-γ-An Jidingsuan ester	Mgabu
				D-Alpha-Methyl L-Ala	Dmala	The Alpha-Methyl Cyclohexylalanine	Mchexa
D-Alpha-Methyl arginine	Dmarg	Alpha-Methyl cyclopentyl L-Ala	Mcpen
				D-Alpha-Methyl l-asparagine	Dmasn	Alpha-Methyl-α-naphthylalanine	Manap
D-Alpha-Methyl aspartic acid	Dmasp	The Alpha-Methyl Trolovol	Mpen
				D-Alpha-Methyl halfcystine	Dmcys	N-(the amino butyl of 4-) glycine	Nglu
D-Alpha-Methyl glutamine	Dmgln	N-(2-amino-ethyl) glycine	Naeg
				D-Alpha-Methyl Histidine	Dmhis	N-(3-aminopropyl) glycine	Norn

Unconventional amino acid	Code	Unconventional amino acid	Code
				D-Alpha-Methyl Isoleucine	Dmile	N-amino-α-Jia Jidingsuan ester	Nmaabu
D-Alpha-Methyl leucine	Dmleu	α-naphthylalanine	Anap
				D-Alpha-Methyl Methionin	Dmlys	N-benzyl glycine	Nphe
D-Alpha-Methyl methionine(Met)	Dmmet	N-(2-carbamyl ethyl) glycine	Ngln
				D-Alpha-Methyl ornithine	Dmorn	N-(carbamoyl methyl) glycine	Nasn
D-Alpha-Methyl phenylalanine	Dmphe	N-(2-propyloic) glycine	Nglu
				D-Alpha-Methyl proline(Pro)	Dmpro	N-(carboxymethyl) glycine	Nasp
D-Alpha-Methyl Serine	Dmser	N-cyclobutyl glycine	Ncbut
				D-Alpha-Methyl Threonine	Dmthr	N-suberyl glycine	Nchep
D-Alpha-Methyl tryptophane	Dmtrp	The N-Cyclohexylglycine	Nchex
				The D-alpha-methyltyrosine	Dmty	N-ring decyl glycine	Ncdec
D-Alpha-Methyl Xie Ansuan	Dmval	N-ring dodecyl glycine	Ncdod
				The D-N-methylalanine	Dnmala	N-ring octyl group glycine	Ncoct
The D-N-methylarginine	Dnmarg	N-cyclopropyl glycine	Ncpro
				The D-N-methylasparagine	Dnmasn	N-ring undecyl glycine	Ncund
The D-N-methylaspartic acid	Dnmasp	N-(2,2-two styroyls) glycine	Nbhm
				D-N-methyl halfcystine	Dnmcys	N-(3, the 3-diphenyl propyl) glycine	Nbhe
D-N-methyl glutamine	Dnmgln	N-(3-guanidine radicals propyl group) glycine	Narg
				D-N-methyl L-glutamic acid	Dnmglu	N-(1-hydroxyethyl) glycine	Nthr
The D-N-methylhistidine	Dnmhis	N-(hydroxyethyl)) glycine	Nser

Unconventional amino acid	Code	Unconventional amino acid	Code
				D-N-methyl Isoleucine	Dnmile	N-(imidazolyl ethyl)) glycine	Nhis
The D-N-methylleucine	Dnmleu	N-(3-indoles ethyl) glycine	Nhtrp
				The D-N-methyllysine	Dnmlys	N-methyl-γ-An Jidingsuan ester	Nmgabu
N-methylcyclohexyl L-Ala	Nmchexa	The D-N-methylmethionine	Dnmmet
				D-N-methyl ornithine	Dnmorn	N-methylcyclopentyl L-Ala	Nmcpen
Sarcosine	Nala	D-N-methylbenzene L-Ala	Dnmphe
				N-methylamino isobutyrate	Nmaib	The D-N-methylproline	Dnmpro
N-(1-methyl-propyl) glycine	Nile	D-N-methyl Serine	Dnmser
				N-(2-methyl-propyl) glycine	Nleu	The D-N-methylthreonine	Dnmthr
The D-N-methyl tryptophan	Dnmtrp	N-(1-methylethyl) glycine	Nval
				The D-N-methyltyrosine	Dnmtyr	N-methyl-α-naphthylalanine	Nmanap
The D-N-methylvaline	Dnmval	N-methyl penicillanate amine	Nmpen
				γ-An Jidingsuan	Gabu	N-(p-hydroxybenzene) glycine	Nhtyr
L-tertiary butyl glycine	Tbug	N-(thiomethyl) glycine	Ncys
				L-ethyl L-Ala	Etg	Trolovol	Pen
The L-hyperphenylalaninemia	Hphe	L-Alpha-Methyl L-Ala	Mala
				L-Alpha-Methyl arginine	Marg	L-Alpha-Methyl l-asparagine	Maon
L-Alpha-Methyl aspartic acid	Masp	L-Alpha-Methyl tertiary butyl glycine	Mtbug
				L-Alpha-Methyl halfcystine	Mcys	L-methylethyl glycine	Metg
L-Alpha-Methyl glutamine	Mgln	N-Alpha-Methyl L-glutamic acid	Mglu

Unconventional amino acid	Code	Unconventional amino acid	Code
				L-Alpha-Methyl Histidine	Mhis	N-Alpha-Methyl hyperphenylalaninemia	Mhphe
L-Alpha-Methyl Isoleucine	Mile	N-(2-methyl sulphur ethyl) glycine	Nmet
				L-Alpha-Methyl leucine	Mleu	L-Alpha-Methyl Methionin	Mlys
L-Alpha-Methyl methionine(Met)	Mmet	L-Alpha-Methyl nor-leucine	Mnle
				L-Alpha-Methyl Xie Ansuan	Mnva	L-Alpha-Methyl ornithine	Morn
L-Alpha-Methyl phenylalanine	Mphe	L-Alpha-Methyl proline(Pro)	Mpro
				L-Alpha-Methyl Serine	Mser	L-Alpha-Methyl Threonine	Mthr
L-Alpha-Methyl tryptophane	Mtrp	The L-alpha-methyltyrosine	Mtyr
				L-Alpha-Methyl Xie Ansuan	Mval	L-N-methyl hyperphenylalaninemia	Nmhphe
N-(N-(2,2-two styroyls) carboxamide methyl) glycine	Nnbhm	N-(N-(3, the 3-diphenyl propyl) carboxamide methyl) glycine	Nnbhe
				1-carboxyl-1-(2,2-hexichol ethylamino) cyclopropane	Nmbc

For example, can use linking agent, use same bi-functional cross-linking agent, as have (CH to stablize the 3D conformation ₂) _nThe difunctional imide acid esters of spacer groups, n=1-n=6 wherein, glutaraldehyde, the N-hydroxy-succinamide ester, and heterobifunctional agent, it generally includes amino active part, as N-hydroxy-succinamide, and other group specificity active parts, as maleimide or dithio part (SH) or carbodiimide (COOH).In addition, can carry out the conformation constraint to peptide, for example, by integrating C _αAnd N _α-methylamino acid is at amino acid whose C _αAnd C _βImport two keys between the atom, and form cyclic peptide or analogue,, forming amido linkage between two side chains or between side chain and N or C-terminal as between N and C-terminal by importing covalent linkage.

Be specially adapted to by antisense-or the machine-processed induced gene silence of justice-mediation is arranged such as the nucleic acid molecule of RNA or DNA.There is the gene silencing of justice-mediation to be known as common inhibition again, and relates to number of mechanisms, comprise inducing of RNAi.

Term " nucleic acid ", " Nucleotide " and " polynucleotide " comprises RNA, cDNA, genomic dna, synthesized form and blended polymkeric substance, include justice and antisense strand, and can be the nucleotide base that maybe can comprise non-natural or derivatize of chemistry or biological chemistry modified, be readily appreciated that as those skilled in the art.For example, described modification comprises mark, methylates, replace one or more naturally occurring Nucleotide with analogue (as the morpholine ring), modify between Nucleotide, as uncharged key (for example, methyl orthophosphoric acid, phosphotriester, phosphamide (phosphoamidate), carbamate etc.), charged key is (for example, thiophosphatephosphorothioate, phosphorodithioate etc.), side group part (for example, polypeptide), intercalator (for example, acridine, psoralene etc.), sequestrant, the key of alkylating agent and modified (for example, α-different nucleic acid etc.).Also comprise synthetic molecules, it can simulate polynucleotide by hydrogen combination and other chemical interactions and specified sequence bonded ability.This molecule is well known in the art, and, comprise, for example replaced the molecule of the phosphate bond on the described molecular backbone chain with peptide bond.

For example, the antisense polynucleotides sequence can be used to make the transcript silence of G-CSF genetic sequence or G-CSFR genetic sequence.In addition, can be with all or part of the polynucleotide carrier that comprises the G-CSF locus along having justice or antisense orientation to place under the control of promotor, and transfered cell.Described have an expression in cell of justice or antisense constructs, can disturb target to transcribe and/or translate.In addition, the mechanism that can also adopt common inhibition (that is, use has justice-inhibition) and induce RNAi or siRNA.In addition, can directly use antisense or adopted molecule is arranged.In a kind of embodiment in back, can or adopted molecule preparation be arranged in composition with antisense, use in several ways then target T cell.

Antisense and have the version of adopted molecule to relate to the use morpholinyl, the oligonucleotide that it is made up of morpholine nucleotide derivative and diamino phosphoric acid salt (phosphoradiamidate) key (for example, Summerton and Weller, Antisense and Nucleic Acid Drug Development7:187-195,1997).For example, described compound injection in the embryo, and is observed interference effect to mRNA.

In one embodiment, the present invention will be used to regulate the function or the effect of the nucleic acid molecule of coding G-CSF or G-CSFR such as the compound of oligonucleotide and analogue, when promptly described oligonucleotide can inducible transcription or PTGS.This purpose is can realize with the oligonucleotide of the nucleic acid molecule specific hybrid of one or more described inhibitor of encoding by providing.Described oligonucleotide directly is provided for cell, perhaps in described cell, produces described oligonucleotide.In this article, term " target nucleic acid " and " nucleic acid molecule of coding G-CSF or G-CSFR " are advantageously used in comprising described coding DNA, the RNA that transcribes by described DNA (before comprising-mRNA and mRNA or its part), and comprise cDNA from described RNA.The hybridization of compound of the present invention and its target nucleic acid is commonly referred to as " antisense ".Therefore, be considered to preferable mechanism included when implementing certain preferred embodiments of the present invention, be known as " Antisense Suppression " here.Described Antisense Suppression is normally based on oligonucleotide chain or segmental hybridization based on hydrogen bond, so that at least one chain or fragment are carried out cracking, degraded or other forms of modification are so that can not operate it.Thus, at present preferably, the function of lead special nucleic acid molecule and their relevant described antisense reagent.

Wanting the function of interferential DNA to comprise duplicates and transcribes.For example, duplicating and transcribe can be from the endogenous cell template, carrier, plasmid construction body or other molecules.Want the function of interferential RNA can comprise such as following function: rna transport is to the protein translation site, rna transport is to the interior site away from the synthetic site of RNA of cell, translate into albumen by described RNA, the described RNA of montage, so that produce the RNA of one or more types, and catalytic activity or mixture formation, it includes and may participate in or be subjected to the promoted RNA of described RNA.

In the present invention, the pairing of the complementary strand of " hybridization " oligomeric compounds.In the present invention, the paired preferable mechanism relates to hydrogen bonded, and it can be the complementary nucleosides of chain of oligomeric compounds or Watson-Crick, Hoogsteen or the anti-Hoogsteen hydrogen bonded between the nucleotide base (nuclear base).For example, adenine and thymine is a complementary nuclear base, and they match by forming hydrogen bond.Hybridization can be carried out under various environment.

Combine the normal function that disturbs described target nucleic acid with described target nucleic acid at described compound, cause reduced activity, and have enough complementary degree, so that avoid described antisense compounds and non-target nucleic acid sequence to combine in specificity under the situation of the non-specific binding under the condition of needs, such antisense compounds is can specific hybrid.

Accurate pairing between the nuclear base of this paper said " complementary " expression oligomeric compounds.For example, if can carry out hydrogen bonded with the nuclear base on the specific position that is positioned at target nucleic acid in the nuclear base on the specific position of oligonucleotide (oligomeric compounds), described target nucleic acid is DNA, RNA, or oligonucleotide molecules, the position of the hydrogen bonded between described oligonucleotide and the described target nucleic acid just is called as the complementary position.Described oligonucleotide and other DNA under following situation, RNA, or oligonucleotide molecules is complimentary to one another promptly can be formed the nuclear base of hydrogen bond when occupied each other when the complimentary positions of the sufficient amount on each molecule.Therefore, " but specific hybrid " and " complementary " are be used to indicate accurate pairing or the complementary term that carries out enough degree on the nuclear base of sufficient amount, so that form stable and specific combination between oligonucleotide and target nucleic acid.

According to the present invention, compound comprises antisense oligomeric compounds, antisense oligonucleotide, ribozyme, external guide sequence (EGS) oligonucleotide, alternative splicing (splicer), primer, probe, and can be at least and other oligomeric compounds of the part hybridization of target nucleic acid.Therefore, can import strand, two strands, the oligomeric compounds of ring-type or hair clip form, and this compound can comprise structural element, as inner or terminal protuberance or ring.In case import system, compound of the present invention just can induce one or more enzymes or structural protein to work, so that enforcement is to the modification of target nucleic acid.A kind of indefiniteness example of described enzyme is RNAse H, can cleaving rna: the cell endonuclease of the RNA chain of DNA duplex.The strand antisense compounds that it is well known in the art that " DNA-sample " can be induced RNAse H.Therefore the intensity of activation of RNase H causes the cracking of RNA target molecule, thereby strengthens the effectiveness of the genetic expression inhibition of oligonucleotide-mediation greatly.Inferred the similar effect of other rnases already, as the RNase III of enzyme and the member of ribonuclease l family.

Although the preferred form of antisense compounds is a single stranded antisense oligonucleotides, but in a lot of species, duplex structure, for example the importing of double-stranded RNA (dsRNA) molecule had been proved weakening of the gene that can induce effective and specific antisense-mediation or its relevant gene product function already.

Within the scope of the present invention, term " oligomeric compounds " expression comprises the polymkeric substance or the oligomer of a plurality of monomer unit.In the present invention, the oligomer or the polymkeric substance of term " oligonucleotide " expression Yeast Nucleic Acid (RNA) or thymus nucleic acid (DNA), or its stand-in, mosaic, analogue and homologue.This term comprises by naturally occurring nuclear base, the oligonucleotide that (main chain) key is formed between sugar and covalency nucleosides, and oligonucleotide with part of non-natural existence, and it can bring into play similar effect.Described modification or the oligonucleotide that replaced are better than the Nucleotide of natural form usually, because it has the characteristic that needs, for example, the cell that has strengthened absorbs, the avidity that has strengthened to target nucleic acid, and strengthened in the stability that exists under the condition of nuclease.

Although oligonucleotide is the compound of preferred form of the present invention, the invention still further relates to the compound of other families, include, but are not limited to oligonucleotide analogs and stand-in, as described herein molecule.

Zone between open reading-frame (ORF) known in the field (ORF) or " coding region " expression translation initiation codon and translation stop codon, it is the zone that can effectively lead.Within the scope of the present invention, the zone is the translation initiation of the open reading-frame (ORF) (ORF) that comprises gene or the territory, intron of terminator codon.

Other target areas comprise 5 ' non-translational region (5 ' UTR), refer to be positioned at the mRNA part of 5 ' direction of translation initiation codon in the art, therefore and comprise in 5 ' cap site of mRNA and the Nucleotide between the translation initiation codon (or on this gene corresponding Nucleotide), and 3 ' non-translational region (3 ' UTR), refer to be positioned at the part of the mRNA of translation stop codon 3 ' direction in the art, and therefore comprise in translation stop codon of mRNA and the Nucleotide between 3 ' end (or on this gene corresponding Nucleotide).5 ' cap site of mRNA comprises the N7-guanosine residue that methylates, and it combines with 5 of mRNA '-outermost residue by 5 '-5 ' triphosphoric acid key.5 of mRNA ' adds the cap zone and is believed to comprise 5 ' add cap structure itself, and near preceding 50 Nucleotide of described cap site.Preferred guiding 5 ' add cap zone.

Although some eukaryote mRNA transcript is directly translation, a lot of transcripts comprise the district of one or more being called as " intron ", and before its translation, these districts are excised from transcript.Remaining (and therefore translating) district is called as " exon ", and by montage together, so that form successive mRNA sequence.The guiding splice site, promptly intron-show outward in contact or exon-intron contact also can be applied to unusual montage and the occasion of disease-related especially, or is applied to and the excess of special montage product produces and the occasion of disease-related.Because resetting or lacking the unusual fusion contact that causes is preferred target site equally.The mRNA transcript that produces by the montage process from two kinds of (or two or more) mRNAs of different gene sources is called as " fusion transcript ".Be known that equally and can use the antisense compounds that is directed at DNA for example or the preceding-mRNA intron that effectively leads.

As known in the field, nucleosides is the composition of base-sugar.The base portion of nucleosides is heterocyclic base normally.Two kinds of modal types of this heterocyclic base are purine and pyrimidine.Nucleotide be also comprise on the nucleosides with the covalently bound phosphate group of sugar moieties on nucleosides.For the nucleosides that comprises furan pentose base sugar, phosphate group can be connected 2 of sugar ', 3 ' or 5 ' hydroxylic moiety.When forming oligonucleotide, described phosphate group is together covalently bound with adjacent nucleosides and another nucleosides, so that form the linear polymerization compound.Conversely, each end of this linear polymerization compound can further combined with so that form ring compound, but, ol cpds is normally preferred.In addition, ol cpds can have the inner core base complement, and therefore folding in some way, so that produce complete or partially double stranded compound.In oligonucleotide, described phosphate group is commonly referred to as main chain between the nucleosides that has formed described oligonucleotide.The normal key of RNA and DNA or main chain are 3 ' → 5 ' phosphodiester bonds.

Carry antisense compounds, described oligonucleotide for the part and can comprise key between the main chain of modified or non-natural nucleoside.As defined in this manual, the Nucleotide with main chain of modified is included in the oligonucleotide that has kept phosphorus atom on the main chain, and the oligonucleotide that does not have phosphorus atom on main chain.For this specification sheets, and, on main chain between their nucleosides, there is not the oligonucleotide of the modified of phosphorus atom can also be called as the oligomerization nucleosides as what sometimes mentioned in the art.

The oligonucleotide main chain that preferably comprises the modified of phosphorus atom comprises, for example, thiophosphatephosphorothioate, the chirality thiophosphatephosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl group phosphotriester, methyl and other alkyl phosphates (comprise 3 '-the alkylidene group phosphoric acid ester, 5 '-alkylidene group phosphoric acid ester and chiral phosphorus acid esters), phosphinate, phosphoramidate (comprise 3 '-amino phosphoramidate and aminoalkyl group phosphoramidate), the thiocarbonyl group phosphoramidate, the thiocarbonyl group alkyl phosphate, thiocarbonyl group alkyl phosphotriester, seleno phosphoric acid ester and boron substituted phosphate, has normal 3 '-5 ' key, their 2 '-5 ' connection analogue, and have the polar of putting upside down compound, wherein, between one or more Nucleotide key be 3 '-3 ', 5 '-5 ' or 2 '-2 ' key.Have put upside down the preferred oligonucleotide of polar comprise be positioned at 3 '-outermost Nucleotide between single 3 '-3 ' key on the key, it is the single nucleosides residue of putting upside down, it can be alkaline (lacked described nuclear base, or have oh group on its position).Also comprise various salt, blended salt and free acid form.

In another embodiment, will comprise that the genetic constructs of DNA " vaccine " is used to prepare antisense or adopted molecule mammalian cell is arranged.In addition, above-mentioned a lot of preferable feature has been applicable to the phosphorothioate odn molecule.

The reagent of identifying according to the inventive method provides easily with the medicinal compositions form.

The invention still further relates to the composition of the conditioning agent that the interactional conditioning agent that comprises between G-CSF activity or G-CSF and the G-CSFR or G-CSF/G-CSFR express, said composition also comprises carrier and/or the thinner that one or more can be medicinal.

Be suitable for the sterilized powder that the composition forms of injecting purposes comprises aseptic aqueous solution (water miscible) and is used for preparing aseptic injectable solution temporarily.Under production and preservation condition, it must be stable, and must be able to prevent the microbiological contamination effect such as bacterium and fungi.Described carrier can be solvent or diluent media, comprises water for example, ethanol, polyvalent alcohol (for example glycerine, propylene glycol and liquid macrogol etc.), their suitable mixture and vegetables oil.Can keep suitable flowability, for example by using tensio-active agent.Can pass through various antibacteriums and antifungal agents realization to the prevention of microbial process, for example, parabens, chlorobutanol, phenol, Sorbic Acid and Thiomersalate etc.In many instances, preferably include etc. and to ooze reagent, for example, sugar or sodium-chlor.The long-term absorption of Injectable composition can be by the reagent with delayed absorption, and for example, aluminum monostearate and gel application realize in described composition.

Sterile injectable solution is to mix in the suitable solvent by the active compound that will need quantity, mixes the activeconstituents that needs simultaneously and randomly mixes other activeconstituentss, carries out then that filtration sterilization or other suitable sterile modes prepare.For the sterilized powder that is used to prepare aseptic injectable solution, appropriate preparation method comprises vacuum-drying and Freeze Drying Technique, has produced the powder that activeconstituents adds the composition that any other needs thus.

When described conditioning agent is subjected to suitable protection; it can orally use; for example; use inert diluent or use assimilable edible carrier; or it is wrapped in the hard or soft shelly gelatine capsule; perhaps it is compressed into tablet, perhaps it is directly mixed in the diet, or use by breast milk.Use for oral administration, described activeconstituents can combine with vehicle, and the tablet taking in, buccal, and tablet, capsule, elixir, suspension, forms such as syrup and thin slice are used.Described composition and preparation should comprise that weight is at least 1% conditioning agent.Certainly, the per-cent of described composition and preparation can change, and accounts for the weight unit of about 5-about 80% easily.The consumption of conditioning agent in this composition that is used for the treatment of is such, so that the dosage that can obtain to be fit to.Preferred compositions of the present invention or preparation have been prepared, so that oral dosage unit form comprises the conditioning agent of about 0.1 μ g-200mg.Other dosage comprise about about 1000mg of 1 μ g-and the about 500mg of about 10 μ g-.These dosage can be each individual or every kg body weight.Using can be per hour, every day, weekly, the moon in week or annual.

Described tablet, tablet, pill, capsule and emulsifiable paste etc. can also comprise following cited composition.Tackiness agent, as natural gum, Sudan Gum-arabic, W-Gum, or gelatin; Vehicle is as Lin Suanergai; Disintegrating agent, as W-Gum, yam starch and alginic acid etc.; Lubricant is as Magnesium Stearate; And sweetener, as sucrose, lactose or asccharin can add, or seasonings, as Mentha arvensis L. syn.M.haplocalyxBrig, and oil of wintergreen or cherry seasonings.When described dosage unit form was capsule, except the material of the above-mentioned type, it can comprise liquid vehicle.Can exist various other materials with making coatings or change the profile of described dose unit with other forms.For example, can use shellac, sugar or the two are to tablet, and pill or capsule carry out dressing.Asccharin or elixir can comprise active compound, and as the sucrose of sweetener, as the methyl and the propyl para-hydroxybenzoate class of sanitas, dyestuff and seasonings are as cherry or orange seasonings.Certainly, any material that is used to prepare any unit dosage form all should be pure on the pharmacology, and nontoxic basically under the amount of using.In addition, described active compound can be mixed in extended release preparation or the prescription.

Carrier that can be medicinal and/or thinner comprise any and all solvents, dispersion medium, and coating, antibacterium and anti-mycotic agent wait to blend absorption delay agent etc.The use on active medicinal matter of described medium and reagent is well-known in the art, and except inconsistent conventional media of any and described conditioning agent or the reagent, their application in described therapeutic composition all can be expected.The active compound that replenishes can also be mixed in the described composition.

Just as noted above, use and can be undertaken by any way.For treatment of arthritis or local inflammation, intraarticular or subcutaneous administration are particularly preferred.

Described composition can also comprise genetic molecule, as carrier that can transformed target cell, wherein, described carrier have can the coding and regulating agent nucleic acid molecule, at this moment, described conditioning agent is the protein molecule.For example, described carrier can be a virus vector.Thus, the present invention relates to the several genes treatment, comprise the separation specific cells, carry out genetic manipulation, and send described cell back to identical subject, or send relevant or similar subject on the genetics back to.

The present invention also provides the animal model of inflammation, can be used for screening and can suppress G-CSF or G-CSFR, and therefore can improve the reagent of inflammatory effect.Relate to the animal model that can produce high or low horizontal G-CSF or G-CSFR.Described animal can be used to screen the reagent that can improve inflammatory symptom or preventing inflammation appearance.In addition, in the animal with lower level G-CSF, other cytokines or endogenous molecule may appear, so that replenish the shortage of G-CSF.They become the target of other treatment molecule.

Therefore, the animal that another aspect of the present invention provides genetic modification to cross, wherein, described animal is compared with the animal that the not genetic modification of same species is crossed, and can produce more a spot of G-CSF or G-CSFR.

Preferably, the animal that described genetic modification is crossed is a mouse, rat, cavy, rabbit, pig, sheep or goat.More preferably, the animal that crosses of described genetic modification is mouse or rat.Preferably, the animal that described genetic modification is crossed is a mouse.

Therefore, the mouse that preferred aspect of the present invention provides genetic modification to cross, wherein, described mouse is compared more a spot of G-CSF of generation or G-CSFR with the mouse that the not genetic modification of identical strain is crossed.

Animal model of the present invention can be the animal form, perhaps can be, for example is used to embryo's form of transplanting.Described embryo preferably preserves under freezing state, and chooses wantonly and sell with working instructions.

Another aspect of the present invention provides the oriented carrier of the gene inactivation that can be used for making coding G-CSF or G-CSFR, described oriented carrier comprises two sections of the described genetic stocks at lateral G-CSF of positive selectable marker or G-CSFR of coding, wherein, described oriented carrier transfection is being done (ES) cell to the embryo, and when selecting described mark, produced the ES cell, wherein, made the gene inactivation of coding G-CSF or G-CSFR by homologous recombination.

Described ES is preferably from mouse, rat, and cavy, pig, sheep or goat, most preferably described ES cell is from mouse.

Another aspect of the present invention relates to above-mentioned oriented carrier is used to produce the animal that the genetic modification that can not produce G-CSF or G-CSFR is basically crossed.

Another aspect of the present invention relates to above-mentioned oriented carrier is used to produce the mouse that the genetic modification that can not produce G-CSF or G-CSFR is basically crossed.

Described carrier is DNA preferably.Selective marker on the described oriented carrier makes it possible to select stably to have integrated already the target cell of described guiding DNA.When using inefficient transformation technology, this point is particularly useful, electroporation for example, calcium phosphate precipitation and liposome merge, wherein, usually in 1000 cells only less than 1 cytotostatic integrated described foreign DNA.Use high-efficiency method, as microinjection in nuclear, have usually 5-25% cellular integration described guiding DNA; Therefore, can directly screen described target cell, and needn't at first screen the stable integration of selective marker.Can use isogenic or non-isogenic DNA.

The example of selective marker comprises that generation to the gene such as the resistance of antibiotic compound, gives the gene of the ability of growing on the substrate of selecting, and coding produces detectable signal, as luminous proteic gene.Multiple such mark is arranged is known and can obtain, and for example, comprises antibiotics resistance gene, as neomycin resistance gene (neo) and hygromycin gene (hyg).Selective marker also comprises the gene of giving the ability of growing on some substratum substrate, as tk gene (thymidine kinase) or hprt gene (hypoxanthine phosphoribosyltransferase), it can give the ability of growing (xanthoglobulin, aminopterin and thymidine) on the HAT substratum; With bacterium gpt gene (guanine/xanthine phosphoribosyl transferase), it allows to go up growth at MAX substratum (mycophenolic acid, VITAMIN B4 and xanthine).Other selective markers that are used for mammalian cell are described in following document: Sambrook etc., Molecular Cloning-ALaboratory Manual, Cold Spring Harbour, New York, USA, 1990 with the plasmid that carries the multiple choices mark.

The optimum position of described marker gene on the guiding construct depends on the purpose of gene targeting.For example, if purpose is to destroy expression of target gene, described selective marker can be cloned on the guiding DNA that is equivalent to the encoding sequence on the described target dna.In addition, if purpose is by the altered product of described expression of target gene, the albumen that for example has amino acid replacement, just can modify described encoding sequence, described the substituting so that encode, and selective marker can be placed on outside, described coding region, for example, place on the nigh intron.

Described selective marker can be depending on it self the expression promoter that is used for, and described marker gene can be from the biology very big with the biological differences that will lead (for example, the protokaryon marker gene being used to the mammalian cell that leads).But, preferably produce described original promotor of generation with the known record changer that in recipient cell, works.There are a large amount of transcription initiation regions to can be used for this purpose, for example, comprise metallothionein promoter, thymidine kinase promoter, beta-actin promotor, immunoglobulin promoter, SV40 promotor and human cytomegalic inclusion disease virus promotor.Generally the example of Shi Yonging is the pSV2-neo plasmid, and it has the bacterium neomycin phosphotransferase gene that is subjected to the control of SV40 early promoter, and can give the resistance of mammalian cell to G418 (microbiotic relevant with Xin Meisu).Multiple other versions can be used for strengthening the expression of described selective marker at zooblast,, and add the synthetic translation initiation sequence as interpolation poly (A) sequence.Composing type and inducible promoter can use.

Preferably modify described DNA by homologous recombination.Target DNA can be present in any organoid of zooblast, comprises nuclear and plastosome, and can be complete gene, exon or intron, regulating and controlling sequence or intergenic any district.

Homologous dna is the dna sequence dna that has at least 70% homogeny with the reference dna sequence.The homologous sign of two kinds of sequences is, they can hybridization each other under stringent condition (Sambrook etc., 1990, the same).

The invention still further relates to common inhibition (promptly having justice to suppress) and Antisense Suppression, so that the expression of downward modulation G-CSF or G-CSFR.This phenomenon appears in the target experimental animal usually, as is used to prepare disease model.

The animal of genetic modification also can produce a large amount of G-CSF or G-CSFR.For example, the overexpression of normal G-CSF or G-CSFR can produce the negative interaction of dominance, and can be changed into useful disease model.

Therefore, another aspect of the present invention relate to can overexpression the animal that crosses of the genetic modification of genetic sequence of coding G-CSF or G-CSFR.

The animal that genetic modification is crossed comprises transgenic animal, or " knocking out " or " knocking in (knock-in) " animal.

The invention will be further described by following indefiniteness embodiment.

Embodiment 1

Mouse

C57BL/6 (B6; Wild-type, [WT]) mouse is to obtain from Walter and Eliza HallInstitute (WEHI) Animal Supplies (Victoria, Australia).G-CSF-defective type (G-CSF ^-/-) mouse is from Ludwig Institute for CancerResearch, Victoria, Australia obtains, and be to do Cysf3 genes produce on (ES) cell by guiding to destroy the 129/OLA embryo, with described gene injection to B6 blastocyst (Lieschke etc., Blood 84:1737-1746,1994).Allow mouse and B6 background backcross and surpassed for 20 generations.All mouse are: when experiment, be for 〉=8 ages in week, and feeding standard rodent feed, and arbitrarily drink water, and stable breeding (≤6 mouse/cage) is in the cage of splendid attire sawdust.All animal methods have obtained the permission of Institutional Ethics Committee.

Embodiment 2

Inducing of mBSA/IL-1-inductive sacroiliitis (acute arthritis)

This method is based on the technology (Lawlor etc., Arthritis andRheumatism 44:442-450,2001) of former description.Mouse is anaesthetized, and (Sigma, St Louis MO) are expelled in the knee joint by the intraarticular approach with the 20mg/ml mBSA of 10 μ l.The vehicle (common salt solution) of equal volume is accepted in the contrast joint.The 12.5 μ g/ml recombinant human IL-1 β (specific activities 5 * 10 that will be present in 20 μ l in common salt solution/0.5% (v/v) the normal mouse serum (vehicle) then by subcutaneous (s.c.) injection ⁸U/mg; Amgen, Thousand Oaks CA) is expelled on the back palmula of mouse, and repeats described injection at the 2nd day.

Slaughter mouse at the 7th day (or on time point of indicating), and the excision knee joint, and in the formalin solution of 10% (v/v) neutral buffered fixing at least 2 days, decalcification and carry out Treating Cuttings with Paraffin Wax.With four kinds of degree of depth cutting front tissue slicies (4 μ m) of about 100 μ m at interval, and with phenodin and eosin (H ﹠amp; E) dyeing is so that the assessment arthropathology.

Described experimental group is carried out the assessment of blind test sacroiliitis.Arthritic five kinds of compositions have been assessed, i.e. joint space transudate, synovitis, pannus formation, cartilage and bone degraded.Severity is divided into grade from 0 (normally) to 5 (seriously).According to the histology score classified in the joint, if transudate provides 1 or surpass 1 score, and must being divided into more than 2 or 2 of synovitis, then the joint is categorized as inflammatory arthritis.Arthritis mutilans is categorized as must being divided into more than 2 or 2 of pannus, and what cartilage and/or bone were degraded must be divided into more than 1 or 1.Also calculated total average tissue seriousness score, the maximum possible in each joint must be divided into 25 (Lawlor etc., 2001, the same).The painted section of preparation safranin O, and to the assessment of chondroproteoglycan minimizing carrying out blind test.

Embodiment 3

Collagen-induced sacroiliitis (CIA) induces

With chicken II Collagen Type VI (CII; Sigma) with the concentration of 2mg/ml in 10mM acetate, dissolving is spent the night under 4 ℃, carry out emulsification in isopyknic Freund's complete adjuvant (CFA), the full adjuvant of this Fu Shi gas is by adding heat-killed mycobacterium tuberculosis (Mycobacterium taberculosis) (bacterial strain H37Ra in Freund's incomplete adjuvant (Difco); DifcoLaboratories, Detroit, MI, the U.S.) be prepared as the concentration of 5mg/ml.By intracutaneous (i.d.) injection the described emulsion of 100 μ l is expelled to some positions of mousetail root, and after 21 days, repeats above-mentioned injection.

Monitor animal erythema and limb swelling, and 3 times each mouse carried out clinical score weekly, up to 40 days.Points-scoring system (Campbell etc., European Journalof Immunology 30:1568-1575) as indicated above, wherein 0=is normal, the slight swelling of 1=, 2=serious swelling, and 3=joint deformity and/or stiff, and the maximum of every mouse must be divided into 12.Clinical assessment be by do not understand two of described experimental group independently the researchist finish.When slaughtering, take out claw, fixing, paraffin embedding is carried out in decalcification and processing, and is as indicated above.Have the fore paw of four mouse of the highest clinical score and the H ﹠amp of rear solid end; E stained (5 μ m) is assessed (Campbell etc., 2000, the same) as stated above.At the 30th day and slaughtering the same day (the 62nd day), gather blood so that measure serum anti--CII Ab.

Embodiment 4

Use G-CSF

Intraarticular G-CSF and IL-1

IL-1 (25ng) or recombinant human g-csf (rHuG-CSF the 0th, 1 and 2 day every day with 10 μ l; 0.1,0.5,1 and 1.5 μ g) or vehicle (salt solution, physiological saline/0.5% (v/v) normal mouse serum (vehicle)) mouse is carried out intra-articular injection.Slaughtered mouse at the 3rd day, and to H ﹠amp; The painted joint of E section carrying out Histological assessment.

Subcutaneous injection G-CSF replaces IL-1 β in acute arthritis

With mBSA mouse is carried out intra-articular injection, and at 0-2 days subcutaneous injection IL-1 (250ng) or rHuG-CSF[filgrastims on palmula] (15 μ g) or vehicle contrast.Slaughtered mouse as stated above at the 7th day.

Consume WT and G-CSF ^-/-The intravital neutrophilic granulocyte of mouse

Before inducing, disease handled WT (B6) and G-CSF by peritoneal injection in 2 days ^-/-Mouse, and at 0-2 days, with 0.6mg neutrophilic granulocyte expendable monoclonal antibody (mAb), RB6.8C5 or isotype contrast mAb GL121 handled.Then, the from the 3rd to the 6th day, every day, the mAb with 0.5mg handled mouse.At the 0th, 2 and 7 day,, analyze the neutrophilic granulocyte counting of peripheral blood by the cell divide analysis of accounts.

Embodiment 6

The T cell proliferating determining

After initial injection 52-62 days, results inguinal lymph nodes (LN) from CIA mice immunized (n＞5 mouse/experiments).Preparation single-cell suspension liquid in the RPMI that contains 2 mercapto ethanol 1 (50 μ M) and 5% (vol/vol) foetal calf serum (FCS).To be present in the LN cell (2 * 10 in the 200 μ l volumes ⁵Cell) paves plate to round bottom 96 hole flat boards (Becton DickinsonLabware, Franklin Lakes, New Jersey, the U.S.), and stimulate with the CII (boiling 10 minutes) of 0-100 μ g/ml sex change.At 37 ℃ of (5%CO ₂) following culturing cell 72 hours, took out supernatant liquor at the 20th and 48 hour, and with 1 μ Ci[ ³H] thymidine carried out last pulse 8 hours.With Inotech Cell Harvester (Inotech) harvested cell, and use small plate scintillometer (Canberra Packard, Victoria, Australia) mensuration [ ³H] thymidine integrates the index as T cell proliferation.The aliquots containig of gathering cell conditioned medium liquid at the 20th and 48 hour.

Soak into leukocytic flow cytometry

Dissected from the B6 mouse of mBSA/ salt solution-processing and the B6 and the G-CSF of mBSA/IL-1-processing at the 3rd day and the 7th day ^-/-Patella of mouse and the soft tissue that is connected with it.Remove bone, fat and muscle fragment, synovial membrane is cut into the fragment of 2-mm, and with the 2.4mg/ml Dispase II (Boehringer, Mannheim, Germany) that is present among the RPMI1640,1mg/ml II Collagen Type VI enzyme (Sigma), be digested to single-cell suspension liquid with 100 μ g/ml DNAse I (BoehringerMannheim, Indianapolis, the U.S.).Down softly stirring described suspension 45 minutes at 37 ℃, use the 10%[v/v that is present among the RPMI then] FCS is by the washing of 70 μ M nylon cell nutsche filters (Falcon).Before cell dyeing, carry out cell counting.Use rat anti-mouse Fc γ RIIb/III (CD16/CD32; Clone 2.4G2; AmericanType Culture Collection (ATCC), Manassas, Virginia, the U.S.) the blocking-up unspecific staining.With biotinylated rat anti-mouse CD45.2 (PharMingen, SanDiego, California, the U.S.) and fluorescence dye chain plain and plain three looks (SA-TRI) (Caltag) and the combination of the Ab that enumerates in the example 12 below white corpuscle is dyeed.Also at 37 ℃, 5%CO ₂In make the synovial membrane peptic cell adherent so that recover by of the expression of Dispase cracked such as the CD4 mark.Non-adherent cell is collected among the 2%v/v FCS that is present among the PBS, and dyeing, carry out fluorescence amplifying cell separator (FACS).

Embodiment 7

Cytokine analysis and white blood cell morphology are quantitative

According to manufacturer's explanation (Pharmingen), use the paired monoclonal antibody by catching IFN-γ, IL-4 and the IL-2 in the ELISAs mensuration T cell conditioned medium liquid.

Joint transudate white blood cell morphology is quantitative

Amplify under (1000x) in high power, by the polymorphonuclear leukocyte in the joint space transudate of 5 focuses being uploaded the barrier, monocyte/macrophage and lymphocyte carry out quantitatively, analyze H ﹠amp; The transudate compositional analysis is carried out in the painted joint section of E (n=2 slice depth/joint).

Embodiment 8

Serum is anti--mensuration of CII antibody (Ab)

Carry out ELISAs stated above, so that the Abs of detection CII (Campbell etc., 2000, the same).The goat that horseradish peroxidase-level is closed resists-mouse IgG (Sigma ChemicalCo.) IgG2b, IgG2c, IgG1, IgG3 or IgM (Southern BiotechnologyAssociates, Birmingham, Alabama, U.S.) antiserum(antisera) is as detecting Abs.Use arbitrary unit, with the serum production standard curve of the hyperimmunization DBA/1 mouse that merges.

Measure serum I g level

With total IgG, IgM, IgG2c, IgG2b, IgG1, the relevant Abs of IgG3 and IgA catch from the not contacted antigenic and hemorrhage serum Abs of the back orbital sinus CII/CFA-mice immunized, and Abs (the Southern BiotechnologyAssociates that puts together with horseradish peroxidase (HRP), Birmingham, Alabama, the U.S.) detect.

Embodiment 9

The peripheral blood leucocyte counting

At the 0th, 3 and 7 day, from the acute arthritis model, obtain peripheral blood by back eye socket clump venous cutdown, and be collected in the test tube of EDTA dressing.BM in the femur of every mouse is flushed to 3%[v/v] in foetal calf serum (FCS)/phosphate-buffered saline (PBS).Use Advia120 Hematology System (Bayer Diagnostics, Tarrytown, New York, the U.S.) to carry out total leukocyte counting and differential analysis.Also on cytospins, carry out manual BM counting (with the speed of 1200rpm with 7 fens clock times with 1 * 10 ⁵Cell centrifugation is to microslide).

Embodiment 10

Because the directly joint inflammation of using G-CSF to cause at intraarticular

At first by intra-articular injection rHuG-CSF (0.1,0.5 and 1 μ g) is expelled in the knee joint of wild-type (WT) C57BL/6 mouse, injected continuously 3 days, whether research granulocyte colony-stimulating factor (G-CSF) has short scorching characteristic in the joint.Contrast comprises intra-articular injection IL-1 (IL-1; 25ng) and vehicle (0.5%[v/v] be present in (normal mouse serum) in the physiological saline.At the 3rd day, take out the joint and carry out Histological assessment.Find that G-CSF inductive inflammation is dosage-dependent form form (Fig. 1), but, described significant reaction is than a little less than reacting with the IL-1 inductive.This result shows that external source G-CSF has short scorching effect in natural joint.

Embodiment 11

More weak IL-1 inductive joint inflammation has appearred in the G-CSF-deficient mice

In order to study the effect of G-CSF in regulating the influence of local I L-1-inductive inflammatory, by G-CSF deficient mice (G-CSF-/-) and B6 mouse being carried out intra-articular injection with IL-1.IL-1 can G-CSF-/-induce the joint inflammation in the mouse, have lighter degree (Fig. 2 A) and compare with normal B6 mouse, show that G-CSF is the downstream media of IL-1 in the compartment of joint.Although G-CSF-/-have weakening of this inflammatory feature in the mouse, do not having difference (Fig. 2 B) aspect the minimizing of joint cartilage proteoglycan, show that IL-1 may directly destroy cartilage.

Peripheral blood, BM and synovial membrane infiltrate are analyzed

In order to check that the G-CSF defective is to the influence by mBSA and the Inflammatory response of IL-1 inductive, from B6 and G-CSF-/-take out peripheral blood the mouse, BM and synovial membrane, described mouse is by intra-articular injection mBSA and at this model the 0th, 3 with carried out subcutaneous injection with IL-1 or salt solution vehicle in 7 days and handled.Result such as table 2 and 3 and shown in Figure 6.The G-CSF defective has caused the blunt neutrophilia reaction to mBSA/IL-1.By using IL-1, B6 and G-CSF-/-BM cellularity in the mouse all reduced (table 2), shown that the BM cell transfer is in blood.Compare with the B6 mouse, G-CSF-/-mouse has the metamyelocyte and the polymorphism of the remarkable quantity that has reduced in the BM compartment, and promyelocyte still less and myelocyte (table 2), and above result is basic and at the reaction of IL-1.Between stage of attack, the B6 mouse has produced significant periphery neutral cytosis in acute arthritis.What form sharp contrast is, in described model process, and G-CSF-/-mouse shows significant neutropenia (table 3), shows that by IL-1 inductive neutrocytophilia be G-CSF dependent form.

The G-CSF-that mBSA/IL-1-is handled/-cell that the synovial membrane of the inflammation of mouse carries out forms and discovers the leukocytic minimizing (Fig. 6 A-C) of soaking at the 3rd and the 7th day.Compare with the synovial tissue that handles from the mBSA/IL-1-of B6 mouse, the cell counting of synovial tissue's Digestive system G-CSF-that mBSA/IL-1-handles/-synovial tissue of joint in, showed total cellularity at the 7th day and reduced about 2 times (1.87 ± 0.07 * 10 ⁵Cell/B6mBSA/ salt solution joint is to 3.13 ± 0.10 * 10 ⁵Cell/B6mBSA/IL-1 joint is to 1.66 ± 0.05 * 10 ⁵Cell/G-CSF-/-the mBSA/IL-1 joint).Specifically, on these two time points, the per-cent of neutrophilic granulocyte and quantity all significantly reduce (GR1hi CD11bhi), and soak into the per-cent and the also significantly minimizing of quantity of monocytes/macrophages (GR1lo CD11bhi).Other phenotypic alternations be included in the G-CSF-that suffers from acute arthritis/-have the M-CSFR of low per-cent in the synovial tissue of mouse ⁺CD16/CD32+ and CD44 ⁺Cell shows that G-CSF induces this activation tagging needed comprehensively on the synovial cell.

The 7th day CD44 also found in the dyeing of after overnight incubation non-adherent white corpuscle being carried out ⁺The minimizing of lymphocytic infiltration shows that G-CSF is not only neutrophilic granulocyte and scavenger cell and transfers in the joint neededly, but also is CD4 ⁺T lymphocyte needed (Fig. 6 C).

Embodiment 12

At the arthritic G-CSF of acute inflammation ^-/-With consume neutrophilic granulocyte in the WT mouse

In order to study at G-CSF ^-/-Whether be only be the result (Lieschke etc., 1994, the same) of neutropenia to mBSA/IL-1-inductive sacroiliitis in the mouse if alleviating, and uses monoclonal antibody (mAb) RB6.8C5 to consume neutrophilic granulocyte.Contrast mAb (GL121) to WT and G-CSF with anti--neutrophilic granulocyte mAb (RB6.8C5) or isotype ^-/-Mouse carries out peritoneal injection (i.p.).At the 0th, 2 and 7 day, analyze the neutrophilic granulocyte level (Fig. 7 A) of peripheral blood by the cell divide analysis of accounts.Compare (it has produced significant neutrophilic granulocyte increase disease) with the animal that isotype contrast mAb handles, in anti--WT animal that neutrophilic granulocyte mAb handled, observed＞90% consumption.Arthritic generation (Fig. 7 B) is not eliminated in the neutrophilic granulocyte consumption of WT mouse, but it has significantly reduced the joint space transudate really.On the contrary, G-CSF ^-/-Mouse is to the better resistance of disease, and further neutrophilic granulocyte consumption fails further to palliate a disease severity.This shows at G-CSF ^-/-Neutrophilia drawing-down born of the same parents' minimizing not only determines the arthritic protection of mBSA/IL-1-inductive in the mouse.

Embodiment 13

External source G-CSF can partly replace IL-1 in acute inflammation sacroiliitis

The joint of the mouse of handling with mBSA and G-CSF (mBSA/G-CSF) has produced inflammatory and arthritis mutilans, although its severity is lower than the animal (Fig. 4 A-B) that mBSA/IL-1-handles.Be mainly the granulocyte infiltrate in the mBSA/IL-1-inductive sacroiliitis and compare, the main cell that soaks into the joint of the animal that mBSA/G-CSF-handles is a monocyte/macrophage.This result shows, uses to general external source G-CSF, can partly replace the IL-1 of whole body at least, be used to start this acute arthritis model, and G-CSF has caused monocyte/macrophage to add in the described joint.

Embodiment 14

The G-CSF defective can weaken collagen--inductive sacroiliitis (CIA)

The G-CSF-deficient mice has more weak acute inflammation sacroiliitis

Consider that intra-articular injection and whole body use the short scorching effect of filgrastim to joint disease, we attempt to use G-CSF-/-mouse determines the absolute dependency of described acute arthritis model to G-CSF.With mBSA to G-CSF-/-and the B6 mouse carry out intra-articular injection (the 0th day), and on palmula, carried out subcutaneous injection with IL-1 at the 0th, 1 and 2 day.Significantly the weakening of inflammatory and disruptive features (Fig. 5 A and B) found in the diseased tissue of carrying out at the 7th day assessment.To the dyeing of the safranin O of chondroproteoglycan content, compare with the B6 mouse, found G-CSF-/-mouse in chondroproteoglycan reduce significantly weaken (Fig. 5 C).Therefore, endogenous G-CSF is inflammation and a destructive important medium in this acute arthritis model.

The G-CSF defective can weaken CIA sickness rate and seriousness

CIA is chronic autoimmunization sacroiliitis, and it is widely used in studying RA.In order to check the effect of G-CSF to CIA, with be present among the CFA CII to B6WT and G-CSF-/-mouse carries out immunity, carried out reinforced immunological injection (Lieschke etc., Blood 84:1737-46 then after 21 day, 1994), and the relatively sickness rate and the seriousness of disease.CIA G-CSF-/-outbreak in the mouse postponed, and compare with the WT mouse, G-CSF-/-sickness rate and the severity of mouse significantly weaken (Fig. 8 A and B).G-CSF-/-mouse in the sickness rate of disease and weakening of seriousness, hinted the vital role of endogenous G-CSF in chronic autoimmunization sacroiliitis.

Embodiment 15

CIA is at G-CSF ^-/-Histologic analysis in the mouse

To four B6 coming to have during the comfortable CIA the highest clinical score and G-CSF-/-H of the claw of mouse; Histological assessment is carried out in the painted section of E.For getting 0 normally to the score of 3 (referring to examples 18) in each joint, and measure the per-cent in normal and arthritis joint.Compare with the WT mouse, G-CSF-/-have the significantly natural joint (Fig. 9 A) of higher per-cent in the mouse, and G-CSF-/-have a spot of affected claw in the mouse, wherein not having an example is serious (Fig. 9 B).On the contrary, from the joint of WT mouse, has the existence of showing slightly to the arthritic multiple histologic characteristics of severe (Fig. 9 A and B).The clinical assessment that described histological observation and this paper proposed coincide.

Embodiment 16

External T cell proliferation and cytokine production at CII

In order to assess cell immune response to CII, G-CSF-/-measured external T cell proliferative response in the mouse and T cell cytokine (IFN γ and IL-2) is produced, and with WT relatively.Be used for preparing single-cell suspension liquid from the inguinal region LN of mouse, described mouse is with being present in CII immunity among the CFA, and stimulates 72 hours at the sex change CII of external use 0-100 μ g/ml.By in last 8 hours that cultivate, measuring tritiate TdR absorption measurement t cell responses.Figure 10 A be illustrated in G-CSF-/-and the WT mouse in observed stimulation index, they are suitable.By G-CSF-/-as if the LN cell produce T cell cytokine IFN-γ and IL-2 relatively normal (Figure 10 B).

Embodiment 17

G-CSF-/-mouse in anti--CII isotype weaken to the conversion of IgG from IgM

Inducing of CIA is (Campbell etc., 2000, the same) that depend on the body fluid of CII and cell immune response.Checked whether the G-CSF defective can change the serum level (the 30th and 62 day) that anti--CII Ab produces during CIA.Although at the 30th and 62 day, WT and G-CSF-/-have quite anti--CII IgM of level in the mouse, have the minimizing (Figure 11) of total anti--CII IgG.All isotype-IgG2b have been found in the analysis that antagonism-CII IgG isotype carries out, IgG2c, and the production of IgG3 and IgG1 reduces.This show G-CSF-/-have isotype conversion defective in the mouse, this may cause the protective effect to CIA.This discovery shows that endogenous G-CSF is playing a role aspect the Ab of B cell generation, and the immunity that the antigen that is present among the CFA is carried out responds at least.

Embodiment 18

Not contacted antigenic G-CSF-/-the Ig basal level that increases in the mouse and the total IgG that in the CII/CFA-mice immunized, increases

Research is to the humoral immune reaction of CII, find G-CSF-/-mouse has from IgM to IgG reaction conversions defective.In order to determine whether this defective has embodied the defective that is pre-existing in circulation A b, analyzed from not total IgG of the serum of contacted antigenic mouse, IgM and IgG isotype.The analysis of serum Ab level has been found to compare with not contacted antigenic B6 mouse, not contacted antigenic G-CSF-/-output of total IgG and isotype IgG2b and IgG2c is significantly higher in the mouse, and the IgM with normal basic output, IgG1, IgG3 and IgA (Figure 12 A).In addition, the G-CSF-of CII/CFA-immunity/-mouse in, the level of non-specific total IgG significantly improves (Figure 12 B).Above-mentioned observation shows that G-CSF plays a role in antibody producing and the isotype conversion at the B cell maturation, and the immunity that the antigen that is present among the CFA is carried out responds at least.

Embodiment 19

G-CSF-/-mouse in normal T-dependent form and not dependent form Ag B cell response of T-

In order to study whether the weakening of Ab production of causing at CII/CFA is owing to lack T-dependent form or not dependent form Ag reaction of T-, with the T-dependent form antigen NP-KLH that is present in the alum, or be present among the PBS T-not dependent form antigen DNP-dextran stimulate B6 and G-CSF-/-mouse.G-CSF-/-mouse produced T-dependent form and the not normal reaction of dependent form Ag (Figure 13) of T-, shows that the B cell response that weakened in CIA is for being special with being present in the attack that the CII among the CFA carries out.

Embodiment 20

G-CSF consumes and has caused the peripheral blood neutrocytopenia, and has alleviated acute (mBSA/IL-1) arthritic inflammation

Be enclosed in therapeutic action in the WT mouse in order to assess G-CSF, before inducing acute arthritis (the 0th day), with rat anti-G-CSF mAb (clone 67604 of 50 or 250 μ g; R ﹠amp; D systems, Minneapolis, Minnesota, the U.S.) or isotype contrast (GL113) mAb injection B6 mouse.Also the 1st, 2, used mAb in 3 and 5 days.Gather peripheral blood at the 4th and 7 day, and carry out differential analysis (Figure 14 A).In this two the sky, compare with the mouse that isotype contrast mAb mBSA/I L-1-handles, accept high dosage (250 μ g) anti--mouse of G-CSF mAb has the remarkable minimizing of peripheral blood neutrophilic granulocyte.The remarkable minimizing of neutrophilic granulocyte only appearred in acceptance at the 7th day than the mouse of the anti--G-CSF mAb of low dosage.BM colony is analyzed, also find the remarkable minimizing (data are not delivered) of marrow pedigree cell in the mouse that the mBSA/IL-1-that G-CSF-consumes handles.To the visual assessment that carry out in the arthritis joint of the mouse of handling with anti--G-CSF or isotype contrast mAb, found alleviate (Figure 14 B) of in the mouse that anti--G-CSFmAb (250 μ g) handles disease.In the mouse of the anti--G-CSF mAb that accepts higher dosage, there is a synovial tissue's cellularity (Figure 14 C) that has reduced, and the infiltration white corpuscle per-cent that has reduced, be neutrophilic granulocyte (GR1hiCD11bhi especially; Figure 14 D).Above result shows that the genius morbi of the acute arthritis that suppresses at G-CSF has dose dependent and weakens in wild-type mice.

Table 3

G-CSF during mBSA/IL-1-inductive sacroiliitis ^-/-The peripheral blood analysis of mouse

Analyze peripheral blood with the Advia counter, and measured cell counting * 10 of every microlitre ³Data are expressed as mean value ± SD.

* the 0th day research is pre-treatment baseline quantity; Numeral in the bracket is the quantity of this group mouse of being used to check.

In P＜0.05 between each group on the time point of checking.

It will be appreciated by persons skilled in the art that invention described herein can carry out variation and the change except the specific descriptions form.Be understandable that, the present invention includes all such variation and changes.The present invention also comprises the independent in this manual or unified institute that mentions or point out in steps, feature, composition and compound, and two or more any and all combinations arbitrarily in described step or the feature.

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Claims (28)

1. the reagent that suppresses the active of granulocyte colony-stimulating factor (G-CSF) or granulocyte colony stimulating factor receptor (G-CSFR) and/or reduce the expression of gene level of described G-CSF of coding or G-CSFR is used for the treatment of or prevents purposes in subject's the arthritic medicine in preparation.

2. purposes as claimed in claim 1, wherein, described sacroiliitis is chronic inflammatory arthritis.

3. purposes as claimed in claim 1, wherein, described situation is rheumatoid arthritis (RA).

4. purposes as claimed in claim 1, wherein, described sacroiliitis is collagen-induced sacroiliitis (CIA).

5. as purposes any among the claim 1-4, wherein, described subject is an animal.

6. purposes as claimed in claim 5, wherein, described animal is a Mammals.

7. purposes as claimed in claim 6, wherein, described Mammals is a primate.

8. purposes as claimed in claim 7, wherein, described primate is the people.

9. purposes as claimed in claim 6, wherein, described Mammals is a rodent.

10. purposes as claimed in claim 9, wherein, described rodent is a mouse.

11. purposes as claimed in claim 1, wherein, described reagent is the antibody of anti-G-CSF or G-CSFR.

12. as the purposes of claim 11, wherein, described antibody is monoclonal antibody.

13. as the purposes of claim 11, wherein, described antibody is polyclonal antibody.

14. purposes as claimed in claim 1, wherein, described reagent is the G-CSFR of soluble form or its G-CSF binding fragment.

15. purposes as claimed in claim 1, wherein, described reagent is albumen.

16. purposes as claimed in claim 1, wherein, described reagent is nucleic acid.

17. as the purposes of claim 16, wherein, described nucleic acid is DNA or RNA, and comprise coding G-CSF or G-CSFR justice or antisense polynucleotides sequence arranged.

18. one kind is used to identify and is used for the treatment of arthritic, as to suppress G-CSF or G-CSFR active compositions and methods, this method comprises allows the inhibitor of inferring contact with described G-CSF or G-CSFR, wherein, by with described G-CSF or G-CSFR combine or other forms of the contact identifies that described reagent is inhibitor.

19. compositions and methods that is used to identify the expression that is used for the treatment of arthritic, as to regulate and control coding G-CSF or G-CSFR genetic sequence, this method comprises allows the adjusting control agent of inferring contact with the genetic sequence of coding G-CSF or G-CSFR, wherein, by with the genetic sequence of described coding G-CSF or G-CSFR combine or other forms of the contact determines that described reagent is adjusting control agent.

20. one kind is used for the treatment of arthritic composition, and comprise G-CSF or G-CSFR antagonist and/or can reduce the reagent of the expression of gene level of coding described G-CSF or G-CSFR, and carrier that can be medicinal or thinner.

21. as the composition of claim 20, wherein, described reagent is the antibody of anti-described G-CSF or G-CSFR.

22. as the composition of claim 21, wherein, described antibody is monoclonal antibody.

23. as the composition of claim 21, wherein, described antibody is polyclonal antibody.

24. as the composition of claim 20, wherein, described reagent is the G-CSFR of soluble form or its G-CSF binding fragment.

25. as the composition of claim 20, wherein, described reagent is albumen.

26. as the composition of claim 20, wherein, described reagent is nucleic acid.

27. as the composition of claim 26, wherein, described nucleic acid is DNA or RNA, and comprise coding G-CSF or G-CSFR justice or antisense polynucleotides sequence arranged.

28. a guiding or a label switched mutagenesis carrier that is used for making the gene inactivation of cell coding G-CSF or G-CSFR, described carrier comprises two sections genetic stockss of coding G-CSF or G-CSFR, or their fragment, the positive or negative marker of its side.

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