CN101384619A - Modulation of bone formation - Google Patents

Abstract

This invention relates to modulating Ror activity (e.g., Ror2 protein activity) and/or 14-3-3 ss to affect bone formation or resorption. The invention further relates to compositions and methods for the screening, diagnosis and development of therapies for bone-related disorders such as osteoporosis and bone fracture. Antibodies and antibody fragments directed to Ror2 protein are particularly useful in causing dimerization of Ror2 proteins, thereby leading to the activation of Ror2.

Description

Osteoplastic adjusting

Related application

The application's case is advocated the right of priority of U.S. Provisional Patent Application case USSN 60/774,534 that on February 17th, 2006 applied for and the USSN 60/844,239 that applied on September 13rd, 2006 down at 35 U.S.C. § 119 (e), and it is incorporated herein by reference.

Technical field

Do not have

Background technology

In several years, the special topic of bone photo related disorders and disease has received sizable concern in the past.The bone photo related disorders is characterised in that the bone that imbalance caused between bone resorption and the bone forming runs off.In whole vital process, exist the skeletal bones that continues to rebuild.In this process of reconstruction, there is delicate balance between the bone forming that bone resorption and scleroblast caused that osteoclast caused.Relating to the interior scleroblast with intramembranous ossification of cartilage is to make the special cells of reaching new osteoplastic stromatin in the osseous tissue.Indispensable the bone amount (bone mass) of bone forming (that is osteogenesis) in keeping bone.Be different from scleroblast, osteoclast is removed with bone resorption and bone and is associated.In normal bone, the regulation and control of the balance between the bone resorption of the bone forming of scleroblast mediation and osteoclast mediation by complexity interact and keep.

There are many deficiencies that are associated with Skeletal system, disease and illness.Some examples include, but is not limited to osteoporosis, osteocarcinoma, sacroiliitis, rickets, fracture, periodontopathy, damaged, the molten bone osteopathia of segmental bone, primary and secondary hyperparathyroidism, handkerchief wise man sick (Paget ' s disease), osteomalacia, hyperostosis and osteopetrosis.The discriminating that relates to the mechanism of Osteoblast Differentiation and renewal process is to understand physiology of bone and such as the key of osteoporotic bone disorders.These illnesss can comprise the bone forming deficiency that is caused by the one-tenth osteoprogenitor cells defectiveness maturation of inferring.

To the method for the research and development disease that is associated with bone resorption and bone forming of treatment or illness, promote osteoplastic method, differentiate the method for regulating (increase or reduce) osteoplastic medicament, differentiate the method for the medicament of regulating (increase or reduce) bone resorption and the gene that discriminating is associated with the bone photo related disorders or the method existence needs of its protein.

The discriminating that relates to the mechanism of bone forming and bone resorption is to understand physiology of bone and such as the key of osteoporotic bone disorders.The gene or its protein that are associated with the bone photo related disorders can be used for illustrating the molecule mechanism of bone forming, bone resorption, screening and research and development new drug, the treatment of diagnosis, prognosis, prevention and treatment bone development and bone loss disorders and assessment such as osteoporotic bone photo related disorders.Gene of being differentiated and protein also may be applicable to explore regulates osteoplastic medical agent.A kind of such protein of having differentiated recently is Ror2 albumen.The downward modulation of Ror2 genetic expression suppresses the Osteoblast Differentiation (Fig. 1) of the human mescenchymal stem cell of dexamethasone (dexamethasone) inductive, and Ror2 crosses Osteoblast Differentiation (the ratio Reed people's such as (Billiard) of application on April 14th, 2004 U.S. patent application case U.S.S.N.10/823,998 of expressing these cells of promotion; It is incorporated herein by reference).Therefore, Ror2 and Ror2 path are to regulate osteoplastic suitable target.

Summary of the invention

The invention provides the osteoplastic system of a kind of adjusting.This system is with the basis that is found to be of the effect of Ror2 in osteoblast differentiation.Specifically, the proteic activation of Ror2 impels the bone forming that mineralizes.Find that the Ror2 expression is the key in the mescenchymal stem cell Osteoblast Differentiation.Find that also Ror2 crosses the expression inhibiting mescenchymal stem cell and is divided into adipocyte.Also find Ror2 proteic activation impelling 14-3-3 β phosphorylation.Find that the matrix that mineralizes that the downward modulation of 14-3-3 β increases in the human mescenchymal stem cell forms.These discoveries about the downward modulation of Ror2 albumen, itself and proteic interaction of 14-3-3 β and 14-3-3 β make Ror2,14-3-3 β and other downstream signal transduction biomolecules become the principal mark target in the exploration of regulating osteoplastic novel medicament.Activation Ror2 albumen, the active medicament that suppresses 14-3-3 β albumen or regulate other downstream target are applicable to treatment and prevention bone photo related disorders, especially with bone loss disorder associated.These medicaments also are applicable to and promote osteoblast differentiation and the promotion matrix that mineralizes to form.Perhaps, these medicaments can be to obtain in the adipocyte to use and may be applicable to treatment of obesity, metabolic disorder or diabetes suppressing differentiation of stem cells also.

The invention provides the proteic medicament of activation Ror2.In certain embodiments, these medicaments cause two polymerizations of Ror2 albumen, thereby cause the Ror2 activation.Proteic two polymerizations of Ror2 cause kinase activity to increase and Ror2 subsequently in conjunction with the phosphorylation of collocation thing (comprising 14-3-3 β albumen).Medicament also causes promote osteogenesis.

On the other hand, the invention provides the medicament that suppresses or reduce 14-3-3 activity (especially 14-3-3 β or 14-3-3 γ).These medicaments can work under DNA or protein level to reduce the activity of 14-3-3 in cell.In certain embodiments, make the medicament target relate to osteoplastic cell, such as any other cell in scleroblast, mescenchymal stem cell, embryonic stem cell, fetal stem cell, osteoprogenitor cells, skeletonization precursor cell (pre-osteoblast), ripe scleroblast or the scleroblast strain.In other embodiments, make the medicament target relate to the cell that adipocyte forms.For example, medicament is partly engaged with the target bone with diphosphonate.Find that the downward modulation increase of the 14-3-3 β matrix that mineralizes forms.The specific medicament with the merit iso series of target 14-3-3 or 14-3-3 can be used in combination with the activation proteic medicament of Ror2 (for example, causing Ror2 albumen two polymeric medicaments).This is combined in and promotes may have synergistic effect in the bone forming.

Others the invention provides the medicament that other downstream components of Ror2/14-3-3 β path very important in the regulation and control bone forming has been found in regulation and control.These medicaments can use separately or be used in combination with described other medicament herein.

Medicament of the present invention can be the compound of any kind, but is preferably protein, peptide, polynucleotide and small molecules.In certain embodiments, medicament is to promote Ror2 albumen two polymeric antibody or its fragment.Antibody can be polyclone or monoclonal antibody; Yet, concerning the treatment human individual, preferably be the peopleization monoclonal antibody usually.Antibody or its fragment can be any isotype; Yet, be preferably the IgG isotype usually.In certain embodiments, medicament is at proteic divalence of Ror2 or multivalent antibody fragment.In other embodiments, medicament is that 14-3-3 β specific RNA i, siRNA or shRNA construct body.

On the one hand, throw and activate the medicament of Ror2 albumen or inhibition 14-3-3 'beta ' activity to promote bone forming to individuality.In certain embodiments, throw and of the combination of the proteic medicament of activation Ror2 to individuality with the medicament that suppresses the 14-3-3 'beta ' activity.Especially, the throwing of medicament forms with promoting osteoblast differentiation and/or the increase matrix that mineralizes.Individuality can suffer from any bone photo related disorders or be in the risk of suffering from these illnesss, and these illnesss comprise osteoporosis, osteocarcinoma, sacroiliitis, rickets, fracture, periodontopathy, damaged, the molten bone osteopathia of segmental bone, primary and secondary hyperparathyroidism, handkerchief wise man disease, osteomalacia and hyperostosis.Medicament is particularly useful for treating the disease that runs off and be associated with bone.On the one hand, medicament promotes two polymerizations of Ror2 albumen, thus activation Ror2 albumen.Medicament can be the compound of any kind; Yet small molecules, polynucleotide, protein and peptide are especially suitable.In certain embodiments, medicament is at proteic antibody of Ror2 or antibody fragment.Suppose successful end userization monoclonal antibody in such as the treatment of the human diseases of Crohn disease (Crohn ' s disease) and multiple sclerosis, preferably be the peopleization monoclonal antibody usually.In certain embodiments, medicament downward modulation 14-3-3 expresses, and especially 14-3-3 β or 14-3-3 γ express.In some specific embodiment, medicament is 14-3-3 β specific RNA i, shRNA or siRNA.Also can use medicament of the present invention to promote osteoblast differentiation to come treatment of obesity, metabolic disorder or diabetes by be divided into cost with adipocyte.

On the other hand, cell is contacted to promote osteoblast differentiation or Osteoblast Differentiation with the medicament of activation Ror2 albumen or inhibition 14-3-3 'beta ' activity.The cell that is contacted is expressed Ror2 albumen or 14-3-3 β albumen usually and can be stood to be divided into osteoblasts in vitro.In certain embodiments, cell is stem cell, for example mescenchymal stem cell.Cell in vivo or is in vitro contacted with medicament.Cell is exsomatized with medicament contact, and then be introduced in the individuality (individuality of the bone photo related disorders that for example, suffer from the bone photo related disorders, especially is associated) that needs with the bone loss.

Find that also Ror2 crosses expression and 14-3-3 β inhibition can be suppressed to the fat differentiation.Therefore, the medicament of activation Ror2 albumen or inhibition 14-3-3 'beta ' activity also is applicable to and is suppressed to the fat differentiation.Therefore, medicament of the present invention is particularly useful for suppressing the one-tenth fat differentiation of stem cell (for example, mescenchymal stem cell).Be found to be the basis with this, Ror2 activator or 14-3-3 β adjust applicable to treatment or obesity prevention, diabetes or other metabolic disorder down.

The present invention also comprises a kind of system that Ror2 is active or express or regulate the medicament of the proteic phosphorylation of 14-3-3 β that regulates that differentiates.Described screening system comprises makes Ror2 albumen contact and detect the effect that this medicament is active to Ror2 or express with medicament.It is active or express and increase the indication medicament and be applicable to and promote bone forming, promote osteoblast differentiation or be suppressed to the fat differentiation to detect Ror2.In certain embodiments, by measuring the proteic activity of Ror2 albumen two polymeric degree calibrating Ror2.In other embodiments, by measuring the phosphorylation state evaluation Ror2 activity of Ror2 albumen itself or 14-3-3 β.In other embodiments, (for example) uses ³²P γ ATP or use immunoprecipitation (immunoprecipiation) the measure R or2 kinase activity of anti-phosphorylated tyrosine antibody.In certain embodiments, examine and determine the calibrating of expressing the proteic cell of Ror2 into using based on cell.In other embodiments, examine and determine acellularly, and use purifying or half purifying Ror2 albumen.Medicament and its medical composition of using sieve method of the present invention to differentiate are particularly useful for described methods of treatment of the present invention herein.

The present invention also provides and differentiates the calibrating that promotes Ror2 albumen two polymeric medicaments.The high-throughput techniques of a large amount of expecting compounds of screening is especially stood in calibrating.Chimerical receptor and the TrkB cell internal area be made up of Ror2 albuminous cell foreign lands are merged.The medicament activation TrkB signal transduction pathway that Ror2 cell foreign lands dimerization is closed, thus make the CRE promoter activity increase.Then, the reporter gene (such as, luciferase) that can use operability to be connected in the CRE promotor is differentiated and is made Ror2 two polymeric compounds.Ror2-TrkB mosaic calibrating has been used and has before been showed the anti-Ror2 antibody of Ror2 two polymeric is verified.When comparing with the cell of handling with non-specific IgG, the Ror2 specific antibody causes that the dose-dependently of viewed uciferase activity increases (referring to Figure 12).Described calibrating provides the high-throughput and the highly sensitive quick calibrating of the medicament of differentiating activation Ror2.It will be understood by one of ordinary skill in the art that other cell internal area that can use except that TrkB prepares mosaic.In calibrating, may need operability to be connected in the different promoters of reporter gene then.Examine and determine to differentiate to be that the activator of Ror2 or the medicament of two polymerizing agents (dimerizer) also are regarded as a part of the present invention by the present invention.

Definition

Provide to give a definition to fully understand term used herein and abbreviation.

Unless context has clearly indication in addition, otherwise as in this paper and the claims of enclosing use, singulative " " and " described " comprise a plurality of reference substances.Therefore, for example, mention that " cell " comprises a plurality of described cells, and mention that " antibody " is to mention one or more antibody and known its Equivalent of those skilled in the art etc.

Abbreviation in the specification sheets is corresponding to following linear module, technology, character or compound: " g " means gram, and " mg " means milligram, and " ng " means nanogram, and " kDa " means kilodalton, ".℃ " mean degree centigrade, " cm " means centimetre, and " s " means second; " min " means minute, and " h " means hour, and " l " means liter; " ml " means milliliter, and " μ l " means microlitre, and " pl " means the skin liter; " M " means volumetric molar concentration; " mM " means millimolar concentration, and " mmole " means mmole, and " kb " means kilobase; " bp " means base pair, and " RT " means room temperature.

" high performance liquid chromatography " is abbreviated as HPLC.

" open reading frame " is abbreviated as ORF.

" mass spectrum " is abbreviated as MS.

" tandem mass spectrum " is abbreviated as MS/MS.

" polyacrylamide gel electrophoresis " is abbreviated as PAGE.

" polymerase chain reaction " is abbreviated as PCR.

" reverse transcriptase-polymerase chain reaction " is abbreviated as RT-PCR.

" sodium lauryl sulphate " is abbreviated as SDS.

" sodium dodecyl sulfate-polyacrylamide gel electrophoresis " is abbreviated as SDS-PAGE.

" adenine nucleotide transfer protein 2 " is abbreviated as the ADP/ATP carrier proteins.

" bmd " is abbreviated as BMD.

" ribosome-RNA(rRNA) " is abbreviated as rRNA.

" non-translational region " is abbreviated as UTR.

In the context of this disclosure, will use a large amount of terms.As used herein, term Ror is meant the family of receptor tyrosine kinase sample orphan receptor." Ror molecule " is meant the nucleic acid of Ror polypeptide, Ror albumen, Ror peptide, its fragment, varient and mutant and coding Ror polypeptide, Ror albumen, Ror peptide and its fragment or varient or mutant." Ror molecule " also refers to Ror polynucleotide, gene and its varient and mutant." Ror molecule " and " Ror " are meant Ror1 and Ror2 molecule.

" target Ror molecule " is meant by medicament of the present invention and regulates active Ror molecule.Target Ror molecule can be Ror polypeptide, its homologue, derivative or fragment or varient or mutant.The Ror molecule of being paid close attention to also can be nucleic acid (oligonucleotide of RNA or DNA or polynucleotide).For example, if the protein of Ror gene receives publicity in experiment, target Ror molecule will be protein so.Should be understood that term target Ror molecule is meant full-length molecule and its fragment, varient and mutant, such as proteinic epi-position.Target Ror molecule can be Ror1 molecule or Ror2 molecule or both.In some specific embodiment, target Ror molecule is a Ror2 albumen.

Term " 14-3-3 " is meant the proteinic family that relates to signal transduction.14-3-3 albumen has a large amount of combination collocation things and relates to large numbers of and multifarious cell processes.14-3-3 albumen is by in conjunction with its target and cause (1) topographical variations; (2) physical blockage of sequence-specific or structural protein feature and/or (3) proping are brought into play its effect.The several pieces of comments about the proteic 26S Proteasome Structure and Function of 14-3-3 are Bu Lizhisi (Bridges) and Mo Hede (Moorhead), " 14-3-3 albumen: the proteinic multiple function of a kind of numbering " (＂ 14-3-3proteins:A Number of Functionsfora Numbered Protein, ＂) signal transduction knowledge environment (Sci.STKE) re10,2004; Macintosh (Mackintosh), " the dynamic interaction regulation and control diversity cell processes between 14-3-3 albumen and the phosphoric acid albumen " (＂ Dynamicinteractionsbetween14-3-3proteins and phosphoproteins regulate diversecellular processes, ＂) journal of biological chemistry (Biovhem.J.) 381:329-42,2004; It is incorporated herein by reference separately." 14-3-3 " can refer to the nucleic acid of 14-3-3 polypeptide, 14-3-3 albumen, 14-3-3 peptide, its 14-3-3 fragment, 14-3-3 varient and 14-3-3 mutant and coding 14-3-3 polypeptide, 14-3-3 albumen, 14-3-3 peptide and its 14-3-3 fragment or 14-3-3 varient or 14-3-3 mutant.Several that differentiated 14-3-3 comprise β, ε, η, γ, τ, ξ and σ with the merit iso series.Find that the proteic activation of Ror2 causes the phosphorylation with merit iso series 14-3-3 β.Find that 14-3-3 β and 14-3-3 γ interact with Ror2.In some cases, especially mention 14-3-3 β.Under some other situation, especially mention 14-3-3 γ.

Term " nucleic acid molecule " is meant the phosphoric acid ester form of ribonucleotide (RNA molecule) or deoxyribonucleotide (dna molecular) or is single stranded form or any phosphodiester analogue of double-stranded spiral form.May be double-stranded DNA-DNA, DNA-RNA and RNA-RNA spiral form.Term nucleic acid molecule and DNA or RNA molecule are meant the primary structure of molecule and secondary structure and it are not confined to any specific three grades of forms specifically.Therefore, this term comprises visible double-stranded DNA in wire (for example, restricted fragment) or ring-shaped DNA molecule, plastid and the karyomit(e).When the structure of specific double chain DNA molecule is discussed, can be according to only providing non-transcribed chain along DNA (that is, have with mRNA homologous sequence chain) 5 ' describe sequence to the normal regulation of the sequence of 3 ' direction.

" recombinant nucleic acid molecules " is the nucleic acid molecule that stands the molecular biosciences operation, that is, and and nucleic acid molecule or genetically engineered nucleic acid molecule that non-natural exists.In addition, term " recombinant DNA molecules " is meant the nucleotide sequence of two fragments of separating (that is, by common discontinuous DNA fragment is linked together) manufacturing that nucleotide sequence that non-natural exists maybe can be by the artificial combination nucleotide sequence." reorganization produces " means artificial combination, it is usually by chemical synthesis process or realizing through isolated fragment by manual operation nucleic acid, for example the genetic engineering technique by using restriction enzyme, ligase enzyme and as following document described in similar recombinant technology realize: mountain nurse Brooker people such as (Sambrook) for example, molecular cloning (Molecular Cloning), the 2nd edition Spring Cold Spring Harbor Laboratory (Cold Spring Harbor Laboratory), Plainview (Plainview), New York (N.Y.), (1989); Or A Su Bel people such as (Ausubel), molecular biology experiment guide (Current Protocols in Molecular Biology), experiment guide (Current Protocols) (1989); And dna clone: hands-on approach (DNA Cloning:A Practical Approach), I volume and II volume (Di Nigaoerfu (D.N.Glover) volume), IREL press (IREL Press), Oxford (Oxford), (1985); It is incorporated herein by reference separately.

Described operation available code redundant code identical or conservative amino acid is replaced codon, introduces usually simultaneously or removes the recognition sequence site and carry out.Perhaps, can combine by the nucleic acid fragment that will have desirable function and comprise in the common natural form the single hereditary entity of sightless desirable function combinations with generation and carry out.Restriction enzyme recognition site is generally described manually-operated target, but can deliberately incorporate other locus specificity target into, for example promotor, dna replication dna site, regulating and controlling sequence, control sequence, open reading frame or other feature that is suitable for.The example of recombinant nucleic acid molecules comprises recombinant vectors, be 5 such as containing ' to 3 ' (justice is arranged) directed or 3 ' to 5 ' (antisense) the directed coding Ror family proteins or the clone or the expression vector of the albuminous dna sequence dna of immune globulin.

Term " polynucleotide ", " nucleotide sequence ", " nucleic acid ", " nucleic acid molecule ", " nucleotide sequence " and " oligonucleotide " are meant a series of nucleotide bases (being also referred to as " Nucleotide ") among DNA and the RNA and mean the chain of any two or more Nucleotide.Polynucleotide can be strand or double-stranded chimeric mixture or derivatives thereof or modified form.Oligonucleotide can be in base portion, sugar moieties or phosphoric acid salt main chain place modified (for example) stability, its hybridization parameter etc. with the improvement molecule.Antisense oligonucleotide can comprise modified base portion; described part is selected from following group; include, but is not limited to 5 FU 5 fluorouracil; 5-bromouracil; the 5-chlorouracil; 5-iodouracil; xanthoglobulin; xanthine; 4-ethanoyl cytosine(Cyt); 5-(carboxyl hydroxymethyl) uridylic; 5-carboxyl methylamino methyl-2-thiouridine; 5-carboxyl methylamino 6-Methyl Uracil; dihydrouracil; β-D-galactosyl Q nucleosides (galactosylqueosine); inosine; the N6-isopentenyl gland purine; the 1-methyl guanine; the 1-methylinosine; 2; the 2-dimethylguanine; the 2-methyladenine; the 2-methyl guanine; the 3-methylcystein; 5-methylcytosine; the N6-VITAMIN B4; the 7-methyl guanine; 5-methylamino 6-Methyl Uracil; 5-methoxyl group amino methyl-2-thiouracil; β-D-mannose group Q nucleosides; 5 '-methoxyl group carboxyl 6-Methyl Uracil; the 5-methoxyuracil; 2-methylthio group-N6-isopentenyl gland purine; fourth oxygen nucleosides (wybutoxosine); pseudouracil (pseudouracil); Q nucleosides (queosine); 2-sulphur cytosine(Cyt); 5-methyl-2-thiouracil; the 2-thiouracil; the 4-thiouracil; methyl uracil; uridylic-5-ethoxyacetic acid methyl esters; uridylic-5-ethoxyacetic acid; 5-methyl-2-thiouracil; 3-(3-amino-3-N-2-carboxyl propyl group) uridylic and 2,6-diaminopurine.Nucleotide sequence carries genetic information usually, comprises that cell mechanism is used to make the information of protein and enzyme.These terms comprise two strands or strand genome and cDNA, RNA, any synthetic and through the polynucleotide of genetic manipulation with adopted polynucleotide and antisense polynucleotide are arranged.It comprises strand and duplex molecule, that is, and and DNA-DNA, DNA-RNA and RNA-RNA hybrid and by making " protein nucleic acid " that alkali and amino acid backbone conjugation form (PNA).It also comprises the nucleic acid that contains modified base (for example, thiouracil, Tioguanine and Fluracil) or contain carbohydrate or lipid.

Polynucleotide of the present invention can be synthetic by known standard method in the affiliated field, for example, by use the automated DNA synthesizer (such as, can search for the synthesizer of (Biosearch), u.s.a. applied biosystem company (AppliedBiosystems) etc. available from biology) synthesize.For instance, can be according to Si Tanyin people such as (Stein), nucleic acids research (Nucl.Acids Res.), 16,3209 (1988) method is synthesized the thiophosphatephosphorothioate oligonucleotide, can be by using controlled micropore glass polymer support thing (sarin people such as (Sarin), periodical (Proc.Natl.Acad.Sci.U.S.A.) 85 of institute of NAS, 7448-7451 (1988)) preparation methyl-phosphonate oligonucleotide etc.Researched and developed multiple method from RNA to cell that transmit antisense DNA or, for example, antisense molecule can be injected directly into tissue site, but or general throw with through designing modified antisense molecule with the desirable cell of target (be connected in specificity and combine the acceptor that is expressed in the target cell surface or the antisense molecule of antigenic peptide or antibody).Perhaps, can produce the RNA molecule by the dna sequence dna of transcribing encoding antisense RNA molecule in vitro and in vivo.Described dna sequence dna can be incorporated in the multiple carrier that is associated with suitable rna polymerase promoter (such as T7 or SP6 polymerase promoter).Perhaps, the Antisense cDNA of deciding composition or inducibility synthesize antisense rna on employed promotor can be constructed body stably introduces in the clone.Yet, be difficult to reach the IC of the antisense molecule that is enough to suppress endogenous mRNA translation usually.Therefore, the preferred method utilization recombinant DNA of placing antisense oligonucleotide under the control of strong promoter is constructed body.Use the described target cell that constructs body transfection patient will reach transcribing of capacity single stranded RNA, these RNA will be right with endogenous target gene transcript formation complementary base, thus and the translation of prevention target gene mRNA.For instance, consequently it is occupied and guides transcribing of sense-rna by cell can in vivo to introduce carrier.Described carrier can be still for free type or become and on karyomit(e), integrate, as long as it can be transcribed to produce desirable sense-rna.Described carrier can be constructed by the standard DNA recombinant technology method in the affiliated field.Carrier can be used for mammalian cell duplicate and plasmid, virus or the affiliated field of expressing in known other analogue.The expression of the sequence of encoding antisense RNA can act on mammalian cell, preferable human cell by known any promotor in the affiliated field.That described promotor can be inducibility or composition.Described promotor includes, but is not limited to: SV40 early promoter district (Bei Nuosite (Bernoist) and look into ripple (Chambon), nature (Nature), 290,304-310 (1981)), contained promotor (Yamamoto people such as (Yamamoto) in 3 ' long terminal repeat of Rous sarcoma virus (Rous sarcoma virus), cell (Cell), 22,787-797 (1980)), herpes thymidine kinase promoter (Wagner people such as (Wagner), institute of NAS periodical, 78,1441-1445 (1981)) regulating and controlling sequence of metallothionein gene (Bu Yinsite people such as (Brinster), nature (Nature), 296,39-42 (1982)) etc.Can use plasmid, coemid (cosmid), yeast artificial chromosome or the virus vector of any kind to prepare the recombinant DNA that directly to introduce in the tissue site and construct body.Perhaps, can use selectivity to infect the virus vector of desirable tissue, under this situation, can pass through another approach (for example, general) and realize dispensing.

Polynucleotide can be by natural regulation and control (expressing control) sequence side joint, or can associate with heterologous sequence, these heterologous sequences comprise promotor, internal ribosome entry site (internal ribosome entry site, IRES) and other ribosome bind site sequence, strengthen son, response element, supressor, signal sequence, gather the VITAMIN B4 sequence, intron, 5 ' and 3 ' non-coding region and its analogue.Nucleic acid also can be modified by known several different methods in the affiliated field.The limiting examples of described modification comprise methylate, " adding cap ", replace between one or more naturally occurring Nucleotide and Nucleotide with analogue and to modify, such as (for example using not charged binding, methyl-phosphonate, phosphotriester, phosphoramidate, carbamate etc.) and the Nucleotide of charged binding (for example, thiophosphatephosphorothioate, phosphorodithioate etc.) between modify.Polynucleotide can contain one or more other covalently bound part, such as protein (for example, nuclease, toxin, antibody, signal peptide, poly-L-Lysine etc.), intercalator (intercalator) (for example, acridine, psoralene etc.), sequestrant (for example, metal, radioactive metal, iron, oxidized metal etc.) and alkylating agent (alkylator).Can be by forming trimethyl phosphite 99 or triethyl phosphate or the phosphamide alkyl ester binding polynucleotide of deriving.In addition, the also available mark of direct or indirect detectable signal that can provide of polynucleotide is herein modified.Exemplary mark comprises radio isotope, fluorescence molecule, vitamin H and its analogue.

" rna transcription thing " is meant the product of being transcribed generation by the RNA polymerase catalysis of dna sequence dna.When the rna transcription thing is the complementary duplicate of dna sequence dna, be referred to as the one-level transcript, or it can be the RNA sequence of transcribing post-treatment that is derived from the one-level transcript and is called mature rna." messenger RNA(mRNA) (mRNA) " is meant intronless and can be translated as the RNA of polypeptide by cell." cRNA " is meant the complementary RNA of being transcribed by the recombinant cDNA template." cDNA " is meant complementary and be derived from the DNA of mRNA template with the mRNA template.The Klenow fragment (Klenow fragment) that cDNA can be single stranded form or use (for example) dna polymerase i is converted into double chain form.

With the sequence of the part " complementation " of RNA be meant have be enough to can with the complementarity of RNA hybridization, stablize double-helical sequence thereby form; Therefore under the situation of double-stranded antisense nucleic acid, can test the formation that the single chain of duplex DNA maybe can be examined and determine triple helical.The hybridization ability will be decided on the length of complementary degree and antisense nucleic acid.Usually, hybrid nucleic acid is long more, and it may contain and to form the base mispairing of the RNA stablize duplex (or triple helical, depend on the circumstances) again many more.The those skilled in the art finds out the tolerable degree of mispairing by using standard program, thereby determines the fusing point of hybridization complex.

Term " nucleic acid " or " nucleotide sequence ", " nucleic acid molecule ", " nucleic acid fragment " or " polynucleotide " can exchange use with " gene ", " mRNA of genes encoding " and " cDNA ".

Polynucleotide that can only comprise encoding sequence and the polynucleotide that can comprise extra coding or non-coding sequence contained in term " polynucleotide of coded polypeptide ".

When the single stranded form of nucleic acid molecule can be under proper temperature and solution ion strength condition be annealed with another nucleic acid molecule (such as cDNA, genomic dna or RNA), nucleic acid molecule and this another nucleic acid molecule " can be hybridized " so, mountain nurse Brooker outstanding person (Sambroo k, J.) etc. the people compiles, molecular cloning (Molecular Cloning): laboratory manual (ALaboratory Manual) (the 2nd edition, 1989), press of cold spring harbor laboratory (Cold Spring Harbor LaboratoryPress), New York, 1-3 rolls up (ISBN 0-87969-309-6)." the strict degree " of temperature and ionic strength conditions decision hybridization.For the preliminary screening homologous nucleic acid, can use corresponding to T _mBe 55 ℃ low stringent hybridization condition, for example, 5 * SSC, 0.1% SDS, 0.25% milk and do not have methane amide; Or 30% methane amide, 5 * SSC, 0.5%SDS.Medium stringent hybridization condition is corresponding to higher T _m, for example, 40% methane amide, 5 * or 6 * SSC.High stringent hybridization condition is corresponding to the highest T _m, for example, 50% methane amide, 5 * or 6 * SSC.Hybridization requires two nucleic acid all to contain complementary sequence, but decides on the strict degree of hybridization, and the mispairing between the base also is possible.The strict degree that is suitable for making nucleic acid hybridization under the length and the complementary degree of the variable nucleic acid known in the field decide.Similarity or homology between two nucleotide sequences are big more, have the T of the nucleic acid hybridization thing of these sequences _mBe worth big more.The relative stability of nucleic acid hybridization is (corresponding to higher T _m) reduce in the following order: RNA:RNA, DNA:RNA, DNA:DNA.For the hybrid of length greater than 100 Nucleotide, derived the equation that calculates Tm, mountain nurse Brooker people such as (Sambrook) compiles, molecular cloning (Molecular Cloning): laboratory manual (A Laboratory Manual) (the 2nd edition, 1989), press of cold spring harbor laboratory, and N Y. 1-3 volume (ISBN 0-87969-309-6,9.50-9.51).For with shorter nucleic acid (promptly, oligonucleotide) hybridization, the position of mispairing becomes more important, and the length of oligonucleotide determines its specificity, and mountain nurse Brooker people such as (Sambrook) compiles, molecular cloning (Molecular Cloning): laboratory manual (A LaboratoryManual) (the 2nd edition, 1989), press of cold spring harbor laboratory, and N Y. 1-3 volume (ISBN 0-87969-309-6,11.7-11.8).

Term " complementation " is used to describe the relation between the nucleotide base that can hybridize each other.For instance, concerning DNA, adenosine and thymus pyrimidine complementation and cytosine(Cyt) and guanine complementation.

As known in the affiliated field, " consistence " or " similarity " is as passing through the relation between determined two or more peptide sequences of comparative sequences or two or more polynucleotide sequences.In affiliated field, consistence depend on the circumstances also mean as by as described in the determined polypeptide of coupling between the chain of sequence or the sequence degree of correlation between the polynucleotide sequence.Consistence and similarity can easily be calculated by currently known methods, the method of described method described in following document: computer molecular biology (Computational Molecular Biology), Lai Sikeami (Lesk, A.M.) compile, Oxford University Press (Oxford University Press), New York (New York), 1988; Biological computation: information science and genome special topic (Biocomputing:Informatics and Genome Projects), Smith's enlightening is fertile, and (Smith D.W.) compiles academic press (Academic Press), New York (New York), 1993; Sequential analysis in the molecular biology (Sequence Analysis in Molecular Biology), and Fan Haiyeji (vonHeinje, G.), academic press (Academic Press), 1987; The Computer Analysis part i of sequence data (Computer Analysis of Sequence Data, Part I), lattice Lifei Yin Ami (Griffin, A.M.) and lattice Lifei Yin Heji (Griffin, H.G.) compile, Hu Mana press (Humana Press), New Jersey (New Jersey), 1994; And sequence analysis primer (Sequence Analysis Primer), (Gribskov, M.) (Devereux J.) compiles lucky cloth koff rice, rice Stockton press (M Stockton Press), New York (New York), 1991 with moral Fu Laikejie.Be generally used for measuring the consistence between the sequence or the method for similarity and include, but is not limited to Ka Liluohe (Carillo, H.) and Li Pimandi (Lipman, D.), SIAM applied mathematics (SIAM JApplied Math.), the method that is disclosed among the 48:1073 (1988).The method of consistence and similarity of measuring is compiled with open available computer program.Measure the consistence between two sequences and the preferred computer program technic of similarity and include, but is not limited to GCG routine package (moral Fu Laikejie (Devereux, people such as J.), nucleic acids research (Nucleic Acids Research), 12 (1), 387 (1984)), ((Atschul S.F.) waits people, molecular biology magazine (J Molec.Biol.) to the Aunar Amlexanox for BLASTP, BLASTN and FASTA, 215,403 (1990)).

" homology " is meant the sequence similarity degree between two polymkeric substance (that is, peptide molecule or nucleic acid molecule).Maximum homology possibility between two polymkeric substance of homology % numeral reflection mentioned herein, that is, and as the homology % of two polymkeric substance when comparison has maximum couplings (homology) position number.

Term " homology % " is meant the degree of the consensus amino acid sequence between the polypeptide.Homology between any two polypeptide is the direct function of the amino acid whose sum of coupling of given position in arbitrary sequence, for example, if half of the amino acid sum in arbitrary sequence is identical, thinks that so two sequences represent 50% homology.

When being meant when mentioning polypeptide, term " fragment ", " analogue " and " derivative " can keep biological function substantially the same or active polypeptide with original polypeptide.Therefore, analogue comprises and can come the activatory precursor protein to produce active mature polypeptide by the cracked precursor protein part.The fragment of polypeptide, analogue or derivative can be wherein one or more amino acid through conservative or non-conservative amino acid residues replace and these amino-acid residues may for or the fragment of the residue of may be not encoding for genetic code, analogue or derivative, or wherein one or more amino-acid residue comprises a substituent fragment, analogue or derivative, or wherein make polypeptide and the fragment that merges such as the compound of polyoxyethylene glycol with the transformation period that increases polypeptide, analogue or derivative, or the fragment that other amino acid and polypeptide are merged, analogue or derivative are such as signal peptide or be used for the sequence (such as the polyhistidyl label) of described polypeptide of purifying or precursor protein.Think that described fragment, analogue or derivative are in category of the present invention.

" guarding " residue of polynucleotide sequence is unaltered those residues in the same position of two or more correlated serieses of being compared.Bao Shou residue is those residues of guarding than the residue that other place occurred in the sequence in correlated series relatively.

Relevant polynucleotide is a polynucleotide of sharing the consistent residue of vast scale.

If a polynucleotide ultimate source is from another polynucleotide, so described not homopolynucleotide is " correspondence " each other.For instance, messenger RNA(mRNA) is corresponding to the gene that is transcribed into described RNA.CDNA corresponding to (such as) by reverse transcription reaction or produce the RNA of described cDNA by DNA chemosynthesis based on the knowledge of RNA sequence.CDNA is also corresponding to the gene of coding RNA.If polynucleotide plays similar action, such as the related polypeptide in the coding different plant species, bacterial strain or the varient that are compared, its " correspondence " also each other so.

" analogue " of DNA, RNA or polynucleotide (for example is meant form and/or function, participate in the ability of the specificity hydrogen bond knot of the base pair on sequence and the complementary polynucleotide sequence) go up similar naturally occurring polynucleotide but the molecule of some aspect and DNA or RNA different (for example, have uncommon or non-natural base or through the change main chain).For example referring to, Liv Ullmann people such as (Uhlmann), chemistry summary (Chemical Reviews) 90,543-584 (1990).

The sequence of " encoding sequence " or " coding " such as RNA, polypeptide or protein expression product is (for example to cause producing described RNA, polypeptide or protein when expressing, enzyme) nucleotide sequence, that is the nucleotide sequence of coding said polypeptide or proteinic aminoacid sequence.

" the substantive part " of amino acid or nucleotide sequence is the enough parts that comprise polypeptid acid sequence or gene nucleotide series, thereby by the those skilled in the art by manual assessment sequence or use algorithm by the comparison of computer automatic sequence with differentiate the part of described polypeptide of inferring property discriminating or gene, described algorithm is such as basic local comparison research tool (Basic Local Alignment Search Tool, BLAST; (Altschul S.F.) waits people, biological molecule magazine (J.Mol.Biol.) 215,403-410 (1993) to Aunar Shu Ersifa; Also referring to www.ncbi.nlm.nih.gov/BLAST).

Therefore, " the substantive part " of nucleotide sequence comprises enough parts of described sequence, thereby the sequence of the nucleic acid fragment that comprises described sequence is differentiated and/or separated to specificity.The those skilled in the art of the sequence right of now, enjoying herein being reported can use the whole sequence that discloses or its substantive part to reach the known purpose of those skilled in the art.

" synthetic gene " can be by using the known program chemistry of those skilled in the art synthetic oligonucleotide to make up packaged joining.Connect these and make up piece and make its annealing to form gene fragment, enzymatic assembles these gene fragments to construct whole gene then." chemosynthesis " means in vitro make-up unit and divides Nucleotide when relevant with dna sequence dna.Can use and know the manual chemosynthesis that program realizes DNA, maybe can use a kind of execution robotics in some available machinery synthetic.Therefore, can repair gene to reach best genetic expression based on the nucleotide sequence optimization of the codon preference that reflects host cell.It will be understood by one of ordinary skill in the art that the possibility that when codon uses those codons of being partial to host's preference, realizes successful genetic expression.Can determine preferred codon based on observation to the gene that is derived from sequence information available host cell.

" gene " is meant the nucleic acid fragment of expressing specified protein, comprises the preposition regulating and controlling sequence (5 ' non-coding sequence) of encoding sequence and the rearmounted regulating and controlling sequence (3 ' non-coding sequence) of encoding sequence." primary gene " is meant that the occurring in nature visible has the gene of autogenous control sequence." mosaic gene " or " the chimeric body of constructing " is meant not to any gene that comprises regulating and controlling sequence that occurring in nature do not find and encoding sequence of primary gene together or constructs body.Therefore, mosaic gene or the chimeric body of constructing can comprise the regulating and controlling sequence that is derived from different sources and encoding sequence or be derived from identical source but regulating and controlling sequence and the encoding sequence arranged in the mode that is different from the occurring in nature visual way." endogenous gene " is meant the primary gene at the natural place place that is arranged in the organism genome." external source " gene is meant in the host organisms usually invisible but introduces gene in the host organisms by transgenosis.Foreign gene can comprise primary gene or the mosaic gene that inserts in the non-protogenous organism." transgenosis " is by the gene in the program introducing transition genome.

" regulating and controlling sequence " is meant the nucleotide sequence that is positioned at encoding sequence upstream (5 ' non-coding sequence), inside or downstream (3 ' non-coding sequence) and influences the transcribing of correlative coding sequence, RNA processing or stability or translation.Regulating and controlling sequence can comprise promotor, translation leader sequence, intron and poly-VITAMIN B4 recognition sequence.

" genetic control sequence " be meant initial gene transcribe required dna sequence dna add regulation and control initial generation the required dna sequence dna of speed.Therefore, genetic control sequence can be made up of promotor, and wherein general transcription factor and polysaccharase add that all regulating and controlling sequences of the speed of these assembling processes in the gene regulating protein institute bonded control promotor assemble together.For instance, be suitable for procaryotic control sequence and can comprise promotor, sequence of operators and ribosome bind site according to circumstances.Eukaryotic cell can utilize promotor, strengthen son and/or poly-VITAMIN B4 signal.

" promotor " is meant the nucleotide sequence of the expression that can control encoding sequence or functional r NA.Encoding sequence is usually located at 3 of promoter sequence ' locate.Promoter sequence is formed by near-end with than the far-end upstream element, and latter's element is commonly referred to strengthens son.Therefore, " strengthen son " is can stimulate promoter activity and can be the congenital element of promotor or through inserting with the expression level that strengthens promotor or the nucleotide sequence of tissue-specific allos element.Promotor can whole be derived from primary gene or comprise the different elements that is derived from occurring in nature visible different promoters or even comprise the synthetic nucleosides acid fragment.It will be understood by one of ordinary skill in the art that in bootable different tissues of different promoters or the cell type or different stages of development or genetic expression that the varying environment condition is reacted.

" 3 ' non-coding sequence " is meant and is positioned at the encoding sequence downstream and comprises poly-VITAMIN B4 recognition sequence and coding can influence the nucleotide sequence of other sequence of the adjustment signal of mRNA processing or genetic expression.The feature of poly-VITAMIN B4 signal is to influence to 3 of mRNA precursor ' end usually adds polyadenylic acid tract (polyadenylic acid tract).

" translation leader sequence " is meant at the promoter sequence of gene and the nucleotide sequence between the encoding sequence.The translation leader sequence appears at the mRNA upstream through processing fully of translation initiation sequence.The translation leader sequence can influence the one-level transcript and be processed as mRNA, mRNA stability or translation efficiency.

Term " operability connection " be instigate two or more nucleic acid fragments associate for the single nucleic acid fragment so that one function is influenced by another.For instance, when promotor can influence the expression of encoding sequence, promotor was connected (that is, encoding sequence is in transcribing under the control of promotor) with this encoding sequence operability.Encoding sequence can have justice or antisense orientation to be connected with the regulating and controlling sequence operability.Term " exercisable promotor in osteocyte " is meant the promotor by the RNA polymerase identification of osteocyte.

" rna transcription thing " is meant the product of being transcribed generation by the RNA polymerase catalysis of dna sequence dna.When the rna transcription thing is the perfect complementary duplicate of dna sequence dna, be referred to as the one-level transcript, or it can be the RNA sequence of transcribing post-treatment that is derived from the one-level transcript and is called mature rna." messenger RNA(mRNA) (mRNA) " is meant intronless and can be translated as the RNA of polypeptide." cDNA " is meant complementary and be derived from the double-stranded DNA of mRNA with mRNA.RNA is meant the rna transcription thing that comprises mRNA and therefore can be translated as polypeptide by cell " justice "." sense-rna " is meant with entire target one-level transcript or mRNA or its part is complementary and the rna transcription thing (referring to United States Patent (USP) the 5th, 107, No. 065, it is incorporated herein by reference) of blocking-up target gene expression.The complementarity of sense-rna can be at any part of specific nucleotide sequence, that is, and and at 5 ' non-coding sequence, 3 ' non-coding sequence, intron or encoding sequence place." functional r NA " is meant adopted RNA, sense-rna, ribose ribozyme or may be translated but still other RNA that the pair cell process exerts an influence.

Term " expression " is meant and is derived from transcribing and stable accumulation of adopted RNA of having of nucleic acid fragment of the present invention (mRNA) or sense-rna.Express and also can refer to mRNA is translated as polypeptide." Antisense Suppression " is meant that generation can suppress the sense-rna transcript that target protein is expressed.

" cross and express " and be meant that generation is above the gene product of the generation level in the normal or non-organism that makes the transition in organism." inhibition " is meant the expression that suppresses external source or endogenous gene or rna transcription thing.

" change level " is meant and produces the amount different with the normal or non-organism that makes the transition or the gene product of ratio in organism.Cross expressing of polypeptide of the present invention can be by at first constructing coding region wherein and can or constructing body at desirable developmental stage guiding gene and be expressed in mosaic gene or the chimeric body of constructing that the promotor operability in the desirable tissue is connected and realize.For easy consideration, mosaic gene or the chimeric body of constructing can comprise promoter sequence and the translation leader sequence that is derived from homologous genes.3 ' non-coding sequence of encoding transcription termination signal also can be provided.Mosaic gene of the present invention or the chimeric body of constructing also can comprise one or more intron to promote genetic expression.Then, can construct and comprise mosaic gene of the present invention or the chimeric plasmid vector of constructing body.The selection of plasmid vector is depended on and will be used to make the host cell method of transition.The those skilled in the art is fully recognized that to successfully making the transition, select and breeding contains mosaic gene or the chimeric host cell of constructing body, and genetic elements must be present on the plasmid vector.The those skilled in the art it should be understood that also incident will produce different expression levels and pattern (Jones people such as (Jones), European molecular biology magazine (EMBOJ.), 4,2411-2418, (1985) different independent transition; De Ameida people such as (De Almeida), MGG (Mol.Gen.Genetics), 218,78-86, (1989)), and therefore must screen a plurality of incidents for the clone that obtains to present desirable expression level and pattern.Described screening can the south by DNA be analyzed the north that (southern analysis), mRNA express and is analyzed the west of (northern analysis), protein expression and analyze (western analysis) or immunocytochemical assay or phenotype analytical and realize.

Term " varient " refers to the variation of the nucleic acid or the aminoacid sequence of Ror molecule.Nucleotide and aminoacid replacement, interpolation or the disappearance of Ror molecule contained in term " varient ".Natural and the synthetic Ror molecule of chemically modified also contained in term " varient ".For instance, varient can refer to be different from the polypeptide of reference polypeptide.Polypeptide different aspect the aminoacid sequence and the difference between the reference polypeptide with reference polypeptide normally limited so that the aminoacid sequence of reference polypeptide and varient is very similar generally, and consistent in some districts.Varient and reference polypeptide aspect the aminoacid sequence may because of one or more can be conservative property or non-conservation and replacement, disappearance, interpolation that can any combination exist, merge with block different.For instance, varient can be wherein with any combination and inserts, replaces or lack several, for example 50 to 30,30 to 20,20 to 10,10 to 5,5 to 3,3 to 2,2 to 1 or 1 amino acid whose polypeptide.In addition, varient can be because of shorter than reference sequences such as terminal or inner disappearance, thus the fragment of of the present invention polypeptide different with the reference polypeptide sequence.The varient of polypeptide of the present invention also comprises and keeps biological function or the active polypeptide substantially the same with described polypeptide, for example can be by cracked precursor activatory precursor protein partly to produce active mature polypeptide.It is that the allelic variation of feature maybe can comprise differentiation montage or posttranslational modification that these varients can be difference with the nucleotide sequence of the structure gene of coded protein.Varient also comprises having identical substantially biological activity but available from the related protein of different plant species.The those skilled in the art can produce the varient with single or multiple aminoacid replacement, disappearance, interpolation or replacement.These varients especially can comprise: (i) wherein one or more amino-acid residue through conservative or non-conservative amino acid residues (preferred conservative amino acid residues) replace and these substituted amino acid residues may for or may not be the varient of genetic code amino acids coding residue, or (ii) wherein peptide or protein lack one or more amino acid whose varient, or (iii) wherein polypeptide or protein are added with one or more amino acid whose varient, or (iv) wherein one or more amino-acid residue comprise a substituent varient, or (the v) varient that merges of mature polypeptide and another compound wherein, described compound is such as the compound that increases the polypeptide transformation period, polyoxyethylene glycol for example, or (the vi) varient that merges of other amino acid and mature polypeptide wherein is such as leader sequence or secretion sequence or be used for the sequence or the precursor protein sequence of purifying mature polypeptide.The varient of polypeptide also can be naturally occurring varient (such as naturally occurring allelic variation body) or its and can be and whether still do not know naturally occurring varient.Think that above all these varients that define are all in the category of the teaching in affiliated field.

Polypeptide of the present invention and polynucleotide are preferably to provide through unpack format and but purifying is a homogeneous.In some cases, polypeptide and polynucleotide are at least 90% pure, at least 95% pure, at least 98% pure or at least 99% pure.

Term " through separating " means the material that shifts out from original or primitive environment (for example, if natural existence is physical environment so).Therefore, the naturally occurring polynucleotide or the polypeptide that are present in the Live Animals are not separated, and are separated by artificial intervention with isolating homopolynucleotide mutually of the some or all coexisting substances in the natural system or polypeptide.For instance, the strand of the nucleotide base of " through separating acid fragment ", non-natural synthetic or change or the polymkeric substance of double-stranded RNA or DNA for containing according to circumstances.Be can the comprising one or more fragment of cDNA, genomic dna or synthetic DNA and can combine of DNA polymer form with carbohydrate, lipid, protein or other material through separating acid fragment.Described polynucleotide can be the part that the part of carrier and/or described polynucleotide or polypeptide can be composition, but it still is separated, because described carrier or composition are not the parts of its residing environment when occurring in nature is visible.Similarly, term " purifying substantially " is meant by artificial intervention with residing direct chemical environment separation when occurring in nature exists or from the material that wherein shifts out.The polypeptide of purifying or nucleic acid can be by any acquisition or the generations in common known some technology and the program in the affiliated field substantially.

Term " purifying " is meant specific activity or the concentration that increases specific polypeptide in the sample.In one embodiment, specific activity is represented with the ratio that sample hits between the concentration of the activity of polypeptide and total polypeptide.In another embodiment, specific activity is represented with the ratio between the concentration of the concentration of target polypeptide and total polypeptide.Purification process includes, but is not limited to dialysis, centrifugal and column chromatography technology, and it is well-known to one skilled in the art program.For example referring to poplar people such as (Young), 1997, " the proteinic generation of biological medicine in the transgenosis milcher milk " (＂ Production of biopharmaceuticalproteins in the milk of transgenic dairy animals, ＂), bio-pharmaceuticals (BioPharm), 10 (6): 34-38.

Term " pure substantially " and " through separating " do not plan to get rid of polynucleotide or polypeptide and occurring in nature not with the mixture of polynucleotide or the associating material of polypeptide.

Term " cell ", " clone " and " cell culture " are used interchangeably.All these terms also comprise its filial generation, and filial generation can be spawn and all offsprings.Should be appreciated that owing to deliberately or accidentally suddenling change, all filial generations may be also inequality.Under the situation of expressing heterologous nucleotide sequence, " host cell " is meant and is positioned in vitro or intravital protokaryon or eukaryotic cell (for example, such as intestinal bacteria (E.coli) bacterial cell, yeast cell, mammalian cell, bird cell, Amphibians cell, vegetable cell, fry cell and insect cell).For instance, the host and can comprise can replicating cell any organism that makes the transition can be arranged in transgenic animal.Host cell can be used as the recipient of carrier vector and/or expresses heterologous nucleic acids by vector encoded.

The general method of expressing and reclaiming the exogenous protein that is produced by the mammal cell line system is by (for example) Ai Zhi li (Etcheverry), " through engineering approaches protein expression in the mammalian cell cultures " (＂ Expression ofEngineered Proteins in Mammalian Cell Culture, ＂), protein engineering: principle and put into practice (ProteinEngineering:Principles and Practice), Cleveland people's (volume) such as (Cleland), the 163rd page of (Willie-(Wiley-Liss of Li Si company, Inc.), 1996) provide.The proteinic standard technique that recovery is produced by bacterial system is by (for example) Ge Lisihanmo people such as (Grisshammer), " by the protein purification of the excessive generation of Bacillus coli cells " (＂ Purification of over-produced proteins from E.coli cells, ＂), dna clone 2: expression system (DNACloning2:Expression Systems), the 2nd edition, Gao Erfu (Glover) (volume), 59-92 page or leaf (Oxford University Press (Oxford University Press), 1995) provides.Insect cell transition reconciling the exterior and interior of the body source polypeptide generation disclose by the open case WO94/06463 of the US5162222 that adds Reno people such as (Guarino) and WIPO.The method of separating recombinant proteins matter is also by Li Chadesen (Richardson) (volume) from rhabdovirus system, " baculovirus expression scheme " (＂ Baculovirus Expression Protocols ＂) ((The Humana Press of company of Hu Mana press, Inc.), 1995) describe.In one embodiment, polypeptide of the present invention can use baculovirus expression system express (referring to, Lu Keyou people such as (Luckow), biology/technology (Bio/Technology), 1988,6,47; " rhabdovirus expression vector: laboratory manual " (＂ Baculovirus Expression Vectors:a Laboratory Manual ＂), (O ' Rielly) waits people's (volume) to Ao Lili, W.H. free man company (W.H.Freeman and Company), New York (New York), 1992; US4,879,236, its mode of quoting in full separately is incorporated herein).In addition, can use the complete baculovirus expression system of MAXBAC.TM. (hero (Invitrogen)) (for example) in insect cell, to produce.

Polypeptide of the present invention also can separate by utilizing special properties.For instance, can use fixing metal ions absorption (IMAC) chromatogram to come the rich histidine protein matter of purifying, comprise that those comprise the protein of polyhistidyl label.In brief, at first make gel carry divalent-metal ion to form inner complex (husky Cowes strange (Sulkowski), the trend in the biological chemistry (Trends in Biochem) 3:1 (1985)).Matter will different avidity be adsorbed in this matrix to rich histidine protein to look employed metal ion, and will or use strong chelating agent and wash-out by competitive wash-out, reduction pH value.Other purification process comprises by lectin affinity chromatography and ion exchange chromatography purifying glycosylation protein matter (Mead bodyguard thorough (volume), Enzymology method (Meth.Enzymol.) 182:529 (1990)).In other embodiments of the invention, can construct the fusions of the polypeptide paid close attention to and avidity label (for example, maltose binding protein, immunoglobulin (Ig) territory) to promote purifying.

Host cell of the present invention can be used in the method for scale operation Ror polypeptide, cell is grown in suitable culture medium, and from cell or from cell is grown residing substratum, separate desirable polypeptide product according to the known purification process in affiliated field, described purification process is the conventional chromatogram method for example, comprises immunoaffinity chromatography, the acceptor affinity chromatography, the hydrophobic interaction chromatography, the lectin affinity chromatography, size exclusion is filtered, positively charged ion or anion-exchange chromatography, high pressure lipuid chromatography (HPLC) (HPLC), reversed-phase HPLC and its similar approach.Other purification process comprises following method, and wherein desirable protein is to have by fusion protein form expression and the purifying of specificity in conjunction with specificity label, mark or the chelating moiety of collocation thing or medicament identification.Can make the protein purification cracking producing desirable protein, or can complete fusion rotein form keep.The cracking of merging component can produce the desirable protein form with other amino-acid residue that is produced by cracking process.

Term " in vitro " is meant reaction or the process that is taken place in artificial environment and the artificial environment.In vitro environment includes, but is not limited to test tube and cell culture.Term " in vivo " is meant process or the reaction that is taken place in natural surroundings (for example, animal or cell) and the natural surroundings.

Method of the present invention can use cell (culturing cell) and cell lysates (comprising nuclear extract) in vitro to carry out.Expection differentiates that the example of the cell of regulating osteoplastic medicament includes, but is not limited to calvaria cell, scleroblast, osteoclast, chondrocyte and multipotency precursor cell, such as the multipotency marrow stromal cell.Analogue (WO01/19855) and the HOB clone described in the following document: the Bo Tingpiwei (Bodine PV) that is provided in MC3T3-E1, C2C12, MG-63 cell, U20S cell, UMR106 cell, ROS17/2.8 cell, SaOS-2 cell and the ATCC catalogue is provided the particular instance of scleroblast and scleroblast precursor cell system, this agree (Vernon SK) Vernon, Kao Mubusi (Komm BS.), incretology (Endocrinology), 137,4592-4604, (1996); Bo Tingpi Wien (Bodine PVN), bent that Smith's rice (TrailSmith M), Kao Mubusi (Komm BS.), bone and mineral substance research magazine (J Bone Min Res), 11,806-819, (1996); Bo Tingpiwei (Bodine PV), Green outstanding person (Green J), the conspicuous Ah (Harris HA) of Harris, cloth hartree Ah (Bhat RA), Si Tanyinjisi (Stein GS), sharp peace Jebb (Lian JB), Kao Mubusi (Komm BS.), cellular biochemistry magazine (J CellBiochem), 65,368-387, (1997); Bo Tingpiwei (Bodine PV), Kao Mubusi (Komm BS.), bone (Bone), 25,535-43 (1999); Bo Tingpi Wien (Bodine PVN), the conspicuous Ah (Harris HA) of Harris, Kao Mubusi (Komm BS.), incretology (Endocrinology), 140,2439-2451, (1999); Pu Linsimi (Prince M), Ban Nayexi (Banerjee C), Jia Weidea (JavedA), Green outstanding person (Green J), sharp peace Jebb (Lian JB), Si Tanyinjisi (Stein GS), Bo Tingpiwei (Bodine PV), Kao Mubusi (Komm BS), cellular biochemistry magazine (J Cell Biochem), 80,424-40, (2001).Method of the present invention also can use cell free system to carry out.

Term " expression system " is meant host cell and the compatible carrier that is under the conditions suitable, and it is entrained and introduce the protein expression of the foreign DNA in the host cell that described condition (for example) is suitable for code carrier.Common expression system comprises e. coli host cell and plasmid vector, insect host cell and baculovirus vector and mammalian host cell and carrier.

" transition " is meant nucleic acid fragment is transferred in the genome of host organisms, go up stable heredity thereby produce heredity.The host organisms that contains the nucleic acid fragment that makes the transition is called " transgenosis " organism.

Term " differentiation " is meant to have the feature or function different with the initial form of tissue or cell.Therefore, " differentiation " is atomization or Differentiation.

Term " osteoblast differentiation " is phalangeal cell is grown dedicated functions during maturation turns to scleroblast a process.Osteoblast differentiation can comprise skeletonization precursor cell, early stage and ripe scleroblast, bone precursor cell and the ripe osteocyte stage (ripple spit of fland people such as (Bodine), Metabolism Vitamins and Hormones 65 (Vitamins and Hormones 65), 101-151 (2002); Si Tanyin people such as (Stein), internal secretion summary (Endocrine Reviews), 14,424-442 (1993) and sharp peace people such as (Lian), Metabolism Vitamins and Hormones 55 (Vitamins and Hormones 55), 443-509 (1999)).

Term " hyperplasia " is meant cytoid growth of class and generation.

Term " phenotype " is the observable feature of phalangeal cell or organism.Described observable feature can comprise that physical appearance and specific physiology composition are present in the content in cell or the organism.Osteoblasts in vitro comprises expresses several marker albumen, such as bone idiosyncratic transcription factor Cbfa1; Type i collagen; Alkaline phosphatase, Bone Gla protein; With bone sialoprotein (bone sialoprotein).

As used herein, term " in conjunction with the collocation thing " or " interaction protein " be meant can specificity in conjunction with the molecule of another molecule, for example antigen and antigen-specific antibodies or enzyme and its inhibitor.Can comprise (for example) vitamin H and avidin or antibiosis protein chain bacterium, IgG and albumin A, acceptor-ligand conjugates, protein-protein interaction and complementaling polynucleotide chain in conjunction with the collocation thing.Term " in conjunction with the collocation thing " also can refer to cell in kinases bonded polypeptide, lipid, small molecules or nucleic acid.Kinases with combine increase that the interactional variation of collocation between the thing itself can be by forming interactional probability or reduction or kinases-combination arrange in pairs or groups the thing mixture concentration increase or reduce to manifest.For instance, Ror1 or Ror2 albumen can combine and form and can cause regulating Ror1 or the active mixture of Ror2 with another protein or polypeptide.

Term " signal transduction pathway " is meant propagates the molecule that becomes signal in the cell by cytolemma with the extracellular signal.But this signal irritation cell reaction then.The peptide molecule that relates to the signal transduction process can be acceptor and non-receptor protein tyrosine kinase.

" acceptor " be in the phalangeal cell or cell surface on be the molecular structure of feature with the selective binding specificity material usually.Illustrative receptors comprises the cell surface receptor of peptide hormone, neurotransmitters, antigen, complement fragment and immunoglobulin (Ig) and the cytoplasmic receptor of steroid hormone.

Term " adjusting " is meant inhibition, enhancing or inducing function.For instance, " adjusting " of genetic expression or " regulation and control " are meant the change of gene activity.The adjusting of expressing can include, but is not limited to gene activation and gene repression." adjusting " or " regulation and control " also refers to increase or reduce bioactive method, the conditioned disjunction medicament of protein, enzyme, inhibitor, signal transduction agent, acceptor, transcriptional activator, cofactor and its analogue.Active this variation can be the mRNA translation, DNA transcribes and/or the increase of mRNA or protein degradation or minimizing, and this may be again corresponding to bioactive increase or reduction.This enhancing or suppress and to decide and/or may only in particular cell types, manifest with the generation of particular event (such as the activation of signal transduction pathway).

" through regulating activity " is meant any activity, condition, disease or the phenotype of being regulated by proteinic biologic activity form.Adjusting may realize by the concentration that influences biologically active proteins matter, for example realize by regulating and expressing or degraded, or suppress by (for example), activation, in conjunction with or discharge and be subjected to matter, chemically or modify direct excitement or the antagonistic effect reached on the structure and realize, or realize by the direct or indirect interaction that may relate to other factor.

" conditioning agent " is meant any medicament of the expression (such as bone forming or Ror developed by molecule) that changes specific activity.For instance, regulate osteoplastic medicament and change (increase or reduce) bone forming.Conditioning agent plans to comprise any compound, for example, and antibody, small molecules, peptide, oligopeptides, polypeptide or protein.

" plasmocyte " is meant and is specifically designed to the ripe bone-marrow-derived lymphocyte that antibody (immunoglobulin (Ig)) produces.Plasmocyte seldom is found in the peripheral blood.It comprises 0.2% to 2.8% myeloplast number.It is oval or fan-shaped that mature plasme cell is generally, through being measured as 8-15 μ m.Being shaped as of nuclear is eccentric circular and oval.

Term " small molecules " is meant synthetic or naturally occurring compound (for example peptide or oligonucleotide), and it can be a deutero-according to circumstances, natural product is natural or organic, biologic inorganic of any other lower molecular weight in synthetic source (usually less than about 5 kilodaltons) or mineral compound.Described small molecules can be the last transferable material of treatment or can further be derived to help transmission.

As used herein, term " inductor " is meant any medicament of inducing, strengthen, promote or increasing specific activity (such as bone forming or Ror developed by molecule).

As used herein, term " inhibitor " or " repressor " are meant inhibition, check or reduce any medicament of specific activity (such as bone forming or Ror developed by molecule).

As used herein, term " medicament " or " test medicament " are meant any compound or molecule to be tested.The example of medicament of the present invention includes, but is not limited to peptide, small molecules and antibody.Medicament can be selected at random or reasonably select or design.As used herein, when not considering that when selecting medicament at random specificity between medicament and target compound or the site interacts, think that medicament is " selecting at random ".As used herein, when considering that medicament interacts with specificity between target compound or the site and/or the nonrandom basis of the configuration relevant with the medicament effect on when selecting medicament, think that medicament is " reasonably selection or design ".

As used herein, term " antibody " is meant immunoglobulin molecules or its immunocompetence part (for example, antigen-binding portion thereof).Antibody is natural generation or synthetic wholly or in part the generation.The example of the immunocompetence part of immunoglobulin molecules comprises can be by using F (ab), Fv and F (ab ') fragment that is produced such as pepsic enzymatic lysis antibody.All its derivatives of keeping specific binding capacity are also included within this term.This term also contain have with the immunoglobulin (Ig) binding domain homologue or basically homologous combine any protein in territory.These protein can be derived from natural origin or partially or completely synthetic the generation.Antibody can be monoclonal antibody or polyclonal antibody.Antibody can be the member of any immunoglobulin class (comprise among human classification IgG, IgM, IgA, IgD and the IgE any).Yet, the common in the present invention preferably derivative of IgG classification.

Term " antibody fragment " is meant any derivative less than total length of antibody.Antibody fragment preferably keeps the significant part of the specific binding capacity that has full length antibody at least.The example of antibody fragment includes, but is not limited to Fab, Fab ', F (ab ') ₂, scFv, Fv, dsFv bifunctional antibody and Fd fragment.Antibody fragment can produce by any way.For instance, antibody fragment can enzymatic or chemistry produce by making the whole antibody fracture, or it can be produced by the gene recombination of encoding part antibody sequence.Perhaps, antibody fragment can synthesize generation wholly or in part.Antibody fragment can be a single chain antibody fragments according to circumstances.Perhaps, fragment can comprise the chain that a plurality of (for example) link together by disulfide linkage or other more stable binding.Fragment can be the polymolecular mixture according to circumstances also.The functional antibodies fragment will comprise usually at least about 50 amino acid, and more generally will comprise at least about 200 amino acid.In certain embodiments, antibody fragment has at least two antigen binding sites.In some preferred embodiment, antibody fragment has 2,3,4 or 5 antigen binding sites definitely.Fragment with two antigen binding sites is particularly useful for the present invention.These medicaments make Ror2 two polymerizations and do not form polycomplex.

Strand Fv (scFv) is only by passing through the covalently bound variable light chain (V of polypeptide connexon each other _L) and variable heavy chain (V _H) recombinant antibody fragment formed.V _LOr V _HCan be NH ₂-terminal territory.The polypeptide connexon can have variable-length and composition, as long as two variable domains bridging under the situation of no severely sterically hindered influence.Connexon mainly comprises usually uses some L-glutamic acid or the Methionin that scatter for solvability that glycine and serine residue are stretched.

Bifunctional antibody is dimerization scFv.The component of bifunctional antibody has the peptide connexon shorter than most of scFv usually, and it shows that preferred the association is dimer.

The V that the Fv fragment is served as reasons and combined by noncovalent interaction _HWith a V _LThe antibody fragment that the territory is formed.Term dsFv is used in reference between the engineering chemoattractant molecule cystine linkage in this article and stablizes V _H-V _LRight Fv.

F (ab ') ₂Fragment for basically with by the antibody fragment of immunoglobulin (Ig) (usually IgG) by the antibody fragment equivalence that under pH4.0-4.5, obtained with the decomposition of enzyme stomach en-.The generation of can recombinating of described fragment.

The Fab fragment is for being combined into F (ab ') with two heavy chain fragments basically with by reduction ₂The antibody fragment of the antibody fragment equivalence that the segmental pair of sulphur bridge key obtained.The generation of can recombinating of Fab ' fragment.

The Fab fragment for basically with antibody fragment by the antibody fragment equivalence that obtained with enzyme papoid decomposition immunoglobulin (Ig) (common IgG).The generation of can recombinating of Fab fragment.The segmental heavy chain fragment of Fab is the Fd fragment.

As used herein, term " reporter gene " is meant any gene that phenotypic expression is easy to monitor.The purposes of reporter gene is particularly useful for screening to determine which test medicament activation signals transduction path.The reporter gene operability is connected in promotor or other controlling element that is subjected to signal transduction pathway control.In certain embodiments, be connected in the promoter region of particular attention given on the preparation reporter gene function or the recombinant DNA of other control region is constructed body, and make this construct the body transfection in cell or organism.The example of normally used reporter gene comprises luciferase (luciferase, LUC), green fluorescent protein (green fluorescent protein, GFP), beta-galactosidase enzymes (β-galactosidase, GAL), GRD beta-glucuronidase (β-glucuronidase, GUS) and E.C. 2.3.1.28 (chloramphenicolacetyltransferase, CAT).

As used herein, term " treatment " be meant therapeutic treatment and prophylactic treatment or preventative operation stimulate the osteocyte differentiation or bone forming, the development of delay bone disorders symptom and/or reduce bone disorders and/or described will or expection by the operation of the severity of the symptom of bone disorders development.These terms further comprise improve existing bone disorders symptom, prevent other symptom, the potential metabolic disease of improvement or prevention symptom because of the metabolic disease of, prevention or reverse symptom because of or prevention or promote osteogenesis.Therefore, the individual advantageous results of suffering from bone disorders or having the potential of the described illness of development represented to give in these terms.In addition, term " treatment " be defined as to may have disease, disease symptoms or a diseases induced inducement individual or individual through chorista clone is used or throwing and medicament (for example, therapeutical agent or therapeutic composition), wherein purpose is to cure, cure, relax, alleviate, change, remedy, improve, improve or influences described disease, disease symptoms or diseases induced inducement.As used herein, " therapeutical agent " is meant and participates in treating any material of disease (for example regulate the bone forming activity or induce new bone forming) or the combination of material.Therefore, therapeutical agent includes, but is not limited to small molecules, peptide, antibody, ribozyme and antisense oligonucleotide.

Therapeutical agent or therapeutic composition also can comprise prevention and/or reduce the compound that is pharmaceutically acceptable form of the symptom of specified disease.For instance, therapeutic composition can be the medical composition of the symptom of prevention and/or minimizing bone photo related disorders.Expect that therapeutic composition of the present invention will provide with any suitable form.The form of therapeutic composition will be decided on multiple factor (comprising the dispensing pattern).Except that other composition, therapeutic composition also can contain thinner, adjuvant and vehicle.

Bone strength can be determined according to bone density (the gram number of the mineral substance of every cubic centimeter volume) and bone mass (mineralize, bone structure, bone conversion, little fracture).As measuring of bone strength, use usually bmd (Bone MineralDensity, BMD).For instance, if BMD surpasses following 2.5 standard deviations of mean value of young white race adult female's BMD, can assert that so bone is the osteoporosis (World Health Organization (World Health Organization), 1994, the application (Assessment of Fracture Risk andit ' s Application to Screening for Postmenopausal Osteoporosis) of risk of bone fracture evaluation and its screening post-menopausal osteoporosis, the 843rd phase of technical report collected works (Technical Report Series 843), Geneva: the World Health Organization (Geneva:World healthOrganization)).

" osseous tissue " is meant calcified tissue's (for example, cranium, shin bone, femur, vertebra, tooth), bone trabecula, medullary space (chamber except that bone trabecula), cortex bone (covering the outer periphery of bone trabecula and medullary space) and its analogue.Osseous tissue also refers to be usually located at the interior osteocyte of collagen stroma that mineralizes; The blood vessel of nutrition is provided to osteocyte; Bone marrow smear (bone marrow aspirate); Synovial fluid; Be derived from the osteocyte of osseous tissue, and can comprise fat marrow.Osseous tissue comprises bone ware, such as section, bone chip, bone meal, osseous tissue biopsy, collagen formulations or its mixture of whole bone, whole bone.For purposes of the present invention, except as otherwise noted, otherwise term " osseous tissue " is used to contain all above-mentioned osseous tissue and goods of the mankind or animal.

As used herein, " bone photo closes active " comprises bone forming activity and bone resorption activity.Can by increase the scleroblast activity, from the osteoblast differentiation and the scleroblast hyperplasia of osteoprogenitor cells, induce the bone forming activity by reducing TNF-a Induced Apoptosis in Osteoblasts and making up by any its.In addition, can make up by increase osteoclast apoptosis with by any its and suppress bone resorption activity by reducing osteoclast activity, osteoclast differentiation and hyperplasia.The bone forming activity can be induced in multiple osseous tissue or cell.

As used herein, phrase " adjusting bone forming " is meant increases or reduces bone forming." increase bone forming " means scleroblast or scleroblast precursor raised position of bone point, and this reaches cytodifferentiation is that ripe scleroblast and ripe scleroblast are secreted the collagen stroma that complete bone material is mineralized and increase the bone amount of site.Ripe scleroblast generation also contained in this term and the excretory collagen stroma increases.Osteoplastic increase can be determined by one or more following situation: fracture speed reduces, regional bone density increases, volume mineral substance bone density increases, the increase of forming property of girder knot, the increase of girder density, cortical density or thickness increases, the bone diameter increases and inorganic bone content increases.Osteoplastic increase can be by the generation that mineralizes of the increase of connection, hyperplasia, survival and/or the differentiation of osteocyte (for example, scleroblast) and bone subsequently.

" bone photo related disorders " comprises bone forming and bone resorption illness.These diseases and symptom include, but is not limited to rickets, osteomalacia, osteopenia, osteosclerosis, renal osteodystrophy, osteoporosis (comprising old age and post-menopausal osteoporosis), handkerchief wise man's disease, bone transfer, hypercalcemia, hyperparathyroidism, osteopetrosis, periodontitis and may follow the ANOMALOUS VARIATIONS of the bone metabolism of rheumatoid arthritis and osteoarthritis.In these diseases some are characterised in that insufficient bone forming or bone run off, and other relates to thickening unusually of osseous tissue or hardens.Include, but is not limited to osteopetrosis and osteosclerosis with benefiting from the example that suppresses the disease that bone thickens unusually.

" bone photo pass medicament " is meant the medicament that influences bone forming or bone resorption." bone photo pass medicament " may induce anabolism or katabolism effect, may suppress bone resorption and make bmd increase, and may increase bone forming and maybe may keep balance between bone forming and the bone resorption.

Term " compound " or " medicament " exchange in this article use with refer to when throw to individual (mankind or animal) and the time induce the compound or the composition of the material of desirable pharmacology and/or physiological effect by part and/or systemic effect.

Term " individuality " is meant any Mammals, comprises the mankind or non-human individuality.The non-human individuality can comprise experiment, test, agricultural, amusement or companion animals.Individuality can be the mankind.Individuality can be performing animal, such as dog, cat, milk cow, goat, sheep, pig etc.Individuality can be laboratory animal, such as mouse, rat, rabbit, monkey etc.

Term " biological sample " extensively defines to comprise any cell, tissue, biofluid, organ, multicellular organisms and its analogue.Biological sample can be derived from (for example) in vitro cell or tissue culture.Perhaps, biological sample can be derived from Living Organism or unicellular organism body colony.Biological sample can be living tissue, such as the bone of living.Term " biological sample " also plans to comprise such as from the sample of individual isolated cells, tissue or biofluid and be present in sample the individuality.That is, detection method of the present invention can be used for detecting in vitro and Ror mRNA, protein, genomic dna or the activity in the biological sample in vivo.For instance, the in vitro technology of detection Ror mRNA comprises that TaqMan analyzes, north hybridization (northern hybridization) and in situ hybridization (in situ hybridization).Detect the proteic in vitro technology of Ror comprise enzyme linked immunological absorption calibrating (enzyme-linked immunosorbent assay, ELISA), western blot (western blot), immuno-precipitation and immunofluorescence technique.The in vitro technology that detects the Ror genomic dna comprises south hybridization (southern hybridization).

Description of drawings

Can understand the present invention more fully with the accompanying drawing of a part that constitutes the application's case by the following detailed description.

Fig. 1 shows the Osteoblast Differentiation of the downward modulation Ror2 expression inhibiting dex human mescenchymal stem cell of inductive (hMSC).Infect human MSC and it is being supplemented with 0.05mM xitix, 10mM β-glycerophosphate (β-GP) and in the MSC growth medium (MSCGM) of 100nM dexamethasone (dex) cultivate with the adenovirus expression carrier that contains Ror2 specificity shRNA or EGFP specificity shRNA (contrast).Cultivate after 9 days, make total cell protein matter extract (50 micrograms/swimming lane) stand the west immunoblotting (A) that contrasts as load with endogenous Ror2 albumen or beta-actin.Cultivate after 11 days, carry out sodium alizarinsulfonate-S dyeing (B) and quantitatively (C) with the evaluation substrate formed degree that mineralizes.In C, the amount of the sodium alizarinsulfonate-S that will incorporate in the presence of EGFP shRNA is set at 100%.Result among B and the C represents three independently experiments (Fig. 1 mentions) in example 1.

Fig. 2 shows that Ror2 crosses the one-tenth fat differentiation of expression inhibiting hMSC.A: (β-gal), Ror2 or Ror2KD infect human MSC and it were cultivated 8 days in being supplemented with into the MSCGM of lipoprotein mixture with beta-galactosidase enzymes.Separate total cell RNA and make it stand to use available from the primer of u.s.a. applied biosystem company (Applied Biosystems) and the real-time RT-PCR analysis that becomes fat transcription factor C/EBP α and PPAR γ of detection.The content standard of mRNA turned to the expression of cyclophilin B in each sample (cyclophilin B) and the relative mRNA of β-gal cells infected expressed and be set at 100%.B: infect human MSC with β-gal, Ror2 or Ror2KD, it was cultivated 21 days in being supplemented with into the MSCGM of lipoprotein mixture, and make it stand oil red O stain (Fig. 2 mentions) then in example 2.

Fig. 3 shows that the proteic total surface of bone of expressing increase newborn mice cranium of crossing of Ror2 amasss, rather than the scleroblast number.The craniums of 4 the biggest brood birth mouse is kept do not infect (contrast) or with the adenovirus infection of encode beta galactosidase enzyme (β) or human Ror2 (R2).After cultivating 7 days in the presence of the adenovirus, with phenodin and eosin cranium is dyeed, evaluate the long-pending and scleroblast number of total surface of bone then.The value that obtains not infecting in the culture is set at 100%.The result is the mean value of every kind of symptom 4-5 cranium

SE ( ^*Compare-P＜0.01 with the beta-galactosidase enzymes infection).Three independently experiments (Fig. 3 mentions in example 3) of this graphical presentation.

Fig. 4 Ror2 protein binding 14-3-3 β is described and with it in tyrosine place phosphorylation.With β-ga1 (β), Ror2 (R2) or Ror2KD (KD) adenovirus infection U2OS cell and after 24-48 hour, prepare total cell lysates and with anti-flag (A), anti-14-3-3 β (B) or anti-phosphorylated tyrosine (C) antibody immunoprecipitation.Analyze immunoprecipitation (Fig. 4 mentions) by carrying out immunoblotting in example 4 with indication antibody.

Fig. 5 shows that endogenous Ror2 albumen in vivo mediates 14-3-3 β phosphorylation.With Ror2 siRNA or non-specific siRNA transient transfection U2OS cell, and after 48 hours, use every swimming lane 50 microgram extracts to make total cell protein matter extract stand the west immunoblotting (A) of endogenous Ror2 albumen or beta-actin (load contrast).In addition, by the identical lysate of 14-3-3 β antibody direct analysis (20 micrograms/swimming lane), or with the anti-phosphorylated tyrosine antibody identical lysate of immunoprecipitation post analysis (B) together (Fig. 5 mentions in example 4).

Fig. 6 shows that the proteic cytosol of Ror2 territory is in vitro in conjunction with 14-3-3 β and make its direct phosphorylation.A: make through the Ror2 of GST mark cytosol territory (GST-R2c) or independent GST is attached to the glutathione agarose bead and under 4 ℃ with in vitro the translation 14-3-3 β cultivated 4 hours.With anti-14-3-3 β antibody analysis binding substance.B: described in " general method ", with the proteic purified reorganization cytosol territory (GST-R2c of Ror2, hero (Invitrogen)) and purified reorganization GST-14-3-3 β (the in vitro kinases calibrating (Fig. 6 mentions in example 4) that biomolecules company (Biomol, Inc.)) carries out.

Fig. 7 illustrates that the Ror2 specific antibody causes two polymerizations and the activation of Ror2 acceptor.A:, be used in the Ror2 expression plasmid transfection U2OS cell of COOH end through Flag (R2-F) or His (R2-H) epi-position label for two polymerizations are described.After 24 hours, handled cell 1 hour with Ror2 specificity goat polyclone IgG or non-specific goat IgG down, and prepare total cell protein matter extract and make its immunoprecipitation on the affine agarose of anti-Flag at 37 ℃.Analyze throw out (last figure) by carrying out immunoblotting with anti-His antibody.Figure below shows that surveying same film again with anti-Flag antibody contrasts to be used for the precipitation level.B: be the explanation activation, with as the U2OS cell of Ror2 specific antibody among the A or contrast IgG processing untransfected, and total cell extract is deposited on the anti-phosphorylated tyrosine antibody and with Ror2 or 14-3-3 β antibody analysis (Fig. 7 mentions in example 5).

Fig. 8 shows that Ror2 antibody causes that the matrix that mineralizes among the hMSC forms.In the MSCGM that contains 0.05mM xitix, 10mM β-GP and 100nM dex of the Ror2 specificity goat IgG that is supplemented with non-specific goat IgG, Ror1 specificity goat IgG (respectively being 50 μ g/ml) or progressive concentration, cultivate human MSC.After cultivating 9 days, by sodium alizarinsulfonate-S dyeing evaluation matrix minerals degree (Fig. 8 mentions in example 6).

Fig. 9 illustrates that it is to mediate by Ror2 that the hMSC of Ror2 antibody induction mineralizes.A: Ror2 or EGFP (contrast) are had the adenovirus expression carrier infection hMSC of specific shRNA and it is cultivated in the MSCGM that is supplemented with 0.05mM xitix, 10mM β-GP and 100nM dex with containing.Cultivate after 9 days, by the degree of sodium alizarinsulfonate-S dyeing evaluation matrix mineralsization.B: use the human MSC of Ror2 adenovirus infection 24 hours, and then it was cultivated 19 days in the MSCGM that contains 0.05mM xitix, 10mM β-GP and Ror2 specificity goat IgG or non-specific goat IgG, afterwards with sodium alizarinsulfonate-S dye (Fig. 9 mentions in example 6).

Figure 10 illustrates that the matrix that mineralizes that downward modulation 14-3-3 β strengthens among the hMSC forms.(β-gal) crosses adenovirus expression carrier that expression cassette or Ror2 cross expression cassette and infects human MSC and it is cultivated in the MSCGM that is supplemented with 0.05mM xitix, 10mM β-GP and 100nM dex with containing messy shRNA (scramble shRNA), 14-3-3 β specificity shRNA, beta-galactosidase enzymes.Cultivate after 9 days, make 50 microgram total cell protein matter extracts stand endogenous 14-3-3 β proteic west immunoblotting (A).Cultivate after 12 days, carry out sodium alizarinsulfonate-S dyeing (B) in example 7 with the evaluation substrate formed degree (Figure 10 mentions) that mineralizes.

Figure 11 illustrates Ror2 Antybody therapy and the new bone forming of the stripped promotion of 14-3-3 β downward modulation.With containing messy shRNA (scr) or 14-3-3 β being had the adenovirus infection mouse cranium of specific shRNA; And after 48 hours, in the presence of fluorexon, handle with anti-Ror2 antibody of 12 μ g/ml or non-specific IgG.After adenovirus and antibody are cultivated 7 days, with hematoxylin-eosin to cranium dyeing and measure total surface of bone long-pending (hollow strips) and scleroblast number (solid bars).The value that is obtained in the culture of handling with IgG that messy shRNA is infected is set at 100%.The result is the mean value of 4 craniums of every kind of symptom

SE ( ^*-p＜0.05) (Figure 11 mentions in example 8).

Figure 12 explanation is used for the generation of the active high-throughput of Ror2, highly sensitive calibrating.A: the synoptic diagram of calibrating that utilizes the signal transduction pathway of TrkB acceptor.Cause the Erk phosphorylation and stimulate equal two polymerizations of the cAMP response element (CRE) in the target gene promoters to activate the TrkB acceptor by the ligand inductive.The Chimerical receptor that the Ror2 cell foreign lands (aa1-407) that our generation is merged by membrane-spanning domain and cell internal area (aa 432-822) with TrkB are formed.When using this mosaic, cause that Ror2 two polymeric medicaments activation TrkB signal transduction pathway and this activation can be fixed by CRE promotor-luciferase reporting health check-up accepted opinion.B: with Ror2-TrkB mosaic and CRE-luciferase plasmids stable transfection HEK293 cell, use the anti-Ror2 antibody of indicatrix or non-specific IgG to handle 24 hours, and the evaluation uciferase activity.To handle the viewed uciferase activity in back with non-specific IgG and be set at 1.The result represents three independently experiment (mean values

SE; N=4; ^*-p＜0.05) (Figure 12 mentions in example 9).

Embodiment

The present invention is based on about Ror family and its downstream signal transduction biomolecules in bone metabolism, the especially discovery of the effect in the osteoblast differentiation.Referring to U.S. patent application case U.S.S.N.10/823,998,60/463,364 and 60/501,340, it is incorporated herein by reference separately.The applicant has found to reduce the Osteoblast Differentiation (Fig. 1) of the human mescenchymal stem cell of Ror2 expression inhibiting induced by dexamethasone.On the contrary, the one-tenth fat differentiation (Fig. 2) of the human mescenchymal stem cell of the mistake expression inhibiting of Ror2.The applicant has showed that also downward modulation 14-3-3 β expresses the matrix that mineralizes that strengthens in the human mescenchymal stem cell and forms (Figure 10).In addition, Ror2 crosses and expresses and 14-3-3 β inhibition any one big matrix mineralsization of induction ratio (Figure 10).Find based on these, under protein level, regulate Ror2 medicament or its medical composition active or adjusting 14-3-3 'beta ' activity and be applicable to treatment osteopathia and/or metabolic disorder, such as obesity or diabetes.In fact, thus the medicament that increases the Ror2 protein-active will promote osteoblast differentiation and the increase bone forming (Fig. 8 and 9) that mineralizes.Again, thus the medicament that suppresses the 14-3-3 'beta ' activity will promote osteoblast differentiation and the increase bone forming (Figure 10) that mineralizes.

On the one hand, the invention provides the medicament of adjusting (increase or reduce) Ror2 protein-active.In certain embodiments, medicament increases the Ror2 protein-active.In other embodiments, medicament reduces the Ror2 protein-active.These medicaments act under protein level usually, increase or reduce the proteic activity level of Ror2.As discussed in this article, the active medicament of increase Ror2 is applicable to and promotes to mineralize bone forming and Osteoblast Differentiation.These medicaments also may be applicable to treatment of obesity (Fig. 2) by being suppressed to the fat differentiation.Under the situation of not wishing to be bound by any particular theory, as if active the increasing of Ror2 promotes Osteoblast Differentiation, is suppressed to the fat differentiation simultaneously.

These regulate the compound that the active medicament of Ror2 can be any kind, comprise small molecules, polynucleotide, protein, peptide etc.In certain embodiments, medicament is a protein.In other embodiments, medicament is a peptide.In other embodiments, medicament is a polynucleotide.In other embodiments, medicament is small molecules (for example, molecular weight is less than 1500g/mol).Preferably, medicament has specificity and other biomolecules of debond to Ror2 albumen.In certain embodiments, medicament other Ror family member of debond especially.In other embodiments, may there be cross reactivity with other biomolecules or Ror family member; Yet, medicament to the avidity of these other molecules less than to the proteic avidity of Ror2.

In some specific embodiment, medicament works by causing two proteic two polymerizations of Ror2.Think that proteic two polymerizations of Ror2 cause the activation of Ror2 acceptor.Activation Ror2 kinase activity causes it to comprise the proteic phosphorylation in conjunction with the collocation thing of 14-3-3 β.Other Ror2 includes, but is not limited to ADP/ATP carrier proteins, UDP-glucose UGCG sample 1,14-3-3 albumen γ, ribophorin I (ribophorin I), arginine N-methyltransgerase 1, apoptosis sensitive protein, NOTCH2 albumen and human skeletal's flesh LIM-albumen 3 (than Reed people such as (Billiard) in conjunction with the collocation thing, the U.S. patent application case U.S.S.N.10/823 of application on April 14th, 2004,998).Medicament generally include at least two proteic at Ror2 in conjunction with the territory.In certain embodiments, medicament have definitely two proteic at Ror2 in conjunction with the territory, promptly medicament is the divalence medicament.For other medicament of multivalence medicament also is applicable to the present invention.In certain embodiments, medicament is for promoting Ror2 albumen two polymeric small molecules or polynucleotides.In other embodiments, medicament is protein or peptide.

In certain embodiments, medicament is at proteic antibody of Ror2 or antibody fragment (for example, bifunctional antibody).Described antibody or antibody fragment can be at the proteic any districts of Ror2; Yet antigen binding site is not preferably at the biological activity that may disturb Ror2 (for example, kinase activity) or disturb two proteic two polymeric districts.Thereby it is proteic in conjunction with promoting Ror2 albumen two polymerizations and promoting its activation by two Ror2 that antibody or antibody fragment are carried out.Antibody can be polyclonal antibody or monoclonal antibody.Antibody can be any isotype; Yet, be preferably the IgG isotype usually.Antibody can be derived from any species; Yet for being used for the mankind, antibody has human origin or usually by the peopleization.If antibody will be used for other species, can make antibody be suitable for this species so.In certain embodiments, antibody behaviourization monoclonal antibody.In other embodiments, antibody is complete human antibodies.In some specific embodiment, antibody is complete human monoclonal antibody.

In certain embodiments, by with purified human Ror2 albumen or be derived from the proteic peptide of Ror2 and make such as rabbit or other rodentine mammalian immune and prepare at Ror2 albumen and antibody.After the immunity, collect the cell (such as B cell or plasmocyte) that produces antibody and use it for preparation and merge knurl, screening is merged knurl to produce at the proteic antibody of Ror2 then.In certain embodiments, make two polymerizations of Ror2 albumen and/or the proteic ability screening of activation Ror2 antibody with regard to antibody.In case differentiate the B cell that produces desirable antibody, just can make the B cell immortality.Then, can use gained to merge knurl and produce desirable monoclonal antibody.Can further characterize and modify by merging the antibody that knurl produces.For instance, in certain embodiments, can make antibody humanization so that not produce untoward reaction to human individual's throwing with antibody, untoward reaction can change to fatal allergy from the clearance rate increase of treatment antibody.In some specific embodiment, use identification Ror2 proteic antibody district (that is complementary determining region) to replace and have the not CDR of homospecific human antibodies.The technology that is used for through engineering approaches and preparation antibody in affiliated field for known and be described in No. the 4th, 816,567, the United States Patent (USP) of on March 28th, 1989 promulgation; No. the 5th, 078,998, the United States Patent (USP) of promulgation on January 7th, 1992; No. the 5th, 091,513, the United States Patent (USP) of promulgation on February 25th, 1992; No. the 5th, 225,539, the United States Patent (USP) of promulgation on July 6th, 1993; No. the 5th, 585,089, the United States Patent (USP) of promulgation on December 17th, 1996; No. the 5th, 693,761, the United States Patent (USP) of promulgation on December 2nd, 1997; No. the 5th, 693,762, the United States Patent (USP) of promulgation on December 2nd, 1997; No. the 5th, 869,619, the United States Patent (USP) of promulgation in 1991; No. the 6th, 180,370, the United States Patent (USP) of promulgation on January 30 calendar year 2001; No. the 6th, 548,640, the United States Patent (USP) of promulgation on April 15th, 2003; No. the 6th, 881,557, the United States Patent (USP) of promulgation on April 19th, 2005; In No. the 6th, 982,321, the United States Patent (USP) of promulgation on January 3rd, 2006, it is incorporated herein by reference.In other embodiments, design and/or modified antibodies are to realize that Ror2 albumen is had antibody than high specific and/or avidity.

In other embodiments, medicament comprises the proteic antibody fragment at Ror2.Can use one or more at the proteic antibody fragment of Ror2.Fragment generally includes is responsible for the complementary determining region (CDR) of antibody to the proteic avidity of Ror2.For making two polymerizations of Ror2 albumen, need at least two and be used for the proteic binding site of Ror2; Therefore, medicament can be two antibody fragments connected to one another.These fragments can covalently or non-covalently link together.For instance, medicament can be two covalently bound F together _AbFragment.Medicament also can be bifunctional antibody.In certain embodiments, medicament can comprise two above antibody fragments.For instance, medicament can comprise 3,4,5 or 6 at the proteic antigen binding site of Ror2.

In some other embodiment, medicament can be protein, peptide or the small molecules of simulation at the antigen binding site of the antibody of Ror albumen (such as Ror2 albumen).These medicaments can be based at the structure of the antigen binding site of the proteic antibody of Ror2 and through board design or discriminating.Then, can in multiple in vitro calibrating, make two polymerizations of Ror2 albumen and/or the proteic ability of activation Ror2 with the evaluation medicament by the test medicament.Also can use the high flux screening method to use small molecules, peptide or polynucleotide library to differentiate medicament.

On the other hand, the invention provides the active method of medicament adjusting Ror of the present invention of using.Regulate the active medicament of Ror albumen (especially Ror2 albumen) and be applicable to that regulating bone photo closes active.These medicaments are also applicable to regulate the adipocyte differentiation in the treatment of obesity, diabetes or other metabolic disorder.The feature of numerous disease and symptom is to need to regulate bone photo to close active (for example, strengthening bone forming).Be apparent that most the situation of fracture, wherein wish stimulation of bone growth and acceleration and finish the bone reparation.For instance, strengthening osteoplastic medicament may potential facial reconstruction algorithm or the orthopedic procedures of being applicable to.Other bone lacks that symptom includes, but is not limited to that the segmental bone is damaged, periodontopathy, metastatic bone disease, molten bone osteopathia and wherein the reticular tissue reparation will be favourable symptom (such as the healing or the regeneration of cartilage defects or damage).Comprise that relevant osteoporosis of age and the osteoporosis symptom such as osteoporosis that are associated with hormone state after the menopause are also significant.To need osteogenesis is that other symptom of feature comprises primary and secondary hyperparathyroidism, the relevant osteoporosis of diabetes, the relevant osteoporosis with glucocorticosteroid of disuse osteoporosis.

Increase the active medicament of Ror2 and can be used for promoting to mineralize bone forming.These medicaments also can be used for promoting osteoblast differentiation.The promotion of osteoblast differentiation may be carried out to become fat to be divided into cost.These medicaments also can be used for promoting to mineralize matrix formation.

On the other hand, the invention provides adjusting (increase or reduce) the active medicament of 14-3-3 (for example, 14-3-3 β, 14-3-3 γ etc.).In certain embodiments, medicament suppresses the activity of 14-3-3.In other embodiments, medicament increases the activity of 14-3-3.Medicament can work under nucleic acid or protein level.In certain embodiments, medicament reduces the expression of 14-3-3 β.As discussed in this article, the medicament of inhibition 14-3-3 'beta ' activity is applicable to and promotes to mineralize bone forming and Osteoblast Differentiation.In certain embodiments, medicament reduces the expression of 14-3-3 γ.These medicaments also may be applicable to by being suppressed to fat and break up to come treatment of obesity, diabetes or other metabolic disorder.As if under the situation of not wishing to be bound by any particular theory, downward modulation 14-3-3 (especially 14-3-3 β) expresses and promotes Osteoblast Differentiation, be suppressed to the fat differentiation simultaneously.

These regulate the compound that the active medicament of 14-3-3 can be any kind, comprise small molecules, polynucleotide, protein, peptide etc.In certain embodiments, medicament is a protein.In other embodiments, medicament is a peptide.In other embodiments, medicament is a polynucleotide.In other embodiments, medicament is a small molecules.In certain embodiments, medicament is a polynucleotide.In certain embodiments, medicament is DNA.In other embodiments, medicament is RNA.In certain embodiments, medicament is 14-3-3 specific RNA i.In some specific embodiment, medicament is 14-3-3 β specific RNA i.In some specific embodiment, medicament is the 14-3-3 specific siRNA.In certain embodiments, medicament is a 14-3-3 β specific siRNA.In some specific embodiment, medicament is 14-3-3 specificity shRNA.In certain embodiments, medicament is 14-3-3 β specificity shRNA.In other embodiments, medicament has specificity to 14-3-3 γ.In certain embodiments, visible 14-3-3 in especially selectively targeted mescenchymal stem cell of medicament or the osteocyte (such as scleroblast).For instance, in certain embodiments, medicament comprises targeting moiety.In certain embodiments, the target medicament is diphosphonate or other bone object official target medicament.

On the other hand, the invention provides the active method of medicament adjusting 14-3-3 of the present invention of using.Regulate the active medicament of 14-3-3 (especially 14-3-3 β) and be applicable to that regulating bone photo closes active.These medicaments also may be applicable to regulates the adipocyte differentiation in the treatment of obesity, diabetes or other metabolic disorder.Existing many is the disease and the symptom of feature with needs adjusting bone photo pass active (for example, strengthening bone forming).Be apparent that most the situation of fracture, wherein will wish stimulation of bone growth and acceleration and finish the bone reparation.For instance, strengthening osteoplastic medicament may potential facial reconstruction algorithm or the orthopedic procedures of being applicable to.Other bone lacks that symptom includes, but is not limited to that the segmental bone is damaged, periodontopathy, metastatic bone disease, molten bone osteopathia and wherein the reticular tissue reparation will be the symptom of favourable (such as the healing or the regeneration of cartilage defects or damage).Comprise that relevant osteoporosis of age and the osteoporotic osteoporosis symptom that is associated with hormone state after the menopause are also significant.To need osteogenesis is that other symptom of feature comprises primary and secondary hyperparathyroidism, the relevant osteoporosis of diabetes, the relevant osteoporosis with glucocorticosteroid of disuse osteoporosis.

Reduce the active medicament of 14-3-3 and can be used for promoting to mineralize bone forming.These medicaments also can be used for promoting osteoblast differentiation.The promotion of osteoblast differentiation may be carried out to become fat to be divided into cost.These medicaments also can be used for promoting to mineralize matrix formation.

The medicament that is used for the inventive method can incorporate into be suitable for to individuality throw and medical composition in.As used herein, medicament can be (for example regulates the Ror molecule, Ror2 albumen) activity or 14-3-3 are (for example, 14-3-3 β, 14-3-3 γ) active any compound (for example, little organic molecule, protein, immunoglobulin (Ig), immunoglobulin fragment, the peptide of Orally active) through differentiating.Common inclusion compound of these compositions and pharmaceutically acceptable supporting agent.Composition of the present invention can contain the medicament that one or more and one or more known adjusting bone photo closes active medicament combination.For instance, promote that Ror is active or suppress the active medicament of 14-3-3 can with similar oestrogenic hormon, diphosphonate or tissue selectivity oestrogenic hormon (promptly, selective estrogen receptor modulators (selective estrogen receptor modulator, the SERM) medicament of) inhibition bone resorption combination.Medicament of the present invention also can make up with promoting osteoplastic other medicament.

One or more medicament is to use with the treatment effective dose.The treatment effective dose is meant the amount of the medicament that is enough to show benefit (for example, reducing symptom and/or the symptom that is associated with the illness of being treated, disease or symptom).When be applied to throw separately and indivedual composition the time, term only refers to this composition.When being applied to make up, term be meant combination, in succession or simultaneously throw and the combined amount of the composition of reaching benefit.For instance, the significant quantity used of therapeutic is to comprise clinical remarkable increase that the fracture repair curative ratio is provided, reverse that bone runs off and the individual fracture of preventing osteoporosis, reverse cartilage defects or illness, prevention or postpone further bone that osteoporosis outbreak, prevention be associated with osteoporosis and run off, stimulate and/or suppress bone forming, the increase in fracture nonunion and the distraction osteogenesis and/or reduce the amount of composition of the medicament of osteogenesis, reparation tooth defective and its similar situation in the prosthetics.These significant quantities will use the optimization routine technology to determine and decide on very pathology to be treated, patient's situation, dosing way, prescription and practitioner's judgement and the other factors that the those skilled in the art understands.The required dosage of The compounds of this invention (for example, in wishing the osteoporosis that bone forming increases) is to guarantee that there is the dosage of statistically evident difference in bone amount between treatment group and the control group.As seen, this difference of bone amount be bone amount in (for example) treatment group increase 5-20% or more than.Cure significant clinically increase other measure and can comprise that (for example) is about the test of forming property of knot increase in breaking strength and tension force, breaking strength and moment of torsion, four-point bending, the bone biopsy and other biomechanics test that the those skilled in the art knows.The experiment that the general guideline of treatment plan is carried out in can the animal model available from the disease of being paid close attention to.

The toxicity of medicament and therapeutic efficiency can be determined in cell culture or laboratory animal according to standard medicine program, for example, determine LD ₅₀(making 50% the lethal dosage of colony) and ED ₅₀(in the treatment to the effective dosage of 50% colony).Dosage rate between toxic action and the therapeutic action is that therapeutic index and its can LD ₅₀/ ED ₅₀Ratio is represented.Be preferably and represent big treatment exponential medicament or compound.The dosage that can be used for allocating certain limit available from the data of cell culture calibrating and zooscopy for human use.The dosage of these medicaments or compound can comprise having minimum toxicity or do not have toxic ED ₅₀The circulation composition scope in.Dosage can change in this scope of deciding on formulation that is adopted and the dosing way that is utilized.

For employed any medicament in the inventive method, the treatment effective dose at first can be by cell culture calibrating estimation.For instance, can in animal model, comprise determined ED in cell culture or the zooscopy with acquisition by allocating dosage ₅₀The circulating plasma concentration range of (that is, realizing half the concentration of test compounds of proteic maximum two polymeric of Ror2).Described information can be used for determining more accurately to be applicable to human dosage.Content in the blood plasma can (for example) be measured by HPLC.Dosage can be selected according to patient's situation by individual doctor.How and when the attending doctor should know stops, interrupts or adjust dispensing.On the contrary, the attending doctor also should know if the clinical response deficiency is adjusted to treatment higher level (eliminating toxicity) so.Throw in the control of the illness of paying close attention to and the value of dosage should change with the severity of symptom to be treated.The severity of symptom can (for example) partly be assessed by standard prognostic appraisal procedure.In addition, dosage and perhaps dose frequency also should change with age, body weight and the reaction of individual patient.The plan that can be equivalent to above institute content of the discussions can be used in the veterinary science.

The suitable dosage that is used for special case fixes in the technical scope in affiliated field really.Treatment is initial by the dosage littler than the optimal dose of compound usually.After this, dosage can little incremental increase up to being issued to best effect in described situation.For instance, total every day of dosage can be divided in case of necessity several parts and one day mid-score part throw with.Dosage every day can be divided into two, three or four parts, each part all in 24 hours time, throw with.

Antibody or antibody fragment as throw and the situation of medicament under, throw and this medicament via intravenous infusion usually.Dosage can be in the scope of every 1-6 week 1-25mg/kg.In certain embodiments, dosage can be in the scope of every 1-6 week 1-10mg/kg.In certain embodiments, every 3-5 week is transmitted the 1-10mg/kg medicament by intravenous infusion.In other embodiments, per 4 weeks are transmitted the 3-6mg/kg medicament by intravenous infusion.

Decide on the very pathology of being treated, but general or local allotment and throwing and medicament.Allocate medical composition of the present invention so that it is compatible with predetermined dosing way.The technology of allotment and dispensing is found in Lei Mingdun medical science (Remington ' s Pharmaceutical Sciences), and the 18th edition, mark publishing company (Mack Publishing Co.), Easton, (Easton is Pa.) in (1990) in the Pennsylvania.Only give some instances, suitable pathways can comprise per os, rectum, vagina, offer medicine in skin, mucous membrane or intestines; Enteron aisle transmits outward, comprises intramuscular, subcutaneous and intramedullary injection; And in the sheath, directly in the ventricle, in the intravenously, intraperitoneal, nose or intraocular injection.More spendable transmission methods include, but is not limited to be packaged in the liposome, incorporate in the prosthetics, by the retroviral vector transduction with subsequently implantation again or throw and through the stripped transfectional cell of transfectional cell.

When pharmaceutically using composition, it makes up with " the pharmaceutically acceptable supporting agent " that use for diagnosis and treatment.The those skilled in the art that is allocated as of these compositions knows.Medical composition of the present invention can comprise one or more other medicament and preferably include pharmaceutically acceptable supporting agent.

Suitable pharmaceutically acceptable supporting agent and/or thinner comprise any and all conventional solvents, dispersion medium, weighting agent, solid carriers, the aqueous solution, dressing, antiseptic-germicide and anti-mycotic agent, isotonic agent and absorption delay agent and its analogue.Term " pharmaceutically acceptable supporting agent " be meant throw and the patient in do not cause allergic reaction or the supporting agent of other undesirable action.Suitable pharmaceutically acceptable supporting agent comprise in (for example) water, salt solution, phosphate buffered saline (PBS), dextrose, glycerine, ethanol and its analogue one or more with and the combination.Pharmaceutically acceptable supporting agent can further comprise a little auxiliary material, such as wetting agent or emulsifying agent, sanitas or buffer reagent, the preservation period of one or more medicament of its enhancing composition or effectiveness.Being used for these media of pharmaceutically acceptable material and the use of medicament is what know in affiliated field.

Be used for that enteron aisle is outer, the solution or the suspension of intradermal or subcutaneous administration can comprise following component: sterile diluent, such as water for injection, salt brine solution, expressed oil, polyoxyethylene glycol, glycerine, propylene glycol or other synthetic; Antiseptic-germicide is such as phenylcarbinol or methyl p-hydroxybenzoate; Antioxidant is such as xitix or sodium bisulfite; Sequestrant is such as ethylenediamine tetraacetic acid (EDTA); Buffer reagent is such as acetate, Citrate trianion or phosphoric acid salt; With the reagent of adjustment of tonicity, such as sodium-chlor or dextrose.The acid or the alkali of available all example hydrochloric acids of pH value or sodium hydroxide are regulated.The enteron aisle external preparation can be packaged in ampoule, disposable syringe or the multiple dose vials of making by glass or plastics.The medical composition that is suitable for injecting generally includes aseptic aqueous solution (wherein water soluble) or dispersion liquid and is used for preparing the sterile powder of aseptic injectable solution or dispersion liquid temporarily.For the intravenously dispensing, suitable supporting agent comprises physiological saline, bacteriostatic water, Sai Mofeiaier (CremophorEL

) (BASF, Pa Sebaini, New Jersey (Parsippany, N.J.)), phosphate buffered saline (PBS).Supporting agent can be solvent or dispersion medium, and it contains (for example) water, ethanol, polyvalent alcohol (for example, glycerine, propylene glycol and liquid macrogol and its analogue) and its suitable mixture.For example, by using dressing, under the situation of dispersion liquid, keep required granularity and, can keep suitable flowability by using tensio-active agent such as Yelkin TTS.Can reach prevention to microbial process, for example p-Hydroxybenzoate, trichloro-butyl alcohol, phenol, xitix, Thiomersalate and its analogue by various antiseptic-germicides and anti-mycotic agent.Under many situations, will preferably in composition, comprise isotonic agent, for example sugar or polyvalent alcohol (such as mannitol, Sorbitol Powder), sodium-chlor.The prolongation of Injectable composition absorbs and can cause by comprise the reagent (for example, aluminum monostearate and gelatin) that postpones absorption in composition.

In addition, other therapeutical agent that also can select of the medicament of the disease differentiated by the present invention of treatment and symptom with the particular utility of the symptom of being treated about opposing throw altogether with.For instance, medicament can with oestrogenic hormon or oestrogenic hormon related compound or the combination of other bone resorption inhibitor.Estrogen compound includes, but is not limited to engage oestrogenic hormon, estradiol and its analogue.Other bone photo closes treatment compound and includes, but is not limited to diphosphonate and related compound (such as United States Patent (USP) the 5th, 312, compound described in No. 814), calcium complement agent (Pu Linsilili (Prince, people such as R.L.), New England Journal of Medicine (N.Engl.J.Med.) 325,1189, (1991)), vitamins D supplement (Cha Pumixi people such as (ChapuyM.C.), New England Journal of Medicine (N.Engl.J.Med.) 327,1637, (1992)), Sodium Fluoride (league (unit of length) Si Buli (Riggs, people such as B.L.), New England Journal of Medicine (N.Engl.J.Med.), 327,620, (1992)), male sex hormone (Nagent de Deuxchaisnes, C, osteoporosis: a multidisciplinary problem, (Osteoporosis, aMulti-Disciplinary Problem), Britain's imperial family's medical association's international conference and the 55th phase of symposium collection of thesis (Royal Society of Medicine International Congress and Symposium Series No.55), academic press (Academic Press), London (London), the 291st page, (1983)) and thyrocalcitonin (Chris Tan Xunxi (Christiansen, C), bone (Bone), 13 (supplementary issue 1): S35, (1992)).

On the other hand, the invention provides a kind of system that differentiates the proteic medicament of activation Ror.Determine method that whether medicament changes the Ror2 protein-active comprises and carry out analysis and the calibrating that the those skilled in the art knows.Example includes, but is not limited to tissue chemical analysis, western-blot analysis, ELISA, enzyme calibrating (for example, kinases calibrating) and functional analysis (degree (higher phosphorylation state reflection greater activity) that comprises measure R or for example or 14-3-3 β phosphorylation).In certain embodiments, evaluate Ror (especially Ror2 albumen) activity by determining to have showed in conjunction with Ror2 albumen and by the phosphorylation state of the 14-3-3 β of Ror2 protein phosphorylation.The known any technology in field was examined and determine under the proteic phosphorylation of 14-3-3 β can be used.Specifically, use the immuno-precipitation of anti-phosphorylated tyrosine antibody to can be used for tracing the proteic phosphorylation of 14-3-3 β.Perhaps, the radio isotope that also can use phosphorus (for example, ³²P-γ-ATP).

The present invention also provides a kind of method that bone photo closes active medicament of regulating of differentiating, wherein the active increase of Ror molecule (for example, Ror2 albumen) or reduction indication medicament adjusting bone photo close active.

In certain embodiments, the invention provides a kind of use Chimerical receptor (for example, Ror2/TrkB) and reporter gene (such as luciferase) by two polymerizations of Ror2 regulation and control differentiate the calibration method that promotes Ror2 two polymeric medicaments.In certain embodiments, in the present invention's calibrating, use the cell of expressing the Chimerical receptor that comprises the Ror2 cell foreign lands of merging with TrkB cell internal area.In some specific embodiment, the amino acid/11-407 of the proteic cell of Ror2 foreign lands merges with the membrane-spanning domain of TrkB and cell internal area (amino acid 432-822).In other embodiments, use different cell internal areas to construct mosaic.For instance, can use any cell internal area of two polymerization postactivated to replace the TrkB territory.Preferably, the cell internal area is from single transmembrane receptor, and signal transduction pathway is known.The limiting examples that can be used for preparing other cell internal area of Chimerical receptor comprises the cell internal area of TrkA, TrkC, EGFR, PDGFR and FGFR.The cell internal area closes signal transduction cascade postactivated and that startup finally causes reporter gene to raise in cell foreign lands dimerization.For instance, under the chimeric situation of Ror2/TrkB, make Ror2 territory, the extracellular two polymeric medicaments of Chimerical receptor cause the activation of TrkB signal transduction pathway.The activation of TrkB signal transduction pathway is by using cAMP response element (CRE) promotor-reporter gene system to evaluate.The activation of another signal transduction pathway (such as EGFR) will need another reporter gene system, such as the system based on the STAT binding member that is started by the EGFR path.The activation of TrkB path causes stimulates the CRE promotor, and this increases the expression of any reporter gene under its control again.The control that can place the CRE promotor such as the Report Body of the easy calibrating of luciferase (LUC), green fluorescent protein (GFP), GRD beta-glucuronidase (GUS) and E.C. 2.3.1.28 (CAT) etc. down and be used for the present invention's calibrating.In certain embodiments, use luciferase as reporter gene.In other embodiments, use green fluorescent protein as reporter gene.In certain embodiments, the CRE promotor-Report Body on the plasmid is constructed the body transfection in the cell of expressing Chimerical receptor.In other embodiments, construct the part that body is a cellular genome.In certain embodiments, will construct the body stable transfection in cell.Having used herein based on the chimeric verification system of the present invention of Ror2/TrkB, displaying is verified Ror2 two polymeric Ror2 specific antibodies.The Ror2 specific antibody causes that the active dose-dependently of viewed Report Body (that is luciferase) increases.Referring to Figure 12.

Chimerical receptor verification system of the present invention can be used with the paired different cell internal areas of corresponding promoter systems and modify.The example of other cell internal area comprises the cell internal area of TrkA, TrkC, EGFR, PDGFR and FGFR.Then, in the Report Body system, use the corresponding promotor of regulating by the cell internal area.For instance, in having the system of Chimerical receptor of EGFR cell internal area, use can use the STAT binding member.

The present invention includes the test kit of carrying out Chimerical receptor calibrating of the present invention.These test kits comprise that some or all of use the present invention examine and determine the necessary component of filler test medicament.In certain embodiments, the component of test kit is used in order to the investigator through packing expediently.Test kit can comprise any or all among following each person: DNA constructs body, clone, damping fluid, enzyme, porous plate, the positive and negative control, medium, microbiotic, Nucleotide, specification sheets etc.In certain embodiments, test kit comprises the clone of expressing the Ror2/TrkB Chimerical receptor.In other embodiments, test kit comprises that the DNA of coding Ror2/TrkB Chimerical receptor constructs body.In other embodiments, test kit comprises the reporter gene that is connected with CRE promotor operability.In certain embodiments, test kit comprises the luciferase gene that is connected with CRE promotor operability.Reporter gene/CRE promotor is constructed body and be can be plasmid.

The present invention includes employed Chimerical receptor in the invention described above calibrating with Ror2 territory, extracellular.The exemplary aminoacid sequence of chimeric Ror2/TrkB acceptor is as follows.Being derived from the proteic aminoacid sequence of Ror2 shows with capitalization; Being derived from the proteic aminoacid sequence of TrkB shows with lowercase.

MARGSALPRRPLLCIPAVWAAAALLLSVSRTSGEVEVLDPNDPLGPLD

GQDGPIPTLKGYFLNFLEPVNNITIVQGQTAILHCKVAGNPPPNVRWLK

NDAPVVQEPRRIIIRKTEYGSRLRIQDLDTTDTGYYQCVATNGMKTITA

TGVLFVRLGPTHSPNHNFQDDYHEDGFCQPYRGIACARFIGNRTIYVD

SLQMQGEIENRITAAFTMIGTSTHLSDQCSQFAIPSFCHFVFPLCDARSR

APKPRELCRDECEVLESDLCRQEYTIARSNPLILMRLQLPKCEALPMPE

SPDAANCMRIGIPAERLGRYHQCYNGSGMDYRGTASTTKSGHQCQPW

ALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRM

ELCDVPSCSPRDSSKMGILYlsvyavvviasvvgfcllvmlfllklarhskfgmkgpasvisn

dddsasplhhisngsntpssseggpdaviigmtkipvienpqyfgitnsqlkpdtfvqhikrhnivlkrelge

gafgkvflaecynlcpeqdkilvavktlkdasdnarkdfhreaelltnlqhehivkfygvcvegdplimvfe

ymkhgdlnkflrahgpdavlmaegnppteltqsqmlhiaqqiaagmvylasqhfvhrdlatrnclvgenll

vkigdfgmsrdvystdyyrvgghtmlpirwmppesimyrkfttesdvwslgvvlweiftygkqpwyqls

nneviecitqgrvlqrprtcpqevyelmlgcwqrephmrknikgihtllqnlakaspvyldilg

(SEQ?ID?NO:4)。

It will be understood by one of ordinary skill in the art that without departing from the invention, can carry out various mutations, disappearance, replacement etc. this chimeric protein.In certain embodiments, chimeric protein and above-mentioned aminoacid sequence at least 99%, 98%, 95%, 90%, 80% or 70% homology.In certain embodiments, Chimerical receptor is at the postactivated signal transduction pathway of caused two polymerizations of two polymerizations by the Ror2 territory, extracellular of Chimerical receptor, such as the TrkB path.It will be understood by one of ordinary skill in the art that under the situation that does not change receptor active and can carry out multiple change above-mentioned protein sequence.Think that these varients of Chimerical receptor are in category of the present invention.The present invention also comprises the polynucleotide sequence of coding Chimerical receptor or its varient.Encoding sequence according to circumstances with the promotor of the expression of regulating chimeric protein and/or translation, strengthen operability such as son, controlling element and be connected.The present invention also comprises cell, and these cells comprise the polynucleotide sequence of the present invention of the Chimerical receptor of encoding.

Method of the present invention can be revised or carry out by any available formats (comprising the high-throughput calibrating).The high-throughput calibrating is applicable to screening substantive test medicament in the given time.In another embodiment, carry out the calibrating of using based on the screening of cell.The United States Patent (USP) of promulgation on August 15th, 2000 the 6th, 103, No. 479 (they are incorporated herein by reference) discloses minicell array approach and the equipment that is used for based on the screening of cell.The method that preparation is used for the homogeneous tiny model cellular array of other application has been described, for example (Mick thinks bodyguard (Mrksich) and White thinks moral (Whitesides) for photochemistry etching method (photochemical resist photolithography), biophysics and biomolecular structure yearbook (Ann.Rev.Biophys.Biomol.Struct.), 25,55-78 (1996)).The United States Patent (USP) the 6th of promulgation on August 1st, 2000,096, No. 509 (it is incorporated herein by reference) provides equipment and the method for real-time measurement cell streaming suspension to the cell response of test medicament, each member's of a series of cell types homogeneous suspension is made up with specific concentrations and test compounds, detection zone is passed in guiding, and measures the cell response of viable cell when the stream of cells in the test mixing thing is distinguished after testing in real time.Described patent announcement equipment is used for the purposes in automatic screening test medicament (for example, small molecules) library.The method that is disclosed in these United States Patent (USP)s be can revise and expression or activity that whether the test medicament regulates the Ror molecule, these cells such as scleroblast (elementary scleroblast determined to use cell; The human osteoblast is such as TE-85, U2OS, SaOS-2 or HOB; Rat osteoblast is such as UMR 106 or ROS 17/2.8; Mouse bone-forming cell is such as MC3T3 or other similar cells), non-scleroblast (COS-7 and other similar cells), stem cell (mescenchymal stem cell, embryonic stem cell), progenitor cell or contain the through engineering approaches cell of Ror nucleotide sequence.In other embodiments, carry out the calibrating of examining and determine (for example, kinases calibrating) based on enzyme.

Then, can further test the test medicament that is applicable to adjusting Ror protein-active through discriminating.In certain embodiments, test medicament at other in based on the calibrating of cell or non-calibrating based on cell.Can be in the animal model (animal model that comprises multiple osteopathia and illness) of multiple disease test compounds.For instance, can in the animal model of fracture, osteoporosis, osteocarcinoma, bone loss etc., test medicament.

The present invention incorporates method and the technology of knowing in molecule and the cytobiology field by reference into.These technology include, but is not limited to the fertile (Old of the technology described in the following discloses case: Ou Deli, R.W.) and Si Bupulimosi (S.B.Primrose), the principle of genetic manipulation: genetically engineered introduction (Principles of Gene Manipulation:AnIntroduction To Genetic Engineering) (the 3rd edition, 1985), Bradley kwell Science Press (BlackwellScientific Publication), Boston (Boston), microbiological research (Studies in Microbiology); The 2nd volume: the 409th page (ISBN0-632-01318-4); Mountain nurse Brooker outstanding person (Sambrook, J.) etc. the people compiles, molecular cloning: laboratory manual (Molecular Cloning:A Laboratory Manual), (the 2nd edition, 1989), press of cold spring harbor laboratory (Cold Spring Harbor Laboratory Press), New York (NY.), 1-3 rolls up (ISBN0-87969-309-6); Outstanding conspicuous (the Miller of Miller, J.H.) and a rice pick up Luo Si (M.P.Calos) compile, the gene transfer vector of mammalian cell (1987), press of cold spring harbor laboratory (Cold Spring Harbor Laboratory Press), New York (NY.), the 169th page (ISBN0-87969-198-0).

Example

Further in following example, define the present invention, wherein except as otherwise noted, otherwise all umbers and per-cent all by weight and the degree for degree centigrade.Should be appreciated that though these example indication the preferred embodiments of the present invention, it only provides in the mode of explanation.According to the above discussion, embodiment and these examples, the those skilled in the art will easily understand, and not break away from novel teaching of the present invention in itself and not break away under the situation of the present invention's spirit and category, may there be multiple correction in exemplary embodiments.In addition, can carry out multiple change and correction so that the present invention adapts to multiple use and situation to the present invention.Therefore, all these revise to plan to be included in the category of the present invention that is defined in claims hereinafter.

The mode that patent mentioned herein, application case, testing method and open case are quoted in full is incorporated herein.

General method

Material and tissue culture

Unless note is arranged in addition, otherwise tissue culture reagent is all available from hero company (Invitrogen Corporation) (Carlsbad, California (Carlsbad, CA)); Other reagent and chemical are available from sigma chemical company (SigmaChemical Co.) (Saint Louis, the Missouri State (St.Louis, MO)) or hero.The cytosol territory of the GST mark of recombinant human Ror2 is that the recombinant human 14-3-3 β available from hero and GST mark is from (the Biomol International of biomolecules international corporation, LP) (the rice court of a feudal ruler, Plymouth, Pennsylvania (Plymouth Meeting, PA)).Anti-Flag M2 mouse monoclonal antibody, the affine agarose of anti-Flag M2 and anti-beta-actin mouse monoclonal antibody are available from Sigma (Sigma); Anti-human Ror2 goat polyclonal antibody is available from peace enlightening biology (R﹠amp; D Systems) (Minneapolis, the Minnesota State (Minneapolis, MN)); Anti-14-3-3 β and anti-His rabbit polyclonal antibody are from Santa Cruz Bioisystech Co., Ltd (Santa Cruz Biotechnology, Inc.) (Santa Cruz, California (Santa Cruz, CA)).Disengaged and agarose engages anti-phosphorylated tyrosine antibody (4G10) available from Upstate cell signalling scheme company (Upstate Cell Signaling Solutions) (Charlottesville, Virginia (Charlottesville, VA)); Fixedly phosphorylated tyrosine antibody P-Tyr-100 is from cell signalling technology company (Cell Signaling Technologies) (Bei Fuli, Massachusetts (Beverly, MA)); And albumin A agarose and glutathione agarose are available from peace agate West Asia Biological Science Co., Ltd (Amersham Biosciences) (Buckinghamshire, Britain (Buckinghamshire, England)).(Horseradish peroxidase, HRP) engage secondary antibody is from Santa Cruz Bioisystech Co., Ltd (Santa Cruz Biotechnology) to horseradish peroxidase.

Human mescenchymal stem cell (hMSC) is available from the Ka Mubu Simon Rex (Cambrex of company, Inc.) (Baltimore, the Maryland State (Baltimore MD)) and use hMSC growth medium (MSCGM, Ka Mubu Simon Rex (Cambrex)) under 37 ℃, to be maintained at 5%CO ₂In-95% humidifying air incubator.With the U2OS human osteosarcoma cell under 37 ℃, remain in contain 10% heat inactivation foetal calf serum (fetal bovine serum, FBS), in the 5A improved culture medium of the McCoy of 1% penicillin-Streptomycin sulphate and 2mMglutaMAX-I.

Plasmid and adenovirus

The previous generation of having described human Ror2-Flag expression plasmid (than Reed (Billiard) He Boting (Bodine), the U.S. patent application case U.S.S.N.10/823 of application on April 14th, 2004,998; Than Reed people such as (Billiard), molecular endocrinology (Mol Endo) 19,90-101,2005).By not replacing Flag epi-position label with the sequence of 6 Histidines of coding and produce Ror2-His and construct body in the end place at the COOH of Ror2-Flag.The GST fusions (GST-Ror2c) in the cytosol territory of Ror2 is to obtain by human Ror2 cell internal area (coded amino acid 428-944) being inserted in the GST label framework afterwards in the pGEX-4T-2 carrier (peace agate West Asia (Amersham)).

The human 14-3-3 β of total length cDNA is available from open biosystem (Open Biosystems) (Huntsville, Alabama (Huntsville, AL)) and be subcloned in the pET28a bacterial expression vector.

Contain human Coxsackie adenovirus receptor (human coxsackie adenovirus receptor, hCAR), the adenovirus of Ror2 specificity shRNA and EGFP specificity shRNA is available from (the Galapagos of Jia Lapage company, Inc.) (Mei Helun, Belgium (Mechelen, Belgium)).(β-gal) generation of adenovirus is (than Reed (Billiard) He Boting (Bodine) to have described Ror2, Ror2KD and beta-galactosidase enzymes, the U. S. application case U.S.S.N.10/823 of application on April 14th, 2004,998, it is incorporated herein by reference).

Cranium organ culture and infection

Excise cranium from 4 the biggest brood birth mouse, radially seam (sagittal suture) cuts and cultivated 24 hours in containing the serum-free BGJ substratum of 0.1%BSA.Then, each half cranium is put in the downward mode of concave surface on the stainless steel grid (widget company (Small Parts Inc), Miami (Miami), Florida State (F1)) in the hole of 12 orifice plates.The BGJ substratum that 1ml has 1%FBS is contained in each hole, can not contain or contain β-gal or Ror2 adenovirus (3.75mln virion/hole).With cranium at 5%CO ₂Cultivate in-95% humidifying air incubator, and after 4 days, change substratum and adenovirus.

Exist under the adenovirus cultivate 7 days after, at room temperature cranium is neutral and fixes 72 hours in the formaldehyde of phosphate buffered 10%, decalcification 6 hours in 10% EDTA that is stored among the PBS then.With each the group in parallel being embedded in the same paraffin mass of cranium, and with hematoxylin-eosin to 4 μ m section statinings.Select consistent bone zone (from frontal suture 200 μ m places) to be used for tissue morphology bibliometric analysis (histomorphometric analysis).In brief, 200 square micron grids are placed on each cranium and with (bone measurement company (Osteometries Inc) of bone measurement system (Osteomeasure System), the Atlanta, the Georgia State (Atlanta, GA)) measures the scleroblast number and total surface of bone is long-pending.All cells on the bone surface is counted as scleroblast.Use calcium diagnostic kit (CalciumDiagnostics Kit) (Sigma (Sigma)), measure substratum calcium according to the experimental program of manufacturers.

Virus infection

With 6,000/cm ²Be inoculated in human MSC in 12 holes or 6 orifice plates and it is adhered to and breed and spend the night.(multiplicity of infection MOI)=750 in the presence of hCAR (MOI=750), uses Ror2, Ror2KD or β-gal adenovirus at 0.4ml/cm to infect multiplying power ²Make cell infection 24 hours to improve efficiency of infection among the MSCGM.After 24 hours, once and add MSCGM, be supplemented with the MSCGM of 0.05mM xitix and 10mM β-glycerophosphate or contain into the MSCGM of fat fill-in (PT-3004, Ka Mubu Simon Rex (Cambrex)) with the PBS washed cell.During indication, in substratum, add 100nM dexamethasone (dex) and/or indicated antibody.Infect for shRNA, with 6,000/cm ²With cell inoculation in 12 holes or 6 orifice plates and it is adhered to and bred 3 days.In the presence of hCAR (MOI=750), use the adenovirus of coding Ror2 specificity shRNA or EGFP specificity shRNA in 0.4ml/cm2 MSCGM, to make cell infection 72 hours with 4,000 virions of each cell (in original inoculum density).After 72 hours, with the PBS washed cell once, and add MSCGM or be supplemented with the MSCGM of 0.05mM xitix, 10mM β-glycerophosphate.During indication, add 100nM dex and/or specific antibody.Every 5 days, replace whole substratum or half substratum with fresh culture.

With 75,000/cm ²With the U2OS cell inoculation in 6 orifice plates and subsequently under MOI=100 with Ror2, Ror2KD or β-gal adenovirus infection 24 hours.Make to infect and carried out 24 hours and collecting cell extract after 24 hours.

Sodium alizarinsulfonate-S histochemical stain

On 12 orifice plates, determine nodular formation that mineralize by hMSC caused by sodium alizarinsulfonate-S histochemical stain.At room temperature use 70% (v/v) ethanol fixed cell and matrix, with deionized water wash and at room temperature used 40mM sodium alizarinsulfonate-S (pH4.2) dyeing 10 minutes.With deionized water wash dyeing matrix and take pictures.For the quantitative painted level of sodium alizarinsulfonate-S, with 10% (w/v) cetylpyridinium chloride eluted dye in 1 milliliter/hole.Under 562nm with the sodium alizarinsulfonate-S in the quantitative elution samples of microplate reader (typical curve of contrast 0-800 μ M dyestuff).

Oil red O histochemical stain

On 12 orifice plates, monitor steatogenesis among the hMSC by oil red O histochemical stain.At room temperature use 10% neutral buffered formalin (formalin) fixed cell, with PBS washing and at room temperature use 18mg/mL oil red O (pH7) dyeing 10 minutes in 60% Virahol.With PBS washing staining cell and take pictures.

RNA separates and the PCR in real time analysis

Use RNeasy test kit (Kai Jie (Qiagen), the Valencia, California (Valencia, CA)), specification sheets according to manufacturers separates total cell RNA and uses ABI PRISM 7700 sequence detection systems (ABIPRISM 7700 Sequence Detection System) (u.s.a. applied biosystem company (AppliedBiosystems), the Foster city, California (Foster City, CA)) makes it stand the real-time RT-PCR analysis.All mRNA level standards are turned to the level of house-keeping gene (housekeeping gene) cyclophilin B.The primer of human C/EBP α and PPAR γ and probe are available from u.s.a. applied biosystem company (Applied Biosystems); Primer and the probe of human cyclophilin B are as follows: 5 '-CACCAACGGCTCCCAGTT-3 ' (forward primer, 438-455,5 SEQ ID NO:1), '-AACACCACATGCTTGCCATCT-3 ' (reverse primer, 486-506, SEQ ID NO:2) and 5 '-TTCATCACGACAGTCAAGACAGCCTGG-3 ' (probe, 457-483, SEQ ID NO:3).

Transient transfection

For the Ror2 plasmid transfection, converge density (confluent density) inoculation U2OS cell and use Fugene6 transfection reagent (Luo Shi applied science (Roche Applied Science) after 24 hours with about 80%, the Indianapolis, the state of Indiana (Indianapolis, IN)) is according to the every 19.6cm of the specification sheets of manufacturers ²The total plasmid DNA transfection of 11 μ g.For the siRNA transfection, with 52,000 cells/square cm is placed on the U2OS cell on 6 orifice plates, (both is from Daxad agate Kanggong department (Dharmacon Inc.) to use 10 μ l Lipofectamine, 2000 reagent to use 25nM Ror2siRNA or non-specific siRNA according to the specification sheets of manufacturers after 24 hours, Lafayette, California (Lafayette, CO)) transfection.

Immuno-precipitation and west immunoblotting

With cytolysis in dissolving damping fluid (the 150mM NaCl that is supplemented with proteolytic enzyme and inhibitors of phosphatases mixture (Sigma (Sigma)), 50mM Tris-HCl (pH7.5), 1mM EDTA, 1% Triton X100 (Triton X100)) in, and by under 4 ℃, under 10,000 * g, making the extract clarification in centrifugal 10 minutes.For the Flag immuno-precipitation, under rotating under 4 ℃, the total cell lysates of 1mg was cultivated 1 hour with the affine agarose of 30 μ l M2 Flag (Sigma (Sigma)).Wash 3 times and wash 3 times by centrifugal collection bead and with the dissolving damping fluid that contains 350mM NaCl with the dissolving damping fluid.For 14-3-3 β precipitation, under 4 ℃, 15 μ l 14-3-3 β antibody are spent the night in the cultivation of 1ml dissolving damping fluid with 30 μ l albumin A agaroses, and, add the total cell lysates of 1mg afterwards by centrifugal collection bead and with the washing of dissolving damping fluid.Under slight the rotation, carrying out association reaction 2 hours under 4 ℃ and collecting bead and as described in the Flag precipitation, washing.For the phosphorylated tyrosine precipitation, in 100 μ l G410 beads, add 1mg (for detecting expressed protein) or 1.5mg (for detecting endogenous protein) cell extract and it was adhered to 3 hours.At this moment, in mixture, add P-Tyr-100 sessile antibody (100 μ l) and last other 3 hours.By all beads of centrifugal collection and as described in the Flag precipitation, washing.When all immunoprecipitations finish, bead is boiled with reductive agent (hero (Invitrogen)) in 30-50 μ l2 * LDS-PAGE damping fluid, and by the protein of SDS-PAGE separate dissolved.Gel is transferred on the 0.45 μ m nitrocellulose membrane, detected with each specific antibody afterwards.

For no sedimentary immunoblotting, under sex change and reductive condition, split total cell lysates of indicatrix by SDS-PAGE, afterwards it is transferred on the 0.45 μ m nitrocellulose membrane and and detect with each specific antibody.

GST mixes and surveys (pool-down) and in vitro kinases calibrating

Use the outer (Expressway of Ai Pusi ^TM) protein synthesis system (hero (Invitrogen)) in vitro, in 50 μ l reaction, in vitro translate 14-3-3 β according to the specification sheets of manufacturers from 14-3-3 β-pET28a.It is colibacillary BL21 (DE3) bacterial strain that GST-Ror2c among pGEX-4T-2 or the pGEX-4T-2 (GST only encodes) is made the transition.Make culture grow into A6 ₀₀Be 0.7 and by adding sec.-propyl-1-sulfenyl-β-D-galactopyranoside (Sigma (Sigma); Ultimate density is 1mM) and cultivate and induced with express recombinant protein in 4 hours.By centrifugal collection bacterium bead, with PBS washing and its resuspending is supplemented with among the PBS of proteolytic enzyme and inhibitors of phosphatases mixture (Sigma (Sigma)) in 30ml.By with 16,000 pound/square inch (p.s.i.) passes French cell press (FrenchPressure Cell Press) (spectral instrument (Spectronic Instruments), Rochester, New York (Rochester, NY)) make cytolysis twice, and by the centrifugal bacterial debris that shifts out.Under 4 ℃, the GST-Ror2c or the GST albumen that are produced were cultivated 4 hours with glutathione agarose.The washing bead makes its resuspending in 1ml PBS, and 4 ℃ add down whole 50 μ l 14-3-3 β in vitro translation reaction last 4 hours.When this cultivates end, bead with PBS washing 3 times, is boiled with reductive agent (hero (Invitrogen)) in 2 * LDS-PAGE damping fluid, and by SDS-PAGE separate dissolved protein.Gel is transferred on the 0.45 μ m nitrocellulose membrane, detected with each specific antibody afterwards.

For in vitro kinases calibrating, recombinant human GST-14-3-3 β (biomolecules (the Biomol)) resuspending of 6.5 μ g purifying is added with or is not added kinase reaction damping fluid (the 10mM MgCl of the recombinant human GST-R2c (hero (Invitrogen)) of 0.9 μ g purifying in 25 μ l ₂, 50mM Tris-HCl (pH7.5), the 1mM dithiothreitol (DTT) (dithiotriethol, DTT), 1mM MATP) in.Kinase reaction was carried out 30 minutes and stopping by in 1 * LDS damping fluid, boiling with reductive agent (hero (Invitrogen)).Split protein by SDS-PAGE, transfer to it on 0.45 μ m nitrocellulose membrane and use the phosphorylated tyrosine antibody test.Then, peel off described film and survey again to verify same even load with 14-3-3 β antibody.

Statistical study

Data present with mean value ± SE form.(Student ' sT-test) determines statistical significance to use one-way analysis of variance (one-way ANOVA) or student t check.When P＜0.05, think that the result is different on statistics.

Example 1

Endogenous Ror2 plays a role in the hMSC differentiation

We before be illustrated in the Osteoblast Differentiation process of hMSC Ror2 express and increase (than Reed (Billiard) people of etc.ing, the U.S. patent application case U.S.S.N.10/823 that on April 14th, 2004 applied for, 998; Than Reed people such as (Billiard), molecular endocrinology (Mol Endo) 19,90-101,2005, it is incorporated herein by reference separately).Whether the rising of expressing for Ror2 in the evaluation hMSC atomization is the crux that scleroblast generates, and we carry out the differentiation of dex inductive when Ror2 expression by inhibitation system.For this purpose, with the adenovirus infection hMSC that contains Ror2 specificity shRNA, when comparing with EGFP specificity contrast shRNA, Ror2 specificity shRNA is the rising (Figure 1A) of strongly inhibited dex inductive Ror2 protein expression in fact.Infect the ability (Figure 1B and Fig. 1 C) that dex induces matrix mineralsization of almost completely abolishing with Ror2 shRNA, thus the osteoblast differentiation that is increased to small part mediation dex inductive hMSC of prompting Ror2.

Example 2

Ror2 crosses the one-tenth fat differentiation of expression inhibiting hMSC

We had before showed also that Ror2 crossed the initial MSC of making of expression and is fixed to the early stage and differentiation in late period of scleroblast strain and the generation of promotion scleroblast (than Reed people such as (Billiard), the U.S. patent application case U.S.S.N.10/823 of application on April 14th, 2004,998; It is incorporated herein by reference).Now, we evaluate Ror2 to by containing indomethacin (indomethacin) and containing becoming of IBMX cultivates the replacement trend (steatogenesis) of the inductive hMSC of institute in the lipoprotein mixture effect.With the human MSC of the adenovirus infection of encoding wild type Ror2 or kinases territory mutant (Ror2KD) (respectively contain COOH and do not hold flag epi-position label).In Ror2KD, replace three Methionins at position 504 (ATP that infers is in conjunction with the territory), 507 and 509 places so that tyrosine kinase activity significantly reduces (day large bamboo hat with a conical crown and broad brim people such as (Hikasa) with different white amino acid, development (Development) 129,5227-5239,2002; Than Reed people such as (Billiard), the U.S. patent application case U.S.S.N.10/823 of application on April 14th, 2004,998; Than Reed people such as (Billiard), molecular endocrinology (Mol Endo), 19,90-101,2005).For contrast, (β-gal) expression cassette infects hMSC with beta-galactosidase enzymes in identical adenovirus background.Ror2 suppresses mainly to become fat transcription factor CCAAT/ to strengthen sub-bindin alpha (CCAAT/enhancer-binding protein α with the Ror2KD mutant, C/EBP α) and peroxisome proliferant agent activated receptor γ (peroxisome proliferator-activated receptor γ, PPAR γ) expression (Fig. 2 A), and make hMSC form ability that the positive lipid of oil red O produces the property adipocyte obviously descend (Fig. 2 B).In conjunction with our previous result (than Reed people such as (Billiard), the U.S. patent application case U.S.S.N.10/823 of application on April 14th, 2004,998; It is incorporated herein by reference), these data indications Ror2 makes its deflection scleroblast generate the cell trend that changes MSC by the balance of mobile transcription factor.

Example 3

Total surface of bone that Ror2 increases the mouse cranium amasss

Then, we test Ror2 whether the in vitro effect of hMSC differentiation are translated into osteoplastic increase in the isolated organ culture.The craniums of 4 the biggest brood birth mouse kept do not infect or with β-gal or Ror2 adenovirus infection.After cultivating 7 days in the presence of the adenovirus, bone is made 200 compatible μ m with hematoxylin-eosin dyeing and use skeletonization measuring system ²Section (from frontal suture 200 μ m places) stands the tissue morphology bibliometric analysis.Contrasting not under the infectious condition 200 μ m ²The cranium section contains 5171 ± 235 μ m ²Long-pending and 84 ± 6.5 scleroblasts of surface of bone.Cause the long-pending increase by 50% of total surface of bone with the Ror2 virus infection, and do not influence the scleroblast number, this indication Ror2 activation scleroblast is to produce more boniness matrix (Fig. 3).

Example 4

14-3-3 β is that the Ror2 kinases of first discriminating is subjected to matter

We before reported by mass spectrum differentiate 9 kinds of potential Ror2 binding factors in the U2OS osteosarcoma cell (than Reed (Billiard) people of etc.ing, the U.S. patent application case U.S.S.N.10/823 that on April 14th, 2004 applied for, 998; It is incorporated herein by reference).In these factors, showed that 14-3-3 albumen plays a role (Macintosh (Mackintosh), journal of biological chemistry (Biochem J) 381,329-342,2004) and we select 14-3-3 β to be used for follow-up study in cell cycle progression and differentiation.We at first confirm to use the immunoprecipitation technology by the observed interaction of mass spectrum.

With β-gal, Ror2 or Ror2KD adenovirus infection U2OS cell, and separate total cell protein matter and make it stand the immunoprecipitation that on the affine agarose of anti-Flag, carries out, then through benefiting from the immunoblotting (Fig. 4 A, last figure) that anti-14-3-3 β antibody carries out.Under collating condition (β-gal cells infected), minute quantity 14-3-3 β precipitation, but Ror2 a large amount of co-precipitation occur after crossing expression.Form with the mixture of Ror2KD mutant even stronger, this indication kinase activity causes complex dissociation.Sedimentary peer-level is verified (Fig. 4 A, figure below) by the immunoblotting that carries out with anti-Flag antibody.Interaction between 14-3-3 β and the Ror2 is by being deposited in 14-3-3 β further to be confirmed on the 14-3-3 β specific antibody and exist (Fig. 4 B) by Ror2 in the anti-Flag immunoblotting checking mixture.

Whether Ror2 causes 14-3-3 β phosphorylation for evaluation, and we survey trace among Fig. 4 A again with anti-phosphorylated tyrosine antibody.This antibody is differentiated the phosphorylated protein (Fig. 4 A, middle figure) that moves and exist only in the cell of expressing wild-type Ror2 rather than β-gal or the nonactive mutant of kinases under the molecular weight identical with 14-3-3 β.This prompting Ror2 directly or indirectly makes 14-3-3 β in tyrosine residues place phosphorylation.This hypothesis by make all tyrosine phosphorylated proteins immunoprecipitations in the U2OS extract on anti-phosphorylated tyrosine antibody and the amount of after Ror2 crosses expression, observing phosphoric acid-14-3-3 β significantly increase be confirmed (Fig. 4 C).

Whether mediate the background phosphorylation of viewed 14-3-3 β among Fig. 4 C for testing endogenous Ror2, we are expressed by the Ror2 that the Ror2 specific siRNA suppresses in the U2OS cell.As shown in Fig. 5 A, when comparing with messy contrast siRNA, the almost completely inhibition of reaching the Ror2 protein expression with the transfection of Ror2 specific siRNA.The reduction that Ror2 expresses can not given birth to influence to the proteic volume production of the 14-3-3 β in the U2OS cell, but causes the remarkable downward modulation (Fig. 5 E) of its tyrosine phosphorylation.Compare with Fig. 4 C, the obvious increase of 14-3-3 β background phosphorylation degree is owing to the longer time shutter as used herein produces.

For evaluation Ror2 and 14-3-3 β combine and whether the phosphorylation of 14-3-3 β is directly, we are with reorganization protein purification execution ex vivo experiment.For in conjunction with experiment, the GST fusions (GST-Ror2c) in the cytosol territory of human Ror2 is expressed in the bacterial cell, it is deposited on the glutathione agarose and cultivates with the 14-3-3 β of in vitro translation.As shown in Fig. 6 A, 14-3-3 β is incorporated into GST-Ror2c, rather than is incorporated into independent GST, and this indication 14-3-3 β is directly in conjunction with the cytosol territory of Ror2.Because in vitro Fan Yi 14-3-3 β contains with kinases and examines and determine the outer (Expressway of inconsistent Ai Pusi ^TM) the synthetic damping fluid of albumen, so we buy purifying reorganization GST mark 14-3-3 β and carry out in vitro kinases with the reorganization GST-Ror2c (hero (Invitrogen)) of purifying and examine and determine.As shown in Fig. 6 B, Ror2c phosphorylation 14-3-3 β and himself, thereby confirm 14-3-3 β be the Ror2 Tyrosylprotein kinase directly be subjected to matter.

Example 5

The Ror2 specific antibody makes the Ror2 receptor dimerization close and activate the Ror2 acceptor

Showed that several receptor tyrosine kinases are by antibody two polymerizations and activation (Si Pajialun people such as (Spaargaren), journal of biological chemistry (J.Biol.Chem.), 266,1733-1739,1991; Good fortune people such as (Fuh), science (Science) 256,1677-1680,1992; It is incorporated herein by reference separately).Therefore, our testing needle makes the ability of two polymerizations of Ror2 receptor tyrosine kinase and activation Ror2 receptor tyrosine kinase to the Ror2 specific antibody of the whole cell foreign lands cultivation of human Ror2.For evaluating two polymerizations, the Ror2 acceptor of Flag mark and His mark is constructed body be expressed in the U2OS cell, and (cultivate the cell foreign lands at human Ror2, peace enlightening biology (R﹠amp with Ror2 specificity goat polyclone IgG down at 37 ℃; D Systems), AF2064) or with non-specific goat IgG contrast (pacify enlightening biology (R﹠amp; D Systems)) handle cell and last 1 hour.After the cultivation, extract total cell protein matter, make it be deposited on the affine agarose of anti-Flag and make it through benefiting from the immunoblotting that anti-His antibody carries out.As shown in the last figure of Fig. 7 A, under the collating condition that non-specific IgG handles, there are some associations between the Ror2 acceptor of His mark and Flag mark, this indication Ror2 is expressed in formation homodimer in back in the U2OS cell crossing.Being formed on sharply strengthening after the Ror2 antibody treatment of this homodimer confirms that this antibody can make the Ror2 receptor dimerization close.Test design is failed immunoprecipitation Ror2-His and anti-His antibody nonrecognition Ror2-Flag proteic true verified (Fig. 7 A, last figure) by anti-Flag antibody under the situation that does not have Ror2-Flag.The sedimentary peer-level of Ror2-Flag is verified (Fig. 7 A, figure below) by the immunoblotting that carries out with anti-Flag antibody.

Whether activate the Ror2 Tyrosylprotein kinase for setting forth antibody, we handle the U2OS cell with Ror2 specific antibody or IgG contrast down at 37 ℃ and last 1 hour, separate total cell protein matter extract and all tyrosine phosphorylated proteins are deposited on the phosphorylated tyrosine antibody.Fig. 7 B explanation with anti-Ror2 antibody treatment reach the kinase whose significantly automatic phosphorylation of Ror2 with and be subjected to the proteic phosphorylation of matter 14-3-3 β.These data provide anti-Ror2 antibody to make the strong evidence of two polymerizations of Ror2 tyrosine kinase receptor and activation Ror2 tyrosine kinase receptor.

Example 6

The specific, activated antibody of Ror2 promotes that hMSC mineralizes

Then, we may well ask whether Ror2 two polymerizations of antibody induction and activation will produce functional outcome to the Osteoblast Differentiation of hMSC.Because unless hMSC is divided into into bone phenotype, its do not express Ror2 (than Reed (Billiard) He Boting (Bodine), the U.S. patent application case U.S.S.N.10/823 of on April 14th, 2004 application, 998; Than Reed people such as (Billiard), molecular endocrinology (Mol Endo) 19,90-101,2005; It is incorporated herein by reference separately), so we are by handling the Ror2 specificity goat IgG or the non-specific goat IgG of inducing Ror2 to express and add incremental change with skeletonization mixture (being supplemented with the MSCGM of 0.05mM xitix, 10mM β-glycerophosphate and 100nM dex).Cultivate after 9 days, evaluate the substrate formed degree that mineralizes with sodium alizarinsulfonate-S histochemical stain.As shown in Figure 8, anti-Ror2 antibody dosage dependency increases the substrate formed degree of calcification among the hMSC.Except that observing the slight inhibition under 100 μ g/ml maximum dose levels, non-specific goat IgG does not exert an influence to matrix mineralsization under all test concentrations.For clarity sake, a dosage (50 μ g/ml) of only showing non-specific IgG among Fig. 8.(pacify enlightening biology (R﹠amp at the goat polyclonal antibody that cultivate total cell foreign lands of human Ror1; DSystems), AF2000) also inoperative (Fig. 8).The effect of anti-Ror2 antibody is receptor-mediated by Ror2, because when the expression of Ror2 in hMSC suppressed by Ror2 specificity shRNA, and this effect disappearance (Fig. 9 A).In addition, even anti-Ror2 antibody also induces calcification matrix to form under the situation that does not have dex, as long as express Ror2 (Fig. 9 B) by the adenovirus infection inducing cell.Therefore, the Ror2 specific antibody can make the Ror2 receptor dimerization close and activate the Ror2 acceptor and promote the calcification matrix of Ror2 mediation in the mescenchymal stem cell to form, and this prompting activation Ror2 antibody can provide effective treatment to osteoporosis and other osteopathia.

Example 7

The inhibition of 14-3-3B strengthens hMSC and mineralizes

Whether 14-3-3 β plays a role in Osteoblast Differentiation for test, and we express not existing and exist Ror2 to cross to suppress 14-3-3 β under the situation of expression.For this purpose, shRNA infects hMSC with 14-3-3 β specificity, and when comparing with messy contrast shRNA, 14-3-3 β specificity shRNA reduces endogenous protein in fact strongly and expresses (Figure 10 A).When the contrast virus infection hMSC that contains messy shRNA and beta-galactosidase enzymes with two kinds, we observe the matrix mineralsization (Figure 10 B) of low degree, and this points out the double infection of himself to mediate slight osteogenesis in the hMSC culture.As before observed (than Reed people such as (Billiard), the U.S. patent application case U.S.S.N.10/823 of on April 14th, 2004 application, 998), with Ror2 adenovirus infection promote forcefully the to mineralize formation of matrix.Exceed us and expect, the downward modulation of 14-3-3 β also increases the degree (Figure 10 B) that mineralizes greatly.Ror2 crosses and expresses and 14-3-3 β suppresses together only institute's inductive matrix minerals intensive matrix mineralsization (Figure 10 B) of induction ratio.This is to point out 14-3-3 β skelemin that the Osteoblast Differentiation of hMSC is played inhibiting first evidence.

Example 8

The Ror2 activation of antibody induction and 14-3-3 β suppress the new bone forming in the stripped braincap culture of promotion

Then, we test the in vitro effect that Ror2 activates and 14-3-3 β suppresses and whether translate into osteoplastic increase in the isolated organ culture.With the adenovirus that contains messy shRNA or 14-3-3 β specificity shRNA with 5 * 10 ⁷Individual virion/milliliter infects the cranium of 4 the biggest brood birth mouse; And after 48 hours, in the presence of 15 μ g/ml fluorexons, handle with anti-Ror2 antibody of 12 μ g/ml or non-specific IgG.After cultivating 7 days with adenovirus and antibody, with hematoxylin-eosin bone is dyeed, and (Nashville, Tennessee State (Nashville, TN)) make compatible 300 μ m elongations (from frontal suture 450 μ m places) stand the tissue morphology bibliometric analysis to use Bioquant-NOVA MR5.50.8.Under the collating condition that messy shRNA infects and IgG handles, the cranium of 600 μ m length contains 9394 ± 1333 μ m ²Long-pending and 39.5 ± 7.4 scleroblasts of surface of bone.Specificity shRNA causes the long-pending increase by 60% of total surface of bone and makes the scleroblast number increase by 50% that to the inhibition of 14-3-3 β indication 14-3-3 β albumen is suppressed to osteocyte number and/or activity (Figure 11).Cause scleroblast number increase by 85% and cause the long-pending increase by 50% of total surface of bone with anti-Ror2 antibody treatment cranium, prompting activation Ror2 antibody can provide effective treatment to osteoporosis and other osteopathia again.Yet, 14-3-3 β shRNA is infected with the combination of Ror2 antibody treatment can't produce synergistic effect, to compare with arbitrary processing only, it causes even smaller reaction (Figure 11).Because in our experiment, the increase of viewed 50-80% does not reach the saturation ratio of this verification system, so attempt inferring that Ror2 and 14-3-3 β are with same path control osteoblast differentiation and/or function.

Example 9

Exploitation is used for the active high-throughput of Ror2, highly sensitive calibrating

As shown in Figure 12 A, described calibrating utilizes the TrkB receptor signal transduction path through fully characterizing.The TrkB acceptor causes the Erk phosphorylation by the ligand inductive and stimulates the equal two polymerizations activation of the cAMP response element (CRE) in the target gene promoters.The Chimerical receptor that the Ror2 cell foreign lands (aa1-407) that generation is merged by membrane-spanning domain and cell internal area (aa432-822) with TrkB are formed.We suppose when using this mosaic, cause that Ror2 two polymeric medicaments will activate the TrkB signal transduction pathway and make the CRE promoter activity increase.For checking this hypothesis, with the Chimerical receptor stable transfection in available from doctor Cao Chengen (Dr.Seongeun Cho) (Wei Si research centre, the Princeton, New Jersey (Wyeth Research, Princeton, NJ)) mistake is expressed in the HEK293A cell (HEK-CRE) of CRE-luciferase plasmids.Use ECM600 electroporation apparatus (BTX, San Diego, California (San Diego, CA)) with the Ror2-TrkB mosaic electroporation among pcDNA3.1 (+)-hygro in the HEK-CRE cell, and cell is grown up to forming isolating hygromycin resistance cell colony with 350 μ g/ml Totomycin.With described colony trypsinized and on 96 orifice plates every hole shift one.Colony is grown down at 37 ℃ with 350 μ g/ml Totomycin, and by west immunoblotting and immunocytochemical method evaluation Ror2-TrkB expression levels.With having showed before that the anti-Ror2 antibody of Ror2 two polymeric (referring to example 5) is handled expressed the chimeric HEK-CRE cell of Ror2-TrkB.As shown in Figure 12, when comparing with the cell of handling with non-specific IgG, the Ror2 specific antibody causes that the consistent dose dependency of viewed uciferase activity increases.Therefore, we have researched and developed high-throughput and highly sensitive quick calibrating that a kind of measurement medicament (including but not limited to small molecules, peptide, protein or antibody) is induced Ror2 two polymerizations and activatory ability.

Claims (59)

1. the method for treatment or prevention bone photo related disorders, its comprise to the individuality of suffering from the bone photo related disorders throw with the treatment significant quantity can activate the proteic medicament of Ror2.

2. method according to claim 1, wherein said bone photo related disorders run off with bone and are associated.

3. method according to claim 1, wherein said illness are selected from by following each sick group that forms: osteoporosis, osteocarcinoma, sacroiliitis, rickets, fracture, periodontopathy, damaged, the molten bone osteopathia of segmental bone, primary and secondary hyperparathyroidism, handkerchief wise man sick (Paget ' s disease), osteomalacia and hyperostosis.

4. method according to claim 1, wherein said individuality are human.

5. method according to claim 1, wherein said medicament cause two polymerizations of Ror2 albumen.

6. method according to claim 1, wherein said medicament are small molecules.

7. method according to claim 1, wherein said medicament are protein.

8. method according to claim 1, wherein said medicament comprise the proteic antibody at Ror2.

9. method according to claim 1, wherein said medicament comprise the proteic monoclonal antibody at Ror2.

10. method according to claim 1, wherein said medicament is the mankind or peopleization monoclonal antibody at Ror2.

11. according to Claim 8,9 or 10 described methods, wherein said antibody has the IgG isotype.

12. method according to claim 1, wherein said medicament comprise the proteic antibody fragment at Ror2.

13. method according to claim 1, wherein said medicament comprise the proteic Fab fragment at Ror2.

14. method according to claim 1, wherein said medicament comprise at least two at the proteic antibody fragment of Ror2, wherein said antibody fragment is together covalently bound.

15. method according to claim 1, wherein said medicament outside enteron aisle, throw with.

16. method according to claim 1, wherein said medicament through intravenously throw with.

17. method according to claim 1, wherein said medicament oral administration with.

18. a method that increases osteoblast differentiation, it comprises makes the cell of expressing Ror2 contact with the medicament that can activate described Ror2.

19. method according to claim 18, wherein said medicament are protein.

20. method according to claim 18, wherein said medicament are small molecules.

21. method according to claim 18, wherein said medicament are antibody.

22. method according to claim 21, wherein said medicament are at the proteic antibody of Ror2.

23. method according to claim 18, wherein said medicament cause two polymerizations of Ror2 albumen.

24. method according to claim 18, wherein said medicament increases the phosphorylation by the 14-3-3 β due to the Ror2 albumen.

25. method according to claim 18, wherein said cell are the human cell.

26. method according to claim 18, wherein said cell are stem cell.

27. method according to claim 18, wherein said cell are mescenchymal stem cell.

28. method according to claim 18, wherein said contact procedure exsomatize and carry out.

29. method according to claim 18, wherein said contact procedure is in vivo carried out.

30. a method that is suppressed to the fat differentiation, it comprises makes cell contact with increase proteic expression of Ror2 or active medicament.

31. one kind is screened the method that increases the active medicament of Ror2, it comprises following steps:

The proteic cell of expression Ror2 is contacted with the test medicament; With

Determine whether the Ror2 activity increases.

32. method according to claim 31, wherein Ror2 is the cell domain of Ror2.

33. method according to claim 31, wherein Ror2 is the kinases territory of Ror2.

34. method according to claim 31, wherein said determining step comprise the proteic kinase activity of evaluation Ror2.

35. method according to claim 34, the step of the proteic kinase activity of wherein said evaluation Ror2 comprise the proteic phosphorylation state of evaluation Ror2.

36. method according to claim 31, wherein said determining step comprise the expression level of evaluation Ror2 albumen or polynucleotide.

37. method according to claim 31, wherein said determining step comprise the proteic phosphorylation state of evaluation 14-3-3 β.

38. method according to claim 31, wherein said determining step comprise the substrate formed level that mineralizes of measuring.

39. medicament of differentiating by method according to claim 31.

40. the antibody at Ror2, wherein said antibody causes the Ror2 protein activation.

41. according to the described antibody of claim 40, wherein said antibody causes two polymerizations of Ror2 albumen.

42. according to the described antibody of claim 40, wherein said antibody is polyclonal antibody.

43. according to the described antibody of claim 40, wherein said antibody is monoclonal antibody.

44. according to the described antibody of claim 40, wherein said antibody is human antibodies.

45. according to the described antibody of claim 40, wherein said antibody is the humanized antibodies.

46. according to the described antibody of claim 40, wherein said antibody has the IgG isotype.

47. according to the described antibody of claim 40, wherein said antibody comprises antibody fragment.

48. according to the described antibody of claim 40, wherein said antibody fragment is the Fab fragment.

49. according to the described antibody of claim 40, wherein said antibody has at least two and is used for the proteic binding site of Ror2.

50. according to the described antibody of claim 40, wherein said antibody has two definitely and is used for the proteic binding site of Ror2.

51. a treatment or the method for preventing the bone photo related disorders, it comprises to the individuality of suffering from the bone photo related disorders throws and the medicament that can suppress the 14-3-3 'beta ' activity for the treatment of significant quantity.

52. according to the described method of claim 51, wherein said medicament downward modulation 14-3-3 β expresses.

53. according to the described method of claim 51, wherein said medicament is 14-3-3 β specific siRNA or shRNA.

54. differentiate the method that promotes Ror2 albumen two polymeric medicaments for one kind, described method comprises following steps:

The cell of expressing the Chimerical receptor comprise Ror2 cell foreign lands and TrkB cell internal area is provided, and wherein said cell comprises the reporter gene that operability is connected in cAMP response element (CRE) promotor and constructs body;

Described cell is contacted with the test medicament; With

Measure the expression level of reporter gene described in the described cell.

55. according to the described method of claim 54, wherein said reporter gene is a luciferase.

56. according to the described method of claim 54, wherein said cell comprises and comprises that operability is connected in the plasmid of gene of the plain enzyme of coding fluorescence of described CRE promotor.

57. a protein, it has following aminoacid sequence:

MARGSALPRRPLLCIPAVWAAAALLLSVSRTSGEVEVLDPNDPLGPLD

GQDGPIPTLKGYFLNFLEPVNNITIVQGQTAILHCKVAGNPPPNVRWLK

NDAPVVQEPRRIIIRKTEYGSRLRIQDLDTTDTGYYQCVATNGMKTITA

TGVLFVRLGPTHSPNHNFQDDYHEDGFCQPYRGIACARFIGNRTIYVD

SLQMQGEIENRITAAFTMIGTSTHLSDQCSQFAIPSFCHFVFPLCDARSR

APKPRELCRDECEVLESDLCRQEYTIARSNPLILMRLQLPKCEALPMPE

SPDAANCMRIGIPAERLGRYHQCYNGSGMDYRGTASTTKSGHQCQPW

ALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRM

ELCDVPSCSPRDSSKMGILYlsvyavvviasvvgfcllvmlfllklarhskfgmkgpasvisn

dddsasplhhisngsntpssseggpdaviigmtkipvienpqyfgitnsqlkpdtfvqhikrhnivlkrelge

gafgkvflaecynlcpeqdkilvavktlkdasdnarkdfhreaelltnlqhehivkfygvcvegdplimvfe

ymkhgdlnkflrahgpdavlmaegnppteltqsqmlhiaqqiaagmvylasqhfvhrdlatrnclvgenll

vkigdfgmsrdvystdyyrvgghtmlpirwmppesimyrkfttesdvwslgvvlweiftygkqpwyqls

nneviecitqgrvlqrprtcpqevyelmlgcwqreph

Mrknikgihtllqnlakaspvyldilg (SEQ ID NO:4); Or

With SEQ ID NO:4 at least 90% homologous aminoacid sequence.

58. according to the described protein of claim 57, wherein said aminoacid sequence is:

MARGSALPRRPLLCIPAVWAAAALLLSVSRTSGEVEVLDPNDPLGPLD

GQDGPIPTLKGYFLNFLEPVNNITIVQGQTAILHCKVAGNPPPNVRWLK

NDAPVVQEPRRIIIRKTEYGSRLRIQDLDTTDTGYYQCVATNGMKTITA

TGVLFVRLGPTHSPNHNFQDDYHEDGFCQPYRGIACARFIGNRTIYVD

SLQMQGEIENRITAAFTMIGTSTHLSDQCSQFAIPSFCHFVFPLCDARSR

APKPRELCRDECEVLESDLCRQEYTIARSNPLILMRLQLPKCEALPMPE

SPDAANCMRIGIPAERLGRYHQCYNGSGMDYRGTASTTKSGHQCQPW

ALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRM

ELCDVPSCSPRDSSKMGILYlsvyavvviasvvgfcllvmlfllklarhskfgmkgpasvisn

dddsasplhhisngsntpssseggpdaviigmtkipvienpqyfgitnsqlkpdtfvqhikrhnivlkrelge

gafgkvflaecynlcpeqdkilvavktlkdasdnarkdfhreaelltnlqhehivkfygvcvegdplimvfe

ymkhgdlnkflrahgpdavlmaegnppteltqsqmlhiaqqiaagmvylasqhfvhrdlatrnclvgenll

vkigdfgmsrdvystdyyrvgghtmlpirwmppesimyrkfttesdvwslgvvlweiftygkqpwyqls

nneviecitqgrvlqrprtcpqevyelmlgcwqreph

Mrknikgihtllqnlakaspvyldilg (SEQ ID NO:4); Or

With SEQ ID NO:4 at least 95% homologous aminoacid sequence.

59. according to the described protein of claim 57, wherein said aminoacid sequence is:

MARGSALPRRPLLCIPAVWAAAALLLSVSRTSGEVEVLDPNDPLGPLD

GQDGPIPTLKGYFLNFLEPVNNITIVQGQTAILHCKVAGNPPPNVRWLK

NDAPVVQEPRRIIIRKTEYGSRLRIQDLDTTDTGYYQCVATNGMKTITA

TGVLFVRLGPTHSPNHNFQDDYHEDGFCQPYRGIACARFIGNRTIYVD

SLQMQGEIENRITAAFTMIGTSTHLSDQCSQFAIPSFCHFVFPLCDARSR

APKPRELCRDECEVLESDLCRQEYTIARSNPLILMRLQLPKCEALPMPE

SPDAANCMRIGIPAERLGRYHQCYNGSGMDYRGTASTTKSGHQCQPW

ALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRM

ELCDVPSCSPRDSSKMGILYlsvyavvviasvvgfcllvmlfllklarhskfgmkgpasvisn

dddsasplhhisngsntpssseggpdaviigmtkipvienpqyfgitnsqlkpdtfvqhikrhnivlkrelge

gafgkvflaecynlcpeqdkilvavktlkdasdnarkdfhreaelltnlqhehivkfygvcvegdplimvfe

ymkhgdlnkflrahgpdavlmaegnppteltqsqmlhiaqqiaagmvylasqhfvhrdlatrnclvgenll

vkigdfgmsrdvystdyyrvgghtmlpirwmppesimyrkfttesdvwslgvvlweiftygkqpwyqls

nneviecitqgrvlqrprtcpqevyelmlgcwqreph

mrknikgihtllqnlakaspvyldilg(SEQ?ID?NO:4).。

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