CN102988957A - Pharmaceutical compositions and methods of using secreted frizzled related protein - Google Patents

Abstract

Pharmaceutical compositions and methods of use in regulation of mammalian bone forming activities of SFRPs (secreted frizzled-related proteins) are disclosed. SFRPs are secreted receptors for Wnts that are important polypeptide growth factors that are known to regulate fundamental biological processes like tissue polarity, embryonic development and tumorigenesis. A SFRP was isolated in human osteoblast cells and identified as SFRP-1 (also known as SARP-2) and shown to be regulated by osteogenic agents in the HOB cells in a differentiation selective manner modulating the life of osteoblasts/preosteocytes.

Description

The Pharmaceutical composition of using secreted frizzled related protein and method

The application is dividing an application of following application: the applying date: on JIUYUE 13rd, 2000; Application number: 00815431.7 (PCT/US00/25035); Denomination of invention: the same.

Invention field

The present invention relates to be adjusted to compositions and the method for bone active.More particularly, the present invention relates to method and the Pharmaceutical composition that secreted frizzled related protein (SFRP), its part and antibody and nucleic acid take it as the basis with the generation of osteoblast system are adjusted to (comprising antisensenucleic acids) bone active.

Background of invention

About skeletonization is regulated and the problem of bone photo related disorders has obtained suitable concern recently; For example in the WomanHealth field, to the special concern of osteoporotic bone photo related disorders.The constant reconstruction of skeletal bones runs through lifelong.In this process of reconstruction, exist fragile balance between osteoblast bone formation and the osteoclast bone resorption subsequently.

As the normal ingredient of aging course, bone matrix experiences various constructive variations, and its character is fully not definite yet.The research great majority of the old and feeble associated change of people's bone are concentrated on illustrate bone in the form level and change or Quantitative Comparison bone loss speed.Distinguish that the osteopathia related mechanism is for understanding physiology of bone and osteopathia is most important.The gene of osteocyte and gene family are regulated in countless participations and by the polypeptide of their codings, because the complexity of skeletonization approach is not so that their function also obtains clear understanding although identified and cloned.

The WNT gene family

Wnt glycoprotein family is one group of gene and encoding proteins thereof of playing an important role in cell development is regulated.Wnt albumen is by the molecular growth factor family of the structurally associated more than 12, participate in regulating basic bioprocess, for example apoptosis, embryo occur, organ occurs, form occurs and (summary is seen Nusse and Varmus 1992 Cell 69:1073-1087) occurs tumor.These polypeptide are the multipotency factor, have similar biological activity to other secreted protein (such as transforming growth factor (TGF)-β, fibroblast growth factor (FGF), nerve growth factor (NGF) and bone morphogenetic protein(BMP) (BMP)).As if a Wnt growth factor family a member that is called Wnt-x is preferentially expressed in osseous tissue and bone derived cell, and participate in being held in maturation osteocyte (bone formation cell) phenotype (PCT/US94/14708; WO 95/17416).

FZ family

Studies show that, some Wnt albumen acts on the protein family that is called " Frizzled ", described " Frizzled " protein family plays the effect of Wnt protein receptor, perhaps (summary is seen Moon etc. for Wnt receptor complex component, 1997 Cell 88:725-728 and Barth etc., 1997Curr.Opin.Cell Biol.9:683-690).FZ comprises the aminoterminal secretory signal sequence, is considered to the cysteine enrichment domain (CRD) in conjunction with Wnt, the membrane spaning domain that is similar to g protein coupled receptor and the kytoplasm c-terminus that 7 are inferred.

1996, Hoang etc. (J.Biol.Chem.271:26131-26137) report was found the first secreted frizzled related protein (SFRP).This albumen forms active ability by cattle articular cartilage extract purification and clone according to it at rat body internal stimulus cartilage owing to the curling motif in bone development is called " Frzb ".Also cloned human homology's thing of described cow genome.Yet different from FZ, Frzb does not contain snakelike membrane spaning domain.Therefore, this newcomer of FZ family secreted receptor of Wnt seemingly.The Frzb cDNA 325aa/36 that encodes, 000 daltonian albumen is mainly expressed in appendicular skeleton.The expression of top level sees the growth long bone, and is corresponding with the epiphysis chondroblast; So express decline to ossification center, the final disappearance.

Recent studies show that, the SFRP apoptosis involvement, and therefore to have identified some SFRP be secreting type apoptosis-related protein (" SARP ").Recently also identify other member of SFRP family, be shown as the antagonist of Wnt effect.There are at present at least 5 known human Serum ferritin RP/SARP genes: SFRP-1/FrzA/FRP-1/SARP-2, SFRP-2/SDF-5/SARP-1, SFRP-3/Frzb-1/FrzB/Fritz, SFRP-4 and SFRP-5/SARP-3 (Leimeister etc., 1998 Mechanisms of Development75:29-42, this list of references content is attached to herein).As if although the accurate effect that SARP/SFRP is play in apoptosis is still clear, these albumen suppress or enhancing programmed cell death process.

In a word, be starved of clearly evaluation treatment human osteopathia (comprising the bone resorption disease, such as osteoporosis) and the osteoplastic target of adjusting.

Summary of the invention

According to the present invention, method and the Pharmaceutical composition of regulating the mammal osteogenic activity are provided, described Pharmaceutical composition contains secreted frizzled related protein (SFRP) or it regulates part.Other compositions of the present invention is used the antibody that is formed by described albumen or its part, perhaps can use the nucleic acid of encoding said proteins or its part, comprises antisense sequences.In a preferred embodiment, SFRP derives from the human osteoblast cell.The osteogenic activity that the present composition is regulated comprises regulates osteogenesis and bone density.SFRP has the aminoacid sequence that SEQ ID NO:2 shows, this sequence is to obtain by the polynucleotide sequence of expressing SEQ ID NO:1 demonstration.In the most preferred embodiment, SFRP is SFRP-1 (SEQ ID NO:2).

On the other hand, method of the present invention comprises the method for the treatment of mammal (particularly people) osteopathia, comprises the step that gives above-mentioned Pharmaceutical composition.Like this, the method for the treatment of osteopathia includes but not limited to following several disease: (a) bone formation disease, (b) bone resorption disease and (c) bone density disease.

In another aspect of this invention, described osteopathia is to be selected from following degeneration osteopathia: neurodegenerative diseases, muscle degenerative disease and degenerative bone disease.Described degenerative bone disease is selected from osteopenia, osteoarthritis, osteoporosis.

In another embodiment, the present invention includes the method for identifying the test-compound of regulating the SFRP activity.In the method, following mensuration is regulated the chemical compound of mammal osteogenic activity: sample incubation in containing the test media of test-compound that at first will contain SFRP, second step is to determine that SFRP is active, wherein active increasing illustrates that described chemical compound is the SFRP activator for independent SFRP, and activity decreased illustrates that described chemical compound is the SFRP inhibitor.

In a further embodiment, the present invention includes the method for the cell signalling of regulating the Wnt mediation, comprise described cell is contacted with above-mentioned SFRP, wherein said Wnt activity is regulated.

In addition, the present invention relates to promote the bone formation of osteocyte culture or the method that bone is repaired, the method comprises separates described cell from the bone culture, import the recombination to construct thing of expressing SFRP (having the aminoacid sequence that SEQ IDNO:2 shows), at last described cell is sent back in the described bone culture.The antisense sequences of the nucleotide sequence of described construction preferred expression coding complete S FRP albumen or its part.

Another embodiment relate to can with the polynucleotide probes of the multi-nucleotide hybrid with nucleotide sequence that SEQ ID NO:1 shows.Such probe comprises whether there is SFRP in the test sample for detection of the diagnostic method of the SFRP polynucleotide in the sample that derives from mammalian hosts.

IV. accompanying drawing summary

Fig. 1Shown the skeletonization RADE result who causes finder's osteoblast (hOB) secreted frizzled related protein (SFRP) cDNA.The figure illustrates the autoradiography of Differential display polymerase chain reaction (DD-PCR) gel of the experiment of carrying out with the three kinds of hOB cell lines (hOB-03-C5, hOB-03-CE6 and hOB-01-C1) that are in three different idiophases (proliferation period, period of maturation and front osteocyte).The hOB SFRP genetic fragment (being the RADE fragment) of arrow 276 base pairs of indication (bp), PGE in hOB-03-C5 and hOB-03-CE6 cell ₂Process and raise this genetic fragment, but TGF-β 1 processes this genetic fragment of downward modulation in the hOB-01-C1 cell.It can also be seen that, the foundation level of this gene of hOB-01-C1 cellular expression is high.Basic local contrast gopher (BLAST) shows this cDNA and mice SFRP-1 and cattle FrzA dna homolog to this genetic fragment of common data library searching.

Fig. 2Show with contrast, PTH, PGE ₂After processing 24 hours with TGF-β 1, by the RNA trace autoradiography of the poly A+RNA of hOB-03-C5 cell separation.In this experiment, the hOB SFRP RADE genetic fragment of excision and clone's Triose phosphate dehydrogenase (GAPDH) cDNA is used as probe.The arrow indication is by PGE ₂Process the hOB SFRP mRNA that described cell thoroughly raises (～4.4kb).

Fig. 3Shown with contrast, PGE ₂After processing 24 hours with TGF-β 1, by the RNA trace autoradiography of the poly A+RNA of hOB-03-C5 cell separation.In this experiment, clone's hOB SFRP RADE genetic fragment and clone's GAPDH cDNA are used as probe.Again show, use PGE ₂Process described cell and thoroughly reduce hOB SFRP mRNA.

Fig. 4Shown with contrast, PTH, PGE ₂Process after 24 hours RNA trace autoradiography by the poly A+RNA of hOB-03-CE6 cell or hOB-01-C1 cell separation with TGF-β 1.In this experiment, the hOB SFRP RADE genetic fragment of excision and clone's GAPDH cDNA are used as probe.Arrow indication PGE ₂Processing hOB-03-CE6 cell thoroughly raises but processes the hOB SFRPmRNA that the hOB-01-C1 cell is reduced with PTH.The foundation level that can also find the described mRNA of hOB-01-C1 cellular expression is high.

Fig. 5Shown with contrast and the TGF-β 1 processing RNA trace autoradiography by the poly A+RNA of hOB-01-C1 cell separation after 24 hours.In this experiment, total length (1.1kb) clone's hOB SFRP cDNA and clone's beta-actin cDNA are used as probe.The result shows that processing described cell with TGF-β 1 reduces hOB SFRP mRNA.In addition, can also find the described mRNA of hOB-01-C1 cellular expression higher baseline level.

Fig. 6Shown the RNA trace autoradiography that separates from the poly of 23 kinds of different people tissues A+RNA.In this experiment, the hOB SFRP RADE genetic fragment of excision and clone's beta-actin cDNA are used as probe.The hOB SFRP mRNA of arrow indication expresses at heart and kidney height, in the medium expression of Placenta Hominis and uterus, organize low expression level and do not express at thymus and lymphocyte at brain, pancreas and other.

Fig. 7Shown with contrast, PTH, PGE ₂After processing 24 hours with TGF-β 1, the RNA trace autoradiography of the poly A+RNA that is separated by the explant culture of SaOS-2 human osteosarcoma osteoblast-like cells and Human osteoblast (hOB).In this experiment, the hOB SFRP cDNA of full-length clone and clone's GAPDH cDNA are used as probe.The result shows the hOB SFRP mRNA of the low foundation level of SaOS-2 cellular expression, and it expresses the adjusting that is not subjected to these factors.By contrast, hOB cellular expression medium level, be subjected to PGE ₂Process the described mRNA that raises.To PGE ₂The TaqMan quantitative RT-PCR of the hOB cell of processing the analysis showed that PGE ₂SFRP mRNA level is raised 10 times.

Fig. 8Shown and used contrast or PGE ₂Process after 24 hours the southern blotting technique autoradiography by the reverse transcription (RT) of total RNA of people's Placenta Hominis or hOB-03-CE6 cell separation-PCR product.Employment FRP-1/SARP-2 specific oligonucleotide primer carries out RT-PCR, and with southern blotting technique and the hybridization of people FRP-1/SARP-2 specificity internal oligonucleotide probe.The expection size of this RT-PCR product is 1.1kb.As desired, the result shows Placenta Hominis expression FRP-1/SARP-2mRNA, and PGE ₂Process the expression of the strong described mRNA of rise of hOB-03-CE6 cell.

Fig. 9HOB-03-C5, the hOB-03-CE6 that demonstration use Coulter cell counter carries out and the result of hOB-01-C1 cell survival experiment.The result be expressed as with respect to the 0th day contrast (i.e. the every hole of 6 orifice plates approximately 200,000 cells) percentage rate.The result of this experiment shows, in 39 ℃ of slow propagation, and the hOB-03-CE6 cell stops division to the hOB-03-C5 cell in containing the culture medium of serum, but survives in 6 days incubation.On the contrary, the hOB-01-C1 cell has experienced the cell death that accelerates, and this is relevant with hOB SFRP mRNA higher baseline horizontal expression in these cells.

Figure 10The result who shows hOB-03-C5 cell (Figure 10 A), hOB-03-CE6 cell (Figure 10 B) and hOB-01-C1 cell (Figure 10 C) the survival experiment of using the Coulter cell counter to carry out.For these experiments, with contrast or PGE ₂(Figure 10 A and 10B) or contrast and TGF-β 1 (Figure 10 C) process described cells 3 or 6 days in 39 ℃ in serum-free medium.The result be expressed as with respect to the 0th day contrast (i.e. the every hole of 6 orifice plates approximately 200,000 cells) percentage rate.The result of this experiment shows, the viability of hOB cell in serum-free medium descends in time.In addition, for hOB-03-C5 cell and hOB-03-CE6 cell, use PGE ₂This fall off rate has been accelerated in processing.The increase of this cell death rate and PGE ₂HOB SFRP mRNA level after processing in these cells raises relevant.On the contrary, the TGF-β 1 with downward modulation hOB SFRPmRNA level processes hOB-01-C1 cell enhancing cell viability.

Figure 11Shown and used PGE ₂Process hOB-03-C5 cell induction apoptosis or programmed cell death.Use annexin V-FITC to detect apoptosis through flow cytometer.Figure A demonstration, neither can be dyeed by annexin V can be by the survivaling cell number of iodate the third ingot dyeing (dead cell stain) with PGE yet ₂Concentration increases and reduces.On the contrary, figure B show dyeed by annexin V but not by the apoptosis cell of iodate the third ingot dyeing with PGE ₂Concentration increases and increases.Equally, figure C shows that both can be dyeed by annexin V also can be by the dead cell number of iodate the third ingot dyeing with PGE ₂Concentration increases and increases.

Figure 12The result of the hOB-03-C5 cell (figure A) that demonstration use Coulter cell counter carries out and another viability experiment of hOB-03-CE6 cell (figure B).Carry out these experiments with the similar manner with Figure 10 description, but, for these experiments, in the situation of the phosphorothioate oligonucleotide of the directed initiation site that justice (contrast) or antisense are arranged that has or do not exist people SARP-2, usefulness vehicle Control (i.e. 0.1% ethanol) or PGE ₂The described cell of coprocessing.The result be expressed as with respect to the 0th day contrast (i.e. the every hole of 6 orifice plates approximately 200,000 cells) percentage rate or be expressed as the percentage rate that contrasts with respect to vehicle treated.The result of this experiment shows, with the antisense oligonucleotide coprocessing hOB cell reversal of SARP-2 PGE ₂Accelerate the ability of cell death rate, and with justice (contrast) oligonucleotide coprocessing being arranged on the not impact of this process.

Figure 13The result (figure A) who shows the hOB-01-C1-PS-09 cell viability experiment that use Coulter cell counter carries out.For these experiments, with hOB SFRP cDNA (being SFRP-1/FRP-1/SARP-2) mammal expression plasmid or the described cell of empty carrier (being pcDNA3.1) stable transfection.The result is expressed as the percentage rate of the 0th day control cells.The result of this experiment shows, and do not have influential empty carrier to compare to this process, and hOB cell overexpression hOBSFRP/SFRP-1/FRP-1/SARP-2 accelerates cell death rate.Figure B display separation is from the result of the RNA of the poly A+RNA of empty carrier cell (V) or SARP-2 overexpression cell (S) hybridization.The SFRP-1/FRP-1/SARP-2mRNA of this analytical proof SARP-2 cellular expression is many more than described carrier cell.

Figure 14Show the result that another cell viability that the sub-clone (SARP-2 clones #1) with the hOB-01-C1-PS-09 cell (pcDNA3.1) of expressing empty carrier and SFRP-1/FRP-1/SARP-2 overexpression cell carries out is tested.The result that figure A display separation is analyzed from the TaqMan of the RNA of described cell quantitative RT-PCR.This analysis showed that, the human Serum ferritin RP-1/FRP-1/SARP-2mRNA of SARP-2 clone #1 cellular expression than the high 50-60 of empty carrier express cell doubly.Equally, shown in figure B, use the Coulter cell counter to detect cell number, fast 3 times than empty carrier control cells of SARP-2 clone #1 cell death rates.

Figure 15For separating from contrast or PGE ₂Process the Western blotting autoradiography of full cell extract of 24 hours hOB-03-CE6 cell.Monoclonal antibody (molecular weight is 92,000) with beta-catenin is surveyed immunoblotting.Result's demonstration PGE ₂Process hOB cell downward modulation beta-catenin white level, this is consistent to the antagonism of Wnt signal transduction pathway with hOB SFRP/SFRP-1/FRP-1/SARP-2.

Figure 16The result who has shown hOB-01-C1-PS-09 cell (figure A) and hOB-02-C1-PS-02 cell (figure B) transient transfection experiment.Wnt signal transduction (comparing with the contrast of pcDNA3.1 empty carrier) is reduced in the testing result demonstration that the TCF-luciferase reporter gene is measured, transfected with human [h] or rat [r] SARP-2 or people Frzb-1 expression plasmid in the hOB cell.This mensuration is the credible detection of Wnt signal transduction and beta-catenin nuclear activity.

Figure 17Summarized the filtering mode that uses the bone anabolism medicine of hOB cell and hOB SFRP/SFRP-1/FRP-1/SARP-2.This filtering mode can be identified the chemical compound of regulating the SFRP-1/FRP-1/SARP-2 effect.

Figure 18Diagram high flux screening (HTS) is measured the example of the chemical compound of the SFRP-1/FRP-1/SARP-2 function that is suppressed to osteocyte/osteocyte.Use the fluorimetric result of CyQuant DNA to show, the hOB-01-C1-PS-09 cell of overexpression SFRP-1/FRP-1/SARP-2 (SARP-2#1 cell) is died an death than the cell (pcDNA3.1) of expressing empty carrier soon.And, the cell death that the described gene overexpression of the anti-peptide antiserum of SFRP-1/FRP-1/SARP-2 (SARP-2AS) blocking-up causes.

Figure 19Summarized the strategy for generation of the SFRP-1/SARP-2 knock out mice.Beta galactosidase and neomycin resistance expression cassette replace the mice SFRP-1/SARP-2 gene extron 1 of the whole cysteine enrichment domain of coding (CRD).

Figure 20Diagram is separated from female and male wild type (WT) and is knocked out the RNA trace of poly A+RNA of the kidney of (KO) SFRP-1 mice.This trace shows the high-caliber SFRP-1mRNA of WT renal expression (4.4kb), and the KO kidney is not expressed this gene.

Figure 21Shown the result of microcomputer x-ray tomography developing (micro-CT) analysis who derives from male (figure A) and female (figure B) wild type (+/+) and knock out the mouse femur of (/-) SFRP-1.When with+/+when control mice was compared, the data of being measured by the method showed,-/-the skeletonization parameter of mice (be BV/TV, Tb.Th., Conn.Den., Tb.N.﹠amp; Tb.Sp.) raise.

Figure 22Be a picture group, shown the cell proliferation of (A) hOB cell; (B) in 39 ℃ of vitamin D ₃Process the hOB cell can/strengthen and raise alkaline phosphatase activities; (C) vitamin D ₃Can not induce hOB emiocytosis osteocalcin in 34 ℃, see that embodiment A introduces in detail.

Figure 23Diagram is about the result of the embodiment A of PTH-1 Receptor mRNA; Wherein remained in 34 ℃ with cell in 48 hours in 39 ℃ of cultivation hOB cells and compare, make the steady-state mRNA level of PTH-1 receptor increase by 7 times.

Figure 24Diagram PTH 1-34 concentration increases the result to the embodiment A of the effect of cyclic adenosine monophosphate (cAMP) in the hOB cell born of the same parents gradually.

Figure 25Diagram raises the effect of the alkaline phosphatase activities of hOB cell when processing with the synthetic glucocorticoid dexamethasone, see that embodiment A introduces in detail.

Figure 26Be shown in the mechanical sexy experimental result of stimulation to prebone hOB cytosis of feeling when using Flexerall Strain Unit.

V. detailed Description Of The Invention

The present invention relates to it and express the gene that in three kinds of different people osteoblast (hOB) cell line, is subject to skeletonization medicine or the adjusting of bone formation vitro Drug.In one aspect of the invention, the expression of this gene was raised in the hOB idiophase, pointed out described gene may participate in osteogenetic process.Dna sequence analysis shows, this genetic fragment has significant sequence homogeneity (Rattner etc., 1997 Proc.Natl.Acad.Sci.USA 94:2859-2863) with the mice cDNA that is called secreted frizzled related protein (SFRP)-1.Subsequently cDNA clone and other sequence analysis show this gene that is called hOB SFRP (Finch etc., 1997Proc.Natl.Acad.Sci.USA 94:6770-6775 identical with people FRP-1/SARP-2; Melkonyan etc., 1997 Proc.Natl.Acad.Sci.USA 94:13636-13641).The sign of hOB SFRP has indicated its purposes and the purposes in the newtype drug screening of identifying bone anabolism medicine as the loose medicine target of Novel bone.

Definition

Term " bone formation " is that bone synthesizes and mineralization process.Osteoblast is regulated this process.

Term " osteogenesis " is the skeleton growth process.This process is one of following two kinds of forms: (1) directly results from the interior bone formation of film of mesenchymal cell or medullary cell; (2) result from cartilage vertically or cartilage (endichindual) bone formation.

The term bone formation synonym of term " skeletonization " and above definition.

Term " secreted frizzled related protein " or " SFRP " are the secreted receptor of Wnt signal transduction pathway, have various features that make it become the useful research tool of Growth of Cells and differentiation.Curling sample gene family coding has the epicyte protein of unknown function membrane spaning domain.

Term secreted frizzled related protein or the SFRP synonym of term " secreting type apoptosis-related protein " or " SARP " and above definition.

Term " albumen ", " peptide " and " polypeptide " are mutual to be used, comprise purification and be connected produce, comprise the amino acid whose SFRP molecule by the connection of peptide bond linearity.Aminoacid of the present invention can be L-type or D type, as long as the biological activity of described polypeptide is kept.SFRP albumen of the present invention can also comprise the albumen by reaction (comprising glycosylation, acetylation and phosphorylation) post translational modification.Described polypeptide also comprises analog, allele and allelic variant, compares with wild-type protein or native protein, and they can contain the biological activity that do not affect SFRP albumen or amino acid derivativges or the non-amino acid moiety of functional activity.Term amino acid refers to naturally occurring aminoacid and derivant (such as TyrMe and PheCl) thereof, and is characterised in that the other parts that both exist available carboxyl also to have amino.The non-amino acid moiety that described polypeptide can comprise comprises for example amino acid analog structure.Model configuration and aminoacid have roughly the same functional group's spatial arrangement, but needn't have simultaneously amino and the carboxyl of amino acid characteristics.

" mutain " for example refer to by direct mutagenesis or other operation, by transcribing or translation error causes SFRP albumen or the polypeptide of aminoacid sequence minor variations, perhaps SFRP albumen or the polypeptide by the synthetic preparation of appropriate design.The biological activity that these minor alterations produce its albumen or polypeptide and wild type or naturally occurring polypeptide or albumen are compared the aminoacid sequence that changes.The example of mutain comprises the SFRP-1 of SEQ ID NO:2 described herein.

Term used herein " hydrophobicity " comprises nonpolar aminoacid, amino acid derivativges, amino acid analog thing and chemical part.Hydrophobic amino acid comprises Phe, Val, Trp, Ile and Leu.Term used herein " positively charged aminoacid " refers to positively charged aminoacid, amino acid derivativges, amino acid analog thing and chemical part.Positively charged aminoacid comprises for example Lys, Arg and His.

When " purification " is used to refer to SFRP albumen or polypeptide, be different from natural or naturally occurring albumen or polypeptide, because they exist with purified form.The variant of these " purification " SFRP albumen described herein or polypeptide or any imagination should refer to substantially not contain usually in its nature or natural surroundings chemical compound or molecule with the pollutant of its combination.Term " roughly pure " or " separation " do not foreclose the mixture of polynucleotide or the same material of being combined with its non-natural of polypeptide.

" natural " SFRP polypeptide, albumen or nucleic acid molecules refer to the SFRP that reclaims by in natural or " wild type " resource.

" compositions " refers to active substance (being SFRP of the present invention) and another kind of inertia (for example detectable substance or label) or the chemical compound of activity or the combination of compositions (for example adjuvant).

" Pharmaceutical composition " refer to comprise as active substance SFRP (particularly SFRP-1) with make described compositions be suitable in external, the body or the in vitro inertia used of diagnosis or treatment or the combination of active carrier.

Term used herein " pharmaceutically acceptable carrier " comprises any standard pharmaceutical carrier, such as phosphate buffered saline(PBS), water and Emulsion (such as oil/water or water/oil emulsion) and various types of wetting agent.Described compositions can also comprise stabilizing agent and antiseptic.The example of carrier, stabilizing agent and adjuvant referring to Martin, Remington ' s Pharm.Sci., the 15th edition (Mack Publ.Co., Easton (1975))

The term " nucleic acid " that relates to SFRP described herein refers to strand and double-stranded DNA, cDNA, genome source DNA and RNA, and normal chain and the minus strand nucleic acid of each other complementation, comprises antisense RNA." nucleic acid molecules " be can with " polynucleotide " the mutual term that uses, all refer to random length nucleotide (perhaps ribonucleotide or deoxyribonucleotide) or its analog of poly form.Described term also comprises the modification of known type, and for example labelling known in the art (for example Sambrook etc. (1989) are the same), methylate, " add medicated cap ", analog replaces one or more naturally occurring nucleotide, modify (for example modification (for example methyl carbamate) of non-electric charge connection) between nucleotide, (for example albumen (comprises nuclease to comprise the modification of overhang, toxin, antibody, signal peptide etc.)), intercalator is modified (acridine for example, psoralen etc.), modification (the metal for example that comprises chelating agen, radioactive metal, boron, oxidized metal etc.), comprise the modification of alkylating agent, the polynucleotide of the modification that modified connects (such as different nucleic acid of α etc.) and unmodified form.Described polynucleotide can carry out chemical modification or biochemical modification, perhaps can contain non-natural or derivation nucleotide base.Described nucleotide can be complementary with the coding mRNA of described polynucleotide.These complementary nucleotides include but not limited to form nucleotide or the antisense nucleotide of triple helix.The present invention also provides and comprises the recombination of polynucleotide that there is sequence in non-natural, as the wild type peptide sequence that changes, include but not limited to owing to disappearance, inserting, replacing one or more nucleotide or by merging the wild type peptide sequence of the change that produces to other polynucleotide sequence.

If the SFRP polynucleotide of native state or use the well-known method of those skilled in the art can be transcribed and/or translate its SFRP polynucleotide that carried out operation to produce polypeptide or maturation protein are then thought SFRP polynucleotide " coding " SFRP polypeptide.Therefore, the term polynucleotide also should comprise not sequence of encoding mature Argine Monohydrochloride of job sequence and other except comprising coded sequence.The antisense strand of described polynucleotide also is considered to the described sequence of encoding.

Term " restructuring " polynucleotide or DNA refer to the polynucleotide that prepare by making up two kinds of sequence sections disconnected from each other, and described combination realizes by the DNA sections that technique for gene engineering or chemosynthesis manually-operated separate.When so operating, people can need the DNA of function sections to link together with having, to produce the function combinations that needs.

" analog " of SFRP DNA, RNA or polynucleotide refers to be similar to naturally occurring polynucleotide in form and/or function (the particularly ability of the base pair on the enhancement sequences specificity hydrogen bonding complementary nucleotide sequence), but at the macromole that is different from DNA or RNA aspect the main chain that for example has rare or non-natural base or change.Referring to for example Uhlmann Deng, (1990) Chemical Reviews 90:543-584

" separation " refers to separate with other cellular component when being used to refer to the SFRP nucleic acid molecules, and described other cellular component usually combines with interior natural or wild type SFRP DNA or the RNA of born of the same parents.

" hybridization " refers to the hybridization that can carry out under different " strictly " conditions.The condition that increases the hybridization stringency is widely known by the people in this area and referring to for example Sambrook etc. are with upperOpen.The example of correlated condition comprises (order that increases according to stringency): the heated culture temperature of 25 ℃, 37 ℃, 50 ℃ and 68 ℃; The buffer concentration of 10 * SSC, 6 * SSC, 1 * SSC and 0.1 * SSC (wherein SSC is 0.15M NaCl and 15mM citrate buffer) and the buffer that is equal to that uses other buffer system; 0%, 25%, 50% and 75% Methanamide concentration, 5 minutes to 24 hours incubative time, wash time prolongs, frequency increases or buffer concentration reduces.

" Tm " is such Celsius temperature: under this temperature, under described experiment condition, the polynucleotide two strands 50% that is comprised of the reverse hydrogen bonding complementary strand of Watson-Crick base pair is dissociated into strand.Tm can calculate according to normalized form, for example:

Tm＝81.5+16.6log[Na ⁺]+0.41(％G/C)-0.61(％F)-600/L

Wherein Na+ be cation concn (being generally sodium ion) (mol/L); (%G/C) be G and C base account for total base in two strands percentage ratio number; (%F) be Methanamide percentage ratio (weight/volume) in the solution; And L is the few nucleotide of double-stranded two chains.

" stablizing duplex " of polynucleotide or " stable complex " that form between two or more components arbitrarily in biochemical reaction refer to keep enough for a long time duplex or complex between duplex or complex formation and its detection subsequently.Described duplex or complex must be able to be stood and form constantly and detecting any condition that exists or introduce between the moment, and these conditions change with mensuration or the reaction that will carry out.Can choose wantonly the intervention condition that exists and can remove duplex or complex comprise cleanings, heating, in reactant mixture adding other solute or solvent (such as denaturant) and compete with other reactant.Stable duplex or complex can be for irreversible or reversible, but must satisfy other requirement of this definition.Therefore, in reactant mixture, can form instantaneous complex, but as long as its nature from separating or dissociating owing to the conditioned disjunction that newly applies detects the front operation of introducing, just can not consist of stable complex.

When two strand polynucleotide (particularly under high stringent condition) form when stablizing duplex with reverse configuration, described chain basic " complementation ".Double-stranded polynucleotide can with another polynucleotide " complementation ", as long as a chain and second polynucleotide of first polynucleotide can form stable duplex.Complementary series by the prediction of strand polynucleotide sequence is that expection forms hydrogen-bonded optimality criterion nucleotide sequence with described strand polynucleotide according to generally accepted basepairing rule.

The chain that " justice arranged " and " antisense " chain refer to the each other strand SFRP polynucleotide of complementation when being used for same situation.They can be the reverse strand of double-stranded polynucleotide, and perhaps a chain can be predicted out by another chain according to generally accepted basepairing rule.Except as otherwise noted or hint, otherwise can arbitrarily a chain or another chain be appointed as " justice is arranged " chain or " antisense " chain.

If the nucleotide sequence of each linear order is identical and do not replace, the displacement of disappearance or material, then SFRP nucleotide linear order and another linear order " identical ".Obviously, according to the Watson-Crick base pairing, the purine with analog structure can be identical on function with the pyrimidine nitrogenous base; And, not constitute displacement of the mutual replacement of similar nitrogenous base (particularly uracil and thymus pyrimidine) or the modification (as methylating) of nitrogenous base.When the RNA sequence has reflected the order of nitrogenous base in the polyribonucleotide, DNA sequence has reflected the order of nitrogenous base in the polydeoxyribonucleotide, and two sequences satisfied this definition other when requiring, then described RNA has identical sequence with the DNA polynucleotide.When at least one sequence is when comprising the degenerate oligonucleotide of ambiguity residue, if the degenerate oligonucleotide of at least a alternative form is identical with its contrast sequence, these two sequences are identical so.For example, if AYAAA (SEQ ID NO:5) is the mixture of ATAAA (SEQ ID NO:6) and ACAAA (SEQ ID NO:7), then AYAAA (SEQ ID NO:3) is identical with ATAAA (SEQ ID NO:4).

When comparing between two kinds of polynucleotide, implicity is known and is easy to obtain complementary strand, can select or predict sense strand or the antisense strand of homogeneity degree maximum between the polynucleotide that contrast.For example, when one or both polynucleotide that will contrast when double-stranded, if when a chain of chain of the first polynucleotide and the second polynucleotide was identical, then described two kinds of sequences were identical.Equally, when polynucleotide probes is described to when identical with its target, obviously it is the complementary strand that participates in the target of hybridization between described probe and the described target.

If two kinds of linear orders can both form duplex, then a kind of described nucleotide linear order and another kind of described linear order " basic identical " or " being equal to " with same complementary polynucleotide hybridization.Should be understood that, although do not relate to the specific nucleic acid molecule always do not explicitly point out the applicant, but also comprise its equivalent.The sequence of more preferably under higher stringent condition, hybridizing.Obviously, hybridization can adapt to insertion, disappearance and the replacement of nucleotide sequence.Therefore, even linear kernel nucleotide sequence part nucleotide residue inaccuracy is corresponding or also can be basic identical during coupling.By contrast more preferably with the sequence of the tightr corresponding or coupling of the present invention disclosed herein.Generally speaking, if approximately the polynucleotide district of 25 residues is identical at least about 80% with another polynucleotide district; More preferably identical at least about 90%; More preferably identical at least about 95%; Even more preferably described sequence 100% is identical, and then described two polynucleotide districts are basic identical.The polynucleotide district of 40 or more residues and another district are after the contrast of homology partial sequence, if identical at least about 75%; More preferably identical at least about 80%; More preferably identical at least about 85%; Even more preferably identical at least about 90%; More preferably described sequence 100% is identical again, and then described two polynucleotide districts are basic identical.

When definite polynucleotide sequence is whether basic identical, particularly preferably keep the functional sequence with the polynucleotide of its contrast.Can be functional by different parametric measurements.For example, if described polynucleotide are used for relating to the reaction with another multi-nucleotide hybrid, so preferred under similar condition with the sequence of identical target hybridization.Generally speaking, the T of DNA duplex _mThe position of depending on the mispairing residue, for the duplex of 200 or more residues, the every reduction by 1% of sequence homogeneity, Tm will descend approximately 10 ℃; For the duplex that is less than 40 residues, the every reduction by 1% of sequence homogeneity, Tm descend approximately 50 ℃ (referring to such as Meinkoth etc.).Approximately 100 residues basic identical or be equal to the general and mutual among of sequence from complementary series be lower than T _mApproximately 20 ℃ temperature forms stable duplex; Preferably be lower than T _mApproximately 15 ℃ temperature forms stable duplex; More preferably be lower than T _mApproximately 10 ℃ temperature forms stable duplex; Even more preferably be lower than T _mApproximately 5 ℃ temperature forms stable duplex; Again more preferably at about T _mTemperature form stable duplex.In another embodiment, if the polypeptide of described polynucleotide encoding is the important component part of its function, sequence of the identical or basic identical polypeptide of optimized encoding so.Therefore, the nucleotide difference that produces the conserved amino acid replacement is better than producing the nucleotide difference of non-conservative aminoacid replacement, and the nucleotide difference that produces non-conservative aminoacid replacement is better than producing the nucleotide difference of non-conservative replacement, more preferably do not change the nucleotide difference of aminoacid sequence, even more preferably identical nucleotide.The polynucleotide that polynucleotide insert or disappearance is better than being lacked of proper care mutually in coding position, downstream that cause inserting in the polypeptide or lacking insert or lack; Even the polynucleotide sequence that does not more preferably comprise insertion or lack.The relative importance of the peptide sequence of hybridization characteristic and polynucleotide encoding depends on application of the present invention.

If two polynucleotide can both form stable duplex with specific the 3rd polynucleotide under similar maximum stringent condition, then these polynucleotide have identical feature with another polynucleotide or are equal to it.Except similar hybridization characteristic, the described polynucleotide essentially identical polypeptide of preferably also encoding.

" guarding " residue of polynucleotide sequence refers to the residue that the same position of two or more correlated serieses in contrast does not change.The more conservative residue of residue that other position occurred during relatively conservative residue referred in correlated series more than sequence.

The polynucleotide of " being correlated with " have the identical residue of larger proportion.

" degeneracy " used herein oligonucleotide sequence is at least two kinds of following implementation sequences that relevant original polynucleotide sequence produces: residue conservative in original series is retained in the degenerate sequence, and not conservative residue can have some selections in degenerate sequence in original series.For example, degenerate sequence AYASA (SEQ ID NO 8) can be definite by original series ATACA (SEQID NO 9) and ACAGA (SEQ ID NO 10), and wherein Y is C or T, and S is C or G.Y and S are the examples of " ambiguity " residue.The degeneracy sections is the polynucleotide sections that comprises degenerate sequence.

Obviously, the synthetic oligonucleotide that comprises degenerate sequence is actually the mixture of the polynucleotide that are closely related that have identical sequence except the ambiguity position.Such oligonucleotide usually synthesize the ambiguity position might nucleotide the mixture of combination.Each oligonucleotide in the mixture is called as " alternative form ".

Polynucleotide used herein " fragment " or " Insert Fragment " generally represent the subprovince of total length form, but also can comprise whole total length polynucleotide.

If polynucleotide derive from another polynucleotide fundamentally, these two different polynucleotide mutually " correspondence " so.For example, messenger RNA is corresponding to its open gene.CDNA corresponding to it for example by reverse transcription reaction or the RNA that produces by chemical synthesising DNA based on the RNA sequence information.CDNA also corresponding to the coding described RNA gene.If polynucleotide have similar function in different species, strain or the variant that contrasts, such as the coding related polypeptide, so described polynucleotide are mutual " correspondence " also.

" probe " refers to the oligonucleotide that provides as reagent when using in the scope in SFRP polynucleotide operations, it is by hybridizing to detect the target that may be present in the target sample with target.Usually, probe comprise label or label can be before hybridization by it or be right after the material of combination after the hybridization.Suitable label includes but not limited to radiosiotope, fluorescent dye, chemiluminescence compound, dyestuff and albumen (comprising enzyme).

" primer " be usually have freely 3 '-oligonucleotide of OH group, by with target hybridization in conjunction with the target that may be present in the target sample, promote thus the multimerization with the polynucleotide of described target complementation.

The method of the identical polynucleotide that production repeats to copy such as PCR or gene clone, is referred to as " amplification " or " copying " at this paper.For example, reproducible strand or double-stranded DNA have other DNA of identical sequence with formation.For example, can then carry out the PCR replicated rna by the RNA polymerase of RNA guidance or by reverse transcription DNA.Under latter event, the RNA of amplification copy is for having the DNA of identical sequence.

" polymerase chain reaction " (" PCR ") is the reaction of using the duplicate copy of one or more primers and polymerization catalyst (such as reverse transcription or archaeal dna polymerase, particularly heat-stabilised poly synthase) preparation herbicide-tolerant polynucleotide.In general, PCR comprises three steps that are concatenated to form: " annealing ", and wherein regulate temperature and form duplex with the polynucleotide that allow oligonucleotide primers and will increase; " elongation " wherein regulates temperature, so that the polynucleotide of use and its formation duplex as template, have formed the oligonucleotide of duplex with the archaeal dna polymerase elongation; And " unwinding ", wherein regulate temperature, in order to dissociate the oligonucleotide of described polynucleotide and elongation.Then repetitive cycling is until obtain the amplifying polynucleotides of requirement.In No. the 4th, 683,202, the USP of No. the 4th, 683,195, the USP of Mullis and Mullis etc., told about the PCR method.

Element in the gene includes but not limited to promoter region, the termination site that strengthens subarea, repressor protein land, transcriptional start site, ribosome binding site, translation initiation site, protein-coding region, intron and exon and transcribe and translate." antisense " copy of specific polynucleotide refers to and can also therefore can regulate the complementary series that described polynucleotide are expressed by the described polynucleotide of hydrogen bonding.It is above-mentioned DNA, RNA or its analog, comprises having the analog that changes main chain.The polynucleotide of antisense copy combination can be single stranded form or double chain form.

Term used herein " effectively connection " means to be transcribed into RNA with respect to necessary adjusting sequence (for example promoter or enhancer) location dna molecular so that described promoter is controlled described dna molecular with stable or instantaneous mode.

" carrier " refers to that the nucleic acid molecules that will insert is transferred to host cell self-replicating nucleic acid molecule inner and/or transfer insertion nucleic acid molecules between host cell.This term comprises the carrier that mainly plays the replicating nucleic acid effect and works the expression vector of transcribing and/or translate DNA or RNA effect.The carrier that also comprises the above-mentioned functions that more than one are provided.

" host cell " comprises any independent cell or cell culture, and they can be or become the exogenous nucleic acid molecule of carrier or adding and/or the receptor of albumen." host cell " also comprises the offspring of individual cells, and described offspring is because natural, accidental or autotelic sudden change, with original parental cell can identical (aspect morphology or genomic DNA complement or total DNA complement).

" antibody " be can conjugated antigen immunoglobulin molecules.Term used herein not only comprises complete immunoglobulin molecules, and comprises anti-idiotype antibody, mutant, fragment, fusion rotein, humanization albumen and comprise the modified immunoglobulin molecule with the specific antigen recognition site of needs.

" antibody complex " is antibody (as defined above) and binding partners or the part of combination.

Be used for the cell that " suitable cell " of the present invention includes but not limited to express SFRP, for example medullary cell, preferably hOB cell.

This paper is defined as the nucleic acid molecules that has basic homogeneity with the reference nucleic acid molecules with " biochemical equivalents " of nucleic acid molecules.The fragment of reference nucleic acid molecules is an example of biochemical equivalents.

" biochemical equivalents " of SFRP polypeptide or albumen is polypeptide or the albumen that keeps reference protein or polypeptide same characteristic features, and comprises the fragment of reference protein or polypeptide.

Can also use commercially available automated peptide synthesizer, such as Applied Biosystems, Inc., Foster City, the model that CA produces is the equipment of 430A or 431A, obtains the SFRP proteins and peptides by chemosynthesis, provides aminoacid sequence in SED ID NO 2.Synthetic albumen or polypeptide can be precipitated and be further purified by for example high performance liquid chromatography (HPLC).Therefore, the present invention also provides the method for following chemosynthesis albumen of the present invention: sequence (for example SEQ ID NO 2) and the reagent (such as aminoacid and enzyme) of described albumen are provided, and with correct direction aminoacid and linear order are linked together.

Perhaps, can for example pass through Sambrook etc., Molecular Cloning:A Laboratory Manual, second edition, (Cold Spring Harbor Laboratory (1989))The well-known recombination method of describing, example as described below and the host cell and the carrier system that exemplify obtain described proteins and peptides.The present invention also provides the host cell that contains coding and need the nucleic acid molecules of albumen by cultivation to produce the method for SFRP, its analog, mutain or fragment, and wherein said nucleic acid effectively is connected to the rna transcription promoter.The gene constructs that can contain by use promoter and the terminator sequence of the nucleotide sequence that needs albumen is incorporated into the albumen of needs in the host cell.Under suitable condition, cultivate host cell, so that albumen is transcribed and be translated as to described nucleic acid.In an independent embodiment, described albumen is further purified.

Albumen of the present invention can also make up with various liquid phase carriers, and described liquid phase carrier is sterile solution or aqueous solution, pharmaceutically acceptable carrier, suspension and emulsion for example.The example of non-aqueous solvent comprises propyl glycol, Polyethylene Glycol and vegetable oil.When for the preparation of antibody, described carrier can also comprise the adjuvant for non-specific enhancing specific immune response.Those skilled in the art can need determine whether at an easy rate adjuvant and select a kind of adjuvant.Yet only for illustration purpose, suitable adjuvant includes but not limited to Freund's complete adjuvant and Freunds incomplete adjuvant, inorganic salt and polynucleotide.

The therapeutic use of SFRP/SARP

Although the SFRP/SARP gene family only just is being found recently, still have its biology many problems to need to understand, these albumen still have several potential therapeutic use.Because known Wnt is relevant with proto-oncogene, so SFRP/SARP is because it is can antagonism Wnt active and can play tumor inhibitor.These albumen can also be used for tissue regeneration.For example, because FrzB-1 stimulates the dystopy cartilage in the body to form activity, so it can be used for the joint healing (number of patent application WO 98/16641 A1) after quickening fracture recovering or hip and knee joint are changed.At last, because as if SFRP/SARP control apoptosis, so these albumen can also be used for the treatment of various degenerative disease, comprise neurodegenerative diseases, flesh degenerative disease and degenerative bone disease.

Pharmaceutical composition

The compositions that the present invention also provides the nucleic acid molecules that comprises any above-mentioned albumen, mutain, fragment, antibody, encoding said proteins, mutain, antibody or its fragment and expresses carrier and host cell and acceptable solid or liquid-carrier buffer or the diluent of described nucleic acid molecules.Use is enough to realize one or more active component to the effective dose that needs regulating action of osteogenic activity or apoptosis activity.Can be by the dose-effect curve determination effective dose of conventional needs activity.When pharmaceutically using described compositions, they can make up for the diagnosis and treatment of purposes with " pharmaceutically acceptable carrier ".The preparation of described compositions is well-known to those skilled in the art.Pharmaceutical composition of the present invention can comprise one or more other active component and preferably include pharmaceutically acceptable carrier.Described other active component can with the effect synergism based on the active component of one or more above-mentioned SFRP.In alternate embodiment, the active component that adds other, because it works to identical disease or disease with SFRP, but model of action is different from active component based on SFRP, and perhaps described other active component can work to Other diseases or the disease that is present in the human or animal body.Suitable pharmaceutically acceptable carrier and/or diluent comprise any and whole conventional solvent, disperse medium, filler, solid carrier, aqueous solution, coating materials, antibacterial and antifungal, isotonic agent and absorption delay agent etc.Term " pharmaceutically acceptable carrier " refers to not cause the carrier of allergy or other inappropriate effect in giving the patient.Suitable pharmaceutically acceptable carrier comprises such as in water, saline, phosphate buffered saline(PBS), glucose, glycerol, the ethanol etc. one or more, and their combination.Pharmaceutically acceptable carrier can also comprise effect duration of one or more active components of the described compositions of enhancing of trace or the auxiliary substance of effectiveness, such as wetting agent or emulsifying agent, antiseptic or buffer agent.Like this medium and the material that are used for pharmaceutically active substances are well known in the art.Conventional medium or the material out of use situation that makes an exception is that it is incompatible with described active component, has also imagined its application in immunogenic composition of the present invention.

These compositionss can also be for the preparation of the medicine of diagnosis and treatment and neurodegenerative diseases (be HuntingtonShi is sick, AlzheimerShi is sick, spinal cord injury), pathology that flesh degenerative disease (being muscular dystrophy, myasthenia gravis, myotonic myopathy) is relevant with degenerative bone disease (being osteoporosis).

In certain embodiments, in described compositions, use all or in part antibody of SFRP albumen of combination, to treat any above-mentioned disease or disease.Can prepare polyclonal antibody and monoclonal antibody by conventional method.In general, the antibody of generation is for the special aminoacid sequence of (a) SFRP albumen and (b) more may have antigenic aminoacid sequence.Can be by carrying out sequence analysis and using any conventional series arrangement and sequence contrast procedure Selection SFRP protein-specific sequence.A general preferred end or a plurality of end (being preferably in two ends) of being used in has hydrophilic aminoacid sequence and produces antibody.Except using hydrophilic amino acid, described hydrophilic amino acid also can be alkalescence (nonacid) in preferred embodiments.Can use the antigenic aminoacid of any increase.For example, proline is through being usually used in the middle body of described sequence.When producing the antibody of anti-SFRP protein-specific initial testing sequence, can or increase according to the decline that produces the antibody amount and come detectable antigens.In certain embodiments of the invention, generation is for the antibody of the sequence of at least 8 continuous amino acids that contain SFRP albumen (preferably at least 10 continuous amino acids).In a more preferred embodiment, produce for containing approximately 15 to the about antibody of 30 amino acid whose aminoacid sequences.In preferred embodiments, generation is for the sequence of the aminoacid 217-231 of the SFRP albumen that contains SEQ ID NO 2 or the antibody of its sequence variant.

Compositions of the present invention can need to promote the individuality of nerve, muscle, cartilage and osteogenesis by all means, and described approach includes but not limited to that intravenous, subcutaneous, intramuscular, sheath are interior, intracranial and part give.Described compositions can directly give to organ or organ cell by method in the body or in vitro.

These compositionss can be solution form or particulate form, perhaps can join in microsphere or the microvesicle, comprise micelle and liposome.

Industrial applicibility

Above-mentioned composition is provided for screening the component into the Screening test of the medicine of suitable cell Wnt receptor stimulating agent or antagonist or medicinal compound.

In addition, expect SFRP polynucleotide of the present invention can be used as diagnostic agent or for detection of the genetic abnormality relevant with the gene of SFRP encoding gene or one or more participation Wnt signal transduction pathways purposes.Described genetic abnormality comprises point mutation, disappearance or the insertion of nucleotide.Some genetic screening methods all are fit to use the available probe of the present invention, and described genetic screening method comprises restriction fragment length polymorphism (RFLP) analysis, ligase chain reaction or PCR.This gene mutation shows the increased risk that appearance is unusual.

According to detailed description provided below, described albumen or its fragment can be used for external acellular and raji cell assay Raji system, the medicine and the medicinal compound that suppress or strengthen Wnt receptor pathway and apoptosis with screening, or the possible therapy of the test disease (for example bone formation disease, carcinogenesis and cardiovascular disease) relevant with this approach.Described albumen or its fragment can also be regulated the embryo and be occured.

Drug screening method can be used for identifying activator or the inhibitor of SFRP albumen.For example, the cartilage-derived growth in the presence of medicine is compared increase and may be shown and activated SFRP with independent SFRP, may show that to have suppressed SFRP active and reduce.

Can use method of the present invention to screen various chemical compounds.They comprise peptide, macromole, micromolecule, chemical mixture and biological mixture.Described chemical compound can be biologic artifact, synthetic compound, organic compound or inorganic compound.

In the present invention, suitable cell is for the preparation of diagnostic assay, expression SFRP or preparation nucleoside acid type diagnostic kit.Described cell can or produce by yeast, antibacterial, fungus or virus preparation.In preferred embodiments, described cell is the hOB cell, particularly be called the hOB-01-C1-PS-09 cell and (be preserved in Manassas, the American type culture collection of Va, preserving number PTA-785) novel immortalization prebone cell line has osteoblast and the osteoblast prepared therefrom (for example daughter cell) of the identification mark of hOB-01-C1-PS-09 cell.Immortalization refers to basic continous and the permanent cell culture with significantly unlimited cell division potential of setting up.That is to say, described cell can substantially ad infinitum be cultivated, and namely cultivates at least about 6 months under the Fast Growth condition, preferably cultivates the longer time under slower growth conditions, and can use the fast continuously breeding of conventional cell culture technology.Change kind of a mode and say, cell of the present invention can be cultivated at least about 100,150 or 200 population doublings.These cells produce the additional albumen with Human osteoblast feature, and can carry out osteoblast differentiation.They can be used for osteoblast to the cell culture studies of various materials (such as hormone, cytokine and somatomedin) sensitivity or are used for tissue treatment.These cells are before by sub-clone after the aging of the disclosed hOB-01-C1 cell lines of 1996Endocrinology 137:4592-4604 such as Bodine.

Therefore, such as our report at this paper, SFRP is osteoporotic newtype drug target, and some embodiment relates to the encode all or part of gene of at least a SFRP albumen or the expression of nucleic acid.The expression in human osteoblast cell (hOB) cell line of described nucleic acid or gene is associated with the acceleration of cell death and apoptosis.HOB-01C1PS-09 cell of the present invention is effective especially with respect to other hOB cell, because the cell of " 09 " expression is ripe osteoblast.In addition, these cells are osteocyte (being ripe cell), and other hOB cell is generally osteoblast by contrast.And, the very low-level FRP-1/SARP-2 mRNA of hOB-01-C1-PS-09 cellular expression.Therefore, hOB-01-C1-PS-09 cell line is that research FRP-1/SARP-2 introduces the unique external model with the overexpression effect again.Another key character of hOB-01-C1-PS-09 cell is that they both can be used for transient transfection research, also can be used for stable transfection research.Below list several with respect in numerous advantages of parental generation hOB-01-C1 cell of this cell: the hOB-01-C1-PS-09 cell is real immortality, and at the fast 2-3 of 34 ℃ divisions doubly, and they also keep many prebone cell characteristics of parental cell.

The hOB-01-C1-PS-09 cell can be used for setting up the stable cell lines of the potential medicine for treating osteoporosis target of overexpression.Described stable cell lines thereby very valuable to characterizing these medicine targets and developing high flux screening and the mensuration of identifying the chemical compound of regulating it.

Embodiment

The present invention is further described by following embodiment.Only in order to reference specific embodiment elaboration the present invention of described embodiment is provided.Although these embodiment have described some concrete aspect of the present invention, do not limit or limit scope of the present invention.

Embodiment A: the generation of hOB cell and analysis

The hOB-01-C1 cell is the condition transformation cell lines that derives from adult bone, presents strictly according to the facts the prebone cell phenotype.These cells transform with the large T-antigen of responsive to temperature type (tsA 209), and the permissive temperature in 34 ℃ is bred when the T-antigenic mutant has activity; Yet when T-antigenic mutant non-activity, described cell stops division in non-permissive temperature (〉=37 ℃).Although the hOB-01-C1 cell is first osteocyte system that sets up, and is suitable for exploratory development, they have some drugs exploitation inferior position.The human cell line of the large T-Antigenic conversion of SV-40 with other is the same, and the hOB-01-C1 cell is twisted and old and feeble by going through to turn cultivating 15-20 generation.Therefore, although often be called as " immortalization ", described cell line in fact only is " extending life ".The hOB-01-C1 cell is also slowly bred when 34 ℃ are cultivated, the doubling time for approximately every 5-6 days once.In order to overcome some such shortcoming, set up hOB-01-C1-PS-09 cell line.

By in the parental generation hOB-01-C1 cell line after cultivating turning point (namely go down to posterity 15-20 generation) of going down to posterity before until propagation is recovered (go down to posterity 20-25 generation), produce the hOB-01-C1-PS-09 cell.Then cell and sub-clone after the amplification cultivation aging.Use reverse transcription-polymerase chain reaction (RT-PCR) analytical characteristic to identify the clone, to detect the expression of parathyroid hormone (PTH)-1 receptor mrna.Select the further characterized of hOB-01-C1-PS-09 cell, because this is cloned in 39 ℃ of PTH-1 receptor mrnas of expressing top level.This cell line goes down to posterity above 50 times subsequently in clone and about 20-25 the population doublings of amplification step process.These cell lines can go down to posterity hundreds of times.Therefore, the hOB-01-C1-PS-09 cell is real immortal cell line.

The same with parental generation hOB-01-C1 cell line, the hOB-01-C1-PS-09 cell can not form monolayer culture thing and slot milling at tissue culture's ware.As if described cell also form cell-cells contacting by long cell projection (long cellular process).According to electron-microscopic analysis, described cell has makes the people associate the finger like cell outthrust (projection) of front osteocyte, and these projections form the room connection when their contact adjacent cells.As parental cell system, according to immunocytochemical determination, the hOB-01-C1-PS-09 cell is also expressed tsA 209 large T-antigens.

Different from parental generation hOB-01-C1 cell line, the hOB-01-C1-PS-09 cell is at the fast 2-3 of the propagation of permissive temperature times, and the doubling time is every 2-3 days 1 time (Figure 22 A).Yet the same with parental cell, the hOB-01-C1-PS-09 cell stops division (Figure 22 A) in non-permissive temperature.This observed result shows, becomes immortality cell although cell has passed through turning point, and they still need active T-antigen to breed.And with parental cell system, the hOB-01-C1-PS-09 cell needs T-antigen inactivation, in order to represent the osteocyte phenotype of enhancing.Alkali phosphatase and osteocalcin are two important symbols of bone cell lineage.Shown in Figure 22 B, when in 39 ℃ of described cells of incubation, vitamin D ₃Process the ability that raises alkaline phosphatase activities and increased approximately 4 times.And, shown in Figure 22 C, vitamin D ₃Can not induce described emiocytosis osteocalcin at 34 ℃.

Yet when described cell during in 39 ℃ of incubations, secosteroid raises 11 times with this bone specificity stromatin output.Should be pointed out that the alkali phosphatase foundation level of hOB-01-C1-PS-09 cellular expression and the osteocalcin foundation level of secretion are similar to parental cell.Therefore, the hOB-01-C1-PS-09 cell all is being similar to parental generation hOB-01-C1 cell in the morphology He on the biochemistry, therefore is the reliable external model of research people prebone cytobiology.

As mentioned above, select to be used for the hOB-01-C1-PS-09 cell line of further characterized according to its high level expression PTH-1 receptor mrna.As shown in figure 23, compare with the cell of keeping at 34 ℃ in 39 ℃ of described cells of incubation 48 hours, the steady-state level of PTH-1 receptor mrna increases by 7 times.Because the PTH-1 expression of receptor is the sign of osteoblast/osteocyte differentiation, so this shows that also described cell has clearer and more definite osteocyte phenotype in non-permissive temperature.Consistent with PTH-1 expression of receptor wild phase, in 39 ℃ of precincubation hOB-01-C1-PS-09 cells 48 hours, then the people PTH 1-34 (hPTH 1-34) that increases gradually with concentration processed 10 minutes in 37 ℃, and making in the born of the same parents cyclic adenosine monophosphate (cAMP) level produce 5-6 dose dependent doubly increases (Figure 24).By contrast, not causing then occuring cAMP concentration in 34 ℃ of described cells of precincubation after hPTH 1-34 processes increases.Therefore, PTH-1 expression of receptor and reactivity all are enhanced behind tsA-209 T-antigen inactivation.The potential use that this cAMP measures is under PTH-1 expression of receptor level condition jumpy, the enough important target cell characterized PTH-analog of energy or the activity of PTH-analogies.

Except vitamin D ₃Outside PTH, the hOB-01-C1-PS-09 cell also responds to other bone active medicine: glucocorticoid and transforming growth factor (TGF)-β 1 for example.Process described cell with the synthetic glucocorticoid dexamethasone in 34 ℃ and make alkaline phosphatase activities raise approximately 2 times (Figure 25), this effect is enhanced again when in 39 ℃ of described cells of incubation.Equally, processing described cell with recombined human (rh) TGF-β 1 in 39 ℃ causes hepatocyte growth factor (HGF) secretion dose dependent to reduce.Confirmed that HGF plays the osteoclast chemotactic factor, and therefore can work aspect the adjusting bone resorption.

A key property of osteocyte is can stimulate to produce to mechanoreception to reply, and stimulates such as the mechanoreception that occurs in the weight training process.A method of in-vitro simulated this stimulation is to use Flexercell Strain Unit (Flexcell International, Hillsborough, NC).For the described experiment of Figure 26, the hOB-01-C1-PS-09 cell is inoculated in the coated BioFlex I type 6 hole tissue culturing plates of collagen protein, and in 34 ℃ of incubations 24 hours.Then at 34 ℃ or the 39 ℃ described cell of incubation 24 hours again in serum-free medium, use afterwards again FX-3000Flexercell Strain Unit to apply physiological correlations stress (3400 μ E, 2Hz, 7200 circulations) in 37 ℃.After stress is processed, the described cell of incubation 4.5 hours, this moment collection condition culture medium and analyze nitric oxide (NO) situation of its existence.Before reported, to external mechanoreception stimulation or the shear stress stimulation NO generation of rodent and chicken osteoblast and osteocyte.As shown in figure 26, the mechanoreception of hOB-01-C1-PS-09 cell stimulates (i.e. " Flex ") to make NO output increase 10-18 doubly.Therefore, these data show, this cell line is effective external model of the mechanical exteroceptive molecular mechanism of research.

Other experiment confirm, hOB-01-C1-PS-09 cell both can be used for transient transfection research, also can be used for stable transfection research.Can use this cell line of Tfx-20 lipofection reagent (Promega, Madison, WI) transfection.In described experiment, described cell is inoculated in the 24 hole tissue culturing plates with different densities, then use beta galactosidase and the luciferase expression plasmid transfection (total DNA=0.5 μ g/ hole) in 0.25 μ g/ hole.After 48 hours, measure beta galactosidase and the uciferase activity of cell pyrolysis liquid at 34 ℃ or 39 ℃ of incubations.Result's confirmation of this experiment, no matter be the beta galactosidase expression, or the luciferase expression level, all increase with cell number.And when in order to control transfection efficiency luciferase expression being expressed normalization to beta galactosidase, the high 2-3 of luciferase expression level of the cell of 34 ℃ of incubations doubly.Because luciferase expression is under the control of SV-40 promoter, thus this observed result during with 39 ℃ tsA 209T-antigen inactivation consistent.Therefore because these cells be immortality cell again can be transfected, so they can be used for producing the stable overexpression cell line of osteocyte background before the people.

Embodiment 1: separate SFRP

RADE (differential expression rapid analysis) the technical appraisement hOB SFRP genetic fragment of using Shiue 1997 Drug Develop.Res.41:142-159 (integral body is attached to herein) to describe.Three kinds of hOB cell lines (hOB-03-C5, hOB-03-CE6 and hOB-01-C1) represent three different idiophases (being respectively proliferation period, period of maturation and front osteocyte), use these three kinds of cell lines to separate and evaluation SFRP.Set up as previously mentioned and cultivate hOB cell line (Bodine etc., 1996 J.Bone Miner.Res.11:806-819; Bodine etc., 1996 Endocrinology137:4592-4604; Bodine etc., 1997 J.Cell.Biochem.65:368-387).These cells responsive to temperature type simian virus (SV) 40 large T-antigen immortalizations, these cell lines show the conversion phenotype in permissive temperature (34 ℃) when the T-antigenic variant has activity.Yet, (Stein and Lian 1993 opposite to osteosarcoma cell Endocrine Rev.14:424-442), hOB cell line has reflected the proliferation/differentiation relation in non-permissive temperature (＞37 ℃) loyalty when T-antigenic mutant inactivation.With described cell line with about 40,000 cells/cm ²Inoculation enters in the 150mm culture dish, and is incubated overnight in 34 ℃, and wherein used growth medium is for containing the D-MEM/F-12 of 10% (v/v) heat inactivation hyclone (FBS), 1% (v/v) penicillin-streptomycin and 2mM GlutaMAX-1.Second day, take out culture medium, clean cell with phosphate-buffered saline (PBS), in culture dish, add the 20ml serum-free medium [without phenol red D-MEM/F-12 (Gibco/BRL), contain 0.25% (w/v) bovine serum albumin (BSA, Pentex crystallized, Bayer), 1% (v/v) penicillin-streptomycin, 2mMGlutaMAX-1,50mM ascorbic acid-2-phosphate (Wako) and 10nM menadione sodium bisulfite (vitamin K ₃)], with described culture dish in 39 ℃ of incubations 24 hours.Second day takes out culture medium, contains carrier (contrast), 8nM Human Parathyroid Hormone 1-34 (PTH), 100nM prostaglandin E with 20ml ₂(PGE ₂) or the fresh serum-free culture of 0.1nM people's transforming growth factor-beta 1 (TGF-β 1) processed again described cell 24 hours based on 39 ℃.PTH, PGE ₂With TGF-β 1 be known osteogenic factor (Whitfield and Morley 1995 Trends Pharmaceut.Sci.16:382-386; Lee and Ma 1997 Bone 21:297-304; Centrella etc., 1994Endocrine Rev.15:27-39).After the processing phase, clean described culture dish with PBS, use TRIzol according to manufacturer's (GibcoBRL) explanation by the total cell RNA of cell separation untreated and that process.Then as above carry out RADE with the RNA sample that separates; To modulated genetic fragment identify, Cloning and sequencing.These experiments identify the gene of 82 differential expressions altogether.The genetic fragment that RADE obtains is carried out BLAST (basic local sequence contrast gopher) common data library searching; Wherein a kind of genetic fragment and mice SFRP-1 the height homology.Use following primer pair during RADE, to identify this genetic fragment: 3 ' terminal 5 '-AAGCTTTTTTTTTTTA-3 ' (HT11A) (reverse primer) and 5 ' end 5 '-AAGCTTGATTGCC-3 ' (H-AP1) (forward primer), its sequence is shown in SEQ IDNO 11 and SEQ ID NO 12.By the expression and regulation of rna blot analysis confirmation with the genetic fragment of mice cDNASFRP-1 homology.

The characterized of embodiment 2:hOB SFRP

Fig. 1 has summarized RADE result.PGE in proliferation period (hOB-03-C5) hOB cell line and period of maturation (hOB-03-CE6) hOB cell line ₂Strong rise hOB SFRP genetic fragment (arrow indication), and TGF-β 1 reduces hOB SFRP genetic fragment strongly in front osteocyte (hOB-01-C1) cell line.And the basal expression of this gene sharply increases in front osteocyte, shows that hOB SFRP gene expression is relevant with the osteoblast differentiation process.

The sequence analysis of embodiment 3:hOB SFRP genetic fragment

The hOB SFRP genetic fragment that contains 276 base pairs (bp) that above embodiment 1 identifies is cloned, checked order, again public database is carried out the BLAST retrieval.Retrieval shows, this gene and previous two other cDNA homologies identifying.Sequence alignment shows, hOB SFRP genetic fragment and mice SFRP-1 gene (GeneBank ^TMAccession number #U88566; Rattner etc. 1997 Proc.Natl.Acad.Sci.USA 94:2859-2863) 3 ' end has 77% sequence homogeneity.HOB SFRP genetic fragment also shows and is called frizzled related protein A (FrzA, GeneBank ^TMAccession number #U85945) the terminal significantly homology (87%) of relevant cattle cDNA 3 '.In addition, hOB SFRP genetic fragment and at least three kinds of Expressed sequence tags (EST) are homology very, and described three kinds of Expressed sequence tags are behaved and cloned TM010 (GeneBank ^TMAccession number #U54715), people CA11 tumor suppressor gene (GeneBank ^TMAccession number #U69122) and human infant brain EST (GeneBank ^TMAccession number #H16753, H16861).

Embodiment 4: osteogenic factor is to the regulating action of hOB SFRP

In order to confirm different osteogenic factor (being the dna fragmentation of identifying among the above embodiment 1) adjusting hOB SFRP gene expression, with PTH, PGE ₂Process hOB cell line with TGF-β 1; Then isolation of RNA carries out RNA hybridization.The results are shown in Fig. 2-5.The following experiment.HOB cell line is inoculated in the 150mm culture dish, processed as described in Example 1, difference is to use Oligotex mRNA maxi test kit described by separating polyA+RNA in total cell RNA according to manufacturer (Qiagen).Use RADE hOB SFRP genetic fragment, clone's hOB SFRP genetic fragment or clone's the total length hOB SFRP cDNA conduct of excision ³²The probe of p-labelling, carry out rna blot analysis (as be attached to Bodine herein etc., 1996, J.Bone.Miner.Res.11:806-819 is described).In these probes each detects the mRNA of 4.4-4.6kb in the hOB cell.The expression of hOB SFRP mRNA is corresponding to using ³²The Triose phosphate dehydrogenase of P-DNA probe (GAPDH) mRNA or beta-actin mRNA normalization.Use 100nM PGE ₂The hOB-03-C5 cell of processing proliferation period thoroughly raised approximately in 24 hours, and 4.6 kilobase prove that to the expression (Fig. 2) of (kb) mRNA this gene is subjected to PGE ₂Regulate.When clone's hOB SFRP genetic fragment is used as probe, at PGE ₂Observe the approximately advantage mRNA of 4.4kb in the cell of processing, confirm that this mRNA is actually SFRP gene (Fig. 3).The sizableness of this mRNA is in people FRP-1/SARP-2 genetic transcription thing (Finch etc., 1997 Proc.Natl.Acad.Sci.USA94:6770-6775; Melkonyan etc., 1997 Proc.Natl.Acad.Sci.USA 94:13636-13641).Rna blot analysis also confirms, uses PGE ₂Process period of maturation hOB-03-CE6 cell and raise hOB SFRP mrna expression (Fig. 4).In addition, the basal expression of this gene raises in front osteocyte hOB-01-C1 cell.After processing with 8nM PTH, this foundation level expression inhibiting 35%.And, PGE ₂Process the basal expression level that period of maturation hOB-03-CE6 cell is increased to the SFRP expression front osteocyte, this hints PGE ₂Raising SFRP in osteoblast strengthens relevant with cell differentiation.At last, process the inhibition (Fig. 5) to the level generation 80% of hOB SFRP mRNA in 24 hours of front osteocyte hOB-01-C1 cell with 100pM TGF-β 1.

For confirming that hOB SFRP mRNA level changes along with cell differentiation increases, by separating total RNA in preosteoblast hOB-03-C5 cell, ripe osteoblast hOB-03-CE6 cell, front osteocyte hOB-01-C1 cell and the mature osteocytes hOB-05-T1 cell.Then detect basic SFRP mRNA level by the TaqMan quantitative RT-PCR.When comparing with the hOB-03-C5 cell, the basic SFRP mRNA level in the hOB-03-CE6 cell increases approximately 4 times, and the basic SFRP mRNA level in the hOB-01-C1 cell increases approximately 23 times.On the other hand, the SFRP mRNA level in the hOB-05-T1 cell drops to approximately 0.5 times.Therefore, as if in the cell of osteoblast pedigree, front osteocyte is expressed the hOB SFRP mRNA of top level.

The expression kinetics of embodiment 5:hOB SFRP

As described in Example 1, the hOB-03-C5 cell of propagation is inoculated in the 150mm culture dish, and the PGE that increases gradually with concentration ₂Processed 24 hours or use 100nM PGE ₂Process different time length.Do with the RADE hOB SFRP genetic fragment of the 276bp that excises or clone's 1.1kb hOB SFRP genetic fragment ³²The P-label probe carries out rna blot analysis to PolyA+RNA.The PGE that increases gradually with concentration ₂Process proliferation period hOB-03-C5 cell and raise hOB SFRP mrna expression, EC in dosage dependence mode ₅₀Be about 8nM.Equally, the PGE that increases gradually with concentration ₂Process ripe hOB-03-CE6 cell and also raise hOB SFRP mRNA level in dosage dependence mode, but the PGE that should react ₂EC ₅₀Approximately higher 10 times than hOB-03-C5 cell.

Use 100nM PGE ₂Process hOB-03-C5 cell 2-24 hour and raise hOB SFRP mrna expression in the time-dependent mode.Observing SFRP gene expression after processing 2-4 hour obviously increases, and is adding PGE to cell culture medium ₂Rear steady-state mRNA level continues to increase and reaches 24 hours.These results suggest, SFRP may be a kind of to PGE ₂Process the gene of hOB cell delayed response, and hint under its secondary control that may be in another kind of gene outcome.Obtain similar result with ripe hOB-03-CE6 cell, but the increase multiple that hOB SFRPmRNA expresses is not high in the hOB-03-C5 cell yet.

In a similar manner the hOB-01-C1 cell is inoculated in the 100mm culture dish, and processed 24 hours with the TGF-β 1 that concentration increases gradually.Then detect hOB SFRP mRNA by the total RNA of separation in the described cell, and by the TaqMan quantitative RT-PCR.TGF-β 1 relies on mode with dosage, and gene expression produces approximately 70% inhibition, IC to SFRP ₅₀Be about 4pM.

Except PGE ₂Outside, also increase hOB SFRP mRNA level with interleukin (IL)-1 β, RETINOIC ACID or complete-anti--retinoic acid treatments hOB-03-C5 cell or hOB-03-CE6 cell.On the other hand, the same with TGF-β 1, with PTH, bone morphogenetic protein (BMP)-2, insulin like growth factor (IGF)-1,17 beta estradiols (17 β-E ₂), vitamin D ₃(VD ₃), dexamethasone (Dex) or hyclone (FBS) process various hOB cell lines, their suppress hOBSFRP mrna expression.In a word, between increasing or reduce the ability of ability that hOB SFRP expresses and their enhancer or inhibitor osteoblast/apoptosis of bone cells, these different factors exist good (although imperfect) related (referring to for example Manolagas 2000 Endocrine Reviews21:115-137).

The tissue distribution of embodiment 6:hOB SFRP

In order to determine the tissue distribution of hOB SFRP gene expression, survey the RNA trace (deriving from Clonetech) that various human is organized poly (A)+RNA with the RADE hOB SFRP genetic fragment of excision as described in Example 4.When using described RADE fragment to survey the RNA trace of poly (A)+RNA, several tissue expressions are the transcript of 4.4kb (Fig. 6) approximately.As these results during to beta-actin normalization, SFRP express arrange as follows:

Kidney＞heart＞Placenta Hominis＞liver=skeletal muscle=stomach=thyroid＞adrenal gland=testis=uterus=small intestinal=pancreas=brain＞trachea=spinal cord=prostate=colon＞spleen＞lung=lymph node=bone marrow;

In thymus and peripheral blood lymphocyte, do not observe expression.This expression pattern and people FRP-1/SARP-2 gene similar (Finch etc., 1997 Proc.Natl.Acad.Sci.USA 94:6770-6775; Melkonyan etc., 1997 Proc.Natl.Acad.Sci.USA 94:13636-13641).

The distribution of embodiment 7:SFRP in osteoblast cell line

Except the hOB cell line that identifies at first SFRP, detect other this gene of vitro human osteoblast model have a situation.Obtain SaOS-2 human osteosarcoma osteoblast-like cells by American type culture collection (ATCC), and it is cultivated in 37 ℃ in containing the McCoy 5A modified form culture medium of 10%FBS, 1% (v/v) penicillin-streptomycin and 2mM GlutaMAX-1.(Bodine etc., 1996J.Bone.Miner.Res.11:806-819, incorporated herein by reference) are set up the explant culture of Human osteoblast (hOB) by the reticulated bone fragment equally, as previously mentioned.Then as described in embodiment 1 and 4 with as described in cell inoculate in the 150mm culture dish and process, difference is that described cell carries out incubation in 37 ℃ rather than 39 ℃.Use ³²The clone's of P-labelling 1.1kb hOB SFRP genetic fragment is carried out rna blot analysis to polyA+RNA.The SFRP mRNA of the relatively low foundation level of SaOS-2 cellular expression, it is not subjected to PTH, PGE ₂Or the adjusting (Fig. 7) of TGF-β 1 processing.In these cells, be difficult to quantize the expression of described gene owing to expression is low.On the contrary, normal hOB cellular expression is than the SFRP mRNA of higher baseline level, and uses 100nM PGE ₂Process the steady-state level (approximately 1.3 times) of 24 hours described mRNA of as if slight rise of described cell.TaqMan quantitative RT-PCR to these RNA samples the analysis showed that PGE ₂In the hOB cell, raise 10 times of SFRP.Because the low foundation level in osteosarcoma cell is expressed, these cells may not be the external models of gratifying research SFRP Gene regulation.Because described gene expresses and is subjected to PGE in the Human osteoblast of cultivating ₂Regulation and control are so the hOB cell line that the present invention describes has confirmed can be used for external osteoblast model.Separation fails to detect hOB SFRP mrna expression from rna blot analysis and the RT-PCT of total RNA of people's giant cell tumor of bone in this tissue.These results show, the osteoclast like cell is expressing said gene not.

Embodiment 8: separate total length hOB SFRP cDNA

Because it is identical with several human EST to derive from the clone hOB SFRP genetic fragment of RADE, so carry out the est database analysis, so that the full-length cDNA of assembling hOB gene, this the analysis showed that, hOB SFRP is actually known human gene FRP-1 (also being called secreting type apoptosis-related protein-2 or SARP-2).Observed result (Rattner etc., 1997Proc.Natl.Acad.Sci.USA 94:2859-2863 according to this analysis and mice SFRP-1 gene and people FRP-1 gene and the obvious homology of people SARP-2 gene; Finch etc., 1997 Proc.Natl.Acad.Sci.USA 94:6770-6775; Melkonyan etc., 1997Proc.Natl.Acad.Sci.USA 94:13636-13641), design a kind of strategy based on RT-PCR, so that by people's Placenta Hominis RNA and PGE ₂The hOB-03-CE6 cell RNA of processing obtains the 1.1kb hOBSFRP cDNA of total length.Use the total RNA of 1 μ g, cross over the hFRP-1/SARP-2 coding region primer (forward primer: 5 '-GCTGGGGACTGCGCCTTTTGT-3 ', SEQ ID NO 13; Reverse primer: 5 '-CCTGCCCCCGGGAGAATCACTTA-3 ', SEQ ID NO 14), PCR and the Advantage-GC PCR test kit (Clonetech) of 35 circulations, carry out RT-PCR according to manufacturer's explanation.In order to detect the expression of mRNA, use with the base 501-530 specific hybrid of hFRP-1/hSARP-2 coding region ³²The P-oligonucleotide probe carries out southern blotting technique analysis (consult among the 1997J.Cell.Biochem.65:368-387 such as Bodine and describe in detail about the experiment of RT-PCR and DNA hybridization) with the RT-PCR product.Separate total length 1.1kb hOB SFRP cDNA, use 100nM PGE ₂Process the hOB-03-CE6 cell and made its rise (Fig. 8) in 24 hours.Equally, separate from PGE ₂The RT-PCR of total RNA of the hOB-03-C5 cell of processing identifies the cDNA of 2.2kb, its by 5 of hFRP-1/SARP-2cDNA '-district across to 3 '-the RADE fragment of the 276bp of end.These cDNA fragments are cloned into pcDNA3.1 (Invitrogen) mammalian expression vector (1.1kb cDNA) or TA (Invitrogen) cloning vehicle (2.2kb cDNA), and order-checking.The sequence analysis of hOB SFRP 1.1kb (SEQ.ID.NO.:1) and 2.2kb cDNA makes the possibility that is assembled into of 2.6kb cDNA, and described 2.6kb cDNA comprises 5 '-terminal transcriptional start site and 3 '-terminal RADE fragment.Use 1.1kb cDNA that public database is carried out the BLAST retrieval and show, it is substantially the same with people FRP-1/SARP-2.The deduction aminoacid sequence of SFRP cDNA coding region is shown in SEQ.ID.NO.:2.The aminoacid that this sequence contains is different from published people SARP-2 sequence: 174 alanine has replaced the proline (Melkonyan etc., 1997 Proc.Natl.Acad.Sci.USA 94:13636-13641) of this position.

The characterized of embodiment 9:hOB apoptosis activity

Because clone's total length hOB SFRP gene is identical with human Serum ferritin RP-1/FRP-1/SARP-2, so studied the biological agent of this gene in hOB, whether regulate hOB cell viability and Wnt signal transduction (Fig. 9-16) to determine this gene outcome.

As shown in Figure 9, the hOB cell is inoculated in 6 well culture plates with 200,000 cells/well, and in 34 ℃ of cultivations.Second day, one group of culture plate is cleaned trypsinization, as previously mentioned (Bodine etc. with PBS, 1996J.Bone Miner.Res.11:806-819 is incorporated herein by reference) usefulness Coulter Multisizer establishment of base line cell number (and mean corpuscular volume).Another group culture plate is placed 39 ℃ non-permissive temperature, measure cell number (the 3rd day replaced medium) after 6 days.Be in the hOB-03-C5 cell of osteoblast differentiation proliferation period in 39 ℃ of slowly divisions, cell number increases 60-80% after 6 days; This cell division speed is similar to the amplification cultivation (Bodine etc., 1996 Endocrinology 137:4592-4604) of normal hOB cell.On the contrary, period of maturation hOB-03-CE6 cell stops division and cell number keeps constant in non-permissive temperature, and front osteocyte hOB-01-C1 cell 39 ℃ slowly dead so that 6 days afterwards the cell of survival be less than 40%.As previously mentioned, the overexpression of SARP-2 in the MCF-7 breast cancer cell accelerated cell death rate.Observed result is consistent therewith, shown in Fig. 1,4 and 5, compares with period of maturation hOB-03-CE6 cell line with proliferation period hOB-03-C5 cell line, and the basic SFRP-1/FRP-1/SARP-2mRNA in the front osteocyte hOB-01-C1 cell expresses sharply and increases.

Next the hypothesis below verifying: SFRP-1/FRP-1/SARP-2 gene expression is raised and is accelerated the hOB cell death, and the SFRP-1/FRP-1/SARP-2 down regulation of gene expression suppresses cell death.These the results are shown in Figure 10.With growth medium hOB-03-C5 cell, hOB-03-CE6 cell or hOB-01-C1 cell are inoculated in 6 well culture plates with 200,000 cells/well, and in 34 ℃ of incubated overnight.Second day cleans described 6 well culture plates with PBS, adds the BSA culture medium, has or do not exist 100nM PGE ₂(in order to raise SFRP-1/FRP-1/SARP-2 steady-state mRNA level; Figure A and B) or 0.01-1.0nMTGF-β 1 (in order to reduce the SFRP-1/FRP-1/SARP-2mRNA level; Figure C) processes in 39 ℃ in the situation.Apoptosis-induced common method is cultured cell (Melkonyan etc. in serum-free medium, 1997Proc.Natl.Acad.Sci.USA 94:13636-13641), hOB-03-C5 cell line, hOB-03-CE6 cell line and hOB-01-C1 cell line all stop division and dead gradually under these conditions.Yet, when using PGE ₂Cell death rate is obviously accelerated when processing hOB-03-C5 cell and hOB-03-CE6 cell, reduces above 40% so that process the cell of surviving afterwards in 6 days.On the contrary, process the hOB-01-C1 cell with TGF-β 1 and increase approximately 2 times of cell viabilities in dosage dependence mode.Use PGE ₂Process described cell and not only accelerate cell death, and mean corpuscular volume is significantly reduced 10-20%.This observed result and the apoptosis induction consistent (Mesner and Kaufmann 1997, Advances in Pharmacology, 41 volumes, 57-88 page or leaf) that knownly causes the kytoplasm vesicles to form, water is lost to be reduced with cell volume.Be PGE with apoptosis induction is also consistent ₂Process the hOB-03-C5 cell and cause producing histone dependency dna fragmentation.At last, detect according to flow cytometer, use PGE ₂Processing the hOB-03-C5 cell increases in conjunction with the annexin V (a kind of specificity marker of apoptosis) of described cell (Figure 11).

Embodiment 10: the reverse that cell death is induced

In Figure 12, confirmed that with hOB-03-C5 cell and hOB-03-CE6 cell use SFRP-1/FRP-1/SARP-2 antisense oligonucleotide reverses cell death.Carry out these experiments in the similar mode of experiment of describing with Figure 10.But, for these experiments, in the situation of the phosphorothioate oligonucleotide of the directed initiation site that justice (contrast) or antisense are arranged that has or do not exist people SARP-2, usefulness vehicle Control (i.e. 0.1% ethanol) or PGE ₂The described cell of coprocessing.The result with respect to the 0th day contrast (i.e. the every hole of 6 well culture plates approximately 200,000 cells) percentage rate or represent with the percentage rate that contrasts with respect to vehicle treated.The result of this experiment shows, with the antisense oligonucleotide coprocessing hOB cell reversal of SARP-2 PGE ₂Accelerate the ability of cell death rate, and with justice (contrast) oligonucleotide coprocessing being arranged on the not impact of this process.In addition, use PGE ₂Blocked PGE with the described cell of anti-peptide antibody coprocessing of SARP-2 ₂Induce the ability of hOB cell death.The sequence of the sense and antisense oligonucleotide of human Serum ferritin RP-1/SARP-2 is as follows:

MODN is arranged: 5 '-GGCATGGGCATCGGGCGC-3 ' (SEQ ID NO.15)

Antisense oligonucleotide: 5 '-GCGCCCGATGCCCATGCC-3 ' (SEQ ID NO.16)

Embodiment 11:SFRP overexpression accelerates the hOB cell death

Figure 13 has shown the cell viability experimental result of using the hOB-01-C1-PS-09 cell of Coulter cell counter.For these experiments, with hOB SFRP cDNA (being SFRP-1/FRP-1/SARP-2) mammal expression plasmid or the described cell of empty carrier (be pcDNA3.1, derive from Invitrogen, Carlsbad, CA) stable transfection.Application standard clone technology (consulting Ausubel etc., 1997 Short Protocols in Molecular Biology, the 3rd edition, Wiley{New York}).The result represents with the percentage rate of the 0th day control cells.The result of this experiment shows, hOB cell overexpression SFRP-1/FRP-1/SARP-2 has accelerated cell death rate, and empty carrier is on the not impact of this process by contrast.Separation shows from the RNA trace autoradiography of total RNA of empty carrier (v) or SFRP (S) express cell, and the hOB cell is the same overexpression SFRP gene as expected.In addition, the quantitative Rt-PCR of TaqMan the analysis showed that, the SFRP mRNA of SFRP overexpression cellular expression than the high 5-6 of empty carrier express cell doubly.

The cell death rate of inducing in order to improve SFRP-1/FRP-1/SARP-2, sub-clone and characterized overexpression hOB-01-C1-PS-09 (Figure 14).The TaqMan quantitative RT-PCR the analysis showed that, the SFRP-1/FRP-1/SARP-2mRNA that a sub-clone (SARP-2 clones #1) is expressed than the high 50-60 of pcDNA3.1 (empty carrier) express cell doubly.Equally, with empty carrier express cell (t _1/2=～120 hours) to compare, the rate of death of the SARP-2 clone #1 cell in the BSA culture medium is greatly accelerated (t _1/2=39.2 hours).

Embodiment 12:hOB SFRP is to the effect of Wnt activity

Raise whether the Wnt signal transduction pathway is produced antagonism in order to measure SFRP-1/FRP-1/SARP-2 gene expression, use PGE ₂Process the hOB-03-CE6 cell, and survey the cell that produces with the beta-catenin monoclonal antibody.The overexpression of Wnt albumen in cell raise the signal transducer be called as beta-catenin (referring to summary: Moon etc., 1997 Cell 88:725-728; Barth etc., 1997 Curr.Opin.Cell Biol.9:683-690; With Nusse 1997Cell 89:321-323).And, the overexpression downward modulation beta-catenin white level of SFRP-1/FRP-1/SARP-2 in the MCF-7 cell, this and antagonism Wnt active consistent (Melkonyan etc., 1997Proc.Natl.Acad.Sci.USA 94:13636-13641).Therefore, plating hOB-03-CE6 cell and use PGE as described in Example 1 ₂Process, the exception part is to extract total cell protein and as previously mentioned (Bodine etc., 1996Endocrinology 137:4592-4604; Melkonyan etc., 1997Proc.Natl.Acad.Sci.USA 94:13636-13641) use the monoclonal antibody (Transduction Laboratories) of anti-described albumen that beta-catenin is carried out western blot analysis.SFRP-1/FRP-1/SARP-2 steady-state mRNA level is consistent with raising, and uses 100nM PGE ₂Process hOB-03-CE6 cell downward modulation in 24 hours beta-catenin white level, show antagonism Wnt active (Figure 15).In addition, the SFRP-1/FRP-1/SARP-2cDNA cotransfection is entered hOB-01-C1-PS-09 cell or hOB-02-C1-PS-02 cell downward modulation TCF-luciferase expression, the TCF-luciferase expression is the reliable basis (Figure 16) that detects the active and beta-catenin nuclear activity of Wnt signal transduction (such as Bafico etc., 1999 J.Biol.Chem.274:16180-16187).The SFRP-1/FRP-1/SARP-2 of people and rat and people Frzb-1/FrzB/Fritz suppress the TCF-uciferase activity of hOB cell.

Generally speaking, these observed results show, Wnt albumen has prolonged human osteoblast cell's antioxidant, and SFRP-1/FRP-1/SARP-2 promotes osteoblast dead to the antagonism of Wnt signal transduction.Therefore, the SFRP-1/FRP-1/SARP-2 depressant of functions can be enhanced to osteocyte/prebone cell survival, and therefore strengthens bone formation in the body.

The method (for example RT-PCR, gene microarray analysis and cDNA clone) that we use several evaluation hOB cell Wnt to express confirms, these cell lines express in varying degrees Wnt-2B/13 ,-3 ,-4 ,-5A and-11.Among these Wnt any or all may participate in prolonging hOB cell life.Wnt-2B/13 is also referred to as Wnt-x.

The application of embodiment 13:hOB SFRP in the anabolism drug screening method

Designed a kind of novel pattern of the SFRP of use screening bone anabolism medicine.As schematically shown in Figure 17, this filtering mode uses hOB cell and SFRP-1/FRP-1/SARP-2 to identify the chemical compound that can prevent or delay osteoblast death.Such chemical compound plays blocking effect to the ability that SFRP-1/FRP-1/SARP-2 accelerates the hOB cell death.These chemical compounds are in conjunction with SFRP-1/FRP-1/SARP-2, and prevent it in conjunction with Wnt albumen, and perhaps they can and prevent that in conjunction with Wnt it is in conjunction with SFRP-1/FRP-1/SARP-2.If SFRP-1/FRP-1/SARP-2 has and is combined irrelevant activity (for example in conjunction with cell surface receptor) with Wnt, these chemical compounds equally also can work to prevent this Wnt independent type function so.

For the initial mensuration of the filtering mode of Figure 17 signal, with chemical compound and hOB cell line and SFRP-1/FRP-1/SARP-2 incubation.This mensuration can be used SFRP-1/FRP-1/SARP-2 albumen purification or partially purified, or comprises conditioned medium or the cell extract of SFRP-1/FRP-1/SARP-2.HOB cell line can be the cell line (for example hOB-01-C1-PS-09 cell) of the cell line (for example hOB-01-C1 cell) of a horizontal SFRP-1/FRP-1/SARP-2 of natural expression higher baseline, instantaneous or stable overexpression SFRP-1/FRP-1/SARP-2 or the cell line of or natural expression SFRP-1/FRP-1/SARP-2 stable in the condition mode (PGE for example ₂The hOB-03-C5 cell of processing).As the detection to the hOB cell death, can use the mensuration (for example MTT or MTS dyeing conversion or CyQuantDNA fluorescence) of quantitative cell number or the mensuration (for example dna fragmentation or annexin V combination) of quantitative apoptosis.The CyQuant test kit is available from Molecular Probes (Eugene, OR).

Figure 18 has described the example of a SFRP-1/FRP-1/SARP-2 inhibitor high flux screening mensuration (HTS).For this mensuration, the hOB-01-C1-PS-09 cell of empty carrier (pcDNA3.1) or stable overexpression SFRP-1/FRP-1/SARP-2 (SARP-2#1) is inoculated in the 96 hole flat boards of use growth medium with 5000 cells/well.After 6 hours, clean each hole with PBS in 34 ℃ of of short duration incubations, and in the BSA culture medium in 39 ℃ of incubations 3 days.When incubation finishes, clean each hole with PBS again, then use CyQuant DNA fluoremetry (Molecular Probes) to measure dna content.When comparing with the empty carrier cell, SARP-2 overexpression cell in 39 ℃ dead faster so that after 3 days, only have the cell of 20-30% still to survive.On the contrary, the empty carrier cell of 50-60% is survived in incubation.When using anti-peptide antiserum (AS) the treatment S ARP-2 overexpression cell that produces for the aminoacid 217-231 of SFRP-1/FRP-1/SARP-2, the cell of 50-60% is afterwards survival at 3 days.This shows, suppresses the SFRP-1/FRP-1/SARP-2 protein function and prevents that it from accelerating the hOB cell death.Preimmune serum does not in contrast act on SARP-2 overexpression cell, and is that preimmune serum or immune serum all do not act on the empty carrier express cell.

The chemical compound of the hOB cell death of then, blocking-up SFRP-1/FRP-1/SARP-2 being induced carries out in the other external test.These measure these chemical compounds are blocked the hOB cell death with SFRP-1/FRP-1/SARP-2 dependency or dependent/non-dependent mode the ability that detects, but also measure ability and the effectiveness of these chemical compounds with regard to these effects.Design other mensuration, to detect these chemical compounds to the cell selective of these effects (for example, using MCF-7 cell or other cell) and these chemical compounds specificity to SFRP-1/FRP-1/SARP-2 and other member of SFRP/SARP family (for example FrzB/Fritz, SARP-1 or SARP-3).Whether other mensuration also can regulate the downstream signal transduction event that relates to apoptosis (for example caspase is active) or Wnt active (for example beta-catenin white level and the function of TCF-luciferase assay) for detection of these chemical compounds.At last, then will in these external tests, have the chemical compound of suitable activity for bone formation, osteopenia or osteoporotic various animal model (for example ovariectomized rat or mice).It is believed that the chemical compound that is suppressed to osteocyte/apoptosis of bone cells is bone anabolism medicine, it prolongs the life of these cells, and therefore increases the bone matrix amount of synthetic and mineralising and/or the integrity of maintenance bone.

Obviously, can be different from the specifically described mode of above stated specification and embodiment and implement the present invention.But according to above guidance modifications and variations of the present invention are, therefore they belong to the scope of appended claims.

Establishment and the use of embodiment 14:SFRP-1 knock out mice

As described in embodiment 13, the chemical compound that is suppressed to osteocyte/apoptosis of bone cells is compellent bone anabolism medicine, and it prolongs the life of these cells, and so increase is synthetic and the integrity of the amount of the bone matrix of mineralising and/or maintenance bone.In order to verify this hypothesis and measure SFRP-1/FRP-1/SARP-2 whether affect skeleton, negative (SFRPneg) mice of preparation SFRP-1 (referring to Wattler etc., 1999, BioTechniques 26:1150-1160).Be similar to medicine and suppress its function by removing the SFRP-1/FRP-1/SARP-2 gene in the mice, this method allows us to confirm that this genes/proteins is osteoporotic potential drug target.

Summarize such as Figure 19, by replace the exons 1 of mice SFRP-1 gene with beta galactosidase reporter gene/neomycin drug tolerant gene expression box, produce the SFRP-1 knock out mice.As shown in figure 20, separation shows from the rna blot analysis of the poly A+RNA of female or male mice kidney (16-18 age in week), SFRP-1mRNA (4.4kb) is high level expression in wild type (WT) control mice, but gene expression complete obiteration in gene knockout (KO) mice.

As shown in figure 21, use the male and female wild type of microcomputer x-ray tomography developing (micro-CT) characterized to contrast the trabecular bone structure of distal femoral of (+/+) and gene knockout (/-) mice (about the summary of this technology, referring to Genant etc., 1999Bone 25:149-152 and Odgaard 1997 Bone 20:315-328).In 20 week age male mices (figure A),-/-mice with+/+control mice compares, trabecular bone volume (BV/TV) exceeds 31%, little cantilever thickness (Tb.Th.) increases by 8%.In age in 26-27 week female mice (figure B),-/-mice with+/+control mice compares, girder engages density (Conn.Den.) increases by 91%, girder number (Tb.N.) increases by 16%, and little case bay (Tb.Sp.) reduces by 16%.Therefore, support these results of hypothesis to prove, mice disappearance SFRP-1 gene causes trabecular bone to form parameter raising (P.J.Meunier 1995 osseous tissue morphologys, be stated from: osteoporosis: etiology, diagnosis and treatment, second edition, B.L.Riggs and L J.MeltonIII write Lippincott-Raven, Philadelphia, the 299-318 page or leaf).

The nucleotide of CPCH0963369D description and aminoacid sequence table

＜110〉American Home Products Corp (American Home Products Corporation)

＜120〉Pharmaceutical composition of using secreted frizzled related protein and method

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gcgcggggag gcggcggagg cagccccgac gtcgcggaga acagggcgca gagccggcat 120

gggcatcggg cgcagcgagg ggggccgccg cggggcagcc ctgggcgtgc tgctggcgct 180

gggcgcggcg cttctggccg tgggctcggc cagcgagtac gactacgtga gcttccagtc 240

ggacatcggc ccgtaccaga gcgggcgctt ctacaccaag ccacctcagt gcgtggacat 300

ccccgcggac ctgcggctgt gccacaacgt gggctacaag aagatggtgc tgcccaacct 360

gctggagcac gagaccatgg cggaggtgaa gcagcaggcc agcagctggg tgcccctgct 420

caacaagaac tgccacgccg gcacccaggt cttcctctgc tcgctcttcg cgcccgtctg 480

cctggaccgg cccatctacc cgtgtcgctg gctctgcgag gccgtgcgcg actcgtgcga 540

gccggtcatg cagttcttcg gcttctactg gcccgagatg cttaagtgtg acaagttccc 600

cgagggggac gtctgcatcg ccatgacgcc gcccaatgcc accgaagcct ccaagcccca 660

aggcacaacg gtgtgtcctc cctgtgacaa cgagttgaaa tctgaggcca tcattgaaca 720

tctctgtgcc agcgagtttg cactgaggat gaaaataaaa gaagtgaaaa aagaaaatgg 780

cgacaagaag attgtcccca agaagaagaa gcccctgaag ttggggccca tcaagaagaa 840

ggacctgaag aagcttgtgc tgtacctgaa gaatggggct gactgtccct gccaccagct 900

ggacaacctc agccaccact tcctcatcat gggccgcaag gtgaagagcc agtacttgct 960

gacggccatc cacaagtggg acaagaaaaa caaggagttc aaaaacttca tgaagaaaat 1020

gaaaaaccat gagtgcccca cctttcagtc cgtgtttaag tgattctccc gggggcaggg 1080

aattctgcag atatccagca tggggaggga gcctcgggtg gggtgggagc gggggggaca 1140

gtgccccggg aacccggtgg gtcacacaca cgcactgcgc ctgtcagtag tggacattgt 1200

aatccagtcg gcttgttctt gcagcattcc cgctcccttc cctccatagc cacgctccaa 1260

accccagggt agccgtggcc gggtaaagca agggccattt agattaggaa ggtttttaag 1320

atccgcaatg tggagcagca gccactgcac aggaggaggt gacaaaccat ttccaacagc 1380

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tttaaatgga aacagactca taccacactt acaattaagg tcaagcccag aaagtgataa 1620

gtgcagggag gaaaagtgca agtccattat gtagtagtga cagcaaaggg accaggggag 1680

aggcattgcc ttctctgccc acagtctttc cgtgtgattg tctttgaatc tgaatcagcc 1740

agtctcagat gccccaaagt ttcggttcct atgagcccgg ggcatgatct gatccccaag 1800

acatgtggag gggcagcctg tgcctgcctt tgtgtcagaa aaaggaaacc acagtgagcc 1860

tgagagagac ggcgattttc gggctgagaa ggcggtagtt ttcaaaacac atagttaaaa 1920

aagaaacaaa tgaaaaaaat tttagaacag tccagcaaat tgctagtcag ggtgaattgt 1980

gaaattgggt gaagagctta cgattctaat ctcatgtttt ttccttttca catttttaaa 2040

agaacaatga caaacaccca cttatttttc aaggttttaa aacagtctac attgagcatt 2100

tgaaaggtgt gctagaacaa ggtctcctga tccgtccgag gctgcttccc agaggagcag 2160

ctctccccag gcatttgcca agggaggcgg atttccctgg tagtgtagct gtgtggcttt 2220

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Val Leu Leu Ala Leu Gly Ala Ala Leu Leu Ala Val Gly Ser Ala Ser

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Glu Tyr Asp Tyr Val Ser Phe Gln Ser Asp Ile Gly Pro Tyr Gln Ser

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Gly Arg Phe Tyr Thr Lys Pro Pro Gln Cys Val Asp Ile Pro Ala Asp

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Leu Arg Leu Cys His Asn Val Gly Tyr Lys Lys Met Val Leu Pro Asn

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Leu Cys Ser Leu Phe Ala Pro Val Cys Leu Asp Arg Pro Ile Tyr Pro

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Pro Cys His Gln Leu Asp Asn Leu Ser His His Phe Leu Ile Met Gly

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Claims (29)

1. Pharmaceutical composition of regulating the mammal osteogenic activity, this Pharmaceutical composition comprises at least a following component: (i) secreted frizzled related protein (SFRP) or its regulating action part; (ii) antibody of described albumen or its part formation; (iii) coding (i) or nucleic acid (ii); And (iv) antisensenucleic acids of any nucleic acid in (iii).

2. according to the Pharmaceutical composition of claim 1, wherein said SFRP derives from the human osteoblast cell.

3. according to the Pharmaceutical composition of claim 1, wherein said osteogenic activity is to regulate osteogenesis.

4. according to the Pharmaceutical composition of claim 1, wherein said osteogenic activity is to regulate bone density.

5. according to the Pharmaceutical composition of claim 1-3, wherein said SFRP has the aminoacid sequence that SED IDNO:2 shows.

6. method for the treatment of the mammal osteopathia, the method comprises the step of the Pharmaceutical composition that gives claim 1-5.

7. the method for the treatment osteopathia of claim 6, wherein said disease comprises (a) bone formation disease; (b) bone resorption disease and (c) bone density disease.

8. the method for claim 6, wherein said disease is the degeneration osteopathia.

9. the method for claim 8, wherein said degeneration osteopathia is selected from neurodegenerative diseases, flesh degenerative disease and degenerative bone disease.

10. the method for claim 9, wherein said degenerative bone disease is selected from osteopenia, osteoarthritis, osteoporosis.

11. the method for claim 6, wherein said mammal is behaved.

12. a method of identifying the test-compound of regulating the SFRP activity, described chemical compound is regulated mammiferous osteogenic activity, and described method comprises that (a) incubation in containing the tested media of described test-compound contains the sample of SFRP; (b) measure the SFRP activity, wherein show that with respect to active the increasing of independent SFRP described chemical compound is the SFRP activator, and activity decreased shows that described chemical compound is the SFRP inhibitor.

13. a method of regulating the cell signalling of Wnt-mediation, the method comprises makes described cell contact with the SFRP of claim 1-5, and wherein said Wnt activity is regulated.

14. the method for the signal transduction of the adjusting Wnt-of claim 13 mediation, wherein said SFRP has the aminoacid sequence that SEQ ID NO:2 shows.

15. method that promotes that osteocyte culture bone formation or bone are repaired, the method comprises by separating described cell in the bone culture, introduce to express coding and have the recombination to construct thing of antisense sequences of nucleotide sequence of the SFRP of the aminoacid sequence that SEQ ID NO:2 shows, more described cell is turned back in the described bone culture.

16. a polynucleotide probes, it can with the multi-nucleotide hybrid with nucleotide sequence that SEQ ID NO:1 shows.

17. a detection derives from the diagnostic method of the SFRP polynucleotide in the sample of mammalian hosts, the method comprises that right to use requires whether to have SFRP in 16 the described sample of probe in detecting.

18. the Pharmaceutical composition of claim 1, wherein said compositions comprises acceptable carrier or diluent.

19. a compositions of regulating the mammal osteogenic activity, said composition comprise at least a antibody for secreted frizzled related protein (SFRP) or its regulating action part.

20. the Pharmaceutical composition of claim 19, wherein said compositions comprises acceptable carrier or diluent.

21. the Pharmaceutical composition of claim 19, wherein said antibody is at least 8 continuous amino acids of SFRP albumen.

22. the Pharmaceutical composition of claim 19, wherein said antibody is at least 10 continuous amino acids of SFRP albumen.

23. the Pharmaceutical composition of claim 19, wherein said antibody is at least aminoacid 217-231 of the SFRP albumen of SEQ ID NO:2.

24. according to the Pharmaceutical composition of claim 1-4 and 18-21, wherein said SFRP has the aminoacid sequence that obtains by the polynucleotide sequence of expressing SEQ ID NO:1 demonstration.

25. a method of identifying the test-compound of regulating the SFRP activity, described chemical compound is regulated the mammal osteogenic activity, and described method comprises that (a) comparative sample is to SFRP ^NegAnimal active and do not have the activity of described sample, wherein active to raise and reduce the described chemical compound of explanation be the regulator of SFRP activity to the SFRP dependency.

26. immortal human skeletonization (hOB) cell of expressing the temperature sensitive mutant of the large T proteantigen of simian virus 40, wherein when described T-antigenic mutant has activity, described cell is in about 34 ℃ of lower propagation, but do not breed surpassing approximately under 37 ℃ the temperature.

27. the hOB cell of claim 26, it expresses the nucleotide sequence of coded polynucleotide, described polynucleotide encoding SFRP or its fragment.

28. the hOB cell of claim 26, wherein said hOB are hOB-01-C1-PS-09 cell or its offspring, the hOB-01-C1-PS-09 cell is preserved in American type culture collection, Manassas, and Va, preserving number are PTA-785.

29. cell population that comprises the hOB cell of claim 26.

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