CN105985426A

CN105985426A - LGR4 protein fragment and application thereof to preparation of drug for treating osteoclast induced bone disease

Info

Publication number: CN105985426A
Application number: CN201510094956.7A
Authority: CN
Inventors: 罗剑; 杨正峰; 刘明耀
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2015-03-03
Filing date: 2015-03-03
Publication date: 2016-10-05
Anticipated expiration: 2035-03-03
Also published as: CN105985426B

Abstract

The invention provides a separated LGR4 protein fragment shown as SEQ ID NO.1. The protein has a protein sequence shown as SEQ ID NO.1. The invention further provides application of the protein fragment to preparation of a drug for treating the osteoclast induced bone disease. The protein fragment can inhibit differentiation of osteoclast in the body. Further, the invention provides application of the protein fragment to study of osteoclast differentiation inhibition in vitro or in vivo.

Description

LGR4 protein fragments and the purposes in the medicine of the osteopathy of preparation treatment osteoclast induction thereof

Technical field

The present invention relates to LGR4 (the Leucine-rich repeat containing G-protein coupled of biological technical field Receptor 4, full asphalt mixture motif g protein coupled receptor 4) protein fragments, in particular it relates to a class with The osteoporosis LGR4 protein fragments related to giant cell tumor of bone, and it is in the osteopathy preparing treatment osteoclast induction Purposes in medicine.

Background technology

Research with regard to osteoclast regulation human body physiological function in recent years is on the increase, and increasing research proves osteoclast Not being only involved in the Equilibrium of organism Bone m etabolism, also occupy a tiny space in immune system (Immunol Rev.2005 Dec；208:19-29.).The more important thing is, under many pathological states, osteoclast function disappearance or overactivity all when have Occur, so that increasing misery and the treatment complexity of disease patient.Such as, rheumatoid arthritis condition subject is often thin with broken bone Born of the same parents' hyperfunction, causes serious bone loss, makes patient's sup sorrow；Often easily there is Bone tumour late period in patient with breast cancer, The cancer cell being transferred to bone can promote osteoclast overactivity, can result in osteoporosis, treatment and the animation to patient All bring very big inconvenience；In osteitis deformans, the overactivity of osteoclast is the main cause of subjects bones's deformity；Bone giant cell In knurl, Osteoclast-like cells acutely activates, and causes patient's sclerotin and seriously loses, teratogenesis of disabling；Osteoclast work(in osteopetrosis patient Can lose, illness bone density is too high, easily deformity, and these are all made troubles to life.Additionally, under normal physiological condition, women Also easily there is bone loss in post menopausal, its main cause is also just being hyperfunction (the Nat Rev Drug Discov.2012 of osteoclast function May；11(5):401-19.doi:10.1038/nrd3705.).Therefore, the research of drug target is developed in bone disease for osteoclast Field especially osteoporosis account for exhausted vast scale.

Can be divided into two big currently for the drug therapy of osteoclast relevant disease (including osteoporosis and giant cell tumor of bone) Class, a class is micromolecular compound, such as bisphosphonates；Two is protein medicaments, such as RANKL inducing receptor RANK-Fc And OPG albumen, RANKL monoclonal antibody ground promise monoclonal antibody (Denosumab).Specifically, micromolecular compound aspect, bis phosphoric acid Salt can promote mature osteoclast apoptosis, thus can have therapeutic action to osteoclast relevant disease.Up to now, bis phosphoric acid Drug class development experience three generations: first generation Diphosphonate is free from the Diphosphonate of nitrogen, including etidronate, chlorine bend phosphine Acid sodium and tiludronate disodium.Owing to their structure is much like with bis phosphoric acid base, therefore when osteoclast absorbs bone surface Mineralized Area After, they can be inserted in newly synthesized adenosine triphosphate (ATP) under the effect of two type RNA transamination synzyme, this The ATP analog of kind non-hydrolysable can suppress the various kinds of cell process being mediated by ATP after intracellular is constantly accumulated, thus to broken bone Cell produces CDCC, causes osteoclast apoptosis；From just for unlike Diphosphonate, the second generation and the double phosphorus of the third generation Hydrochlorate (Alendronate sodium, risedronate sodium, aiben sodium phosphonate, Sodium Pamidronate and zoledronic acid) is at R²Exist at side chain Nitrogen-atoms, they cause the mechanism of osteoclast apoptosis from just different for Diphosphonate.Research shows that this two generations Diphosphonate can be tied Merge suppression farnesyl pyrophosphate synzyme, and the regulation mevalonic acid metabolism of this enzyme is cholesterol and other sterols and class isoamyl Diene lipid, thus, the albumen of regulation osteoclast core active (such as stress fiber assembling, film gauffer and survival) (includes Rab, Rac and Rho) posttranslational modification (isoprenylation) can be suppressed and finally cause osteoclast apoptosis.Clinical On, Diphosphonate this effect to osteoclast makes it be widely used in multiple related disease hyperfunction to osteoclast function, Including polytype osteoporosis (such as the osteoporosis after hebetic, postclimacteric or old, glucocorticoid The osteoporosis of induction, the osteoporosis of organ transplant and long-time motionless initiation and go the osteoporosis that androgen is related), Pei Jiteshi osteopathy, osteogenesis imperfecta, hypercalcinemia and malignant metastatic tumor of bone (Mayo Clin Proc.2008 Sep；83(9):1032-45.doi:10.4065/83.9.1032.).

Protein medicaments aspect, current basic scientific research and clinical practice are mainly for the RANKL needed for differentiation of osteoclast and activation Albumen is researched and developed, and more well-known is RANKL competitive type receptor protein and the ground promise having been used for partial clinical illness in recent years Monoclonal antibody.Existing two kinds of competitive type receptor proteins for RANKL exploitation are seen in report: one is to comprise RANK acceptor portion knot The RANK-Fc polypeptide in structure territory, it is made up of the extracellular fragment CRD domain of RANK and the Fc domain of IgG1.RANK-Fc Can specifically combine RANKL and not interact with other TNF family parts, thus play in some preclinical models The effect of suppression bone information；Two is OPG-Fc polypeptide.OPG is natural RANKL Decoy receptors, can be subject to RANK Body competition binding RANKL thus block RANKL-RANK signal path.The research and development of OPG-Fc polypeptide experienced by multiple stage, The heavy dose of (> 10-30mg/kg of in early days research discovery) restructuring OPG full-length polypeptide can effectively suppress Mice Body in bone information now As the structure comparison solidification of this OPG full-length polypeptide, therefore its pharmacokinetic parameter is very pessimistic.As research is goed deep into, Have developed again the OPG polypeptide only comprising N-terminal CRD domain and lacking C terminal hairpin structure, the medicine generation of this peptide species Kinetics level is significantly improved, and after carrying out Large-scale Screening to OPG truncate, researcher finds mankind OPG The OPG-Fc polypeptide being formed after the 22-194 amino acid polypeptide fragment of albumen and the Fc domain fusion of IgG1 can be than total length OPG Albumen has the activity raising of nearly 200 times, and this polypeptide also has the longer half-life in vivo.Prokaryotic expression system is utilized to obtain To the phase clinical trial of OPG-Fc polypeptide show that it concentration gradient can quickly promote bone with relying on and update in vivo, this is real It is effective for testing explanation first and blocking RANKL signal and update mankind's bone.Meanwhile, another kind of by mammalian cell expression OPG-Fc polypeptide (AMGN-0007) also there is this effect, and its half-life longer (nearly 10 times of prokaryotic expressions OPG-Fc polypeptide), effect also will good 2-3 times, this be possibly due to OPG in eukaryotic system also there occurs glycosylation therefore.Always For, effectively suppressed the activity of internal osteoclast for the competition of RANKL design by physical efficiency, thus improve bone loss and show As.Removing outside competition acceptor, also research team have developed RANKL specific antibody ground promise monoclonal antibody in recent years, opens The Antybody therapy approach of osteoclast.Ground promise monoclonal antibody is a class IgG2 monoclonal antibody, has high with people's RANKL albumen Affinity, its dissociation equilibrium binding constant (Kd) is 3pM, and the RANKL of it and soluble form and film combining form can Interact, but the RANKL albumen of nonrecognition mouse and rat.Clinical research shows, promise monoclonal antibody efficacy and safety in ground is held concurrently Standby, the bone loss and the osteoclasia that cause osteoporosis and cancer are respectively provided with significant curative effect.Study also discovery ground promise list further Resisting can also the sluggish generation with prevention prostate cancer and Bone of Breast Cancer transfer.Additionally, also there are some researches show that ground promise monoclonal antibody is big and small to bone Born of the same parents knurl patient also effective in cure (Nat Rev Drug Discov.2012May；11(5):401-19.doi:10.1038/nrd3705.).These Study and show effectively suppress for the medicine of RANKL design research and development formation osteoclast activity, and osteoclast is related to Osteoporosis and giant cell tumor of bone have remarkable result.Therefore, the drug development of RANKL molecule is suppressed to have extensive use With value.

Chinese patent application the 201310047174th, the denomination of invention " preparation method of mother-of-pearl albumen N16 and at preparation preventing and treating bone Application in section's disease medicament " discloses one and utilizes method of gene recombination to express mother-of-pearl albumen N16 in bacterium, establishes N16 Isolated and purified scheme.Preparation-obtained N16 can suppress the propagation of precursor osteoclast, and suppression RANKL induction precursor breaks Bone cell differentiation becomes ripe osteoclast, the activity of suppression osteoclast significant enzyme acid phosphatase TRAP, and suppression is broken The formation of osteocyte, the expression of suppression differentiation of osteoclast related gene, suppression differentiation of osteoclast is ripe.Test result indicate that, N16 albumen can not only promote osteoblast differentiation, more can suppress differentiation of osteoclast, thus reach anti-osteoporosis and treatment bone The purpose of folding.This imply that this albumen is possibly used for strengthening osteoblastic mineralization function, the sclerotin that treatment removal ovary causes is dredged Pine disease.

Chinese patent application the 201210232648th, denomination of invention " comprises fused protein and the preparation side thereof of cFms extracellular segment Method and application " discloses the fused protein that one comprises macrophage stimulation factor acceptor (cFms) extracellular segment, and this albumen has There is bind interleukin-34 (IL-34) and combine macrophage stimulation factor (M-CSF), blocking IL-34/M-CSF part with altogether It with the effect of the combination of acceptor cFms, is suitable under treatment abnormal conditions by the disease caused by IL-34/M-CSF.Self is exempted from Epidemic disease disease, diseases associated with inflammation, osteoporosis and tumour have potential treatment prospect.

International patent application WO2010/117011, invention entitled " anti-Siglec 15 antibody " disclosing one can target Antibody to the albumen of the gene code by strong expression in osteoclast.This antibody is selectively targeted special in giant-cell tumor Property Siglec 15 gene expressed, and suppress the activity of osteoclast formation, obtain good effect.

As described above, be currently used for suppression or the protein medicaments of sclerotin class disease that treatment causes because of osteoclast, focus mostly on Prepare specific antibody, inhibitor or RANKL competitive type acceptor aspect, and for g protein coupled receptor (GPCR) target Point is designed and developed the related osteoporosis for the treatment of osteoclast and is seldom had been reported that with giant cell tumor of bone.In recent years with different kind organism Gene order-checking complete the development with comparative genomics, it has been found that in vertebrate and spinal animal, there is also many Similar to above-mentioned glycoprotein hormone receptor structure or that there is homology receptor protein.In vertebrate, according to these acceptor eggs The time order and function order being found in vain, named LGR4, LGR5, LGR6, LGR7 and LGR8 etc. successively.These knots The similar albumen of structure constitutes a subfamily in GPCR superfamily, i.e. the G-protein coupling rich in leucine repetitive sequence is subject to Body (LGR, Leucine-rich G-protein Coupled Receptor).Phylogenetic analysis display LGRs is segmented into three Individual subgroup: first group is classical glycoprotein hormone receptor, including follicular stimulating hormone (follicle-stimulating hormone, FSH) Acceptor, thyrotropic hormone (thyroid stimulating hormone, TSH) acceptor and lutropin (1uteinizing Hormone, LH) acceptor, it each combines with glycoprotein hormones FSH, TSH and LH/hCG.Second group by LGR4-6 group Become, once because its function and cognate ligand are failed to understand and belonged to orphan receptor, studies have found that Rspondin family member's energy in the recent period Occur to interact with the extracellular fragment structure of LGR family and activate WNT signal path, therefore, Rspondin family member's quilt It is considered the native ligand of LGR family.3rd group includes LGR7 and LGR8, it has recently been found that be relaxain and Lai Dixi respectively Insulin-Like peptide (Leydig insulin-like peptide or relaxin-like factor 3, INSL3) acceptor.Up to now, LGR Known physiologic function is mainly concerned with eye or kidney development and reproduction.

With regard to LGR4, (Am J Med Genet is A.2011 on 11p14～13 of chromosome for mankind's LGR4 assignment of genes gene mapping Jun；, belong to g protein coupled receptor (GPCR) A family, again belong to 155A (6): 1272-80.doi:10.1002/ajmg.a.33878.) In full asphalt mixture motif g protein coupled receptor (LGR) family (Development 136,2747-2756 (2009) Doi:10.1242/dev.033571), the cell-membrane receptor albumen containing 951 amino acid is encoded.The mouse Lgr4 assignment of genes gene mapping On No. 2 chromosomes, also encode the cell-membrane receptor albumen containing 951 amino acid.LGR4 is distributed quite in vivo Extensively, including reproductive system, kidney, adrenal gland, bone etc., and as far back as the 7th day of mice embryonic, LGR4 was just Starting to express, LGR4 is at the developmental important function of mouse (Katoh-Fukui Y, Owaki A, Toyama Y, et al. in prompting Mouse Polycomb M33 is required for splenic vascular and adrenal gland formation through regulating Ad4BP/SF1 expression.Blood,2005,106(5):1612-20.).In addition, LGR4 knock out mice Intrauterine growth retardation and postnatal high mortality also illustrate that LGR4 has very important impact to the embryonic development of mouse (Mendive F,Laurent P,Van Schoore G,et al.Defective postnatal development of the male Reproductive tract in LGR4 knockout mice.Dev Biol, 2006,290 (2): 421-34.), but up to now for LGR4 concrete organ embryonic period, embryonic phase developmental function there is not yet document report.And studying more deep at present is LGR4 Impact on mouse propagation system postnatal development.LGR4 knock out mice efferent duct and epididymis postnatal development are abnormal, Extend and extension obstacle, and cause line clogging and testis ponding, ultimately result in male mice sterile.Trace it to its cause and be probably LGR4 Affect propagation and differentiation (Hoshii T, the Takeo T, Nakatata N, et al.LGR4 Regulates the of genital tract cells Postnatal Development and Integrity of Male Reproductive Tracts in Mice.Biol Reprod,2007, 76(2):303-13.).Additionally, LGR4 also assists in the growth of kidney, LGR4 defect cause glomerulus number reduce (Lala DS, Rice DA,Parker KL,et al.Steroidogenic factor I,a key regulator of steroidogenic enzyme expression,is the mouse homolog of fushi tarazu-factor I.Mol Endocrinol,1992,6(8):1249-58.)。 Also discovery LGR4 can promote the transfer of tumour cell recently, but the propagation on cell do not appear to impact (Honda S, Morohashi K,Nomura M,et al.Ad4BP regulating steroidogenic P-450gene is a member of steroid hormone receptor superfamily.J Biol Chem,1993,268(10):7494-502.).At present LGR4 function is recognized Know and be only limitted to the above.

Content of the invention

The principle of the invention is, inventor be first innovatively study discovery LGR4 protein fragments (1aa-504aa, LGR4-ECD) with RANK competition binding RANKL, thus the molecule mechanism of differentiation of osteoclast and function can be suppressed, say Bright LGR4 protein fragments can be used for developing RANKL competition polypeptide drugs thus the possibility that suppresses osteoclast activity.In view of More than finding, inventor utilizes prokaryotic expression system, and with the protein fragments of recombinant expressed LGR4, discovery Primary bone marrow monokaryon gulps down Phagocyte is after the process of this albumen, and its ability to mature osteoclast differentiation induced by RANKL or Gegenbaur's cell can be complete Full suppression, and present concentration gradient dependence.Additionally, this protein fragments can also effectively suppress pathological state sending down the fishbone huge knurl patient to swell Cytomegalic differentiation in oncocyte；Meanwhile, the research in two kinds of mouse osteoporosis animal models is it has furthermore been found that this albumen Fragment can be prevented and can treat the bone loss phenomenon due to osteoclast overactivity.

Therefore, first purpose of the present invention is to provide the protein fragments with the outer segment structure of LGR4 protein extracellular of a kind of separation, The sequence of described protein fragments is selected from any one of protein sequence as follows:

(1) protein sequence as shown in SEQ ID NO.1；

(2) there is at least 90% homogeneity with the protein sequence shown in SEQ ID NO.1 and there is the albumen of identical function；

(3) occur in the protein sequence shown in SEQ ID NO.1 one or several amino acid residue replacement and/or disappearance and/ Or add but there is the albumen of identical function；

(4) on the basis of the coded sequence of the protein sequence shown in SEQ ID NO.1 through one to several bases replace and/ Or one to the nucleotide sequence of the insertion of several bases and/or disappearance and large fragment insert, disappearance, after displacement or inversion, Expressed and that there is identical function protein fragments；Or

(5) can hybridize with the coded sequence of the protein sequence shown in SEQ ID NO.1 under moderate stringency and coding obtains The protein fragments with identical function.

In one embodiment, described protein sequence preferably also include be occur 1 to 5 sudden change and with SEQ ID NO.l Sequence has other protein sequences of the albumen of at least 99% homology, and wherein this albumen has identical function.Implement preferred In scheme, described sudden change is 1-4, more preferably 1-3, or more preferably 1 or 2, most preferably occurs 1 Sudden change.Wherein, described sudden change is preferably to replace, inserts or deletion mutation.It should be pointed out that, albumen involved in the present invention Sequence, it is by having the peculiar space conformation that formed of albumen of certain sequence composition, and RANK competition binding RANKL, Thus suppressing differentiation of osteoclast and function, it not directly acts on related locus or function suppression osteoclast Differentiation and function.And protein sequence total length 504aa shown in SEQ ID NO.1, its sequence with 99% homology and described SEQ Protein sequence shown in ID NO.1, the sudden change less than 5 aa for the difference.And to those skilled in the art, for phase With multiple protein sequences in source (human genome), there is the homology of 99% and the sequence suddenlyd change less than 5 each other, Its more situations are proven to have identical function, rather than are proved its afunction.Therefore those skilled in the art can be pre- Seeing, on the premise of limiting above-mentioned sequence, limited homology and sudden change quantity, other described protein sequences have phase equally With or similar function.

In one embodiment, the LGR4-ECD in the protein fragments behaviour source as shown in SEQ ID NO:1 described in above-mentioned (1) (hereinafter referred to as h4), it can combine with receptor activation of Nuclear factor kappa B part.

In other embodiments, described protein fragments has suppression RANKL or the combination of suppression RANKL-RANK Function, thus suppression or the sclerotin class disease that causes because of osteoclast for the treatment of.

Second purpose of the present invention is to provide gene coded sequence or its degenerate sequence of above protein fragments.

The 3rd purpose of the present invention is to provide the use in the medicine of the osteopathy in preparation treatment osteoclast induction for the above-mentioned protein fragments On the way.Wherein, described protein fragments is used for suppressing internal differentiation of osteoclast.Further, including described protein fragments is at sclerotin Osteoporosis and the application in giant cell tumor of bone disease treatment, including described protein fragments is big and small with bone in preparation treatment osteoporosis Application in the medicine of born of the same parents' knurl disease.

In one embodiment, described protein fragments can suppress the differentiation of osteoclast that Gegenbaur's cell is induced.

In another embodiment, described protein fragments can suppress to be reduced by the internal sclerotin caused by RANKL overstimulation.

In another embodiment, described protein fragments can alleviate the sclerotin minimizing suffering from osteoporosis mouse, alleviates due to bone The loosen sclerotin that causes of matter reduces.

In another embodiment, described protein fragments can suppress the osteoclast activity of bone huge knurl patient.

In the above-described embodiment, above-mentioned pharmaceutical applications include the medicine of osteoporosis that preparation suppression osteoclast causes or The medicine of suppression growth and metastasis of tumours.In one specific embodiment, wherein, the bone that described suppression osteoclast causes The medicine of matter osteoporosis is for treating rheumatoid arthritis, caused by bone tissue that arthritis, metastases cause absorbs too much The medicine of osteoporosis；Wherein, the medicine of growth and metastasis of tumours is suppressed to refer to suppress the medicine of the huge knurl of bone or giant cell tumor of bone, Including the medicine of the optimum invasive tumor that collagen is sent out is treated in preparation.

In the above-described embodiment, described medicine also includes pharmaceutically acceptable salt, carrier or adjunct ingredient.

In another embodiment, said medicine also can prepare the medicine of above-mentioned disease with other drug active ingredient combination.

The 4th purpose of the present invention is the use providing above-mentioned protein fragments for studying external or internal suppression differentiation of osteoclast On the way.

In one embodiment, described purposes utilizes above-mentioned protein fragments research external or internal suppression differentiation of osteoclast Purposes.For example, protein fragments of the present invention is used for preparing the medicine suppressing differentiation of osteoclast in vitro or in vivo.

In another embodiment, described purposes is to utilize above-mentioned protein fragments research external or internal suppression Gegenbaur's cell induction The purposes of differentiation of osteoclast.For example, protein fragments of the present invention is used for preparing suppression Gegenbaur's cell in vitro or in vivo The medicine of the differentiation of osteoclast of induction.

In another embodiment, described purposes is to utilize above-mentioned protein fragments to study external or internal suppression into by RANKL mistake Degree stimulates caused internal osteopenic purposes.For example, protein fragments of the present invention is used for preparing in vitro or in vivo Suppression is by the internal osteopenic medicine caused by RANKL overstimulation.

In another embodiment, described purposes is to utilize the external or internal alleviation of above-mentioned protein fragments research to suffer from osteoporosis The osteopenic purposes of mouse.For example, protein fragments of the present invention is used for preparing alleviates in vitro or in vivo due to sclerotin The osteopenic medicine that osteoporosis causes.

In another embodiment, described purposes is to utilize breaking of above-mentioned protein fragments research external or internal suppression bone huge knurl patient The purposes of bone cell activity.For example, protein fragments of the present invention is used for preparing and suppresses broken bone in the huge knurl of bone in vitro or in vivo The medicine of cytoactive.

In embodiments above, more specifically purposes is to utilize above-mentioned protein fragments, is used for screening suppression or alleviates above-mentioned disease Or the purposes of the medicine of symptom.Above-mentioned disease or symptom include, the differentiation of osteoclast of Gegenbaur's cell induction, by RANKL mistake Degree stimulates caused internal sclerotin to reduce, and the sclerotin causing due to osteoporosis reduces, in the huge knurl of bone and osteoclast activity.

Term and definition

Term " g protein coupled receptor (G-protein Coupled Receptor, GPCR) ", is that a class has the conservative of ancient origin Cell surface receptor, multiple environmental stimuluses are reacted by it, cause intracellular response, play in many physiological processes Important function and be the important target spot of drug research.

Term " protein fragments ", refers to the Partial Fragment in LGR4 activated protein.

Term " people source LGR albumen " refers to g protein coupled receptor (the leucine rich repeat in people source with full asphalt mixture motif Containing G prtein coupled receptor) family.

Term " people source LGR4 protein fragments " refers to the protein fragments with the amino acid of sequence shown in SEQ ID NO:1, and it is long Degree is 504aa.Wherein, the total length of people LGR4 albumen is 951aa, and its NCBI accession number is NP_060960.2, this total length The function of albumen is to have regulated and controled eye, kidney, testis, ovary, the internal organs in uterus and intelligence development, and can be as regulation and control white adipose To the switch of brown fat conversion, activate Wnt signal path the generation promoting the cancers such as colon cancer.

Term " RANK ", refers to receptor activation type nuclear receptor factor κ B (Receptor activator of nuclear factor κ B), Its NCBI accession number is NP_033425.3, and its function, for causing breast cancer to occur, promotes osteoclast formation and regulates sclerotin, Promote that lymph node is grown, strengthen the function of T cell growth and BMDC.

Term " RANKL ", refers to receptor activation type nuclear receptor factor κ B part (Receptor activator of nuclear factor κ B Ligand), its NCBI accession number is NP_035743.2, and its function, for promoting osteoclast formation, promotes BMDC Survival, strengthens the function of T cell growth and BMDC.

Term " RANKL-RANK ", refers to the interaction of RANKL and rank protein.

Term " Opg knock-out mice ", refers to Opg knock out mice, is currently a kind of generally acknowledged osteoporosis animal model.

Term " at least 90% homogeneity ", refers to the ratio of sequence same loci number each other more than 90%.In the present invention, The 91%th, described protein sequence preferably has the 92%th, 93% or 94% homogeneity, more preferably have the 95%th, 96% or 97% homogeneity, most preferably has 98% or 99% homogeneity.

Term " medium stringency condition " refers in the solution of 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS (w/v), 65 DEG C Under the conditions of hybridize and wash the condition of film.

Brief description

Fig. 1 show the purifying expression figure of LGR4 protein fragments, wherein,

Fig. 1 a show LGR4 protein fragments SDS-PAGE Coomassie brilliant blue result figure, and arrow show and purifies the LGR obtaining Protein fragments.

Fig. 1 b show LGR4 protein fragments protein immunoblot result figure, and the purpose band of h4 group is for through the inspection of His tag antibody Survey the LGR4 protein fragments confirming.NC, negative control.

Fig. 2 show LGR4 protein fragments and RANKL interaction diagram, its display LGR4 protein fragments (LGR4 ECD) Process LAN 293T cell pyrolysis liquid and RANKL protein immunization precipitation result, RANKL can be co-precipitated with LGR4 ECD, RSPO-1 is positive control.

Fig. 3 show LGR4 protein fragments to scheme with RANK competition binding RANKL, and it shows LGR4 protein fragments (LGR4 ECD) process LAN 293T cell pyrolysis liquid, RANK process LAN 293T cell pyrolysis liquid and RANKL protein immunization precipitation knot Really.The interaction of RANKL and RANK adds the raising of concentration constantly to weaken with LGR4 ECD.

Fig. 4 show LGR4 protein fragments and in vitro suppresses differentiation of osteoclast figure, wherein,

Fig. 4 a shows anti-tartaric acid alkaline phosphatase (TRAP) Enzyme activity assay result figure, and claret giant cell is osteoclast, This result shows that LGR4 protein fragments (LGR4-ECD) can suppress the differentiation of osteoclast induced by RANKL.

The differentiation of osteoclast continuous data of Fig. 4 b display LGR4 protein fragments (LGR4-ECD) suppression RANKL induction divides Analysis figure, wherein * * * p < 0.001, n=3, p value is checked through student T-test and is obtained, and p value is defined as * * * less than 0.001, with Show that LGR4 protein fragments suppression differentiation of osteoclast has that there was no significant difference；N=3 represents that this experiment one group devises 3 repetitions Hole.

Fig. 5 show LGR4 protein fragments suppression Gegenbaur's cell induction differentiation of osteoclast figure, wherein,

Fig. 5 a shows anti-tartaric acid alkaline phosphatase (TRAP) coloration result figure, and claret giant cell is osteoclast.This knot Fruit shows that LGR4 protein fragments (LGR4-ECD) can suppress the differentiation of osteoclast induced by Gegenbaur's cell.

The differentiation of osteoclast continuous data of Fig. 5 b display LGR4 protein fragments (LGR4-ECD) suppression Gegenbaur's cell induction divides Analysis figure, wherein * p < 0.05, n=3.P value is checked through student T-test and is obtained, and p value is defined as *, to show LGR4 less than 0.05 Protein fragments suppresses differentiation of osteoclast to have, and there was no significant difference；N=3 represents that this experiment one group devises 3 repeating holes.

Fig. 6 show the animal reality that LGR4 protein fragments suppression RANKL injects the differentiation of osteoclast causing and bone loss Test complex chart, wherein

Fig. 6 a-1 shows anti-tartaric acid alkaline phosphatase (TRAP) coloration result figure, and claret signal is osteoclast；This knot Fruit shows the internal acute differentiation of osteoclast that LGR4 protein fragments (LGR4-ECD) can suppress RANKL to induce.Fig. 6 a-2 The internal acute differentiation of osteoclast osteoclast table of display LGR4 protein fragments (LGR4-ECD) suppression RANKL induction Area metering data analysis figure, wherein * * * p < 0.001, n=6.P value is checked through student T-test and is obtained, and p value is less than 0.001 It is defined as * * *, to show that the LGR4 protein fragments internal differentiation of osteoclast of suppression has that there was no significant difference；N=6 represents this experiment one Group employs 6 mouse.

Fig. 6 b-1 show microCT result display injection LGR4 Extracellular domain protein coloration result figure, and arrow show bone injury And the reparation district after protein fragments process, this result shows that injection LGR4 protein fragments (LGR4-ECD) can alleviate RANKL The caused bone loss of injection；Fig. 6 b-2 display LGR4 protein fragments alleviates the bone that RANKL injects the bone loss causing Gauge amount data analysis figure, wherein * P < 0.05, * * P < 0.01, n=6.P value is checked through student T-test and is obtained, and p value is little Being defined as * in 0.05, p value is defined as * * less than 0.01, to show that the internal differentiation of osteoclast of LGR4 protein fragments suppression has or not Significant difference；N=6 represents that this experiment one group employs 6 mouse.

Fig. 7 show LGR4 protein fragments and alleviates the overactivity of Opg knock-out mice osteoclast and the dynamic of osteoporosis symptoms Thing tests synthesis result figure

Fig. 7 a show anti-tartaric acid alkaline phosphatase (TRAP) coloration result display injection LGR4 protein fragments (LGR4-ECD, i.e. h4 see table 1) can suppress the overactivity of osteoclast in Opg knock-out mice skull, claret Signal is osteoclast.Fig. 7 a-1 is coloration result figure, and Fig. 7 a-2 is data analysis figure.* P < 0.01, * * * P < 0.001, N=6.P value is checked through student T-test and is obtained, and p value is defined as * * less than 0.01, and p value is defined as * * * less than 0.001, with In showing LGR4 protein fragments suppression Opg knock-out mice body, differentiation of osteoclast has that there was no significant difference；N=6 represents this experiment one Group employs 6 mouse.

Fig. 7 b show microCT result display injection LGR4 protein fragments (LGR4-ECD, i.e. h4 see table 1) energy Alleviating the osteoporosis symptoms of Opg mouse skull, arrow show the reparation district after bone injury and protein fragments are processed.Fig. 7 b-1 For coloration result figure, Fig. 7 b-2 is data analysis figure.* P < 0.05, * * P < 0.01, n=6.P value is examined through student T-test Testing and obtaining, p value is defined as * less than 0.05, and p value is defined as * * less than 0.01, to show that LGR4 protein fragments is alleviated Opg and struck Except mouse bone loss has, there was no significant difference；N=6 represents that this experiment one group employs 6 mouse.

Fig. 7 c show anti-tartaric acid alkaline phosphatase (TRAP) coloration result display injection LGR4 protein fragments (LGR4-ECD, i.e. h4 see table 1) can suppress the overactivity of osteoclast in Opg knock-out mice shin bone, claret Signal is osteoclast.Fig. 7 c-1 is low power lens display result, and Fig. 7 c-2 is high power lens display result, and Fig. 7 c-3 is that data are divided Analysis figure.* * P < 0.001, n=11.P value is checked through student T-test and is obtained, and p value is defined as * * *, to show less than 0.001 In LGR4 protein fragments suppression Opg knock-out mice body, differentiation of osteoclast has that there was no significant difference；N=11 represents this experiment one group Employ 11 mouse.

Fig. 7 d show microCT result display injection LGR4 protein fragments (LGR4-ECD, i.e. h4 see table 1) energy Alleviate the osteoporosis symptoms of Opg mouse tibia.Fig. 7 d-1 is coloration result figure, and Fig. 7 d-2 is data analysis figure.* P < 0.05, N=11.P value is checked through student T-test and is obtained, and p value is defined as * less than 0.05, to show that LGR4 protein fragments alleviates Opg Knock-out mice bone loss has that there was no significant difference；N=11 represents that this experiment one group employs 11 mouse.

Fig. 8 show LGR4 protein fragments and in vitro suppresses giant cell activation results figure in giant cell tumor of bone patient, wherein,

Fig. 8 a show anti-tartaric acid alkaline phosphatase (TRAP) coloration result figure, and claret giant cell is osteoclast；Should Result shows that LGR4 protein fragments (LGR4-ECD) can suppress the differentiation of osteoclast in giant cell tumor of bone primary cell.Figure The continuous data of differentiation of osteoclast in 8b display LGR4 protein fragments (LGR4-ECD) suppression giant cell tumor of bone primary cell Analysis chart, wherein * p < 0.05, * * p < 0.01, n=3.P value is checked through student T-test and is obtained, and p value is defined as less than 0.05 *, p value is defined as * * less than 0.01, to show that in LGR4 protein fragments suppression giant cell tumor of bone, differentiation of osteoclast has or not conspicuousness Difference；N=3 represents that this experiment one group devises 3 repeating holes.

Detailed description of the invention

In conjunction with specific examples below and accompanying drawing, the present invention is described in further detail, the protection content not office of the present invention It is limited to following example.Under the spirit and scope without departing substantially from inventive concept, those skilled in the art it is conceivable that change and excellent Point is all included in the present invention, and with appending claims as protection domain.Implement the process of the present invention, condition, Reagent, experimental technique etc., outside the lower content mentioned specially, be universal knowledege and the common knowledge of this area, this Bright content is not particularly limited.

The purifying of embodiment one: LGR4 protein fragments is expressed

PCR expands LGR4 gene, amplified production is building up to PET-28a (+) on prokaryotic expression carrier, PET-28a-ECD turns Changing Rosetta bacterium (Quan Shijin, cd801-03), building process is with reference to table 1.

Table 1

The general prokaryotic expression method reported according to " Acta Biophysica Sinica in September, 2010 volume 26 the 9th phase: 790-798 ", By above-mentioned vector Rosetta bacterium, abduction delivering LGR4 protein fragments, experimental result is shown in Fig. 1.

Fig. 1 a:LGR4 protein fragments (LGR4 ECD, h4) SDS-PAGE Coomassie brilliant blue result figure, arrow show pure Change the LGR protein fragments obtaining.

Fig. 1 b:LGR4 protein fragments (LGR4 ECD, h4) protein immunoblot result figure, the purpose band of h4 group is warp The LGR4 protein fragments that the detection of His tag antibody confirms.NC, negative control.

Embodiment two: LGR4 protein fragments combines RANKL experiment

By the plasmid containing above-mentioned LGR4 fragment, (building process sees down according to manufacturer's scheme to use lipofectamine2000 Literary composition) process LAN is in 293T cell, and after transfecting 24 hours, cell is washed one time with PBS, scrapes with cell scraper, through 12000rpm After centrifugal one minute, cell block is cracked 15 minutes on ice with RIPA slack melt liquid, centrifuge 10 minutes through 12000rpm 4 degree After take supernatant in new EP pipe, be separately added into 500ngRANKL and 200ngRSPO-1 (positive control)；EP pipe is put quiet mixed 4 degrees Celsius of overnight incubation in clutch (< 12 hours), next day, often pipe added 5 microlitre FLAG-M2 pearls, continued at 4 Celsius Degree hatch 3 hours, after with 3000rpm in 4 degree centrifuge 1 minute, remove supernatant, with 1XSDS loading after washing 3 times with PBS Buffer solution crack pearl, 100 degrees Celsius boil 10 minutes after carry out immunoblot experiment.Immunoblot experiment idiographic flow is as follows:

1) 10% separation gel is configured

2) by whole samples in glue hole, 60 volts are run 30 minutes, and 120 volts are continued to run to bromophenol blue to (about 75 minutes) at the bottom of glue

3) transferring film, 100 volts turn, and below 40KD albumen turns 45 minutes, and 40KD～130KD albumen turns 90 minutes

4) 5% skim milk room temperature is closed 1 hour

5) one resists 4 degrees Celsius to combine overnight

6) PBST washs 4 times, and 5 minutes every time, rear corresponding fluorescence two resisted, room temperature 1 hour

7) PBST washs 3 times, 5 minutes every time, after sweep film with Licor Oddessy loom, record result

Experimental result is shown in Fig. 2

Fig. 2: LGR4 protein fragments (LGR4 ECD) process LAN 293T cell pyrolysis liquid precipitates with RANKL protein immunization As a result, RANKL can be co-precipitated with LGR4 ECD, and RSPO-1 is positive control.

Table 2

Visible according to experimental result, protein fragments of the present invention can be combined with RANKL, has the effect of suppression RANKL Really, protein fragments of the present invention can be used for suppression or treats the sclerotin class disease causing because of osteoclast.

Embodiment three: LGR4 protein fragments suppresses RANKL-RANK Binding experiment

Use lipofectamine2000 according to manufacturer's scheme by 10ug LGR4 fragment of plasmid (seeing table 2), 6ugRank, Process LAN is in 293T cell respectively for 4ug FLAG-Vector and 2ugEGFP-N3, and after transfecting 24 hours, cell is respectively Wash one time with PBS, scrape with cell scraper, after centrifuging one minute through 12000rpm by cell block with RIPA slack melt liquid in ice Upper cracking 15 minutes, takes supernatant after centrifugal 10 minutes in new EP pipe through 12000rpm4 degree Celsius, by following design mixing Protein lysate: 2ugEGFP-N3+2ugFLAG, 2ugRANK+2ugFLAG, 2ugRANK+2ugLGR4 fragment, 2ugRANK+8ugLGR4 fragment, is then separately added into 100ngRANKL again；EP pipe puts in Mute mixer 4 degrees Celsius Overnight incubation, next day, often pipe was initially charged 4 microlitres RANK antibody (1:50), hatches 3 hours, adds 20 for 4 degrees Celsius Microlitre proteinA/G pearl, continues at 4 degrees Celsius and hatches 3 hours, after centrifugal 1 minute in 4 degrees Celsius with 3000rpm, Remove supernatant, after washing 3 times with PBS with 1XSDS sample-loading buffer crack pearl, 100 degrees Celsius boil 10 minutes after exempt from Epidemic disease Blot experiment.Immunoblot experiment idiographic flow is as follows:

1) 10% separation gel is configured

4) 5% skim milk room temperature is closed 1 hour

5) one resists 4 degrees Celsius to combine overnight

6) PBST washs 4 times, and 5 minutes every time, rear upper fluorescence two resisted, room temperature 1 hour

7) PBST washs 3 times, 5 minutes every time, after sweep film with LicorOddessy loom, record result

Experimental result is shown in Fig. 3

Fig. 3: LGR4 protein fragments (LGR4 ECD) process LAN 293T cell pyrolysis liquid, RANK process LAN 293T cell Lysate and RANKL protein immunization precipitation result.The interaction of RANKL and RANK adds dense with LGR4 ECD Degree raising and constantly weaken.

Visible according to experimental result, protein fragments of the present invention has the effect of the combination of suppression RANKL-RANK, this Bright described protein fragments can be used for suppression or treats the sclerotin class disease causing because of osteoclast.

Embodiment four: LGR4 protein fragments in vitro suppresses differentiation of osteoclast

Take 6 weeks big wild types and knock out type mouse femur and shin bone tissue, reject non-bone tissue, through alcohol disinfecting and PBS washing After, cut off the two ends of femur and shin bone with scissors, use 10 milliliters of syringes and 27G syringe needle to cultivate by 2～3ml α-MEM Base/femur goes out bone marrow mononuclear phagocyte to 50 milliliters of centrifuge tubes, and cell suspension goes after centrifuging 8 minutes through 1000rpm Except supernatant, with 5 milliliters containing dual anti-serum-free α-MEM culture medium re-suspended cell, 20 milliliters of erythrocyte cracked liquids of rear addition, Stand 2 minutes after mixing up and down, add 25 milliliters of α-MEM culture mediums containing 10% serum and terminate cracking, through 1000rpm Remove supernatant after centrifugal 8 minutes, with the appropriate α-MEM culture medium re-suspended cell containing 10% serum, add 5ng/ml phagocyte Colony stimulating factor, overnight takes afterwards suspension cell in new culture dish until cell attachment, adds 10ng/ml phagocyte colony thorn Swash the factor to cultivate, after cell attachment covers with, with Versene vitellophag and by 10⁴Individual/hole is accessed in 24 orifice plates, with core because of The sub-part (100ng/ml) of sub-κ B receptor activation and phagocyte colony stimulating factor (10ng/ml)) stimulate bone marrow mononuclear phagocytosis thin Born of the same parents to differentiation of osteoclast, add while adding 100ng/ml RANKL induction differentiation concentration gradient (20ng/ml, 100ng/ml, 200ng/ml) LGR4 protein fragments (LGR4-ECD, i.e. h4) (seeing table 1) process cell, through 6 After its differentiation, according to TRAP staining kit specification, (Sigma company) carries out TRAP dyeing counting statistics cell Differentiation situation.

Experimental result is shown in Fig. 4.

Visible according to experimental result, protein fragments of the present invention is inhibited to the differentiation of osteoclast.Of the present invention Protein fragments can suppress the differentiation of the osteoclast induced by RANKL.

The differentiation of osteoclast of embodiment five: LGR4 protein fragments suppression Gegenbaur's cell induction

Obtain Primary bone marrow MNP (BMM) according to embodiment four method；Meanwhile, the birth suckling mouse of latter 4 days is chosen Obtain Gegenbaur's cell, method particularly includes: separate mouse skull in 4 day age, scraped totally by scalpel after drying through 75% ethanol wash Skull surface is in immersion PBS, surface of bone is white；Thereafter skull is put in 12 orifice plates, add 3.5 milliliters of regular tenacities to disappear Change liquid (3.5 milliliters of α-MEM culture mediums, 35 microlitre 10mg/ml collagenase P, 88 microlitre 0.05% pancreatin) Celsius in 37 Digestion 30 minutes in degree incubator, with in aseptic nipper transfer skull to 6 orifice plates, adds 800 microlitre high intensity digestive juices (1 Milliliter α-MEM culture medium, 20 microlitre 10mg/ml collagenase P, 25 microlitre 0.05% pancreatin), by sterile scissors by craniotomy scissors Become fractionlet, be cut into uniform 30～40 with a piece of intact skulls and be advisable, in 37 degrees Celsius of incubators digestion 60 minutes, Rear 2 milliliters of culture mediums (42 milliliters of α-MEM culture mediums, 7.5 milliliters of hyclones, 500 microlitre 100X are dual anti-) of addition are eventually Only digestion treats that it is adherent；Attached cell is Gegenbaur's cell.

In 96 orifice plates, 1X10 is accessed in every hole⁴Individual BMM cell and 1X10³Individual Gegenbaur's cell, adds after 24 hours adhere-wall culture LGR4 protein fragments (the LGR4-ECD of concentration gradient；20ng/ml, 50ng/ml, 100ng/ml) process cell, through 7 days After cultivation, according to TRAP staining kit specification, (Sigma company) carries out TRAP dyeing and counting statistics cell divides Change situation.

Experimental result is shown in Fig. 5.

Visible according to experimental result, protein fragments of the present invention is inhibited to the differentiation of osteoclast.Of the present invention Protein fragments can suppress the differentiation of the osteoclast induced by Gegenbaur's cell.

Differentiation of osteoclast and the animal of bone loss that the suppression RANKL injection of embodiment six: LGR4 protein fragments causes are real Test

Set up the single injection of reference protein group, RANKL mono-injection group, LGR4 protein fragments (LGR4-ECD, i.e. h4) respectively Group and RANKL, LGR4 protein fragments (LGR4-ECD, i.e. h4) injection group altogether；Expressing protein is injected in 6 week old C57BL/6 mouse skull is subcutaneous, and injection continuously took mouse skull after 14 days, after fixing through 4% formaldehyde, dyes according to TRAP (Sigma company) described in kit specification integrally carries out TRAP dyeing to skull, dyes 2 hours in 37 degrees Celsius；Meanwhile, Skull sample is carried out micro-CT detection (SKYSCAN company) by Shanghai institute of oncology.Above-mentioned experimental result is shown in Fig. 6.

Fig. 6 a-1: anti-tartaric acid alkaline phosphatase (TRAP) coloration result figure, claret signal is osteoclast；This result table The internal acute differentiation of osteoclast that bright LGR4 protein fragments (LGR4-ECD) can suppress RANKL to induce.Fig. 6 a-2 shows Show the internal acute differentiation of osteoclast osteoclast surface of LGR4 protein fragments (LGR4-ECD) suppression RANKL induction Long-pending continuous data analysis chart, wherein * * * p < 0.001, n=6.P value is checked through student T-test and is obtained, and p value is less than 0.001 It is defined as * * *, to show that the LGR4 protein fragments internal differentiation of osteoclast of suppression has that there was no significant difference；N=6 represents this experiment one Group employs 6 mouse.

Fig. 6 b-1:microCT result display injection LGR4 Extracellular domain protein coloration result figure, arrow show bone injury and egg Reparation district after the process of white tiles section, this result shows that injection LGR4 protein fragments (LGR4-ECD) can alleviate RANKL note Penetrate caused bone loss；Fig. 6 b-2 display LGR4 protein fragments alleviates the bone amount that RANKL injects the bone loss causing Continuous data analysis chart, wherein * P < 0.05, * * P < 0.01, n=6.P value is checked through student T-test and is obtained, and p value is little Being defined as * in 0.05, p value is defined as * * less than 0.01, to show that the internal differentiation of osteoclast of LGR4 protein fragments suppression has or not Significant difference；N=6 represents that this experiment one group employs 6 mouse.

Visible according to experimental result, protein fragments of the present invention can suppress the osteoclast being caused by RANKL overstimulation Differentiation and internal sclerotin reduce.

Embodiment seven: LGR4 protein fragments alleviates the overactivity of Opg knock-out mice osteoclast and the dynamic of osteoporosis symptoms Thing is tested

Reference protein group, LGR4 protein fragments are set up respectively to the wild-type mice and Opg knock-out mice at 5 monthly ages (LGR4-ECD, i.e. h4) injection group；Expressing protein is injected in respectively wild-type mice and Opg knock-out mice skull is subcutaneous, Injection continuously took mouse skull after 14 days, after fixing through 4% formaldehyde, and (Sigma according to TRAP staining kit specification Company) TRAP dyeing is integrally carried out to skull, dye 2 hours in 37 degrees Celsius, experimental result is shown in Fig. 7 a；Meanwhile, skull Sample is carried out micro-CT detection (SKYSCAN company) by Shanghai institute of oncology, and experimental result is shown in Fig. 7 b；

With right leg, PBS and LGR4 protein fragments h4 is injected respectively to the left leg of the Opg knock-out mice at 5 monthly ages；Egg will be expressed The shin bone being injected in Opg knock-out mice in vain respectively is subcutaneous, and injection continuously took mouse tibia after 14 days, fixed 24 through 4% formaldehyde After little Shi, carrying out decalcification embedded section, idiographic flow is as follows: shin bone sample fixes 6 again with fresh 4% formaldehyde after clear water washing Hour, clear water washs, and sets to 0 .5MEDTA (PH8.0) respectively normal temperature decalcification 7 days, after clean 10 times with distilled water, put PBS Middle soak at room temperature 3 hours, carries out tissue paraffin embedding process afterwards, and process is as follows: sample puts the 50%th, the 75%th, 75% respectively (4 C overnight), the 85%th, the 95%th, dehydration 2 hours in 95% (4 C overnight), the 100%th, 100% alcohol, after Put 50% dimethylbenzene (alcohol is retarder thinner), 100% dimethylbenzene, saturatingization 2 hours in 100% dimethylbenzene respectively, then put thawing Paraffin oil in 60 C overnight, in fresh wax oil 2 hours, sample embeds；Embedded samples is through Leica slicer (RM2235) Carrying out TRAP dyeing after being cut into 6uM section, dyeing idiographic flow is as follows:

1) section is changed cured 1 hour through 60 degrees Celsius；

2) section respectively through 100% dimethylbenzene, 100% dimethylbenzene, 50% dimethylbenzene (alcohol is retarder thinner), 100% alcohol, 95% alcohol, 85% alcohol, 75% alcohol, distilled water, distilled water aquation, per pass flow process all processes 5 minutes；

3) according to TRAP staining kit specification, (Sigma company) carries out TRAP dyeing to section, in 37 degrees Celsius Dye 2 hours；

4) section is put and is terminated dyeing in distilled water, redyes, mounting in 0.1% methyl green；

5) cut into slices collection result under Olympus microscope, utilize osteomeasure to analyze software (charles's bone measures company) Carrying out osteoclast Parameter analysis, experimental result is shown in Fig. 7 c；Meanwhile, shin bone sample is carried out micro-CT by Shanghai institute of oncology Detection (SKYSCAN company), experimental result is shown in Fig. 7 d.

Fig. 7 a: anti-tartaric acid alkaline phosphatase (TRAP) coloration result display injection LGR4 protein fragments (LGR4-ECD, I.e. h4, sees table 1) overactivity of osteoclast in Opg knock-out mice skull can be suppressed, claret signal is osteoclast. * P < 0.01, * * * P < 0.001, n=6.P value is checked through student T-test and is obtained, and p value is defined as * * less than 0.01, p Value is defined as * * * less than 0.001, has or not significantly with differentiation of osteoclast in showing LGR4 protein fragments suppression Opg knock-out mice body Sex differernce；N=6 represents that this experiment one group employs 6 mouse.

Fig. 7 b:microCT result display injection LGR4 protein fragments (LGR4-ECD, i.e. h4 see table 1) can be alleviated The osteoporosis symptoms of Opg mouse skull, arrow show the reparation district after bone injury and protein fragments are processed.* P < 0.05, * P < 0.01, n=6.P value is checked through student T-test and is obtained, and p value is defined as * less than 0.05, and p value is less than 0.01 definition For * *, to show that LGR4 protein fragments is alleviated Opg knock-out mice bone loss and had that there was no significant difference；N=6 represents this experiment one Group employs 6 mouse.

Fig. 7 c: anti-tartaric acid alkaline phosphatase (TRAP) coloration result display injection LGR4 protein fragments (LGR4-ECD, I.e. h4, sees table 1) overactivity of osteoclast in Opg knock-out mice shin bone can be suppressed, claret signal is osteoclast. Fig. 7 c-1 is low power lens display result, and Fig. 7 c-2 is high power lens display result, and Fig. 7 c-3 is data analysis figure.* * P < 0.001, N=11.P value is checked through student T-test and is obtained, and p value is defined as * * *, to show that LGR4 protein fragments suppresses less than 0.001 In Opg knock-out mice body, differentiation of osteoclast has that there was no significant difference；N=11 represents that this experiment one group employs 11 mouse.

Fig. 7 d:microCT result display injection LGR4 protein fragments (LGR4-ECD, i.e. h4 see table 1) can be alleviated The osteoporosis symptoms of Opg mouse tibia.* P < 0.05, n=11.P value is checked through student T-test and is obtained, and p value is less than 0.05 is defined as *, to show that LGR4 protein fragments is alleviated Opg knock-out mice bone loss and had that there was no significant difference；N=11 represents This experiment one group employs 11 mouse.

Visible according to experimental result, protein fragments of the present invention can alleviate osteoporosis and sclerotin reduces, and can suppress brokenly The overactivity of osteocyte, can suppress owing to Opg knocks out the osteoclast overactivity causing, and treat owing to Opg knocks out The osteoporosis causing osteoclast overactivity and causing.

Embodiment eight: LGR4 protein fragments in vitro suppresses giant cell activation in giant cell tumor of bone patient

In the giant cell tumor of bone of the primary separation of patient add concentration gradient LGR4 protein fragments (LGR4-ECD, i.e. h4, See table 1；100ng/ml, 500ng/ml, 2000ng/ml) process cell, after processing through 4 days, dye examination according to TRAP (Sigma company) described in agent box specification carries out TRAP dyeing counting statistics cell differentiation situation.Experimental result is shown in Fig. 8.

Fig. 8 a: anti-tartaric acid alkaline phosphatase (TRAP) coloration result figure, claret giant cell is osteoclast；This result Show that LGR4 protein fragments (LGR4-ECD) can suppress the differentiation of osteoclast in giant cell tumor of bone primary cell.Fig. 8 b shows Show the continuous data analysis of differentiation of osteoclast in LGR4 protein fragments (LGR4-ECD) suppression giant cell tumor of bone primary cell Figure, wherein * p < 0.05, * * p < 0.01, n=3.P value is checked through student T-test and is obtained, and p value is defined as * less than 0.05, p Value is defined as * * less than 0.01, to show in LGR4 protein fragments suppression giant cell tumor of bone that differentiation of osteoclast has that there was no significant difference； N=3 represents that this experiment one group devises 3 repeating holes.

Visible according to experimental result, protein fragments of the present invention can suppress giant cell tumor of bone, can suppress in giant cell tumor of bone The activity of osteoclast, thus suppress growth and metastasis of tumours.

Claims

1. a protein fragments with the outer segment structure of LGR4 protein extracellular, it is characterised in that the sequence of described protein fragments is tool There is a sequence as follows:

(1) protein sequence as shown in SEQ ID NO.1；

(3) there is replacement and/or the disappearance of one or several amino acid residue at the protein sequence shown in SEQ ID NO.1 and/or add Add but there is the albumen of identical function；

(4) on the basis of the coded sequence of the protein sequence shown in SEQ ID NO.1 through one to several bases replace and/or One inserts to the insertion of several bases and/or the nucleotide sequence of disappearance and large fragment, lack, shifts or after inversion, institute That express and that there is identical function protein fragments；Or

2. encode coded sequence or its degenerate sequence of protein fragments as claimed in claim 1.

3. the protein fragments coded by sequence described in the protein fragments described in claim 1 or claim 2 is thin at the broken bone of preparation treatment Purposes in the medicine of the osteopathy of born of the same parents' induction.

4. purposes as claimed in claim 3, it is characterised in that described protein fragments can suppress the osteoclast that Gegenbaur's cell is induced Differentiation, or can suppress to be reduced by the internal sclerotin caused by RANKL overstimulation, or can alleviate owing to osteoporosis causes Sclerotin reduces, and or can suppress the osteoclast activity in the huge knurl of bone.

5. purposes as claimed in claim 3, it is characterised in that described medicine includes the osteoporosis suppressing osteoclast to cause Medicine or suppression growth and metastasis of tumours medicine.

6. purposes as claimed in claim 5, it is characterised in that the medicine of the osteoporosis that described suppression osteoclast causes is For treating rheumatoid arthritis, bone tissue that arthritis, metastases cause absorb too much caused by the medicine of osteoporosis Thing；The medicine of described suppression growth and metastasis of tumours is the medicine of the suppression huge knurl of bone or giant cell tumor of bone, preferably includes preparation treatment The medicine of the optimum invasive tumor that collagen is sent out.

7. the purposes as described in any one of claim 3-6, it is characterised in that described medicine also include pharmaceutically acceptable salt, Carrier or adjunct ingredient, and/or the medicine that described medicine is prepared with other drug active ingredient combination.

8. the protein fragments coded by sequence described in the protein fragments described in claim 1 or claim 2 in preparation in vitro Or internal suppression differentiation of osteoclast medicine in purposes.

9. purposes as claimed in claim 8, it is characterised in that described protein fragments suppresses skeletonization in vitro or in vivo for preparation The medicine of the differentiation of osteoclast of cell induction；Or, described protein fragments suppresses by RANKL in vitro or in vivo for preparation Internal osteopenic medicine caused by overstimulation；Or, described protein fragments for preparation alleviate in vitro or in vivo due to The osteopenic medicine that osteoporosis causes；Or, described protein fragments suppresses in the huge knurl of bone in vitro or in vivo for preparation The medicine of osteoclast activity.

10. purposes as claimed in claim 9, it is characterised in that described protein fragments be used for screening suppression or alleviate above-mentioned disease or The purposes of the medicine of symptom.