WO2007066708A1 - Induction de la formation de tissu dur sur la base d’un systeme de signalisation wnt5a/sfrp4 - Google Patents

Induction de la formation de tissu dur sur la base d’un systeme de signalisation wnt5a/sfrp4 Download PDF

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Publication number
WO2007066708A1
WO2007066708A1 PCT/JP2006/324416 JP2006324416W WO2007066708A1 WO 2007066708 A1 WO2007066708 A1 WO 2007066708A1 JP 2006324416 W JP2006324416 W JP 2006324416W WO 2007066708 A1 WO2007066708 A1 WO 2007066708A1
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WIPO (PCT)
Prior art keywords
wnt5a
hard tissue
sfrp4
tissue formation
cells
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PCT/JP2006/324416
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English (en)
Japanese (ja)
Inventor
Shinya Murakami
Satoru Yamada
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Osaka Industrial Promotion Organization
Osaka University
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Priority to JP2007549165A priority Critical patent/JPWO2007066708A1/ja
Publication of WO2007066708A1 publication Critical patent/WO2007066708A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • 000 1 a method of screening a substance that induces growth, a step of using a substance as an active ingredient, and a step of screwing a substance.
  • the method of weaving which was destroyed by a bone fracture in 2000, has come to be used in clinical settings, and has achieved certain results.
  • n is known as a guana involved in morphogenesis. It is known that the gnuna of n is transmitted via the 7-volume (below, zze-d volume) which is carried by the zzed (ZD) gene. However, the set is diverse, and it is known that there are at least 19 or more Wn. It is known that the involvement of Wn is complicated and that there are at least 3 or more Gna pathways activated by Wn, and which Wn activates which pathway. Not visible in minutes (2).
  • RP7 is also known as a molecule having an effect on Wn, and five or more men have been known so far. However, there is a choice for it, and it is not clear enough which RP member acts on which Wn.
  • the main purpose is to obtain proper induction of growth using the GN4 system.
  • the main purpose is to obtain a method and a stage for screwing the substance that is the active ingredient of.
  • test compound evaluating the test compound as a function of promoting and suppressing the use of the shift selected from Wn5a and RP4.
  • Wn5a Selected from Wn5a The above is the active ingredient
  • Propellant a product obtained by the screening method described in 3, RP4 Inbinant RP4 selected as an active ingredient
  • Wn5a having the amino series described in (1), which contains the white matter described in 002 5 () or (w2) as an active ingredient
  • a white matter having sex which has a part of the ano sequence described in (w2), which is substituted and / or added.
  • the active ingredient is Wn5a or Wn5a Agents are included.
  • (w2) part of the ano sequence has a substituted and / or added ano sequence, and has a white matter
  • Items such as 7 include agents containing RP4 or recombinant RP4 as the active ingredient.
  • 8 Wn5a element and / or RP4 element is introduced.
  • 8C Wn5a or RP4 sRNA is introduced.
  • g Wn5a and / or RP4 gene-carrying cells and a skunk kit containing components that retain the cells in the state.
  • woven means a biological tissue, and includes tissues forming the case of the skull and trunk, and alveolar bone and cement constituting the tissue.
  • 0027 is a tissue existing around and supporting the teeth.
  • the cementum is a weave that exists on the root surface of the tooth, and is a layer that strengthens
  • the end is buried.
  • 002 The mechanism of intercellular cells and cement cells is generally known as a stain. It is believed that the stains in the weave are similar to those in other weaves, and cells present in the root form cells or cement to form the weave. 002, we will guide, in other words, provide what we call this transformation.
  • induction refers to promotion of induction of growth, and control of suppression of induction of growth. You can
  • a method for measuring aquatase activity (a) a method for measuring aquatase activity, (b) a method for measuring (for limestone), (c) a method for measuring white matter, ( d) Mention the method of measuring genes, the above method selected from the group consisting of.
  • the Bessa method can be used.
  • white matter contains white matter that promotes and suppresses growth. Physically, it includes cells such as Os eopon n Os eocacn Co agen y e Pe os n P AP and children such as R nx2 Os e x.
  • Os eopon n Os eocacn Co agen pe Pe os for the 003 (d) gene It contains genes such as nPAP and the child-bearing genes such as Rnx Os ex.
  • the means for measuring the amount of white matter in (c) and the gene in (d) can be selected and set by the following method.
  • RP4 (sec e ed m zzed ea edpo en4) is an RP and has a stain domain (CRD).
  • RP4 acts inhibitory to the components. From this, it is considered that the compound that promotes and suppresses the growth of RP4 has a function of controlling the growth process of RP4, in other words, promoting and suppressing the growth.
  • the substance can be further squeezed.
  • a substance having a function of promoting the growth of RP4 can be selected as a substance.
  • a substance having a function of suppressing the growth of RP4 can be selected as a substance.
  • the method by which the substance controls the function of promoting and suppressing the growth of RP4 can be selected from the following methods and is not particularly limited.
  • it can be adjusted by contacting () with the root material that has expressed the RP4 gene, and analyzing the cell performance before and after ().
  • the cells that have expressed the RP4 gene include the RP4 gene.
  • Wn5a is a W-type, and has a high quality of stain.
  • the substance can be further squeezed.
  • a substance that promotes Wn5a production can be used as a hardening compound.
  • a substance that suppresses Wn5a growth is possible to be used as a hard substance and use it for sintering.
  • Wn5a gnuna is performed through the zzed container. Furthermore, it is known that Guna via the zzed container by Wn5a passes through NK. In addition, Wn5a knockout mice are observed to fail. These results suggest that the Wn5a guna system is hardened. From this, it is considered that it exerts the effects of promoting and suppressing the use of Wn5a and zzed volume, controlling the involvement of Wn5a in the development process, and inducing the growth in an accelerated and suppressive manner.
  • a compound that promotes the use of Wn5a and a zzed container may exert a growth promoting effect.
  • a compound that suppresses the use of Wn5a and zzed volume can exert an effect of suppressing the growth.
  • the substance can be further squeezed.
  • an object that suppresses the use of Wn5a and the zzed body can be used as a hard material.
  • the substance can promote and suppress the growth of Wn5a by any of the following methods and is not particularly limited.
  • Wn5a white matter loss can be determined by analyzing the Wn5a gene loss using public methods.
  • the method for controlling the test substance to promote and suppress the use of Wn5a and zzed container can also be selected from these methods and is not particularly limited.
  • the substance that improves calcosis and / or increases calcification is selected as a substance. can do.
  • a substance that reduces the cellular susceptibility and / or the ash dust of the cell can be selected as the substance.
  • test substance can be directly squeezed in the liquid, or a plurality of substances can be simultaneously squeezed on an appropriate chip or equate.
  • RP4 is an antagonist of Wn5a and that Wn5a and RP4 have direct molecular uses. Furthermore, it was also revealed that the composition was controlled by use.
  • the substance can be further screened. For example, by evaluating an object that promotes the use of Wn5a and RP4, it can be used as an object. Also, an object that suppresses the use of Wn5a and RP4 can be used as an object.
  • the method for controlling the use of Wn5a and RP4 can be selected from the following methods. It is also possible to perform the squanning by allowing Wn5a and RP4 to coexist under and without the test compound and comparing the use of Wn5a and RP4 with and without the test compound.
  • the method to be used is not particularly limited, and can be set by the method of. For example, co-precipitation, EA, and competition.
  • the order of screening is also not particularly limited, and can be determined by the method within the range in which the effect is clear. For example, multiple objects can be simultaneously squeezed on a suitable chip or an equal.
  • 007 includes a promoter that promotes growth
  • the products include W n5a RP4 and the like obtained by the above-mentioned Sung method.
  • Wn5a includes recombinant Wn5a.
  • RP4 includes the recombinant RP4. It may be a seed or 2 or more as long as it is an active ingredient and within the range that produces the clear results.
  • Nbinant Wn5a and Nbinant RP4 can be obtained according to the method of.
  • recombinant Wn5a can be obtained by introducing an expression vector having the human Wn5a gene into the large intestine of an animal and refining the produced protein.
  • recombinant RP4 can be obtained by introducing an expression vector having the human RP4 gene into an animal or the large intestine and refining the produced protein.
  • the progressive agent can be obtained by using, as an active ingredient, the progressive compound obtained by the above-mentioned Sung method, Wn5a or Wn5a. Further, the antagonism can be obtained by using the substance obtained by the above-mentioned Sung method, RP4, or recombinant RP4 as an active ingredient.
  • the progressing agent can be obtained by using the white matter described in the following () or (w2) as an active ingredient.
  • the progressing agent via the use of Wn 5a and RP4 is as follows (
  • control agent can be obtained by using the white matter described in () or (52) as an active ingredient.
  • the product itself as the above-mentioned active ingredient may be prepared, or the product may be prepared by blending the product as an active ingredient and naturally grown.
  • 008 No particular limitation is imposed on 008 and it can be optionally prepared depending on the method of use.
  • it can be prepared in the form of gum, gum, powder.
  • the administration method can also be determined according to the administration. For example, for the purpose of weaving raw material, it is possible to give alveolar to.
  • alveolar drugs that have been destroyed by-reinjection of cementum alveolar drugs used for dental implant treatment, and ointment drugs applicable to dental implants, etc. It can be used as an active ingredient of
  • tissue drug for fractures In addition, in the medical field, it can be used as a tissue drug for fractures, a drug for pathological lime, etc.
  • Wn 5a RP4 could be obtained as a target for hardening. From this, it is considered that cells that retain the Wn 5a or RP4 gene in an expressible state, in other words, cells that have the Wn 5a gene and / or the RP4 gene, can be suitably used as a substance clone. Moreover, it is considered that a kit containing a component that retains cells in a chemically modified state can be suitably used as a screening kit.
  • a cell having the Wn 5a gene and / or the RP4 gene means that the cell has the Wn 5a gene and / or the RP4 gene, but all of them are not completely expressed or are very actual.
  • the stem cells that have the potential to compete are maintained.
  • a tooth root is particularly preferably used as the screw.
  • the cells retain the ability to express the Wn5a gene and / or the RP4 gene, and can easily control the expression of Wn5a or RP4.
  • the cell is not particularly limited, and may be, for example, human or human.
  • 0097 cells can be determined, for example, root root, first generation
  • somatic embodiments include strains such as Uskun, human, MC3T31, etc., which have been cloned by the limit. 0099 and Uskun are suitable for use because they are excellent in foreign gene transfer, and when it is easy to distinguish genes and daughters in Usus, it is easy to distinguish them.
  • 0100 it may be a cell having a skung or a gene introduced therein.
  • 010 may be a cell into which the Wn5a gene and / or the RP4 gene have been introduced. It can be obtained by transforming Uskun.
  • 0103 also has a RP4 gene, for example, the sequence described in Sequence 2.
  • it may be the one into which the sRNA of Squan, Wn5a or RP4 is introduced.
  • the 0105 SRNA eg, RP4 sRNA directly introduced into the cell, can be prepared by incorporating it into the sRNA vector and introducing it into the cell.
  • Examples of the method of using 0107 cells include a method including analyzing the ability of the roots to function in the root roots. To improve the ability of root roots to
  • the method of squeezing can be mentioned as a method of contacting the RP4 gene product to promote and suppress the growth of RP4.
  • a substance that suppresses the growth of RP4 is selected as a hard product
  • a substance that promotes the growth of RP4 is selected as a hard product.
  • a kit suitable for use in the cells is provided.
  • the 0110 kit contains cells having the Wn5a gene and / or the RP4 gene,
  • Squag can be carried out by separating the components that retain the cells in the state during the squeezing, and then contacting the test substance with the squeezing cell.
  • Cytokines can be cited as examples of components that have cells. Examples of cytokines include G 2 and the like.
  • kits containing Uskun G2 are kits containing Uskun G2.
  • the Uskin is in a chemical state, in other words, has a Wn5a gene and / or an RP4 gene for a long period of time. It is held as.
  • the kit of Akira can contain other components or means, if necessary, within the range where the effects of Akira are achieved.
  • Other ingredients include Wn 5a or RP4.
  • the Ming sung cells and kits have adequately retained the markings for squeezing the objects and allow efficient squeezing.
  • Caco2 is a human colon cancer.
  • the root tissue was collected from a mouse and 24 cells were subjected to 37, 5 C 2 feeding in 0 g and 60 gm canine MEM) and used as a mouse.
  • MPD MPD 22
  • the human RP4 vector was constructed by the following method. First, cDNA was obtained from RNA extracted from human cells by reverse transcription, and the position of RP4 was cloned by polymerase chain (PCR) reaction using it as a template. The CD site was incorporated into the p3x AG CMV vector to construct the human RP4 vector.
  • PCR polymerase chain
  • the 0127 RP4 vector was introduced into MPD22 using EceneTansconReagen and cultured at 400 gm of neoin to establish human RP4 normal expression.
  • This normal expression was cultivated in 10 MEM medium supplemented with M-gousine and 50 gm asbestic acid (below, both), and the A Pase activity and lime formation were compared with those of emp Veco introduced. I killed it.
  • Fig. 2 shows the results of the zan color on the 12th and 15th.
  • Each of the 0131-generated DNAs was incorporated into the pence SRNA vector and transferred into MPD22.
  • MPD 22 RNA 1 and MPD 22 RNA 2 were obtained.
  • MPD 22 RNA 1 and MPD 22 RNA 2 were obtained.
  • sRNA vector incorporating a random sequence of 19 were obtained.
  • RNA vector incorporating a random sequence of 19 was obtained.
  • Analysis was carried out by the RP4 transcription polymerase chain (RT PCR) method on the endogenous, RP4 gene, and RNA extracted from the cell line in each of the cells.
  • RT PCR RP4 transcription polymerase chain
  • RNA and MPD 22 RNA 2 showed a statistically significant increase in A Pase activity as compared with that of the mouse (3).
  • the more proliferating cells were treated with 0,05 top and 0,02 EDTA to release the adherent cells, and the cells were cultivated in a 00 mm x 20 mm plate with a tube, and the cells from the 7th to 13th cells were released.
  • 0,05 top and 0,02 EDTA to release the adherent cells
  • the cells were cultivated in a 00 mm x 20 mm plate with a tube, and the cells from the 7th to 13th cells were released.
  • Wn gene was analyzed by RT PCR method using 3 human species established from Donna.
  • Caco which is a human colon cancer, expresses many kinds of Wn genes, and was used as a positivity for RT PCR analysis. The results are shown in Fig. 5.
  • the cells containing the Wn5a gene were cultivated in a cultivated area, collected, and stored frozen. Furthermore, fresh medium was added to the cells, the cells were triturated, collected, frozen, and mixed to obtain recombinant Wn5a. It was confirmed by the Western method using the upper Wn5a and Wn5a bodies. On the other hand, the wild cells were collected by the same method as above, and used as the agent for the recombinant Wn5a in the following experiments.
  • Human 293 cells were cotransfected with the Us Wn5a vector and the Us RP4 vector from AG. Then, we carried out (commmnopecp a onassa) using the specific body for each offspring and analyzed the Wn5a RP4 binding. I used a gene, E ceneTans c on Reagen. Cells were harvested 24 hours after the gene.
  • the NK gnuna by Wn5a was examined by the stimulating method using the re-JNK body stimulated with Libina Wn5 MPD 22.
  • the recombinant Wn5a or PD described in 0145 was stimulated with PD to 30 cells, and the cells were recovered and solubilized. Transcription was performed at 0 D and men.
  • nbnant RP4 purchased from RD s em
  • RP4 against NK guna caused by Wn5a was increased.
  • the sound was examined in the same way as in Implementation 6, using the body and NK body. The results are shown in the figure. As a result, it was revealed that the concentration of nbinant RP4 suppressed the NK of NK activated by Wn5a in the root follicles, and that RP4 was shown to inhibit the NK gn of Wn5a.

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Abstract

L’invention divulgue : un procédé de criblage d’un composé pouvant induire la formation d’un tissu dur comprenant l’évaluation d’un composé voulu par l’utilisation en guise d’indicateur d’une fonction de promotion ou d’inhibition d’une activité quelle qu’elle soit choisie parmi les suivantes (1) à (4) : (1) une aptitude à produire SFRP4 ; (2) une aptitude à produire Wnt5a ; (3) une interaction entre Wnt5a et un récepteur Frizzled et (4) une interaction entre Wnt5a et SFRP4 ; un inducteur de formation de tissu dur comprenant un composé obtenu par le procédé de criblage, Wnt5a ou SFRP4 en constituant un ingrédient actif ; une cellule destinée à servir au criblage d’un composé pouvant induire la formation d’un tissu dur, ladite cellule pouvant exprimer un gène Wnt5a et/ou un gène SFRP4, et un kit de criblage comprenant une cellule pouvant exprimer un gène Wnt5a et/ou un gène SFRP4 et un composant pouvant maintenir la cellule dans un état indifférencié.
PCT/JP2006/324416 2005-12-07 2006-12-07 Induction de la formation de tissu dur sur la base d’un systeme de signalisation wnt5a/sfrp4 WO2007066708A1 (fr)

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JP2007549165A JPWO2007066708A1 (ja) 2005-12-07 2006-12-07 Ｗｎｔ５ａ／ＳＦＲＰ４シグナル系に基づく硬組織形成誘導

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111548989A (zh) * 2020-05-25 2020-08-18 西安医学院 一种通过sfrp4促进棕色脂肪分化的方法及应用

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WO2002092128A1 (fr) * 2001-05-11 2002-11-21 Genzyme Corporation Compositions et procedes permettant de reguler le phosphate de serum
WO2004018669A1 (fr) * 2002-08-21 2004-03-04 Proteinexpress Co., Ltd. Kinases 2 induites par des sels et leur utilisation
WO2004022082A1 (fr) * 2002-09-04 2004-03-18 Proteinexpress Co., Ltd. Inhibiteur de l'implantation d'embryons
WO2004072298A1 (fr) * 2003-02-12 2004-08-26 G2 Therapies Ltd Methodes de diagnostic et de traitement de maladies inflammatoires au moyen de pac-1 (dusp2)

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CN111548989A (zh) * 2020-05-25 2020-08-18 西安医学院 一种通过sfrp4促进棕色脂肪分化的方法及应用
CN111548989B (zh) * 2020-05-25 2023-04-14 西安医学院 一种通过sfrp4促进棕色脂肪分化的方法及应用

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