JPWO2007066708A1 - Induction of hard tissue formation based on Wnt5a / SFRP4 signal system - Google Patents

Abstract

Promote or inhibit any action selected from (1) SFRP4 production, (2) Wnt5a production, (3) interaction between Wnt5a and Frizzled receptor, and (4) interaction between Wnt5a and SFRP4 A method for screening a hard tissue formation-inducing compound, comprising evaluating a test compound using a function as an index. A hard tissue formation-inducing agent comprising a compound, Wnt5a, or SFRP4 obtained by the screening method as an active ingredient. A cell for screening a hard tissue formation-inducing compound having the ability to express Wnt5a gene and / or SFRP4 gene. And a screening kit comprising a cell having the ability to express Wnt5a gene and / or SFRP4 gene and a component for retaining the cell in an undifferentiated state.

Description

  The present invention relates to a method for screening a compound that induces hard tissue formation, a hard tissue formation-inducing agent containing the compound as an active ingredient, and a means for screening the compound.

  In recent years, regenerative therapy of bone defects caused by fractures and periodontal tissues destroyed by periodontal disease has been carried out at clinical sites, and has achieved certain results.

  However, current therapies still have problems to overcome, such as being only partially reproducible and causing hard tissue hyperplasia. Therefore, the development of a hard tissue regeneration therapy based on a new idea is still eagerly desired.

  On the other hand, among the periodontal tissues, the periodontal ligament, which is a soft tissue, plays an important role as a reservoir of cell groups essential for periodontal tissue regeneration accompanied by the renewal of alveolar bone and cementum. When periodontal ligament cells are cultured in calcification-inducing medium for a long time in vitro, alkaline phosphatase (ALPase) activity increases and formation of calcified nodules is observed. That is, the periodontal ligament maintains a soft tissue state in vivo, but also has a function of retaining cells having a hard tissue forming ability. Various factors are thought to be involved in maintaining the homeostasis of such periodontal ligament, and gene level analysis relating to the periodontal ligament is underway (see Non-Patent Document 1).

  However, until now, it has not been fully clarified which factors are involved in how hard tissues such as periodontal ligament cells are formed.

  On the other hand, the Wnt family is known as a signal molecule involved in morphogenesis. Wnt signals are known to be transmitted to target cells via the 7-transmembrane receptor (hereinafter also referred to as “Frizzled receptor”) encoded by the Frizzled (FZD) gene. . However, the mechanism is diverse and it is known that there are at least 19 Wnts. In addition, Wnt morphogenesis is complex, and it is known that there are at least three signaling pathways activated by the Wnt family, and it is clear which Wnt activates which pathway. (See Non-Patent Document 2).

The SFRP family is known as a molecule having an inhibitory effect on Wnt, and five or more members have been known so far. However, its action is selective and it is not fully clear which SFRP member acts on which Wnt.
Gene, 275 (2001), 279-286 Annu. Rev. Cell Dev. Biol., 2004, 20: 780-810

  The main object of the present invention is to obtain a hard tissue formation inducing agent that appropriately induces hard tissue formation using the Wnt5a / SFRP4 signal system. In addition, the main object is to obtain a method and screening means for efficiently screening a compound which is an active ingredient of the same agent.

  As a result of intensive studies on the process of inducing hard tissue formation, the present inventor has found that the induction of hard tissue formation is controlled by the involvement of SFRP4 and Wnt5a. It came to be completed.

  That is, the present invention relates to the following matters.

  Item 1: Promote any action selected from (1) SFRP4 production, (2) Wnt5a production, (3) interaction between Wnt5a and Frizzled receptor, and (4) interaction between Wnt5a and SFRP4 Alternatively, a test method for a hard tissue formation-inducing compound, comprising evaluating a test compound using an inhibitory function as an index.

  Item 1A: The screening method according to Item 1, wherein the effects of (1) to (4) are effects on periodontal ligament cells.

  Item 1B: The screening method according to Item 1, comprising: (i) a step of bringing a periodontal ligament cell into which SFRP4 gene is introduced into contact with a test compound; and (ii) a step of analyzing a change in a hard tissue forming ability of the periodontal ligament cell.

  Item 1C: (i) a step of bringing a periodontal ligament cell into contact with a test compound in the presence and absence of Wnt5a, and (ii) a hard tissue forming ability of the periodontal ligament cell after the contact in the presence and absence of Wnt5a Item 2. The screening method according to Item 1, comprising the step of comparing in (1).

Item 1D: (i) a step of bringing a periodontal ligament cell into contact with a test compound in the presence of Wnt5a; and
(Ii) The screening method according to Item 1, comprising a step of analyzing a change in the ability of a periodontal ligament cell to form a hard tissue.

  Item 1E: (i) a step of allowing Wnt5a and SFRP4 to coexist in the presence and absence of a test compound, (ii) a step of comparing the interaction between Wnt5a and SFRP4 in the presence and absence of the test compound The screening method of claim | item 1 containing.

  Item 2: Any one selected from (1) inhibition of SFRP4 production, (2) promotion of Wnt5a production, (3) promotion of interaction between Wnt5a and Frizzled receptor, and (4) inhibition of interaction between Wnt5a and SFRP4 A method for screening a hard tissue formation-promoting compound, comprising evaluating a test compound using the function of

  Item 3: Any one selected from (1) promotion of SFRP4 production, (2) inhibition of Wnt5a production, (3) inhibition of interaction between Wnt5a and Frizzled receptor, and (4) promotion of interaction between Wnt5a and SFRP4 A method for screening a hard tissue formation-inhibiting compound, comprising evaluating a test compound using the function of

Item 4: A hard tissue formation inducer comprising one or more compounds selected from the compounds obtained by the screening method according to Item 1, Item 1A to 1E, Item 2 or Item 3, Wnt5a and SFRP4 as an active ingredient.
Item 4 includes a hard tissue formation-promoting compound obtained by the screening method according to Item 2, a hard tissue formation accelerator comprising one or more selected from Wnt5a and recombinant Wnt5a as an active ingredient, and the screening method according to Item 3. A hard tissue formation inhibitor containing one or more selected from the obtained hard tissue formation inhibitor compound, SFRP4, and recombinant SFRP4 as an active ingredient is included.

Item 5: Hard tissue formation promoter containing the protein according to (w1) or (w2) below as an active ingredient:
(W1) Wnt5a having the amino acid sequence set forth in SEQ ID NO: 1,
(W2) A protein having an amino acid sequence in which a part of the amino acid sequence shown in SEQ ID NO: 1 has been deleted, substituted and / or added, and having a hard tissue formation promoting activity.
The embodiment of Item 5 includes a hard tissue formation promoter containing Wnt5a or recombinant Wnt5a as an active ingredient.

Item 6: The hard tissue formation promoter according to Item 5, through an intermolecular interaction between Wnt5a and SFRP4.
In the embodiment of Item 6, a hard tissue formation promoter containing the protein according to (w1) or (w2) below as an active ingredient through an intermolecular interaction between Wnt5a and SFRP4:
(W1) Wnt5a having the amino acid sequence set forth in SEQ ID NO: 1,
(W2) A protein having an amino acid sequence in which a part of the amino acid sequence set forth in SEQ ID NO: 1 has been deleted, substituted and / or added and having a hard tissue formation promoting activity is included.

Item 7: Hard tissue formation inhibitor containing the protein according to (s1) or (s2) below as an active ingredient:
(S1) SFRP4 having the amino acid sequence set forth in SEQ ID NO: 2,
(S2) A protein having an amino acid sequence in which a part of the amino acid sequence shown in SEQ ID NO: 2 has been deleted, substituted and / or added, and having hard tissue formation inhibitory activity.
The embodiment of Item 7 includes a hard tissue formation inhibitor containing SFRP4 or recombinant SFRP4 as an active ingredient.

Item 8: A cell for screening a hard tissue formation-inducing compound, which has a Wnt5a gene and / or SFRP4 gene expression ability.
Item 8A: The cell according to Item 8, which is a periodontal ligament cell.
Item 8B: The cell according to Item 8, wherein the Wnt5a gene and / or the SFRP4 gene is introduced.
Item 8C: The cell according to Item 8, wherein siRNA of Wnt5a and / or SFRP4 is introduced.

Item 9: A kit for screening a hard tissue formation-inducing compound, comprising a cell having the ability to express Wnt5a gene and / or SFRP4 gene, and a component that retains the cell in an undifferentiated state.
Item 9A: The kit according to Item 9, wherein the component that retains the cells in an undifferentiated state is a cytokine.
Item 9B: The kit according to Item 9, wherein the component that retains cells in an undifferentiated state is FGF-2.

  Hereinafter, the present invention will be described in more detail.

I. Hard Tissue Formation In the present invention, hard tissue means living hard tissue, and includes bone tissue that forms the skeleton of the skull and trunk, alveolar bone that forms periodontal tissue, cementum, and the like.

  Alveolar bone is bone tissue that exists around the teeth and supports and maintains the teeth. Cementum is a hard tissue existing on the surface of the root of the tooth, and is embedded with sharpy fiber ends that firmly connect the teeth and the alveolar bone.

  As a mechanism of hard tissue formation, a differentiation mechanism from mesenchymal stem cells to osteoblasts and cementoblasts is generally known. The mechanism of hard tissue formation in periodontal tissue is thought to be similar to hard tissue formation in other living tissues, and mesenchymal stem cells present in periodontal ligament cells are divided into osteoblasts and cementoblasts. To form a hard tissue.

  The present invention provides compounds that induce, in other words, control such differentiation of hard tissue formation.

  In this specification, hard tissue formation induction includes hard tissue formation promotion that promotes hard tissue formation and hard tissue formation suppression that induces hard tissue formation in a suppressive manner.

  Analysis of the ability to form hard tissue in cells can be carried out by appropriately selecting from known methods.

  For example, as a method for analyzing hard tissue forming ability, (a) a method for measuring alkaline phosphatase activity, (b) a method for measuring the amount of calcified product (or calcified nodules) formed, (c) a hard tissue-related protein One or more methods selected from the group consisting of a method for measuring the production amount and (d) a method for measuring the expression level of the hard tissue-related gene can be mentioned.

  For the measurement of (a), for example, the Bessay method can be used.

  The formation amount of the calcified product (or calcified nodule) in (b) can be measured by, for example, alizarin staining.

  The hard tissue-related protein (c) includes a protein that promotes or suppresses hard tissue formation. Specifically, extracellular matrix proteins such as Osteopontin, Osteocalcin, Collagen type I, Periostin, PLAP-1, and transcription factors such as Runx2 and Osterix are included.

  The hard tissue-related genes in (d) include extracellular matrix proteins such as Osteopontin, Osteocalcin, Collagen type I, Periostin and PLAP-1, and genes encoding transcription factors such as Runx2 and Osterix.

  The means for measuring the protein production in (c) and the gene expression in (d) can be appropriately selected and set from known methods.

  Using the analytical method as described above, when the alkaline phosphatase activity is improved and the amount of calcified (or calcified nodules) is increased, it can be evaluated that the hard tissue forming ability of the cells is promoted. .

  Moreover, when alkaline phosphatase activity falls and the formation amount of a calcification thing (or calcification nodule) has decreased, it can be evaluated that the hard tissue formation ability of the cell was suppressed.

  Furthermore, the ability of cells to form hard tissue can be evaluated using as an index the change in the production amount of a hard tissue-related protein or the change in the expression level of a gene encoding a hard tissue-related protein.

II. Method for screening hard tissue formation inducing compound
II-1. Hard tissue formation of SFRP4
SFRP4 (secreted frizzled-related protein 4) is one of the SFRP families and has a cysteine-rich domain (CRD).

  SFRP4 acts to suppress hard tissue formation differentiation. From this, it is considered that a compound that promotes or suppresses the production of SFRP4 controls the participation of SFRP4 in the hard tissue formation process, in other words, has a function of promoting or suppressing the formation of hard tissue.

  For example, a compound that promotes the production of SFRP4 can exert an effect of suppressing hard tissue formation. On the other hand, a compound that suppresses the production of SFRP4 can exert an effect of promoting hard tissue formation.

  Therefore, a hard tissue formation-inducing compound can be efficiently screened by evaluating a test compound using the function of promoting or suppressing the production of SFRP4 as an index. For example, a compound having a function of promoting the production of SFRP4 can be selected as a hard tissue formation inhibiting compound. In addition, a compound having a function of suppressing the production of SFRP4 can be selected as a hard tissue formation promoting compound.

  The method for examining the function of the test compound to promote or suppress the production of SFRP4 can be appropriately selected from known methods, and is not particularly limited.

  For example, (i) contacting a periodontal ligament cell whose SFRP4 gene expression is controlled with a test compound, and (ii) examining the periodontal ligament cell's ability to form a hard tissue before and after contact. it can.

  Examples of the cells in which the expression of the SFRP4 gene is controlled include SFRP4 gene overexpression periodontal ligament cells and SFRP4 gene expression-suppressed periodontal ligament cells.

  The screening procedure can be appropriately selected and set from known methods within the scope of the effects of the present invention. For example, a plurality of test compounds can be simultaneously screened on an appropriate chip or microarray.

II-2. Involvement of Wnt5a in hard tissue formation and interaction between Wnt5a and Frizzled receptor
Wnt5a is one of the Wnt family and is a hydrophobic signal protein rich in cysteine.

  In the present invention, it has been clarified that Wnt5a promotes hard tissue formation. From this, it is considered that a compound that promotes or suppresses the production of Wnt5a controls the involvement of Wnt5a in the process of hard tissue formation and acts to induce hard tissue formation either in a proactive or inhibitory manner.

  For example, a compound that promotes Wnt5a production or Wnt5a gene expression has an effect of promoting hard tissue formation. On the other hand, a compound that suppresses Wnt5a production or Wnt5a gene expression has an effect of suppressing hard tissue formation.

  Therefore, a hard tissue formation inducing compound can be efficiently screened by evaluating the test compound using the function of promoting or suppressing the production of Wnt5a as an index.

  For example, a compound that promotes Wnt5a production can be screened as a hard tissue formation promoting compound. In addition, a compound that suppresses Wnt5a production can be screened as a hard tissue formation-inhibiting compound.

  It is also known that the Wnt5a signal is transmitted via the Frizzled receptor. Furthermore, it is known that the signal via the Frizzled receptor by Wnt5a passes through the JNK transmission system. In addition, skeletal dysplasia is observed in Wnt5a knockout mice, suggesting that the Wnt5a signal system is involved in hard tissue formation.

  Based on this, it is considered that compounds that promote or suppress the interaction between Wnt5a and Frizzled receptor control the involvement of Wnt5a in the hard tissue formation process and promote the action of promoting or suppressing hard tissue formation. It is done.

  For example, a compound that promotes the interaction between Wnt5a and the Frizzled receptor can exert an effect of promoting hard tissue formation. On the other hand, a compound that suppresses the interaction between Wnt5a and the Frizzled receptor can exert an effect of suppressing hard tissue formation.

  Therefore, a hard tissue formation-inducing compound can be efficiently screened by evaluating the test compound using the function of promoting or suppressing the interaction between Wnt5a and the Frizzled receptor as an index.

  For example, a compound that promotes the interaction between Wnt5a and the Frizzled receptor can be screened as a hard tissue formation promoting compound. In addition, a compound that suppresses the interaction between Wnt5a and the Frizzled receptor can be screened as a hard tissue formation-inhibiting compound.

  Whether the test compound promotes or suppresses the production of Wnt5a can be appropriately selected from known methods, and is not particularly limited. For example, the increase or decrease in the production amount of Wnt5a protein or the increase or decrease in the expression level of Wnt5a gene can be examined by analyzing using a known method.

  The method for examining whether the test compound promotes or suppresses the interaction between Wnt5a and the Frizzled receptor can be appropriately selected from known methods, and is not particularly limited. For example, the interaction or intermolecular binding between Wnt5a and Frizzled receptor can be examined by using a known protein-protein interaction analysis means.

  In addition, the periodontal ligament cells are brought into contact with the test compound in the presence and absence of Wnt5a, and the hard tissue forming ability of the periodontal ligament cells in the presence and absence of Wnt5a is compared and analyzed, thereby performing screening. It can also be done.

  For example, in the presence of Wnt5a, periodontal ligament cells can be brought into contact with the test compound, and screening can be performed by analyzing changes in the alkaline phosphatase activity and / or calcified nodule formation amount of periodontal ligament cells.

  Specifically, a compound that compares changes in alkaline phosphatase activity and / or calcified nodule formation in periodontal ligament cells, and improves alkaline phosphatase activity and / or increases the amount of calcified nodule formation, as a hard tissue formation promoting compound You can choose. Conversely, a compound that decreases the alkaline phosphatase activity of the periodontal ligament cells and / or reduces the amount of calcified nodules can be selected as a hard tissue formation-inhibiting compound.

  The screening procedure is not particularly limited, and can be set as appropriate from known methods within the scope of the effects of the present invention. For example, test compounds can be screened one by one in a solution, or a plurality of compounds can be screened simultaneously on an appropriate chip or microarray.

II-3. Interaction between Wnt5a and SFRP4 In the present invention, it was revealed that SFRP4 is an antagonist of Wnt5a and that Wnt5a and SFRP4 have a direct intermolecular interaction. Furthermore, it became clear that the hard tissue formation was controlled by the interaction.

  From this, it is considered that a compound that inhibits or promotes the interaction between Wnt5a and SFRP4 (in other words, the intermolecular binding between Wnt5a and SFRP4) plays a role of promoting hard tissue formation in an accelerated or suppressive manner. .

  For example, a compound that promotes the interaction between Wnt5a and SFRP4 has an effect of suppressing hard tissue formation. On the other hand, a compound that suppresses or inhibits the interaction between Wnt5a and SFRP4 has an effect of promoting hard tissue formation.

  Therefore, a hard tissue formation-inducing compound can be efficiently screened by evaluating the test compound using the function of promoting or suppressing the interaction between Wnt5a and SFRP4 as an index. For example, by evaluating a compound that promotes the interaction between Wnt5a and SFRP4, it can be screened as a hard tissue formation-inhibiting compound. In addition, a compound that suppresses the interaction between Wnt5a and SFRP4 can be screened as a hard tissue formation promoting compound.

  A method for examining whether a test compound promotes or suppresses the interaction between Wnt5a and SFRP4 can be appropriately selected from known methods.

  In addition, screening can be performed by allowing Wnt5a and SFRP4 to coexist in the presence and absence of a test compound and comparing the interaction between Wnt5a and SFRP4 in the presence and absence of the test compound. . The method for examining the protein-protein interaction is not particularly limited, and can be set by appropriately selecting from known methods. Examples thereof include a coprecipitation method, an ELISA method, and a competitive inhibition method.

  The screening procedure is also not particularly limited, and can be appropriately set from known methods within the scope of the effects of the present invention. For example, a plurality of test compounds can be simultaneously screened on an appropriate chip or microarray.

III. Hard tissue formation inducing agent According to the present invention, a hard tissue formation inducing agent that appropriately induces the formation of hard tissue is provided.

  The hard tissue formation inducing agent includes a hard tissue formation accelerator that promotes hard tissue formation and a hard tissue formation inhibitor that suppresses hard tissue formation.

  The hard tissue formation-inducing compounds include compounds obtained by the above screening methods, Wnt5a, SFRP4, and the like. Wnt5a includes recombinant Wnt5a. SFRP4 includes recombinant SFRP4. As long as the compound which becomes an active ingredient exists in the range with the effect of this invention, 1 type may be sufficient and you may use 2 or more types.

  Recombinant Wnt5a and recombinant SFRP4 can be appropriately obtained according to known methods.

  For example, recombinant Wnt5a can be obtained by introducing an expression vector having a human Wnt5a gene into animal cells or E. coli and purifying the produced protein. Similarly, recombinant SFRP4 can be obtained by introducing an expression vector having a human SFRP4 gene into animal cells or E. coli and purifying the produced protein.

  The hard tissue formation-inducing agent of the present invention can be obtained by appropriately formulating these hard tissue formation-inducing compounds as active ingredients.

  The hard tissue formation promoter can be obtained by using a hard tissue formation promoting compound, Wnt5a or recombinant Wnt5a obtained by the screening method as an active ingredient.

  The hard tissue formation inhibitor can be obtained by using a hard tissue formation inhibitor compound, SFRP4, or recombinant SFRP4 obtained by the screening method as an active ingredient.

Further, the hard tissue formation promoter can be obtained by using the protein described in (w1) or (w2) below as an active ingredient.
(W1) a Wnt5a protein having the amino acid sequence set forth in SEQ ID NO: 1,
(W2) A protein having an amino acid sequence in which a part of the sequence shown in SEQ ID NO: 1 has been deleted, substituted and / or added, and having a hard tissue formation promoting activity.

For example, a hard tissue formation promoter through an intermolecular interaction between Wnt5a and SFRP4 can be obtained by using a protein described in (w1) or (w2) below as an active ingredient.
(W1) a Wnt5a protein having the amino acid sequence set forth in SEQ ID NO: 1,
(W2) A protein having an amino acid sequence in which a part of the sequence shown in SEQ ID NO: 1 has been deleted, substituted and / or added, and having a hard tissue formation promoting activity.

Further, the hard tissue formation inhibitor can be obtained by using the protein described in the following (s1) or (s2) as an active ingredient.
(S1) a SFRP4 protein having the amino acid sequence set forth in SEQ ID NO: 2,
(S2) A protein having an amino acid sequence in which a part of the sequence shown in SEQ ID NO: 2 has been deleted, substituted and / or added, and having a hard tissue formation inhibitory activity.

  The hard tissue formation inducing agent may be prepared by formulating the compound as the active ingredient itself, or formulated by blending a pharmaceutically or sanitary acceptable carrier with the compound as an active ingredient It may be.

  The hard tissue formation-inducing agent can be blended with other medicinal ingredients and additives as long as the effects of the present invention are not impaired.

  The type and blending amount of such a carrier or additive can be appropriately set as long as the effects of the present invention are not impaired.

  The form of the hard tissue formation inducer is not particularly limited, and can be arbitrarily prepared according to the method of use and the like. For example, it can be prepared in the form of liquid, cream, gel, powder, granule and the like.

  Also, the administration method can be appropriately set according to the administration purpose, administration site and the like.

  For example, for the purpose of regenerating periodontal tissue, it is conceivable to locally administer a hard tissue formation inducing agent to the alveolar bone defect during periodontal surgery.

  In addition, for the purpose of bone tissue regeneration of bone disease, local administration to a bone defect site during orthopedic surgery is considered. In this case, since it is assumed that the defect site is larger than that at the time of periodontal surgery, it is preferable to use a carrier having gelation or shaping.

  The hard tissue formation-inducing agent of the present invention can be used as a therapeutic agent or a component of a therapeutic agent for various diseases that can cure, alleviate, or prevent symptoms by promoting or suppressing hard tissue formation.

  For example, in the case of dentistry, a drug for periodontal tissue regeneration including regeneration of alveolar bone and cementum destroyed by periodontal disease, a drug for alveolar bone augmentation used for pretreatment of dental implant treatment, etc. It can be used as a pharmaceutical for promoting osteointegration that can be applied to dental implants or the like, or an active ingredient thereof.

  In the orthopedic field, it can be used as a bone tissue regeneration drug for bone diseases such as fractures, a drug for suppressing pathological ectopic calcification, and the like.

IV. Cells for screening hard tissue formation-inducing compounds It has been clarified that the action or function of Wnt5a and SFRP4 can be used as an indicator of hard tissue formation according to the present invention. Therefore, a cell that retains Wnt5a and / or SFRP4 gene in an expressible state, in other words, a cell that has the ability to express Wnt5a gene and / or SFRP4 gene is suitable as a screening cell for a hard tissue formation-inducing compound. It can be used. Moreover, it is thought that the kit containing the cell and the component that holds the cell in an undifferentiated state can be suitably used as a hard tissue formation-inducing compound screening kit.

IV-1. Screening cells
In other words, the cell having the ability to express the Wnt5a gene and / or the SFRP4 gene is a cell having the Wnt5a gene and / or the SFRP4 gene, but a part or all of which is not completely expressed or very low in expression. The cells are maintained in an undifferentiated state of stem cells having a hard tissue forming ability.

  Periodontal ligament cells are particularly preferably used as screening cells. Periodontal ligament cells retain the ability to highly express the Wnt5a gene and / or the SFRP4 gene, and thus the action or expression variation of Wnt5a and / or SFRP4 can be easily examined.

  The origin of the cell is not particularly limited, and for example, a mouse-derived or human-derived cell can be used.

  Although the kind of cell can be set suitably, for example, a periodontal ligament cell clone, a primary cultured periodontal ligament cell, a subcultured cell, etc. are contained.

  Examples of specific embodiments include mouse-derived periodontal ligament cell clones cloned by limiting dilution, human primary cultured periodontal ligament cells, and osteoblast cell lines such as MC3T3-E1.

  Among these, mouse-derived periodontal ligament cell clones are excellent for introducing foreign genes, and are mouse-derived cells that can be easily distinguished from endogenous genes and molecules when analyzing human genes and molecules. Therefore, it is preferably used.

  The screening cell may be a cell into which a gene has been introduced.

  For example, it may be a cell into which a Wnt5a gene and / or SFRP4 gene has been introduced.

  Wnt5a gene-introduced cells can be obtained, for example, by preparing an expression vector for animal cells using the human Wnt5a gene having the sequence shown in SEQ ID NO: 1, and transforming mouse periodontal ligament cell clones with the vector.

  SFRP4 gene-introduced cells can be obtained, for example, by preparing an animal cell expression vector using the human SFRP4 gene having the sequence shown in SEQ ID NO: 2 and transforming a mouse periodontal ligament cell clone with the vector. it can.

  The screening cells may be ones into which Wnt5a and / or SFRP4 siRNA has been introduced.

  The siRNA-introduced cell can be prepared, for example, by directly introducing SFRP4 siRNA into a periodontal ligament cell or by incorporating it into a siRNA expression vector and introducing it into a periodontal ligament cell.

IV-2. Method for Using Screening Cells By using the above-described screening cells, it is possible to efficiently screen for a hard tissue formation-inducing compound in vitro.

  Examples of the method for using the screening cells include a method comprising contacting a periodontal ligament cell clone with a recombinant Wnt5a or a recombinant SFRP4 protein and a test compound, and analyzing a change in the hard tissue forming ability of the periodontal ligament cell clone Is mentioned. By the method, a compound that improves the hard tissue formation ability of the periodontal ligament cell clone is selected as a hard tissue formation promoting compound, and a compound that decreases the hard tissue formation ability of the periodontal ligament cell clone is selected as a hard tissue formation inhibiting compound. Can do.

  In addition, the screening cell of the present invention can be suitably used in the screening method described above. For example, (i) a method of performing screening using as an indicator whether SFRP4 gene-introduced periodontal ligament cells and a test compound are brought into contact with each other to promote or suppress the production of SFRP4. In the method, a compound that suppresses the production of SFRP4 can be selected as a hard tissue formation promoting compound, and a compound that promotes the production of SFRP4 can be selected as a hard tissue formation inhibiting compound.

IV-3. Screening kit By combining a screening cell and a component that maintains the cell in an undifferentiated state, a kit that can be suitably used for screening for a hard tissue formation-inducing compound is provided.

  The kit allows cells having the ability to express Wnt5a gene and / or SFRP4 gene to be maintained in an undifferentiated state for a long period of time. Thereby, the cells are maintained in a state suitable for screening.

  At the time of screening, screening can be carried out by separating screening cells and components that retain the cells in an undifferentiated state, and then bringing the test compound into contact with the screening cells.

  Cytokines are mentioned as a component which maintains a cell in an undifferentiated state. Examples of cytokines include FGF-2 and LIF.

  An example of a specific embodiment includes a kit containing a mouse periodontal ligament cell clone and FGF-2. In the kit, mouse periodontal ligament cell clones are maintained as cells that can be passaged for a long time in an undifferentiated state, in other words, in a state having the ability to express the Wnt5a gene and / or the SFRP4 gene.

  In the kit of the present invention, other components and means can be appropriately included within the scope of the effects of the present invention as necessary. Examples of other components include Wnt5a and / or SFRP4 detection reagent.

  By applying the hard tissue formation inducing mechanism clarified by the present invention, a hard tissue formation inducing agent is provided that enables tissue regeneration in a form having the original form and function.

  In physiological situations, bone tissue maintains its homeostasis by the simultaneous formation and suppression of permanent hard tissue, which lacks flexibility if it is not properly maintained. Pathological hard tissue regeneration occurs, such as bone tissue formation, excessive calcification, and fusion with soft tissue. Conventionally used cytokines such as FGF-2 and BMP-2 sometimes have a too strong function of promoting the formation of hard tissue. However, if the action of Wnt5a and SFRP4 in the present invention is used, physiological effects can be obtained. A hard tissue formation feedback mechanism can induce hard tissue formation in a properly controlled manner.

  Therefore, according to the present invention, moderate hard tissue formation and excessive calcification can be suppressed, and the formation of bone tissue lacking flexibility and the formation of pathological hard tissue are suppressed, which is harmonious. It is possible to obtain a hard tissue formation inducing agent capable of inducing regeneration of bone tissue and periodontal tissue.

  Due to such characteristics, the hard tissue inducer of the present invention is suitable for the reconstruction of a long bone defect due to a fracture or the like or the regeneration of periodontal tissue including alveolar bone and cementum destroyed by periodontal disease. It can also be used as an agent.

In addition, the present invention enables in vitro screening of compounds suitable for induction of hard tissue formation.
Thus, it is possible to carry out efficiently.

  Furthermore, the screening cells and kits of the present invention appropriately hold an index for screening for a hard tissue formation-inducing compound, making it possible to efficiently screen for a hard tissue formation-inducing compound.

  As described above, the present invention provides a compound that appropriately induces hard tissue formation and a means for efficiently screening the compound.

It is the figure which showed the time-dependent change of ALPase activity in SFRP4 gene transfer MPDL22. It is a figure which shows the result of having investigated the mineralization nodule formation in the culture | cultivation 12th day and the 15th day by the alizarin staining in SFRP4 gene transfer MPDL22. It is the figure which showed the time-dependent change of ALPase activity in siRNA introduction | transduction MPDL22. It is a figure which shows the result of having investigated the calcification nodule formation in culture | cultivation 9-18 days in siRNA introduction | transduction MPDL22 by alizarin staining. It is the figure which showed the analysis result of the expression of Wnt family gene in a human periodontal ligament cell. Lka-PDL, Yod1-PDL and YY-PDL represent three human periodontal ligament cell lines established from different donors. Caco2 is a human colon cancer-derived cell line. It is the figure which showed the result of having analyzed the influence which it has on hard tissue differentiation in MPDL22 by Wnt5a addition by ALPase activity. It is the figure which showed the result which analyzed the influence which it has on the hard tissue differentiation in MPDL22 by Wnt5a addition by the calcification nodule formation by alizarin staining. The left figure shows the stained image obtained by analyzing the concentration of alizarin staining with a densitometer. Moreover, the right figure shows the density | concentration at the time of adding mWnt5a when the density | concentration of control alizarin staining is set to 1 in the graph. It is the figure which showed the detection result of Wnt5a and SFRP4 interaction by co-immunoprecipitation assay (co-immunoprecipitation assay). The result of having analyzed the influence which Wnt5a exerts on JNK activation using the anti-phosphorylated JNK antibody is shown. Moreover, the result of having analyzed the same sample using the anti-JNK antibody is shown. The result of having analyzed the influence of SFRP4 with respect to JNK activation by Wnt5a using the anti- phosphorylated JNK antibody is shown. Moreover, the result of having analyzed the same sample using the anti-JNK antibody is shown.

  EXAMPLES Hereinafter, examples will be shown to describe the present invention more specifically, but the present invention is not limited to the examples.

[Materials and methods]
(1) Creation of periodontal ligament cell clone
2. Periodontal ligament tissue was collected from the root surface of mandibular molar teeth of 5-week-old BALB / c mice, and in alpha modified Eagle medium (α-MEM medium) containing 10% bovine serum and 60 μg / ml kanamycin in a 24-well culture plate The cells were cultured at 37 ° C. in a gas phase of 5% CO 2, and the migrated and proliferated cells were used as mouse periodontal ligament cells (MPDL). After subculture for 12 passages, MPDL was cloned twice by limiting dilution in a passage medium containing 1 μg / ml FGF-2. The alkaline phosphatase (ALPase) activity of the obtained cell line was measured, and MPDL22 having the highest ALPase activity and capable of expressing Wnt5a gene and SFRP4 gene in vitro was used in this experiment.

(2) Analyzing method of hard tissue forming ability In the following examples, hard tissue forming ability was analyzed by measuring ALPase activity and calcified nodule formation. The Bessay method was used for comparative examination of ALPase activity. Moreover, the alizarin dyeing | staining was used for the comparative examination of calcification nodule formation.

Example 1: Analysis of SFRP4 involved in hard tissue formation (1) SFRP4 analysis using SFRP4 gene-introduced cells A human SFRP4 expression vector was prepared by the following method. First, cDNA was obtained from the total RNA extracted from human periodontal ligament cells by reverse transcription, and the protein coding site of human SFRP4 was cloned by polymerase chain (PCR) reaction using it as a template. The CDS site was incorporated into the p3xFLAG-CMV vector to construct a human SFRP4 expression vector.

  A human SFRP4 expression vector was introduced into MPDL22 using Effectene Transfection Reagent, and drug selective culture was performed with neomycin at 400 μg / ml to establish a human SFRP4 constant expression strain. This constitutively expressing strain was cultured in a medium (hereinafter, also referred to as “calcification induction medium”) in which 10 mM β-glycerophosphate and 50 μg / ml ascorbic acid were added to α-MEM medium containing 10% bovine serum, and ALPase activity and Calcified nodule formation was compared with a control strain into which an empty vector was introduced.

  The results of measuring the ALPase activity in MPDL22 on the third day of culture and the sixth day of culture are shown in FIG. As a result, human SFRP4 overexpressing MPDL22 showed statistically significant suppression compared to a control strain in which only the vector was introduced.

  Moreover, the results of alizarin staining on the 12th day of culture and the 15th day of culture are shown in FIG. As a result, it was shown that human SFRP4 overexpressing MPDL22 suppressed the formation of calcified nodules as compared to the control strain in which only the vector was introduced.

Example 2: Functional analysis of SFRP4 using siRNA-introduced cells
Based on the report of Reynolds et al. (Nat Biotechnol. 2004 Mar; 22 (3): 326-30), two types of 19mer base sequences specific to the mouse SFRP4 gene were selected and synthesized.

  Each synthesized DNA was incorporated into a pSilencer siRNA expression vector and introduced into MPDL22. Drug selection culture was performed with neomycin at 400 μg / ml to obtain siRNA constant expression strains, MPDL22 / RNAi-1 and MPDL22 / RNAi-2. In addition, a control strain was obtained by constructing an siRNA expression vector incorporating a random 19 base pair nucleic acid sequence and introducing the gene into MPDL22. Endogenous SFRP4 gene expression in each cell line was analyzed by the SFRP4-specific reverse transcription polymerase chain (RT-PCR) method in RNA extracted from the cell line. Each cell line was cultured in a calcification induction medium, and ALPase activity and calcification nodule formation were compared.

  As a result of semi-quantitative RT-PCR analysis using the housekeeping gene GAPDH, the expression of endogenous SFRP4 gene in PDL22 / RNAi-1 and MPDL22 / RNAi-2 was reduced to about 1/2 that of the control strain. It was confirmed that

  ALPase activity was examined on the 3rd, 6th, and 9th days of culture. Both MPDL22 / RNAi-1 and MPDL22 / RNAi-2 were statistically significant compared to the control strain. (See FIG. 3).

  Furthermore, on day 9, day 12, day 15, day 15 and day 18 of culture, both PDL22 / RNAi-1 and MPDL22 / RNAi-2 showed higher calcification nodules than control strains. Formation was shown (see FIG. 4).

Example 3: Expression of Wnt family in periodontal ligament cells Primers for human Wnt family genes (Wnt1, Wnt3, Wnt4, Wnt5a, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt11, Wnt14) were designed. Using the primers, the expression of Wnt family genes in the established human periodontal ligament cell line was analyzed.

The human periodontal ligament cell line was collected from the center of the root of the first premolar, which was provided by a patient undergoing orthodontic treatment who accepted the purpose of the experiment and accepted the tissue provision. After chopping with 3 and washing 3 times, it was transferred to a Layton tube and cultured in α-MEM medium supplemented with 10% bovine serum. Culturing was performed under conditions of 5% CO ₂ , 37 ° C., and humidity 95%. Cells grown from minced tissue pieces are treated with phosphate buffer containing 0.05% trypsin and 0.02% EDTA to release adherent cells, washed with Hanks buffer, and then subcultured in a 100mm x 20mm cell culture plate. After subculture, cells from passage 7 to passage 13 were established.

  Using three human periodontal ligament cell lines established from different donors, the expression of Wnt family genes was analyzed by RT-PCR. In addition, Caco2, which is a human colorectal cancer-derived cell line, was used for RT-PCR analysis as a positive control because it expressed many types of Wnt genes. The results are shown in FIG.

  As a result, it was clarified that only Wnt5a was expressed in all cell lines, indicating high expression. On the other hand, Wnt3 and Wnt7b gene expression was also observed in one cell line, but expression in the other two cell lines was not detected.

Example 4: Analysis of Wnt5a participation in hard tissue formation
Mouse L cells incorporating the Wnt5a expression gene were cultured in Dulbecco's modified Eagle medium for 4 days, and the culture supernatant was collected and stored frozen. Further, a new medium was added to the cells, and the cells were cultured for 3 days. The supernatant was recovered, and mixed with the cryopreserved culture supernatant to obtain recombinant Wnt5a. The presence of recombinant Wnt5a in the culture supernatant was confirmed by Western blotting using an anti-Wnt5a antibody. On the other hand, the culture supernatant of wild-type L cells was collected by the same method, and this was used in the following experiment as a control for recombinant Wnt5a.

  ALPase activity was examined by adding recombinant Wnt5a to the long-term culture system of MPDL22. The results on the third day of culture, the sixth day of culture, and the ninth day of culture are shown in FIG. As a result, it was shown that the addition of Wnt5a significantly promoted the ALPase activity of MPDL22 as compared with the control.

  Furthermore, the formation of calcified nodules by adding recombinant Wnt5a to the long-term culture system of MPDL22 was examined by alizarin staining. FIG. 7 shows the results of alizarin staining on day 12 after culture. The left figure shows the result of analyzing the concentration of alizarin staining with a densitometer as a stained image. Moreover, the right figure shows the density | concentration at the time of adding mWnt5a when the density | concentration of control alizarin staining is set to 1 with a graph. As a result, it was shown that the addition of Wnt5a significantly promotes the formation of calcified nodules of MPDL22 as compared with the control.

Example 5: Analysis of interaction between Wnt5a and SFRP4 A mouse Wnt5a expression vector and a mouse SFRP4 expression vector with a FLAG tag were simultaneously introduced into a human fetal kidney cell line 293 cells. From the cell solubilized fraction, a co-immunoprecipitation assay using specific antibodies against each molecule was performed to analyze the mutual binding between Wnt5a / SFRP4 molecules. Gene transfer was performed using Effectene Transfection Reagent. Cells were collected and solubilized 24 hours after gene transfer. From the cell solubilized fraction, immunoprecipitation is performed using an anti-FLAG antibody, the precipitate is developed on a reduced 10% SDS gel, the migrated protein is transferred to a membrane, and then the anti-FLAG antibody and anti-mouse Wnt5a antibody are applied. Western blot analysis was performed to analyze the binding between SFRP molecules and Wnt5a molecules. In addition, from the cell solubilized fraction, immunoprecipitation was performed using an anti-mouse Wnt5a antibody, the precipitate was developed on a reduced 10% SDS gel, and the migrated protein was transferred to a membrane, followed by anti-FLAG antibody. In addition, Western blot analysis was performed using an anti-mouse Wnt5a antibody to examine whether or not the SFRP4 molecule co-precipitates when the Wnt5a molecule was immunoprecipitated. The protein was detected by Western blot using a secondary antibody labeled with horseradish peroxidase (HRP) and ECL as a substrate. The detection result is shown in FIG.

  As a result, it was found that when SFRP4 was immunoprecipitated, Wnt5a was coprecipitated, whereas when Wnt5a was immunoprecipitated, SFRP4 was coprecipitated. From this, it became clear that Wnt5a and SFRP4 show a direct intermolecular mutual bond.

Example 6: Analysis of activation of JNK signaling system by Wnt5a MPDL22 was stimulated with recombinant Wnt5a, and activation of JNK signaling system by Wnt5a was examined by Western blotting using anti-phosphorylated JNK antibody.

  MPDL was stimulated with the above recombinant Wnt5a or control for 30 minutes, and the cells were collected and solubilized. The solubilized fraction was run on a 10% SDS gel and transferred to a membrane. Next, Western blot analysis using an anti-phosphorylated JNK antibody and an HRP-labeled secondary antibody was performed, and the amount of phosphorylated JNK when MPDL was stimulated with recombinant Wnt5a was compared with a control group stimulated with a control. Further, the same sample was subjected to Western blot analysis using an anti-JNK antibody, and it was confirmed that there was no difference in the amount of protein migrated on the SDS gel in the recombinant Wnt5a-stimulated cells and the control group. As the anti-phosphorylated JNK antibody and JNK antibody, specific antibodies that recognize both the JNK protein of p54 and p46 were used. The analysis results are shown in FIG.

  As a result, it was revealed that the amount of phosphorylated JNK was increased in the recombinant Wnt5a-stimulated MPDL compared with the control group, and it was found that the JNK signal was activated by Wnt5a in periodontal ligament cells.

Example 7: Analysis of the effect of SFRP4 on JNK activation by Wnt5a Further, when stimulating MPDL with recombinant Wnt5a under the same conditions as in Example 6, various concentrations of recombinant SFRP4 (purchased from R & D System) In the same manner as in Example 6, the effect of SFRP4 on JNK signal activation by Wnt5a stimulation was examined using anti-phosphorylated JNK antibody and JNK antibody. The results are shown in FIG.

  As a result, it became clear that recombinant SFRP4 inhibited phosphorylation of JNK activated by Wnt5a in periodontal ligament cells in a concentration-dependent manner, and that SFRP4 inhibited JNK signaling activation by Wnt5a. It was done.

Claims (9)

(1) SFRP4 production, (2) Wnt5a production, (3) interaction between Wnt5a and Frizzled receptor,
And (4) a screening method for a hard tissue formation-inducing compound, wherein a test compound is evaluated using as an index a function that promotes or suppresses any action selected from the interaction between Wnt5a and SFRP4. (1) Suppression of SFRP4 production, (2) Promotion of Wnt5a production, (3) Promotion of interaction between Wnt5a and Frizzled receptor, and (4) Suppression of interaction between Wnt5a and SFRP4 A screening method for a hard tissue formation-promoting compound, which comprises evaluating a test compound as an index. (1) Promote SFRP4 production, (2) Suppress Wnt5a production, (3) Suppress the interaction between Wnt5a and Frizzled receptor, and (4) Promote the interaction between Wnt5a and SFRP4. The screening method of the hard tissue formation inhibitory compound characterized by evaluating a test compound as a parameter | index. The hard tissue formation inducer which uses as an active ingredient 1 or more chosen from the compound obtained by the screening method in any one of Claims 1-3, Wnt5a, and SFRP4. Hard tissue formation promoter containing the protein according to (w1) or (w2) below as an active ingredient:
(W1) Wnt5a having the amino acid sequence set forth in SEQ ID NO: 1,
(W2) A protein having an amino acid sequence in which a part of the amino acid sequence shown in SEQ ID NO: 1 has been deleted, substituted and / or added, and having a hard tissue formation promoting activity. The hard-tissue formation promoter of Claim 5 through the intermolecular interaction of Wnt5a and SFRP4. Hard tissue formation inhibitor comprising the protein according to (s1) or (s2) below as an active ingredient:
(S1) SFRP4 having the amino acid sequence set forth in SEQ ID NO: 2,
(S2) A protein having an amino acid sequence in which a part of the amino acid sequence shown in SEQ ID NO: 2 has been deleted, substituted and / or added, and having hard tissue formation inhibitory activity. A cell for screening a hard tissue formation-inducing compound having the ability to express Wnt5a gene and / or SFRP4 gene. A kit for screening a hard tissue formation-inducing compound, comprising a cell having the ability to express Wnt5a gene and / or SFRP4 gene, and a component that retains the cell in an undifferentiated state.

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