Peptides and cell proliferation promoting agents

Abstract

To provide novel peptides, and cell proliferation promoting agents comprising a peptide having cell proliferation promoting activity.SOLUTION: A cell proliferation promoting agent comprises a peptide of (1) or (2): (1) a peptide having an amino acid sequence CX1a from the N terminus in this order; (2) a peptide having an amino acid sequence X0bCX1b from the N terminus in this order. The peptide is (I) or (II): (I) a peptide having an amino acid sequence CX1a from the N terminus in this order, and a length of amino acid residues is from 2 to 21; (II) a peptide having an amino acid sequence X0bCX1b from the N terminus in this order, and the length of amino acid residues is from 3 to 22, where X1a and X1b are independently K, A, G, F, W, Y, S, T, N, Q, C, D, E, R or P, and X0b is Y, F or W.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a peptide and a cell proliferation promoter. [Background technology]

[0002] It is important to activate cell proliferation in order to inhibit cell loss, increase cell numbers, form tissues, etc. Furthermore, today, technologies for tissue formation through in vitro cell proliferation are advancing in fields such as medicine and food and beverages.

[0003] For example, skeletal muscles account for about 40% of the body weight in humans, and contribute greatly to bodily functions such as maintaining motor function, maintaining posture, and energy metabolism. Skeletal muscles have the ability to regenerate when damaged by exercise or injury, and satellite cells, a type of muscle stem cell, play a major role in this regeneration. Satellite cells are usually in a quiescent state, but are activated by stimuli such as muscle damage to resume the cell division cycle, self-replicate, or differentiate into myoblasts, and differentiated myoblasts fuse with existing muscle fibers or with each other to form new muscle fibers, thereby regenerating damaged skeletal muscles. However, it is known that satellite cells decrease with age, and their proliferation is reduced by diabetes, for example, and delayed muscle regeneration causes a decrease in muscle mass (sarcopenia), etc.

[0004] Furthermore, in view of food shortages and other issues, the development of cultured meat produced by culturing cells is also progressing. For example, attempts have been made to produce cultured meat by mixing muscle cells obtained by proliferating and inducing differentiation of cells collected from cows, pigs, sheep, fish, etc., or artificial pluripotent stem cells (iPS cells), with lipids, etc. For example, Patent Document 1 reports the production of cultured meat by culturing cells in a cell culture gel containing adult cow blood plasma and a coagulant to form three-dimensional tissue.

[0005] For this reason, it is important to provide new techniques for promoting cell proliferation, regardless of the field, such as medicine or food and beverage. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2023-47559 A Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present disclosure is to provide a novel peptide and to provide a cell proliferation-promoting agent containing a peptide having cell proliferation-promoting activity. [Means for solving the problem]

[0008] As a result of extensive investigations, the present inventors have found that: (1) the amino acid sequence CX 1a or (2) a peptide having the amino acid sequence X in order from the N-terminus. 0b CX 1b A peptide having the formula (wherein X 1a and X 1b are each independently K, A, G, F, W, Y, S, T, N, Q, C, D, E, R, or P; X 0b is Y, F, or W), cell proliferation can be promoted. The present invention was completed through further investigation based on this finding, and the present disclosure includes, for example, the following representative aspects. Item 1. A cell proliferation promoter comprising the following peptide (1) or (2): (1) The amino acid sequence CX, in order from the N-terminus 1a having (2) Amino acid sequence X in order from the N-terminus 0b CX 1b have (where X 1a and X 1b are each independently K, A, G, F, W, Y, S, T, N, Q, C, D, E, R, or P; X 0b is Y, F or W). Item 2. The peptide (1) is a peptide having a length of 2 to 21 amino acid residues, or The peptide (2) is a peptide having a length of 3 to 22 amino acid residues. Item 1. A cell proliferation promoter as described in Item 1. Item 3. The peptide (1) comprises or consists of the following amino acid sequence (1) in order from the N-terminus: The peptide (2) comprises or consists of the following amino acid sequence (2) in order from the N-terminus: Item 3. The cell proliferation promoter according to item 1 or 2, Amino acid sequence (1) CKNGGFF (SEQ ID NO: 17), CK, CKN, CKNG (SEQ ID NO: 16), CKNAGFF (SEQ ID NO: 18), CKNGAFF (SEQ ID NO: 19), CKNGGAF (SEQ ID NO: 20), CKNGGFA (SEQ ID NO: 21), CKKGGFF (SEQ ID NO: 34), CKNGGFFLR (SEQ ID NO: 37), CKNGGFFLRIH (SEQ ID NO: 39), or CKNGGFFLRIHPD (SEQ ID NO: 41) Amino acid sequence (2) YCKNGGF (SEQ ID NO: 45), YCK, YCKN (SEQ ID NO: 1), YCKNG (SEQ ID NO: 44), YCKNAGFF (SEQ ID NO: 46), YCKNGAFF (SEQ ID NO: 47), YCKNGGAF (SEQ ID NO: 48), YCKNGGFA (SEQ ID NO: 49), YCKNGGFF (SEQ ID NO: 50), YCKKGGFF (SEQ ID NO: 51), YCKNGGFFLR (SEQ ID NO: 52), YCKNGGFFLRIH (SEQ ID NO: 53), or YCKNGGFFLRIHPD (SEQ ID NO: 54). Item 4. The cell proliferation promoter according to any one of Items 1 to 3, which is used in combination with a basic fibroblast growth factor. Item 5. The cell proliferation promoter according to any one of Items 1 to 4, wherein the cells are at least one type selected from the group consisting of tissue stem cells, induced pluripotent stem cells, embryonic stem cells and myoblasts. Item 6. The cell proliferation promoter according to any one of Items 1 to 5, wherein the cells are at least one type selected from the group consisting of mesenchymal stem cells, satellite cells, induced pluripotent stem cells, embryonic stem cells, and myoblasts. Item 7. A peptide according to the following (I) or (II): (I) Amino acid sequence CX in order from the N-terminus 1a and having an amino acid residue length of 2 to 21. (II) Amino acid sequence X in order from the N-terminus 0b CX 1b and has a length of 3 to 22 amino acid residues. (where X 1a and X 1b are each independently K, A, G, F, W, Y, S, T, N, Q, C, D, E, R, or P; X 0b is Y, F or W). Item 8. The peptide (I) comprises or consists of the following amino acid sequence (I) in order from the N-terminus: The peptide (II) comprises or consists of the following amino acid sequence (II) in the order from the N-terminus: Item 7. The peptide according to item 7: Amino acid sequence (I) CKNGGFF (SEQ ID NO: 17), CK, CKN, CKNG (SEQ ID NO: 16), CKNAGFF (SEQ ID NO: 18), CKNGAFF (SEQ ID NO: 19), CKNGGAF (SEQ ID NO: 20), CKNGGFA (SEQ ID NO: 21), CKKGGFF (SEQ ID NO: 34), CKNGGFFLR (SEQ ID NO: 37), CKNGGFFLRIH (SEQ ID NO: 39), or CKNGGFFLRIHPD (SEQ ID NO: 41) Amino acid sequence (II) YCKNGGF (SEQ ID NO: 45), YCK, YCKN (SEQ ID NO: 1), YCKNG (SEQ ID NO: 44), YCKNAGFF (SEQ ID NO: 46), YCKNGAFF (SEQ ID NO: 47), YCKNGGAF (SEQ ID NO: 48), YCKNGGFA (SEQ ID NO: 49), YCKNGGFF (SEQ ID NO: 50), YCKKGGFF (SEQ ID NO: 51), YCKNGGFFLR (SEQ ID NO: 52), YCKNGGFFLRIH (SEQ ID NO: 53) or YCKNGGFFLRIHPD (SEQ ID NO: 54). Item 9. The peptide according to Item 7 or 8, wherein the peptide (I) has the amino acid sequence CKN in this order from the N-terminus. Effect of the Invention

[0009] According to the present disclosure, a novel peptide can be provided. In addition, according to the present disclosure, a cell proliferation-promoting agent containing a peptide having cell proliferation-promoting activity can be provided. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows that the peptide of Example 1 and the peptide of Example 2 have cell proliferation-promoting activity. [Diagram 2] FIG. 2 shows the cell proliferation activity when each amino acid constituting the peptide of Example 1 is substituted with A (alanine). [Diagram 3] FIG. 3 shows the cell proliferation activity when the first to third amino acids from the N-terminus of the peptide of Example 1 were substituted with other amino acids. [Figure 4] FIG. 4 shows that the peptides shortened from the peptide of Example 1 (Examples 3, 4, and 6) and the peptides shortened from the peptide of Example 2 (Examples 5 and 7) also have good cell proliferation-promoting activity. [Diagram 5] FIG. 5 shows that the peptide obtained by lengthening the C-terminus of the peptide of Example 1 has cell proliferation-promoting activity equal to or greater than that of the peptide of Example 1. [Figure 6] FIG. 6 shows that the cell proliferation-promoting activity is peptide dose-dependent. [Figure 7] FIG. 7 shows the results when the peptide of the present disclosure was allowed to coexist with FGF2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, the embodiments included in the present disclosure will be described in more detail. In the present disclosure, "containing" also includes the meaning of "consisting essentially of" and "consisting of".

[0012] Cell proliferation promoter The present disclosure encompasses a cell proliferation-promoting agent comprising the following peptide (1) or (2): (1) The amino acid sequence CX, in order from the N-terminus1a having (2) Amino acid sequence X in order from the N-terminus 0b CX 1b have (where X 1a and X 1b are each independently K, A, G, F, W, Y, S, T, N, Q, C, D, E, R, or P; X 0b is Y, F or W).

[0013] peptide The amino acid residue length of the peptide (1) (hereinafter sometimes referred to as "peptide (1)") and the peptide (2) (hereinafter sometimes referred to as "peptide (2)") is not limited. The amino acid residue length of peptide (1) is preferably 2 to 50, more preferably 2 to 30, even more preferably 2 to 25, particularly preferably 2 to 21, particularly preferably 2 to 20, particularly preferably 2 to 19, etc. are exemplified. In these ranges, the lower limit of the amino acid residue length is not limited as long as it is 2 or more, and more preferably, the lower limit is 2, 3, 4, 5, 6, or 7 amino acid residues. In these ranges, the upper limit of the amino acid residue length is also not limited, and more preferably, the upper limit is 19, 18, 17, 16, 15, 14, or 13 amino acid residues. The lower limit and the upper limit may be any combination, for example, the amino acid residue length may be any of 2 to 18, 2 to 17, 2 to 16, 3 to 18, 3 to 17, 3 to 16, etc.

[0014] The amino acid residue length of peptide (2) is preferably 3 to 51, more preferably 3 to 31, even more preferably 3 to 26, particularly preferably 3 to 22, particularly preferably 2 to 21, particularly preferably 3 to 20, etc. In these ranges, the lower limit of the amino acid residue length is not limited as long as it is 3 or more, and more preferably, the lower limit is 3, 4, 5, 6, 7, or 8 amino acid residues. In these ranges, the upper limit of the amino acid residue length is also not limited, and more preferably, the upper limit is 20, 19, 18, 17, 16, 15, 14, or 13 amino acid residues. The lower limit and the upper limit may be any combination, for example, any combination of amino acid residue lengths of 3 to 19, 3 to 18, 3 to 17, 4 to 19, 4 to 18, 3 to 17, etc.

[0015] Peptide (1) has the amino acid sequence CX 1a That is, the first amino acid from the N-terminus is C and the second amino acid is X. 1a Peptide (2) is composed of the amino acid sequence X from the N-terminus. 0b CX 1b That is, the first amino acid from the N-terminus is X 0b , the second amino acid is C, and the third amino acid is X. 1b Here, X 1a and X 1b are each independently K, A, G, F, W, Y, S, T, N, Q, C, D, E, R, or P. 1a and X 1b are each independently preferably K, A, F, S, N, D, R or P, more preferably K, S, D, R or P, and even more preferably K. 0b is Y, F or W, and preferably Y is exemplified.

[0016] In peptide (1) (for example, in the amino acid sequence represented by sequence 1 described below), there is no further amino acid on the N-terminal side of the first amino acid C. Similarly, in peptide (2) (for example, in the amino acid sequence represented by sequence 2 described below), there is no further amino acid on the N-terminal side of the first amino acid X. 0bThere are no further amino acids on the N-terminal side of the amino acid sequence CX 1a "The amino acid sequence X in order from the N-terminus 0b CX 1b " means that peptides (1) and (2) of the present disclosure each have the amino acid sequence CX 1a "The amino acid sequence X starting from the N-terminus 0b CX 1b " can be rephrased as having.

[0017] Although not limiting the present disclosure, examples of the peptide (1) that constitute the first and second amino acids from the N-terminus (with the first amino acid being C) are preferably CK, CA, CF, CS, CN, CD, CR, or CP, more preferably CK, CS, CD, or CR, and even more preferably CK.

[0018] Although not limiting the present disclosure, examples of the amino acid sequence constituting the first, second and third positions in order from the N-terminus of peptide (2) include preferably YCK, YCA, YCF, YCS, YCN, YCD, YCR or YCP, more preferably YCK, YCS, YCD or YCR, and even more preferably YCK.

[0019] When the peptide (1) has three or more amino acid residues, the first C and the second X from the N-terminus 1a The amino acids constituting the third to C-terminus following the above are not particularly limited as long as they are amino acids capable of constituting a peptide, and may be either natural amino acids or non-natural amino acids. 0b , 2nd C, 3rd X 1aThe amino acids constituting the fourth to C-terminus following the above are not particularly limited as long as they are amino acids capable of constituting a peptide, and may be either natural or non-natural amino acids. Examples of amino acids include hydrophobic amino acids (aliphatic amino acids with a small side chain volume) such as G (glycine) and A (alanine); hydrophobic amino acids (aliphatic amino acids with a large side chain volume) such as V (valine), L (leucine), I (isoleucine), and M (methionine); aromatic amino acids such as F (phenylalanine), Y (tyrosine), and W (tryptophan); polar uncharged amino acids such as C (cysteine), T (threonine), S (serine), Q (glutamine), and N (asparagine); negatively charged amino acids such as D (aspartic acid) and E (glutamic acid); positively charged amino acids such as H (histidine), K (lysine), and R (arginine); and imino acids such as P (proline).

[0020] Without being limiting to the present disclosure, when the number of amino acid residues in peptide (1) is 3 or more, examples of peptide (1) include a peptide that comprises or consists of the sequence from the 1st to the 3rd, the 1st to the 4th, the 1st to the 5th, the 1st to the 6th, the 1st to the 7th, the 1st to the 8th, the 1st to the 9th, the 1st to the 10th, the 1st to the 11th, the 1st to the 12th, the 1st to the 13th, the 1st to the 14th, or the 1st to the 15th of the N-terminus of the amino acid sequence shown in the following sequence 1. Amino acid sequence: CX 1a X 2a X 3a X 4a X 5a X 6a X 7a X 8a X 9a X 10a X 11a X 12a X 13a X 14a (Array 1)

[0021] Without being limiting to the present disclosure, when the number of amino acid residues in peptide (2) is 4 or more, examples of peptide (2) include a peptide that comprises or consists of the sequence from the 1st to the 4th, the 1st to the 5th, the 1st to the 6th, the 1st to the 7th, the 1st to the 8th, the 1st to the 9th, the 1st to the 10th, the 1st to the 11th, the 1st to the 12th, the 1st to the 13th, the 1st to the 14th, the 1st to the 15th, or the 1st to the 16th of the N-terminus of the amino acid sequence shown in the following sequence 2. Amino acid sequence: X 0b CX 1b X 2b X 3b X 4b X 5b X 6b X 7b X 8b X 9b X 10b X 11b X 12b X 13b X 14b (Array 2)

[0022] In the amino acid sequence represented by sequence 1, the first amino acid C and the second amino acid X from the N-terminus 1a is the same as described above. In addition, in the amino acid sequence represented by sequence 2, the first amino acid from the N-terminus, X 0b , the second amino acid C, the third amino acid X 1b is explained in the same manner as above.

[0023] The third amino acid from the N-terminus of the amino acid sequence represented by sequence 1 (X 2a) is preferably exemplified by N, A, G, S, T, Q, C, D, E, K, R, H or P, more preferably by N, S, T, Q or K, and even more preferably by N or K. Although not limiting the present disclosure, examples of the amino acid sequence constituting the first to third amino acids from the N-terminus of peptide (1) are preferably exemplified by CKN, CAN, CFN, CSN, CNN, CDN, CRN, CPN, CKA, CKS, CKT, CKQ, CKD, CKK or CKP, and more preferably by CKN or CKK.

[0024] The fourth amino acid from the N-terminus of the amino acid sequence represented by sequence 2 (X 2b ) are preferably exemplified by N, A, G, S, T, Q, C, D, E, K, R, H or P, more preferably by N, S, T, Q or K, and even more preferably by N or K. An example of the amino acid sequence constituting the first to fourth positions in order from the N-terminus of peptide (2) is a peptide in which Y, F or W has been added to the N-terminus of the amino acid sequence constituting the first to third positions of peptide (1). Preferred examples of the amino acid sequence constituting the first to fourth positions from the N-terminus of peptide (2) include YCKN (SEQ ID NO: 1), YCAN (SEQ ID NO: 2), YCFN (SEQ ID NO: 3), YCSN (SEQ ID NO: 4), YCNN (SEQ ID NO: 5), YCDN (SEQ ID NO: 6), YCRN (SEQ ID NO: 7), YCPN (SEQ ID NO: 8), YCKA (SEQ ID NO: 9), YCKS (SEQ ID NO: 10), YCKT (SEQ ID NO: 11), YCKQ (SEQ ID NO: 12), YCKD (SEQ ID NO: 13), YCKK (SEQ ID NO: 14) and YCKP (SEQ ID NO: 15), more preferably YCKN (SEQ ID NO: 1) or YCKK (SEQ ID NO: 14), and even more preferably YCKN (SEQ ID NO: 1).

[0025] The fourth amino acid from the N-terminus of the amino acid sequence represented by sequence 1 (X 3a ) as the amino acid at the 5th position from the N-terminus of the amino acid sequence represented by sequence 2 (X 3b ) as an amino acid, X 3a and X 3bare each independently preferably exemplified by G or A, more preferably exemplified by G. As an amino acid sequence constituting the first to fourth positions in order from the N-terminus of peptide (1), preferably an amino acid sequence in which the fourth amino acid in the amino acid sequence constituting the first to third positions of peptide (1) is G or A, more preferably G. For example, when the first to third amino acid in peptide (1) is CKN and the fourth amino acid is G, the amino acid sequence constituting the first to fourth positions can be written as CKNG (SEQ ID NO: 16). As an amino acid sequence constituting the first to fifth positions in order from the N-terminus of peptide (2), preferably an amino acid sequence in which the fifth amino acid in the amino acid sequence constituting the first to fourth positions of peptide (2) is G or A, more preferably G.

[0026] The fifth amino acid sequence (X 4a ) as the amino acid at the 6th position (X 4b ) as an amino acid, X 4a and X 4b are each independently preferably exemplified by G or A, and more preferably exemplified by G.

[0027] The sixth amino acid sequence (X 5a ) as the seventh amino acid of the amino acid sequence represented by sequence 2 (X 5b ) as an amino acid, X 5a and X 5b are each independently preferably F, W, Y, A or G, more preferably F or A, and particularly preferably F.

[0028] The seventh amino acid in the sequence represented by sequence 1 (X 6a ) as the amino acid at position 8 (X 6b ) as an amino acid, X 6a and X 6b are each independently preferably F, W, Y, A or G, more preferably F or A, and particularly preferably F.

[0029] The eighth amino acid sequence (X 7a ) as the amino acid at position 9 (X 7b ) as an amino acid, X 7a and X 7b are each independently preferably L, V, I or M, more preferably L.

[0030] The ninth amino acid in the sequence represented by sequence 1 (X 8a ) as the amino acid at position 10 (X 8b ) as an amino acid, X 8a and X 8b are each independently preferably exemplified by R, H or K, and more preferably exemplified by R.

[0031] The 10th amino acid sequence (X 9a ) as the amino acid at position 11 (X 9b ) as an amino acid, X 9a and X 9b are each independently preferably I, L, V or M, and more preferably I.

[0032] The 11th amino acid sequence (X 10a ) as the amino acid at position 12 (X 10b ) as an amino acid, X 10a and X 10b are each independently preferably H, K or R, and more preferably H.

[0033] The 12th amino acid sequence (X 11a ) as the amino acid at position 13 (X 11b ) as an amino acid, X 11a and X 11b are each independently preferably exemplified by P.

[0034] The 13th amino acid sequence (X 12a ) as the amino acid at position 14 (X 12b ) as an amino acid, X 12a and X 12b are each independently preferably exemplified by D or E, and more preferably exemplified by D.

[0035] The 14th amino acid in the sequence represented by sequence 1 (X 13a ) as the amino acid at position 14 (X 13b ) as an amino acid, X 13a and X 13b are each independently preferably exemplified by G or A, and more preferably exemplified by G.

[0036] The 15th amino acid sequence (X 14a ) as the amino acid at position 16 (X 14b ) as an amino acid, X 14a and X 14b are each independently preferably exemplified by R, H or K, and more preferably exemplified by R.

[0037] The amino acids constituting the 16th amino acid from the N-terminus to the C-terminus of peptide (1) may be appropriately determined so long as they do not interfere with the effects of the present disclosure. Similarly, the amino acids constituting the 17th amino acid from the N-terminus to the C-terminus of peptide (2) may be appropriately determined so long as they do not interfere with the effects of the present disclosure.

[0038] When peptide (1) is a peptide having 4 or more amino acid residues in length, a preferred embodiment of peptide (1) is, without being limited to the present disclosure, a peptide having the following amino acid sequence in order from the first amino acid at the N-terminus: When peptide (1) is a peptide having 4 or more amino acid residues in length, peptide (1) may be a peptide consisting of the following amino acid sequence: CKNG (SEQ ID NO: 16), CKNGGFF (SEQ ID NO: 17), CKNAGFF (SEQ ID NO: 18), CKNGAFF (SEQ ID NO: 19), CKNGGAF (SEQ ID NO: 20), CKNGGFA (SEQ ID NO: 21), CANGGFF (SEQ ID NO: 22), CFNGGFF (SEQ ID NO: 23), CSNGGFF (SEQ ID NO: 24), CNNGGFF (SEQ ID NO: 25), CDNGGFF (SEQ ID NO: 26), CRNGGFF (SEQ ID NO: 27), CPNGGFF (SEQ ID NO: 28), CKAGGFF (SEQ ID NO: 29), CKSGGFF (SEQ ID NO: 30), CKT GGFF (SEQ ID NO: 31), CKQGGFF (SEQ ID NO: 32), CKDGGFF (SEQ ID NO: 33), CKKGGFF (SEQ ID NO: 34), CKPGGFF (SEQ ID NO: 35), CKNGGFFL (SEQ ID NO: 36), CKNGGFFLR (SEQ ID NO: 37), CKNGGFFLRI (SEQ ID NO: 38), CKNGGFFLRIH (SEQ ID NO: 39), CKNGGFFLRIHP (SEQ ID NO: 40), CKNGGFFLRIHPD (SEQ ID NO: 41), CKNGGFFLRIHPDG (SEQ ID NO: 42), CKNGGFFLRIHPDGR (SEQ ID NO: 43)

[0039] When peptide (2) is a peptide having a length of 5 or more amino acid residues, without limiting the present disclosure, preferred embodiments of peptide (2) include peptides having or consisting of an amino acid sequence in which Y, F or W is added as the first amino acid to the N-terminus of the amino acid sequence of peptide (1) shown in any one of SEQ ID NOs: 16 to 43, and more preferably peptides having or consisting of an amino acid sequence to which Y is added. For example, the amino acid sequence in which Y is added as the first amino acid to the N-terminus of the amino acid sequence shown in SEQ ID NO: 16 is represented as YCKNG (SEQ ID NO: 44).

[0040] Furthermore, although not limiting the present disclosure, a more preferred embodiment of peptide (1) is, for example, a peptide having or consisting of the following amino acid sequence, in order from the first amino acid at the N-terminus: CKNGGFF (SEQ ID NO: 17), CK, CKN, CKNG (SEQ ID NO: 16), CKNAGFF (SEQ ID NO: 18), CKNGAFF (SEQ ID NO: 19), CKNGGAF (SEQ ID NO: 20), CKNGGFA (SEQ ID NO: 21), CKKGGFF (SEQ ID NO: 34), CKNGGFFLR (SEQ ID NO: 37), CKNGGFFLRIH (SEQ ID NO: 39), or CKNGGFFLRIHPD (SEQ ID NO: 41)

[0041] Furthermore, although not limiting the present disclosure, a more preferred embodiment of peptide (2) is, for example, a peptide having or consisting of the following amino acid sequence, in order from the first amino acid at the N-terminus: YCKNGGF (SEQ ID NO: 45), YCK, YCKN (SEQ ID NO: 1), YCKNG (SEQ ID NO: 44), YCKNAGFF (SEQ ID NO: 46), YCKNGAFF (SEQ ID NO: 47), YCKNGGAF (SEQ ID NO: 48), YCKNGGFA (SEQ ID NO: 49), YCKNGGFF (SEQ ID NO: 50), YCKKGGFF (SEQ ID NO: 51), YCKNGGFFLR (SEQ ID NO: 52), YCKNGGFFLRIH (SEQ ID NO: 53), or YCKNGGFFLRIHPD (SEQ ID NO: 54).

[0042] Peptides (1) and (2) of the present disclosure (hereinafter, peptides (1) and (2) may be collectively referred to as "peptides of the present disclosure") can be produced according to procedures known in the art. For example, they can be produced by general peptide chemical synthesis methods (e.g., the azide method, the acid chloride method, the acid anhydride method, the mixed acid anhydride method, the DDC method, the active ester method, the carboimidazole method, the oxidation-reduction method, etc.) based on information on the target amino acid sequence. The synthesis can be performed by either solid-phase synthesis or liquid-phase synthesis. In addition, the peptides of the present disclosure can be produced by genetic engineering techniques using polynucleotides encoding the peptides.

[0043] The peptide of the present disclosure may be chemically modified at its terminal amino acid residue, so long as it has cell proliferation-promoting activity.

[0044] For example, the C-terminus of the peptide of the present disclosure may be a carboxyl group (-COOH), a carboxylate (-COO-), an amide group (-CONH2) or an ester (-COOR). Here, R in the ester is a C group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1-6 Alkyl groups: cyclopentyl, cyclohexyl, etc. 3-8 Cycloalkyl groups: phenyl, α-naphthyl, etc. 6-12 Aryl groups; phenyl-C such as benzyl and phenethyl 1-2 Alkyl group; α-naphthyl-C such as α-naphthylmethyl 1-2 C, such as alkyl group 7-14 Examples include an aralkyl group; a pivaloyloxymethyl group, etc. Although not limiting the present disclosure, when the C-terminus is chemically modified, preferred examples include a carboxyl group and an amide group, and more preferred examples include an amide group.

[0045] For example, the N-terminal amino group of the peptide of the present disclosure may be protected with a protecting group (e.g., a formyl group, a C1-6 acyl group such as a C1-6 alkanoyl group such as an acetyl group, etc.), or may be myristoylated, pyroglutamylated, methylated, etc.

[0046] The peptide of the present disclosure may have amino acid residues other than the terminals chemically modified as long as it has cell proliferation promoting activity. Examples of chemical modifications include amidation, esterification, etc. of carboxyl groups; protection of amino groups with protective groups, etc. The esterification and protective groups are explained in the same manner as the modification of the terminals described above. The peptide of the present invention is preferably one in which amino acid residues other than the terminals are not chemically modified.

[0047] The peptide of the present disclosure may be in the form of a salt with an acid or a base. The salt is not particularly limited, and either an acid salt or a basic salt can be used. Examples of acid salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0048] The peptide of the present disclosure may be used in a cyclic state in which the aforementioned N-terminal amino acid and C-terminal amino acid constituting the peptide of the present disclosure are bonded together. The peptide of the present disclosure may also be used in a cyclic state in which the aforementioned N-terminal amino acid and non-C-terminal amino acid constituting the peptide of the present disclosure are bonded together, or in a cyclic state in which a non-N-terminal amino acid and a C-terminal amino acid are bonded together, or in a cyclic state in which a non-N-terminal amino acid and a non-C-terminal amino acid are bonded together. As long as the effects of the present disclosure can be obtained, the bond is not limited, and examples of the bond include an amide bond (peptide bond, etc.), an ester bond, a disulfide bond, etc., and the peptide can be made to form a cyclic structure in its entirety or in part according to a conventionally known procedure.

[0049] When the peptide of the present disclosure is used in a cyclic form, an amino acid may be bound to the N-terminal position of the N-terminal amino acid (for example, when the N-terminal amino acid and the C-terminal amino acid are bound to form a cyclic form), but the peptide of the present disclosure, when in a linear form, is preferably bound to the first amino acid C or X. 0b It can be defined as a sequence in which there is no further amino acid on the N-terminal side of the amino acid C or X (the N-terminus constituting the peptide is the amino acid C or X). 0b That is, the amino acid sequence is from the first amino acid to the C-terminus as a linear main chain, and when the ring is cyclic, there is no restriction that an amino acid may be located on the N-terminal side of the first amino acid.

[0050] The peptide of the present disclosure may be in the form of a solvate. The solvent is not particularly limited, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0051] The peptides of the present disclosure may be used alone or in combination of two or more.

[0052] The cell proliferation promoter of the present disclosure contains at least one of peptide (1) and peptide (2). The content of the peptide of the present disclosure in the cell proliferation promoter is not limited, and the total amount of the peptide of the present disclosure is, for example, 0.0001 to 100% by mass, preferably 0.001 to 80% by mass. The lower limit of the content is, for example, 0.01% by mass, 0.1% by mass, 1% by mass, 5% by mass, 10% by mass, 20% by mass, 30% by mass, 40% by mass, 50% by mass, 60% by mass, 70% by mass, 80% by mass, or 90% by mass.

[0053] The dosage form of the cell proliferation promoter of the present disclosure is not limited, and may be any of liquid forms such as liquids, emulsions, and suspensions, and semi-solid or solid forms such as powders, granules, tablets, capsules, gels, and lyophilized liquids. The cell proliferation promoter of the present disclosure may further contain any component within a range that does not interfere with the effects of the present disclosure, and examples of such components include carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, colorants, fragrances, chelating agents, pH adjusters, and preservatives. The components may be used alone or in combination of two or more types, and the amount of each component may be appropriately determined within a range that does not interfere with the effects of the present disclosure. The cell proliferation promoter of the present disclosure can be produced by using the peptide of the present disclosure according to the conventionally known normal procedures according to the dosage form, etc., and further mixing any of the above-mentioned components as necessary.

[0054] The cell growth promoter of the present disclosure can be used by adding it to a conventionally known cell culture medium, etc. Examples of cell culture medium, etc. include cell culture medium, etc., and preferably include the medium, etc. used in the test examples described below. The amount of the promoter added to the medium, etc. may be appropriately determined. Although not limiting the present disclosure, examples of the content (total amount) of the peptide of the present disclosure in the cell culture medium include 1 to 1000 μM, 20 to 800 μM, 80 to 600 μM, etc.

[0055] The cell proliferation promoter of the present disclosure may be applied to a subject (subject animal), whether administered orally or parenterally (percutaneous (including mucosal), enteral, intravenous, pulmonary, subcutaneous, intramuscular, etc.). The subject (subject animal) is also not limited, and examples include humans, non-human mammals (dogs, cats, cows, pigs, horses, etc.), and poultry (chickens, quail, turkeys, ducks, geese, etc.). When the cell proliferation promoter of the present disclosure is applied to a subject (subject animal), the amount of the promoter to be applied (administered (ingested) amount) may be appropriately set depending on the dosage form, mode of use, symptoms, physique, age, expected degree of effect, etc. of the subject (subject animal).

[0056] In the present disclosure, the type of cells to be targeted for cell proliferation promotion is not limited. Examples of such cells include tissue stem cells (somatic stem cells) such as mesenchymal stem cells (bone marrow stromal cells, adipose tissue-derived stem cells, etc.), muscle stem cells (skeletal muscle stem cells such as satellite cells, etc.), skin stem cells, corneal stem cells, hematopoietic stem cells, and neural stem cells; induced pluripotent stem cells (iPS cells); embryonic stem cells (ES cells); myoblasts, muscle cells (skeletal muscle cells, etc.), epithelial cells, endothelial cells, fibroblasts (skin fibroblasts, etc.), hair cells, liver cells, gastric mucosa cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, adipocytes, and blood cells (peripheral blood cells, umbilical cord blood cells, etc.). The cells may be primary culture cells, passage cells, or established cells. The cells may be of human or non-human origin (mouse, rat, etc.). These cells may be used alone or in combination of two or more types. In addition, for example, methods for producing induced pluripotent stem cells are known in the art, and they can be produced by introducing reprogramming factors into any somatic cells. The somatic cells may be, but are not limited to, fetal (offspring) somatic cells, neonatal (offspring) somatic cells, mature healthy or diseased somatic cells, etc. Examples of reprogramming factors include genes and gene products such as Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1, and the reprogramming factors may be used alone or in combination of two or more types. Preferable examples of the cells include mesenchymal stem cells, muscle stem cells, induced pluripotent stem cells, embryonic stem cells, myoblasts, muscle cells, etc., and more preferable examples include mesenchymal stem cells, satellite cells, induced pluripotent stem cells, embryonic stem cells, myoblasts, etc. For example, satellite cells and myoblasts are known to exist in skeletal muscles and play important roles in repairing skeletal muscles, maintaining their functions, etc.

[0057] In the test examples described below, basic fibroblast growth factor (FGF2) was used as a positive control. FGF2, also called bFGF, has been known to have cell proliferation promoting activity and is used in the culture of various cells such as mesenchymal stem cells and induced pluripotent stem cells for the purpose of promoting cell proliferation. From the results of the test examples described below, and further investigations by the present inventors, it was suggested that the peptide of the present disclosure may activate cell proliferation not only through the FGFR pathway known in FGF2, but also through another signaling pathway. In other words, it can be said that the peptide of the present disclosure does not only promote cell proliferation through the same mechanism as FGF2. It can be said that the peptide of the present disclosure is useful for promoting the proliferation of various cells, and is also useful for promoting the proliferation of cells whose proliferation can be promoted by FGF2.

[0058] In addition, in the test examples described below, it was confirmed that the cell proliferation promoting activity of FGF2 can be enhanced by allowing the peptide of the present disclosure to coexist with FGF2. From this, it was confirmed that the peptide of the present disclosure not only replaces FGF2, but also tends to be able to effectively enhance the cell proliferation promoting effect of FGF2 by using them together. From this, the peptide of the present disclosure can be used in combination with FGF2. Although this disclosure is not limited, for example, the peptide of the present disclosure and FGF2 can be mixed and used in a cell culture medium, and the content of FGF2 in the culture medium can be exemplified as 0.05 to 100 ng / mL, 0.05 to 50 ng / mL, 0.1 to 20 ng / mL, etc., and the content of the peptide of the present disclosure in the culture medium can be exemplified as 1 to 1000 μM, 20 to 800 μM, 80 to 600 μM, etc. FGF2 may be of human or non-human origin (mouse, rat, etc.), and may be extracted from organs such as the pituitary gland, brain, retina, corpus luteum, adrenal gland, kidney, placenta, prostate, thymus, etc., or may be produced by genetic engineering techniques such as recombinant DNA technology, and FGF2 is commercially available.

[0059] In addition, in the test examples described below, in addition to the cell proliferation promoting activity, it was also examined whether cell proliferation in an undifferentiated state (paired box 7 (Pax7) positive) can be promoted. In the test examples described below, when CKN, CKNG (SEQ ID NO: 16), CKNGGFF (SEQ ID NO: 17), CKNGGFFLR (SEQ ID NO: 37), CKNGGFFLRIH (SEQ ID NO: 39) or CKNGGFFLRIHPD (SEQ ID NO: 41) was used, it was confirmed that undifferentiated satellite cells could be effectively proliferated. Satellite cells are a type of muscle stem cell, and from the viewpoint of more efficiently repairing skeletal muscles and maintaining their functions, it can be said that the proliferation of undifferentiated satellite cells is important. For this reason, the peptides of the present disclosure, for example, peptide (1) which contains or consists of CKN, CKNG (sequence number 16), CKNGGFF (sequence number 17), CKNGGFFLR (sequence number 37), CKNGGFFLRIH (sequence number 39) or CKNGGFFLRIHPD (sequence number 41) in that order from the first amino acid at the N-terminus, and peptide (2) which has or consists of an amino acid sequence in which Y, F or W is added as the first amino acid further N-terminal to the sequence of peptide (1), can be said to be particularly useful for effectively promoting the proliferation of satellite cells and, ultimately, for effectively repairing skeletal muscle, etc.

[0060] For these reasons, the cell proliferation promoter of the present disclosure can be used as, for example, peptide medicines; food and beverage compositions, pharmaceutical compositions, quasi-drug compositions, feed compositions; additives to food and beverage compositions, pharmaceutical compositions, quasi-drug compositions, feed compositions, etc. In addition, in recent years, in various fields including the production of cultured meat using tissue engineering technology, it is important to efficiently obtain desired cells, tissues, etc. by promoting cell proliferation. For this reason, the cell proliferation promoter of the present disclosure can be used regardless of the field of medicine, food and beverage, etc., and for example, the production of cultured meat can also contribute to the improvement of food shortage problems.

[0061] In view of the above, the present disclosure also encompasses providing the following peptides: A peptide represented by the following formula (I) or (II): (I) Amino acid sequence CX in order from the N-terminus1a and having an amino acid residue length of 2 to 21. (II) Amino acid sequence X in order from the N-terminus 0b CX 1b and has a length of 3 to 22 amino acid residues. (where X 1a and X 1b are each independently K, A, G, F, W, Y, S, T, N, Q, C, D, E, R, or P; X 0b is Y, F or W).

[0062] The peptide (I) (hereinafter sometimes referred to as "peptide (I)") has an amino acid sequence and the like similar to those of peptide (1), except that the length of the amino acid residues is 2 to 21. In this respect, the length of the amino acid residues of peptide (I) is not limited. The length of the amino acid residues of peptide (I) is preferably 2 to 20, more preferably 2 to 19, etc. Within these ranges, the lower limit of the amino acid residue length is not limited as long as it is 2 or more, and more preferably, the lower limit is 2, 3, 4, 5, 6, or 7 amino acid residues. Within these ranges, the upper limit of the amino acid residue length is also not limited, and more preferably, the upper limit is 19, 18, 17, 16, 15, 14, or 13 amino acid residues. The lower limit and the upper limit may be any combination, for example, the amino acid residue length may be any of 2 to 18, 2 to 17, 2 to 16, 3 to 18, 3 to 17, 3 to 16, etc.

[0063] The peptide (II) (hereinafter sometimes referred to as "peptide (II)") has an amino acid sequence and the like similar to those of peptide (2), except that the length of the amino acid residues is 3 to 22. In this respect, the length of the amino acid residues of peptide (II) is not limited. The length of the amino acid residues of peptide (II) is preferably 3 to 21, more preferably 3 to 20, etc. Within these ranges, the lower limit of the amino acid residue length is not limited as long as it is 3 or more, and more preferably, the lower limit is 3, 4, 5, 6, 7, or 8 amino acid residues. Within these ranges, the upper limit of the amino acid residue length is also not limited, and more preferably, the upper limit is 20, 19, 18, 17, 16, 15, 14, or 13 amino acid residues. The lower limit and the upper limit may be any combination, for example, any combination of amino acid residues 3 to 19, 3 to 18, 3 to 17, 4 to 19, 4 to 18, 3 to 17, etc.

[0064] Preferred examples of peptide (I) and the amino acid sequence constituting peptide (I) are the same as those listed for peptides (1) and (2) above, except that the length of the amino acid residues is 2 to 21 or the length of the amino acid residues is 3 to 22, respectively.

[0065] Although not limiting the present disclosure, more preferred examples of peptide (I) include peptides having or consisting of the following amino acid sequences (starting from the N-terminus) in order from the first amino acid at the N-terminus. CKNGGFF (SEQ ID NO: 17), CK, CKN, CKNG (SEQ ID NO: 16), CKNAGFF (SEQ ID NO: 18), CKNGAFF (SEQ ID NO: 19), CKNGGAF (SEQ ID NO: 20), CKNGGFA (SEQ ID NO: 21), CKKGGFF (SEQ ID NO: 34), CKNGGFFLR (SEQ ID NO: 37), CKNGGFFLRIH (SEQ ID NO: 39), or CKNGGFFLRIHPD (SEQ ID NO: 41)

[0066] Although not limiting the present disclosure, more preferred examples of peptide (II) include peptides having or consisting of the following amino acid sequences (starting from the N-terminus) in order from the first amino acid at the N-terminus. YCKNGGF (SEQ ID NO: 45), YCK, YCKN (SEQ ID NO: 1), YCKNG (SEQ ID NO: 44), YCKNAGFF (SEQ ID NO: 46), YCKNGAFF (SEQ ID NO: 47), YCKNGGAF (SEQ ID NO: 48), YCKNGGFA (SEQ ID NO: 49), YCKNGGFF (SEQ ID NO: 50), YCKKGGFF (SEQ ID NO: 51), YCKNGGFFLR (SEQ ID NO: 52), YCKNGGFFLRIH (SEQ ID NO: 53), or YCKNGGFFLRIHPD (SEQ ID NO: 54).

[0067] As described above, peptides (I) and (II) are similar to peptides (1) and (2) contained in the cell proliferation promoter, except that they each have an amino acid residue length of 3 to 21 or 3 to 22, respectively, and can be used in any field, including medicine, food and beverages, etc. EXAMPLES

[0068] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0069] Test Example 1 1-1) Test procedure The cell proliferation-promoting activity of each of the synthesized peptides was examined according to the following procedure. Peptide synthesis In this test example, peptides having the following amino acid sequences were used: Each peptide was synthesized according to a conventional solid phase synthesis procedure. Example 1: Peptide CKNGGFF (SEQ ID NO: 17) Example 2: Peptide YCKNGGF (SEQ ID NO: 45) Comparative Example 1: Peptide KNGGFFL (SEQ ID NO:55) Comparative Example 2: Peptide NGGFLR (SEQ ID NO: 56)

[0070] · Cell preparation Satellite cells were used as a model cell. Specifically, mouse-derived muscle satellite cells (isolated from female C57BL / 6 mice) were used. The cells were taken from a frozen stock and pre-grown in Dulbecco's Modified Eagle Medium Low-Glucose (DMEM Low-Glu), 08456-36, Nacalai tesque, Japan, supplemented with 20% fetal bovine serum (FBS), F7524, SIGMA-ALDRICH, USA), 1% penicillin / streptomycin (PS, 168-23191, Wako, Japan), 2 mM L-glutamine (073-05391, Wako, Japan), and 2.5 ng / mL FGF2 (pre-grown medium). As an FGF2 solution, recombinant human FGF-basic (154 aa) (100-18B, PeproTech, USA) was dissolved in 0.1% albumin ((derived from bovine serum) 012-23381, Wako, Japan) in PBS at 2.5 μg / mL to prepare a solution, which was then dissolved in the preliminary medium to achieve the above composition. When reviving from a frozen stock, the frozen cells were dissolved in a growth medium, centrifuged, and seeded in order to remove the cell banker. For the subculture procedure, after washing twice with PBS, trypsin was added at 1 mL / 75 cm. 2 The cells were then added to a culture vessel coated with Matrigel and incubated at 37°C for 2 minutes at 5% CO2. The cells detached by trypsin were collected in growth medium and centrifuged at 1000 rpm for 4 minutes. The obtained cell pellet was suspended in an arbitrary volume of medium, growth medium was added to a culture vessel coated with Matrigel, and the cells were seeded at a medium volume of 10 mL / T75 flask (seeding density: 3 × 10 5 ~6×10 5 ), 37°C, and 20% O2 (atmospheric oxygen concentration). During the culture, the growth medium was replaced every two days.

[0071] Matrigel (354234, Corning, USA) and serum-free DMEM Low-Glu were mixed at 1:100 on ice to prepare a 100x Matrigel solution, which was then added to any flask or dish used for culture or proliferation (T-25 flask: 1.5-2.5mL, T-75 flask: 4.5-5mL, 10cm dish: 4mL, 24well plate: 120μL / well, 96well plate: 80μL / well). After incubation for 30 minutes in a CO2 incubator at 37℃, the Matrigel solution was removed and the cells were air-dried in a clean bench for 30 minutes or more to perform Matrigel coating.

[0072] · Cell proliferation activity evaluation (WST-8 assay) Satellite cells prepared (preliminarily cultured) as described above were plated on a Matrigel-coated 96-well plate at 7 × 10 3The cells were seeded at 184 μL per well and incubated at 37°C, 5% O2, and 5% CO2. The medium was composed of DMEM Low-Glu, 20% FBS, 1% PS, and 2 mM L-glutamine. After 1 hour of incubation, 16 μL of peptide solution (prepared by mixing the peptide with HBSS buffer) was added, and the cells were incubated again at 37°C, 5% O2, and 5% CO2. After 48 hours from the peptide addition, the solution was removed so that the total medium volume was 100 μL, and 10 μL / well of cell counting Kit-8 (CK04, DOJINDO, Japan) solution (WST-8 solution) was added according to the instruction manual, and the cells were incubated for 2 hours in a 5% O2, 5% CO2 environment. The cell viability of each well was then calculated by measuring the absorbance at 450 nm. HBSS buffer was prepared by diluting 10xHBSS (calcium, magnesium, no phenol red, 14065056, Thermo Fisher Scientific, USA) 10 times with pure water, and dissolving NaHCO3 (144-55-8, Wako, Japan) at 350 mg / L and HEPES (342-01375, Wako, Japan) at 10 mM. As a positive control, FGF2 (Recombinant Human FGF-basic (154 aa) (100-18B, PeproTech, USA)), which is known to have cell proliferation promoting activity, was used. The cells were cultured in the same manner except that FGF2 was added instead of the peptide, and the absorbance was measured. The cell proliferation rate was calculated according to the following formula described in the instruction manual. For "FGF2 0 ng / mL," the medium was incubated in the same manner except that the peptide and FGF2 were not added, and the absorbance was measured. Calculations were based on the average value of three plates (n=12).

[0073]

number

[0074] 1-2) Results The results are shown in Figure 1. As shown in Figure 1, when the peptide of Example 1 or the peptide of Example 2 was added, a higher cell proliferation rate was observed than the control (in the figure, FGF 0 mg / mL (no peptide added)), and a cell proliferation rate was observed that was equal to or higher than that when FGF2 (1.25 ng / mL), which is the positive control, was added. In contrast, when the peptide of Comparative Example 1 or the peptide of Comparative Example 2 was added, the cell proliferation rate was equal to that of the control. This demonstrated that the peptide of Example 1 and the peptide of Example 2 have desirable cell proliferation promoting effects.

[0075] Test Example 2 2-1) Test procedure The following test was carried out to verify whether all of the amino acid sequences of the peptide of Example 1 are necessary for cell proliferation-promoting activity. A total of seven types of peptides were synthesized in which only one residue in the amino acids constituting the peptide of Example 1 was replaced with A (alanine), and proliferation-promoting activity was evaluated in the same manner as in Test Example 1. A peptide in which the first amino acid residue C from the N-terminus in the peptide of Example 1 was replaced with S was also synthesized in the same manner, and proliferation-promoting activity was evaluated. In this test example, when WST-8 solution was added 48 hours after the addition of the peptide, the entire medium was removed, and a mixture of DMEM:WST-8 = 10:1 was added at 100 μL per well. This was to reduce the error in the amount of WST-8 added per well when adding 10 μL / well as much as possible.

[0076] 2-2) Results The results are shown in Figure 2. In Figure 2, "C1A" means a peptide in which the first amino acid from the N-terminus, "C," in the peptide of Example 1, is replaced with the amino acid "A," and "K2A" means a peptide in which the second amino acid from the N-terminus, "K," in the peptide of Example 1, is replaced with the amino acid "A." The same explanation applies to other peptides.

[0077] As shown in FIG. 2, it was revealed that when the amino acid on the N-terminus side was replaced with A, the cell proliferation rate was reduced compared to the original sequence (peptide of Example 1), and when the amino acid on the C-terminus side was replaced with A, the cell proliferation rate of the original sequence was maintained or improved. In particular, G4A showed a cell proliferation rate similar to that of the original sequence, and G5A, F6A, and F7A showed higher cell proliferation rates than the original sequence. From this, it was considered that the residues on the N-terminus side, especially the three N-terminus residues (CKN), are largely involved in the activity. It was considered that the residues on the C-terminus side, especially the two C-terminus residues (FF), are not directly involved in the activity, or that the cell proliferation rate would increase with further substitution.

[0078] Test Example 3 3-1) Test procedure From the results of Test Example 2, it was believed that the three residues (CKN) at the N-terminus in particular were involved in the proliferation-promoting activity. Therefore, further residue modification was carried out, focusing on the three residues at the N-terminus. For the modification, representative amino acids from groups of 20 natural amino acids grouped according to their physical properties and volume as shown in Table 1 below were mainly used. The cell proliferation activity of each modified peptide was evaluated in the same manner as in Test Example 2. The experimental results are shown as the average value and standard deviation. Dunnett's test vs FGF 0 ng / mL (n=12), *: 0.01≦p<0.05, **: 0.001≦p<0.01, ***: p<0.001

[0079] [Table 1]

[0080] 3-2) Results

[0081] As shown in (C1) of Figure 3, for the first residue, the proliferation-promoting activity of all peptides with amino acid substitutions was reduced compared to the original sequence, and the proliferation rate was equal to or lower than that of the control (no FGF2 added, "0" (ng / mL) in the table). From this, it was considered that the first residue must be C, and that the peptide of Example 1 exerts a proliferation-promoting effect due to the characteristics of C.

[0082] As shown in FIG. 3 (K2), the growth-promoting activity of the peptides substituted with L or H at the second residue was significantly reduced compared to the original sequence. Focusing on the peptides substituted with L (hereinafter sometimes referred to as L-substituted), the differences from the amino acid K in the original sequence are hydrophobicity and charge. Moreover, the R-substituted peptide, which is in the same group as K, had the same activity as the original sequence. Furthermore, since the activity was reduced in H, which is in the same group as K, it was considered essential that the second residue be an amino acid that is hydrophilic, positively charged, and has a bulky side chain.

[0083] As shown in Figure 3 (N3), the growth-promoting activity of the third residue was particularly reduced in those with highly hydrophobic amino acid substitutions such as L and F. In addition, since activity was maintained in S, T, and Q, which have the same physical properties as the original sequence N, it was inferred that the hydrophilicity of the third residue is the main factor in the interaction. Furthermore, the activity of the K substitution was significantly increased compared to the original sequence. This was thought to be due to the fact that K has a positive charge compared to the original sequence N and has a longer side chain.

[0084] Test Example 4 4-1) Test procedure Peptides shortened by appropriately increasing or decreasing the amino acid sequence at the N-terminus or C-terminus of the peptide of Example 1 or the peptide of Example 2 (shortened residue peptides) or the reverse sequence of the shortened residue peptides (reverse sequence peptides) were synthesized, and the cell proliferation activity of each peptide was evaluated in the same manner as in Test Example 3. The amino acid sequences of the synthesized shortened residue peptides and reverse sequence peptides are shown in Figure 4.

[0085] 4-2) Results The results are shown in Figure 4. As shown in Figure 4, strong proliferation-promoting activity was observed in peptide CK shortened to 2 residues (Example 3), peptide CKN shortened to 3 residues (Example 4), YCK (Example 5), peptide CKNG shortened to 4 residues (SEQ ID NO: 16 (Example 6)), and YCKN (SEQ ID NO: 1 (Example 7)). These peptides can be said to be shortened peptides having the N-terminal amino acid sequence CK of the peptide of Example 1, or having Y further added to the N-terminal side of the sequence (i.e., a sequence having the N-terminal amino acid sequence YCK of the peptide of Example 2). From these findings, it was found that in both the peptide of Example 1 and the peptide of Example 2, the N-terminal 2 or 3 residues are the sequences that mainly affect the cell proliferation-promoting activity.

[0086] In Table 4, the shortened peptides of 4 amino acids in length are shown as follows: RLYC is shown as SEQ ID NO: 57, LYCK is shown as SEQ ID NO: 58, KNGG is shown as SEQ ID NO: 59, NGGF is shown as SEQ ID NO: 60, GGFF is shown as SEQ ID NO: 61, GFFL is shown as SEQ ID NO: 62, FFLR is shown as SEQ ID NO: 63, and GNKC is shown as SEQ ID NO: 64.

[0087] Test Example 5 5-1) Test procedure From the above test example, it was found that the sequence that mainly affects the cell proliferation-promoting activity is 2 or 3 residues at the N-terminus of the peptide of Example 1 or the peptide of Example 2. In this test example, therefore, an evaluation was performed in the same manner as in Test Example 3 to determine whether a peptide lengthened by adding amino acids to the C-terminus while retaining the amino acid sequence at the N-terminus of the peptide of Example 1 would also exhibit the desired cell proliferation-promoting activity. Note that in this test example, the following peptides were used as the residue-lengthened peptides of the peptide of Example 1. Example 8: CKNGGFFL (SEQ ID NO:36) Example 9: CKNGGFFLR (SEQ ID NO: 37) Example 10: CKNGGFFLRI (SEQ ID NO: 38) Example 11: CKNGGFFLRIH (SEQ ID NO: 39) Example 12: CKNGGFFLRIHP (SEQ ID NO: 40) Example 13: CKNGGFFLRIHPD (SEQ ID NO:41)

[0088] 5-2) Results The results are shown in Figure 5. The results using the peptide of Example 1 (7 residues), the peptide of Example 9 (9 residues), the peptide of Example 11 (11 residues), and the peptide of Example 13 (13 residues) are shown in Figure 5. As shown in Figure 5, it was confirmed that the desired cell proliferation-promoting activity was observed even when the peptide of Example 1 was lengthened by adding an amino acid to the C-terminus. Furthermore, although not shown in the results, the desired cell proliferation-promoting activity was also observed when the peptide of Example 8 (8 residues), the peptide of Example 10 (10 residues), and the peptide of Example 12 (12 residues) were used.

[0089] Test Example 6 6-1) Test procedure The peptide of Example 1, the peptides of Examples 4 and 6 shortened in Test Example 4, and the peptides of Examples 9, 11, and 13 lengthened in Test Example 5 were used to evaluate the effects of these peptides on cell differentiation according to the following procedure.

[0090] Satellite cells precultured in the same manner as in Test Examples 4 and 5 were seeded in a 96-well plate, incubated, and the peptide solution was added. 48 hours after the addition of the peptide solution, 4% paraformaldehyde-phosphate buffer (PFA, 163-20145, Wako, Japan) was added in an amount equal to that of the medium, and the cells were allowed to stand at room temperature (25°C) for 5 minutes. Then, all of the solution was removed, PFA was added, and the cells were allowed to stand at room temperature for 20 minutes for fixation. After fixation, the PFA was removed and the cells were washed three times with PBS (first time: immediately after addition, second time: 5 minutes, third time: 10 minutes. The following three PBS washes for immunostaining were performed in this order).

[0091] Next, immunofluorescence staining with antibodies was performed as follows. After fixation, PBS was removed, 0.3% TritonX-100 in PBS (T8787, Sigma Aldrich, USA) was added to the wells, and membrane penetration was performed at room temperature for 5 minutes. Then, washing with PBS was performed three times, and blocking was performed by adding a base solution and leaving it at room temperature for 1 hour. The composition of the base solution was 10% goat serum (S-1000, Vector Laboratories, Burlingame, CA, USA) and 0.009% TritonX-100 in PBS. After blocking, a primary antibody solution prepared by dissolving it in the base solution was added, and the reaction was allowed to proceed overnight at 4°C. The primary antibody solution was prepared by mixing anti-Pax7 mouse IgG1 monoclonal antibody (DSHB, USA) and anti-MyoD1 rabbit IgG polyclonal antibody (18943-1-AP, proteintech, Rosemont, IL, USA) with the base solution at a ratio of 1:150 and 1:150, respectively, with an equal volume of glycerol (075-00616, Wako, Japan). The solution was removed the next day, and the cells were washed three times with PBS. A solution of the secondary antibody and DAPI (340-07971, DOJINDO, Japan) diluted 1000-fold with the base solution was added, and the reaction was allowed to proceed at room temperature for 1 hour in the dark. The secondary antibodies used were CF555 Goat Anti-Mouse IgG (H+L) (20231, biotium, Fremont, CA, USA) and CF488A Goat anti-rabbit IgG (H+L) (20012, biotium, Fremont, CA, USA). After washing three times with PBS, the wells were filled with PBS and observed for fluorescence using a BZ-X710 (KEYENCE, Osaka, Japan). Images were taken in the combined mode, and three points were taken from the center of each well in the vertical and horizontal directions, for a total of nine points. The nine overlay images were combined into a single image using a BZ-X Analyzer (KEYENCE, Osaka, Japan). When combining, the images were selected to be uncompressed and position-corrected.

[0092] The immunostained images obtained in this way were used to count the Pax7-positive areas (undifferentiated cells) among the areas stained with DAPI (total cells) using the hybrid cell count function of a BZ-X Analyzer (KEYENCE, Osaka, Japan), and quantitative evaluation was performed by calculating the percentage and number. Specifically, blue (indicating DAPI) was set as the mask area, and red (indicating Pax7) was used as the extracted area for counting. Red was considered positive when its brightness value was 10 or higher. The number of Pax7-positive / negative cells was calculated for all samples in each condition, and the average value was calculated, and then normalized by the average cell number in the condition to calculate the percentage of Pax7-positive / negative cells.

[0093] 6-2) Results As a result, the ratio of Pax7 positive (Pax7+) cells to the total number of cells was 64.0% when the shortened peptide (CKN) of Example 4 was used, and 69.2% when the shortened peptide (CKNG) of Example 6 was used. In this case, the ratio was 66.3% when the peptide of Example 1 was used, 66.7% when the control (no addition of FGF2 or peptide) was used, and 82.8% when FGF2 was added. In addition, the ratio of Pax7+ cells to the total number of cells was 70.2% when the long residue peptide (9 residues) of Example 9 was used, 65.6% when the long residue peptide (11 residues) of Example 11 was used, and 77.2% when the long residue peptide (13 residues) of Example 13 was used. In this case, the ratio was 70.7% when the peptide of Example 1 was used, 69.0% when the control (no addition of FGF2 or peptide) was used, and 82.8% when FGF2 was added.

[0094] Thus, it was found that the use of these peptides also resulted in the production of Pax7-positive satellite cells, i.e., undifferentiated satellite cells that had not differentiated into myoblasts. This confirmed that these peptides could promote cell proliferation while also adequately maintaining the undifferentiated state of satellite cells.

[0095] Test Example 7 7-1) Test procedure

[0096] · Dose-dependent proliferation The ability of the peptide to promote cell proliferation in a dose-dependent manner was confirmed by the following procedure. In this test example, the peptides of Example 1 and Example 13 were used. Each peptide was adjusted to a final concentration of 400 μM, 200 μM, 100 μM, or 10 μM in the medium, and an assay using WST-8 was performed in the same manner as in Test Example 3. The results are shown in FIG.

[0097] · Co-addition of peptide and FGF2 The peptide of Example 13 and FGF2 were added simultaneously (co-addition), and an assay using WST-8 was performed in the same manner as in Test Example 3. The final concentrations in the medium were adjusted to 100 μM and 400 μM for the peptide of Example 13, and 0.1 ng / mL, 1 ng / mL, 2.5 ng / mL, and 10 ng / mL for FGF2. The results are shown in FIG.

[0098] 7-2) Results The results regarding dose dependency are shown in Figure 6. In Figure 6, "10 μM", "100 μM", "200 μM", and "400 μM" represent the results when the peptide of Example 1 or the peptide of Example 13 was added to the medium at each concentration, "1 ng / mL" and "10 ng / mL" represent the results when FGF2 was added at each concentration, and "0 ng / mL" represents the result of the test without adding the peptide or FGF2. As shown in Figure 6, a peptide concentration-dependent increase in cell proliferation rate was observed for both the peptide of Example 1 and the peptide of Example 13.

[0099] The results of co-addition are shown in Figure 7. In Figure 7, for example, "p100" indicates the result of adding the peptide of Example 13 at 100 μM, "0.1 ng / mL" indicates the result of adding FGF2 at 0.1 ng / mL, and "F0.1+p100" indicates the result of co-addition of 0.1 ng / mL FGF2 and 100 μM of the peptide of Example 13. The result of using a medium containing neither the peptide of Example 13 nor FGF2 (control) is indicated as "0 ng / mL."

[0100] As shown in FIG. 7, the cell proliferation rate was improved even when the peptide of Example 13 and FGF2 were added together. Since the proliferation rate tended to be improved more than when each was added alone, it was considered that the peptide of Example 13 and FGF2 do not interfere with each other's proliferation promoting effects. In addition, even at a low concentration of FGF2, for example, the addition of the peptide of Example 13 resulted in a proliferation rate equivalent to or exceeding that of a proliferation rate of 10 ng / mL, so it was confirmed that the peptide of Example 13 in particular may enhance the action of FGF2. In addition, from these facts, it was found that according to the peptide of the present disclosure, such as the peptide of Example 13, when used in combination with the peptide of the present disclosure, it is also possible to reduce the amount of FGF2 used.

Claims

1. A cell proliferation promoter comprising the following peptide (1) or (2): (1) The amino acid sequence CX １ａ having (2) Amino acid sequence X in order from the N-terminus ０ｂ C.X. １ｂ have (Here, X １ａ and X １ｂ are each independently K, A, G, F, W, Y, S, T, N, Q, C, D, E, R, or P; X ０ｂ is Y, F or W).

2. The peptide (1) is a peptide having a length of 2 to 21 amino acid residues, or The peptide (2) is a peptide having a length of 3 to 22 amino acid residues. The cell proliferation promoter according to claim 1.

3. The peptide (1) comprises or consists of the following amino acid sequence (1) in order from the N-terminus: The peptide (2) comprises or consists of the following amino acid sequence (2) in the order from the N-terminus: The cell proliferation promoter according to claim 1 or 2: Amino acid sequence (1) CKNGGFF (SEQ ID NO: 17), CK, CKN, CKNG (SEQ ID NO: 16), CKNAGFF (SEQ ID NO: 18), CKNGAFF (SEQ ID NO: 19), CKNGGAF (SEQ ID NO: 20), CKNGGFA (SEQ ID NO: 21), CKKGGFF (SEQ ID NO: 34), CKNGGFFLR (SEQ ID NO: 37), CKNGGFFLRIH (SEQ ID NO: 39), or CKNGGFFLRIHPD (SEQ ID NO: 41). Amino acid sequence (2) YCKNGGF (SEQ ID NO:45), YCK, YCKN (SEQ ID NO:1), YCKNG (SEQ ID NO:44), YCKNAGFF (SEQ ID NO:46), YCKNGAFF (SEQ ID NO:47), YCKNGGAF (SEQ ID NO:48), YCKNGGFA (SEQ ID NO:49), YCKNGGFF (SEQ ID NO:50), YCKKGGFF (SEQ ID NO:51), YCKNGFFLR (SEQ ID NO:52), YCKNGGFFLRIH (SEQ ID NO:53) or YCKNGGFFLRIHPD (SEQ ID NO:54).

4. The cell proliferation promoter according to claim 1 or 2, which is used in combination with a basic fibroblast growth factor.

5. The cell proliferation promoter according to claim 1 or 2, wherein the cells are at least one type selected from the group consisting of tissue stem cells, induced pluripotent stem cells, embryonic stem cells and myoblasts.

6. A peptide represented by the following formula (I) or (II): (I) The amino acid sequence CX １ａ and having an amino acid residue length of 2 to 21. (II) Amino acid sequence X in order from the N-terminus ０ｂ C.X. １ｂ and having a length of 3 to 22 amino acid residues. (Here, X １ａ and X １ｂ are each independently K, A, G, F, W, Y, S, T, N, Q, C, D, E, R, or P; X ０ｂ is Y, F or W).

7. The peptide (I) comprises or consists of the following amino acid sequence (I) in order from the N-terminus: The peptide (II) comprises or consists of the following amino acid sequence (II) in the order from the N-terminus: The peptide of claim 6: Amino acid sequence (I) CKNGGFF (SEQ ID NO: 17), CK, CKN, CKNG (SEQ ID NO: 16), CKNAGFF (SEQ ID NO: 18), CKNGAFF (SEQ ID NO: 19), CKNGGAF (SEQ ID NO: 20), CKNGGFA (SEQ ID NO: 21), CKKGGFF (SEQ ID NO: 34), CKNGGFFLR (SEQ ID NO: 37), CKNGGFFLRIH (SEQ ID NO: 39), or CKNGGFFLRIHPD (SEQ ID NO: 41). Amino acid sequence (II) YCKNGGF (SEQ ID NO:45), YCK, YCKN (SEQ ID NO:1), YCKNG (SEQ ID NO:44), YCKNAGFF (SEQ ID NO:46), YCKNGAFF (SEQ ID NO:47), YCKNGGAF (SEQ ID NO:48), YCKNGGFA (SEQ ID NO:49), YCKNGGFF (SEQ ID NO:50), YCKKGGFF (SEQ ID NO:51), YCKNGFFLR (SEQ ID NO:52), YCKNGGFFLRIH (SEQ ID NO:53) or YCKNGGFFLRIHPD (SEQ ID NO:54).

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