KR101348096B1 - Polypeptide with chondrogenic activity of stem cell - Google Patents

Abstract

The present invention relates to a polypeptide having cartilage differentiation-inducing activity of a stem cell in which a self-assembled peptide and a collagen mimetic peptide in which hydrophilic and hydrophobic amino acids are sequentially linked, have no immune response to the material itself, and are harmless after restoration of a living body. Collagen mime peptides as a material to be degraded to create a suitable environment for stem cell differentiation of cartilage, thereby promoting cartilage differentiation activity, thereby achieving the regeneration of cartilage tissue.

Description

Polypeptide with chondrogenic activity of stem cell

The present invention relates to a biocompatible peptide material having cartilage differentiation inducing activity of stem cells.

Articular cartilage is the most heavily loaded tissue of human tissue and is easily exposed to damage caused by wear. Articular cartilage is a free cartilage (hyaline cartilage) consisting of chondrocytes and abundant extracellular matrix. Cartilage cells occupy less than 10% of total cartilage volume, and play an important role in the maintenance of articular cartilage because it synthesizes and secretes extracellular matrix. Reduction of chondrocyte number and chondrocyte metabolism by aging is considered to be one of the etiologies of osteoarthritis, a high prevalence of senile disease (Bobacz K et al, Ann Rheum Dis, Dec, 63 (12), pp.1618-1622 , 2004). Additionally, articular cartilage is a tissue that is unable to regenerate itself after injury because it lacks blood vessels, nerves and lymphoid tissue. Current medical methods have a very limited method of regenerating articular cartilage normally.

The regeneration of cartilage tissue by autologous chondrocyte transplantation has been recently tried and has been known to be a clinically effective surgical method. . Efforts to solve these problems and to develop more predictable and safer surgical methods are histological regeneration of cartilage (kang et al. Tissue Engineering, 11 (3-4), pp.438-447, 2005 ).

In order to manufacture cartilage using stem cells in tissue engineering, conditions for the support, cells, and growth factors should be appropriately set.

Prior art related to differentiation of mesenchymal stem cells into cartilage cells is described in Korean Patent No. 1012869 for the formation of mesenchymal stem cells in BMP-2 or BMP-7. Method for culturing in ~ 500 ng / ㎖ added cartilage forming medium, cartilage cell differentiation method of culturing mesenchymal stem cells in the presence of DKK-1 or sFRP-1, which is a wnt signaling inhibitor, in Korea Patent No. 990436, Korean Patent Publication No. 2009-69013 discloses mesenchymal stem cells focusing on compounds that affect stem cell differentiation, such as culturing mesenchymal stem cells in the presence of parathyroid hormone-related protein (PTHrP) or PD 98059. The prior art regarding the support used in three-dimensional culture for differentiation into chondrocytes is rare. As to a support used for differentiation of such chondrocytes, Korean Patent No. 684940 discloses a mixed support of fibri and hyaluronic acid.

The present invention does not have an immune response to the material itself, while harmlessly decomposed after restoration of the living body, by creating an environment suitable for stem cell differentiation of cartilage, thereby promoting cartilage differentiation activity, thereby enabling to achieve regeneration of cartilage tissue It is an object to provide a polypeptide.

The present invention provides a polypeptide having cartilage differentiation inducing activity of stem cells to which self-assembled peptides composed of the amino acid sequence represented by the following Formula 1 and collagen mimetic peptides are bound:

[Formula 1]

(αβ) _n

Α is any one hydrophobic amino acid selected from alanine (A), leucine (L) and phenylalanine (F), and β is arginine (R), aspartic acid (D), lysine (K), glutamic acid (E) And histidine (H) is any one of a hydrophilic amino acid, n is an integer of any one of 4 to 16.

According to one embodiment of the present invention, the self-assembled peptide of Formula 1 is RADARADARADARADA, RARADADARARADADA, RARARARADADADADA, DADADADARARARARA, RADARADA, RARADADA, KLDLKLDLKLDL, AEAKAEAKAEAKAEAK, AEAKAEAK, RAEARAEARAEAAEAAEAAEAAEAA It may consist of any one amino acid sequence selected from EFEFKFKF, EAEAEAEAKAKA, EAEAKAKA, KAKAKAKAEAEAEAEA and EAEAEAEAKAKAKAKA.

In addition, according to another embodiment of the present invention, the collagen mime peptide may be a polypeptide having the cartilage differentiation inducing activity of the stem cells comprising the amino acid sequence represented by the following general formula (2):

[Formula 2]

(-Glycine-proline-hydroxyproline-) _m

M is an integer of any one of 1-10.

In addition, according to another embodiment of the present invention, the collagen mime peptide of the general formula 2 may be (-glycine-proline-hydroxyproline-) ₇ .

In addition, according to another embodiment of the present invention, the cartilage differentiation inducing polypeptide may be composed of the amino acid sequence of SEQ ID NO: 3.

The present invention also provides a biodegradable support comprising the polypeptide.

The present invention is a self-assembled peptide in which a hydrophilic amino acid and a hydrophobic amino acid are sequentially combined is a biomaterial for forming a support having excellent biocompatibility, but in order to overcome the limitation that the cartilage differentiation activity of stem cells is extremely insignificant, The present invention provides a polypeptide conjugated with collagen mime peptides. The polypeptide of the present invention has no immune response to the material itself, and is a material that is harmlessly decomposed after restoration of the body. By the composition, the cartilage differentiation activity is enhanced, thereby making it possible to achieve regeneration of cartilage tissue.

FIG. 1A shows cartilage gene expression of stem cells in peptide gels prepared with KDL12 peptide solution, and FIG. 1B shows peptides prepared with 50:50 weight ratio mixed peptide solution of KLD12 and KLD12-CMP7. Electrophoresis picture confirming the cartilage gene expression of stem cells.
Figure 2 confirms the cartilage differentiation activity according to the mixing ratio of KDL12 and KDL12-CMP7, a graph showing the relative expression rate of Collagen type II according to the mixing concentration and the culture period of KLD12-CMP7.
Figure 3 confirms the cartilage differentiation activity of KLD12-CMP7 in the lactide / ε-caprolactone copolymer support, a graph showing the relative expression rate of Collagen type II and Aggrecan according to the mixing of KLD12-CMP7.
It is an immunochemical staining photograph confirming the generation of cartilage tissue by cartilage differentiation of KLD12-CMP7 in the lactide / ε-caprolactone copolymer support of Fig.
Figure 5 Quantitative analysis of the amount of cartilage specific extracellular matrix glycosaminoglycans (GAG) by cartilage differentiation of KLD12 or KLD12-CMP7 in the lactide / e-caprolactone copolymer support and transplanted into mouse subcutaneous to be.

The present invention provides a polypeptide having cartilage differentiation inducing activity of stem cells to which self-assembled peptides composed of the amino acid sequence represented by the following Formula 1 and collagen mimetic peptides are bound:

[Formula 1]

(αβ) _n

Α is any one hydrophobic amino acid selected from alanine (A), leucine (L) and phenylalanine (F), and β is arginine (R), aspartic acid (D), lysine (K), glutamic acid (E) And histidine (H) is any one of a hydrophilic amino acid, n is an integer of any one of 4 to 16.

When n exceeds the upper limit, it is not preferable in terms of peptide synthesis and purification, and when it is lower than the lower limit, it is not preferable in terms of generation of a higher order self-assembled structure.

For reference, representative amino acids and their abbreviations are alanine (Ala, A), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), Tryptophan (Trp, W), Valine (Val, V), Asparagine (Asn, N), Cysteine (Cys, C), Glutamine (Gln, Q), Glycine (Gly, G), Serine (Ser, S ), Threonine (Thr, T), tyrosine (Try, Y), aspartic acid (Asp, D), glutamic acid (Glu, E), arginine (Arg, R), histidine (His, H), lysine (Lys, K) ) In the case of hydroxyproline, Hyp or P * is abbreviated.

In the present invention, the peptide refers to a linear molecule formed by binding amino acid residues to each other by peptide bonds.

Peptides of the invention can be prepared according to chemical synthesis methods known in the art, in particular solid-phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85: 2149-54 (1963) Stewart, et al., Solid Phase Peptide Synthesis, 2nd.ed., Pierce Chem. Co .: Rockford, 111 (1984)).

Self-assembled peptide consisting of the amino acid sequence represented by the general formula 1 of the present invention is a repeating hydrophilic amino acid and hydrophobic amino acid from 8 to 32 by the sequential bond, RADA16-I has the amino acid sequence of RADARADARADARADA, RADA16- II is RARADADARARADADA, RAD16 is RARARARADADADADA, DAR16 is DADADADARARARARA, RADA8-I is RADARADA, RADA8-II is RARADADA, KLD12 is KLDLKLDLKLDL, EAKA16-1 is AEAKAEAKAEAKAEAK, EAKA8-1 is RAAEAEAIA, RAEAEAAEA RAEARAEA, KADA16-I for KADAKADAKADAKADA, KADA8-I for KADAKADA, EAH16-II for EAEAHAHAEAEAHAHA, EAH8-II for EAEAHAHA, EFK16-II for EFEFKFKFEFEFKFKF, EFK8-II for EFEFKFKF, EKAKA, EAKAKA KAKAKAKAEAEAEAEA, EAK16 is the amino acid sequence of EAEAEAEAKAKAKAKA.

Self-assembled peptides consisting of the amino acid sequence represented by the general formula (1) of the present invention is an amphiphilic peptide, forms a beta sheet structure, can be gelled in the presence of a bioion.

In the polypeptide having the cartilage differentiation inducing activity of the stem cell of the present invention, a collagen mimetic peptide is bonded to a self-assembled peptide consisting of the amino acid sequence represented by the general formula (1).

Collagen is a unique structure that contains three strands twisted in triple helix, each strand containing -glycine-proline-hydroxyproline-in repeat units. Wherein hydroxyproline is (2S, 4R) -4-hydroxyproline.

The collagen mimetic peptide of the present invention comprises an amino acid sequence represented by the following general formula (2), so that the stem cells are differentiated into cartilage because the polypeptide of the present invention creates a similar environment as the actual collagen is present in the stem cells. It can increase activity, which can promote the regeneration of cartilage tissue:

[Formula 2]

(-Glycine-proline-hydroxyproline-) _m

M is an integer in any one of 1-10, Preferably it is an integer in any one of 4-8. If m exceeds the upper limit, it is not preferable in terms of peptide synthesis and purification. For example, the collagen mime peptides of Formula 2 may be (-glycine-proline-hydroxyproline-) ₇ .

The present invention may be a biodegradable support comprising a polypeptide having cartilage differentiation inducing activity of stem cells to which self-assembled peptides composed of the amino acid sequence represented by Formula 1 and collagen mimetic peptides are bound.

The biograft scaffolds used in tissue engineering are largely made of synthetic materials such as polyglycolic acid, polylactic acid, polylactic acid-glycolic acid copolymer (PLGA), and agarose, cellulose, chitosan, hyaluronic acid, fibrin, and the like. It can be classified into a support using natural materials and a self-assembled peptide support. The present invention is a synthetic biomaterial, cartilage of a stem cell in which a highly elastic porous support PLCL having a mechanical activity similar to that of supernatural bone and a self-assembled peptide and a collagen mimetic peptide composed of the amino acid sequence represented by Formula 1 above are combined. It may be a biodegradable support comprising a polypeptide having differentiation inducing activity.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It will be apparent, however, to those skilled in the art that these embodiments are for further explanation of the present invention and that the scope of the present invention is not limited thereby.

Manufacturing example : Induced cartilage differentiation activity of stem cells Of the polypeptide (KDL12-CMP7)  Produce

One example of the self-assembled peptides of Formula 1, KDL12, that is, KLDLKLDLKLDL (SEQ ID NO: 1) and collagen mime peptides (GPP *) _{7 in} KDL12, ie GPP * GPP * GPP * GPP * GPP * GPP * GPP * Polypeptide KDL12-CMP7 (SEQ ID NO: 3) having a stem cell cartilage differentiation inducing activity (SEQ ID NO: 2) bound thereto was synthesized as follows by a solid-phase method.

Specifically, 0.06 mmol of glutamic acid-Wang resin was added to a standard reaction vessel, Fmoc of the resin was removed with a base (20% piperidine), and the carboxy terminus of the peptide to be synthesized was added with Fmoc-amino acid. 1-hydroxy-benzotriazole (HOBT) and diisopropyl carbodiimide (DIC) were mixed and activated to start synthesis. After the synthesis in the same order of amino acid sequence in the same way, after coupling was washed several times with N-methylpyrrolidone and dichloromethane and dried. Trifluoroacetic acid: phenol: thioanisole: water: ethanedithiol (Trifluoroacetic acid: Phenol: Thioanisole: H2O: Ethandithiol) 82.5: 5: 5: 5: 2.5 (v / v) After reacting for a time, the protecting group of the peptide was removed, the peptide was separated from the resin, and cold ethyl ether was added to the solution to obtain a white precipitated peptide. The peptide thus obtained was purified by HPLC (High Performance Liquid Chromatography), where the column was a C4 semi-prep column (Phenomenex), buffer A was equilibrated with water and 0.1% TFA, and buffer B was Peptides were eluted with acetonitrile and 0.1% TFA to prepare KDL12 and KDL12-CMP7, respectively.

Experimental Example  One: KLD12 - CMP7 Cartilage differentiation activity

KLD12 and KLD12-CMP7 prepared in Preparation Example 1 were dissolved in sucrose solution to make 1% peptide solution, and KLD12 peptide solution and 50:50 weight ratio mixed peptide solution of KLD12 and KLD12-CMP7 were used to differentiate cartilage differentiation of stem cells. The effect on the was confirmed.

The cultured human adipose tissue-derived stem cells were suspended in PBS at a concentration of 1 × 10 ⁷ cells / mL, and mixed with the prepared peptide solution to form a gel at room temperature while adjusting the final concentration of the peptide to 0.5%. The thickness of the prepared gel was fixed at 1 mm, DMEM-HG, Sodium Pyruvate, 100 nM Dexamethasone, 20 μg / mL Proline, 37.5 μg / mL ascorbic 2-phosphate, 1% PS, 10ng / mL TGF-β1 for 21 days After incubation in cartilage differentiation medium containing 1% FBS, 1X insulin-transferrin-selenium (ITS +), cartilage differentiation was evaluated.

Each of the peptide gel complexes was disrupted using TRIzol reagent, and total RNA was extracted. The extracted RNA was reverse transcribed into cDNA and amplified by polymerase chain reaction. Collagen type II was used as a gene for confirming cartilage differentiation. PCR products of the Collagen type II, Collagen type I, Collagen type Ⅹ, and hActin genes were confirmed by electrophoresis using TBE buffer at 1% agarose gel concentration, and the results are shown in FIG. 2.

Figure 1 A is to confirm the cartilage gene expression of stem cells in the peptide gel prepared with KDL12 peptide solution, B is the stem cell of the peptide gel prepared with 50:50 weight ratio mixed peptide solution of KLD12 and KLD12-CMP7 Cartilage gene expression is confirmed.

Actin was expressed in common and Collagen type I and X were expressed more strongly in KLD12 peptide gels over time, but collagen type II, a cartilage specific gene, was expressed in KLD12 and KLD12-CMP7 mixed peptide gels than KLD12 peptide gels. It was confirmed to express strongly.

When the KLD12 peptide gel was compared with the KLD12 and KLD12-CMP7 mixed peptide gels, the cartilage specific gene Collagen type II was significantly expressed in the KLD12-CMP7 mixed peptide gel.

Experimental Example  2: KLD12 - CMP7 Cartilage differentiation activity according to the mixing ratio of

In order to confirm the cartilage differentiation activity according to the mixing ratio of KDL12 and KDL12-CMP7, the peptide solutions of KLD12 and KLD12-CMP7 of Experimental Example 1 were mixed at a weight ratio of 95: 5, 90:10, and 70:30, respectively. Check the cartilage differentiation activity of the peptide gel prepared with a mixed peptide solution of the results are shown in FIG.

As the mixing ratio of KDL12-CMP7 increased, the expression level of Collagen type II was increased.On the 14th day, the expression level of Collagen type II was higher than that of KDL12 peptide gel only when 30% by weight of KDL12-CMP7 was mixed. Even when 10% by weight of CMP7 was mixed, it was confirmed that the expression level was significantly improved than that of the KDL12 peptide gel.

Therefore, when preparing a peptide gel using the self-assembled peptide of Formula 1, in order to enhance the cartilage differentiation activity, at least 10% by weight of the polypeptide having the cartilage differentiation inducing activity of the stem cells to which collagen mimetic peptides such as KDL12-CMP7 are bound, Preferably it was found that the peptide gel should be prepared using a mixed polypeptide of at least 30% by weight.

Experimental Example  3: Lactide / ε- Caprolactone  On the copolymer support KLD12 - CMP7 Cartilage differentiation activity

First, a highly elastic porous scaffold with mechanical activity similar to that of supernatural bone was synthesized. Biodegradable PLCL was synthesized using lactide, caprolactone and stannous octate as a catalyst. The PLCL polymer was dissolved in a chloroform solvent, mixed with a salt of 300-500 μm size, and then compressed into a mold to prepare a support in the form of a sheet. After the salt was eluted with distilled water, a lactide / ε -caprolactone copolymer support for disk-shaped cartilage regeneration having a diameter of 9 mm and a thickness of 3 mm was prepared.

From the KLD12 and KLD12-CMP7 peptide solution of Experimental Example 1, a mixed peptide solution of KLD12 and KLD12-CMP7 was prepared in a 70:30 weight ratio, and cultured human adipose tissue-derived stem cells were added to PBS at a concentration of 1 × 10 ⁷ cells / mL. The final concentration was brought to a state by mixing with the suspension, and the final concentration was adjusted to 0.5% by mixing with the prepared peptide solution.

The cartilage regeneration lactide / ε-caprolactone copolymer support was inoculated at a concentration of 150 μl / scaffold , and then in vitro for three weeks outside of cartilage differentiation medium (DMEM-HG, Sodium Pyruvate, 100 nM Dexamethasone, 20 μg / mL Proline). , Cartilage differentiation capacity was evaluated by incubating in 37.5 μg / mL ascorbic 2-phosphate, 1% PS, 10ng / mL TGF-β1, 1% FBS, 1X insulin-transferrin-selenium (ITS +). .

Collagen type II and Aggrecan expression levels were confirmed by real-time PCR as a gene for confirming cartilage differentiation.

Experimental Example  4: In vivo cartilage differentiation activity in animal test

In the same manner as in Experiment 3, KLD12 and KLD12-CMP7 were prepared in a mixed peptide solution at a 70:30 weight ratio, and stem cells were mixed to inoculate the lactide / ε-caprolactone copolymer support for cartilage regeneration for 2 weeks. After incubation in cartilage differentiation medium and transplanted subcutaneously in 7-week-old Balb / C nude mice.

The implanted constructs were removed after 5 weeks to evaluate the cartilage differentiation ability. It was fixed in formalin solution for histological analysis, dehydrated by changing ethanol concentration, all water was removed, embedded in paraffin, and cut into 6μm tissue using a cutting machine. Hematoxylin and Eosin (H & E) staining was performed, and Masson Trichrome (MT) staining and alcian blue staining were performed to evaluate mature cartilage tissue formation. In addition, immunohistochemical staining (Collagen type II; green, DAPI; blue) was performed using Collagen type II antibody to evaluate the cartilage differentiation ability of the injected cells, and immunochemical staining pictures are shown in FIG. 4.

The histological analysis of the immunochemical staining photograph of FIG. 4 confirmed the racona structure in the cartilage protein GAG and chondrocytes.

On the other hand, as shown in Example 3, the expression of Collagen type II and Aggrecan genes was confirmed by real-time PCR, and the cartilage differentiation substrate secretion was increased in the group inoculated with KDL12 and KLD12-CMP7 compared to the group inoculated with KLD12 only. Confirmed.

In addition, in order to measure the total GAG content of the peptide complex using the 1,9-dimethylmethylene blue (DMB) method, after the color reaction using DMB dye was measured at 530nm wavelength, the group inoculated with KDL12 and KLD12-CMP7 In the cartilage matrix protein GAG secretion was found to increase (Fig. 5).

Attach an electronic file to a sequence list

Claims (6)

delete delete delete delete A polypeptide having cartilage differentiation inducing activity of a stem cell, characterized in that consisting of the amino acid sequence of SEQ ID NO: 3.
A biodegradable support comprising the polypeptide of claim 5.

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