KR101584146B1 - Compositions containing periostin or its fragment for the prevention or treatment of ischemia - Google Patents

Abstract

The present invention relates to a composition for preventing or treating an ischemic disease comprising a ferri-austin protein or a fragment thereof as an active ingredient. The ferri-austin protein or fragment thereof according to the present invention promotes migration, adhesion and angiogenesis of vascular endothelial cells and has excellent neovascularization-promoting effect in an ischemic limb animal model, which is useful for prevention or treatment of ischemic diseases Can be used.

Description

[0001] The present invention relates to a composition for preventing or treating ischemic diseases comprising a ferri-austin protein or a fragment thereof as an active ingredient,

The present invention relates to a composition for preventing or treating an ischemic disease comprising a ferri-austin protein or a fragment thereof as an active ingredient.

Angiogenesis is a process in which new blood vessels are formed in existing blood vessels. The process of migration of vascular endothelial cells constituting blood vessels, infiltration through extracellular matrix (ECM) an invasion process, a proliferation process of a blood vessel, and a tube formation process. This series of reactions is known to be tightly regulated by a balance between angiogenesis factors and angiogenesis inhibitors (Schott RJ and Morrow LA, Cardiovasc. Res., 1993, 27, 1155-1161 ). These angiogenic responses are rarely observed under normal conditions, but they usually occur when early embryonic development, wound healing, and periodic female genital system changes. The endothelial cells of the capillary vessels in the normal tissues are in a quiescent state (Folkman J and Shing Y, J. Biol. Chem., 1992, 267 , 10931-10934).

On the other hand, ischemic disease refers to a disease in which various types of pathological abnormalities are generated in blood vessels supplying blood to the organs of the body, resulting in a local disorder of normal blood flow. The supply of blood through the blood vessels is an essential condition for wound healing and tissue regeneration, and diseases such as arteriosclerosis, myocardial infarction and angina are caused by an inadequate blood supply. Accordingly, studies on angiogenesis factors have been continued, and recently, there has been a tendency to develop and therapeutic applications of the angiogenesis factors have also been attempted.

Periostin, an extracellular matrix (ECM), is a 90 kDa protein that is overexpressed in a variety of human cancers and is involved in tumor growth and metastasis. It has been reported that periostein is mainly studied in cancer, and recently, it has been reported that the over-expression of periostein in mesenchymal stem cells and its recovery in rat myocardial infarction model is better than that of mesenchymal stem cell transplantation (Cho YH, Biomaterials, 2012, 33 (5), 1376-85). However, the expression of periostein is more difficult than that of other proteins, so it has a disadvantage in that it is more expensive than other proteins in the commercial market.

 Accordingly, the inventors of the present invention have continued to develop a therapeutic agent for a new ischemic disease. As a result, it has been confirmed that a fragment of the ferri-austin protein can be used for the prophylactic or therapeutic effect of ischemic diseases due to the angiogenesis promoting effect Respectively.

It is an object of the present invention to provide a composition for preventing or treating ischemic diseases comprising a ferri-austin protein or a fragment thereof as an active ingredient.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating ischemic diseases comprising ferri-austin protein or a fragment thereof as an active ingredient.

The present invention also provides a food composition for preventing or ameliorating an ischemic disease comprising a ferri-austin protein or a fragment thereof as an active ingredient.

The ferri-austin protein or fragment thereof according to the present invention promotes migration, adhesion and angiogenesis of vascular endothelial cells and has excellent neovascularization-promoting effect in an ischemic limb animal model, which is useful for prevention or treatment of ischemic diseases Can be used.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing a region of the periostin protein fragments 1 to 5; FIG.
FIG. 2 is a diagram showing the expression and purification process of the regions 1 to 5 of the ferri-austin protein fragments using the pET-30a vector.
FIG. 3 is a diagram showing the result of SDS-PAGE of the separated and purified periostin protein fragments 1 to 5 (fragments 1 and 2 of the lane are fragments 2 and 3, fragments 3 and 4 of the fragments 1 and 5 are fragments thereof, The lane represents the fragment 4 and the lane 5 represents the fragment 5.).
FIG. 4 is a graph showing the results of measuring cell migration after treating fragments 1 to 5 of periaustin protein on vascular endothelial progenitor cells. FIG.
FIG. 5 is a graph showing the results of measurement of migration of vascular endothelial progenitor cells after different treatment concentrations of periaustin protein fragment 1. FIG.
6 is a graph showing the results of measurement of degree of adhesion of cells after treating fragments 1 to 5 of periaustin protein with vascular endothelial progenitor cells.
FIG. 7 is a graph showing the results of measurement of adhesion of vascular endothelial progenitor cells after different treatment concentrations of periaustin protein fragment 1. FIG.
Fig. 8 is a graph showing microscopic observation of the degree of angiogenesis after treatment of periostein protein fragments 1 to 5 with vascular endothelial progenitor cells.
FIG. 9 is a graph showing the results of measuring the degree of vascularization after treatment of periostein protein fragments 1 to 5 with vascular endothelial progenitor cells. FIG.
FIG. 10 is a graph showing the treatment of periosteal progenitor cells with periosteine protein fractions 1 by concentration and observation of angiogenesis with a microscope.
11 is a graph showing the degree of vascularization after treatment of periosteine protein fragment 1 with vascular endothelial progenitor cells at different concentrations.
Fig. 12 is a graph showing the results of measurement by laser Doppler at day 0 and day 28 after administering HBSS, ferri-austine or ferri-austin protein fragment 1 to an ischemic limb model.
FIG. 13 is a graph showing blood vessel regeneration measured when HBSS, ferri-austin, or periostin protein fragment 1 is administered to an ischemic limb model.
FIG. 14 is a graph showing the degree of blood vessel necrosis when HBSS, ferri-austin, or periostin protein fragment 1 is administered to an ischemic limb model.
FIG. 15 is a diagram showing results obtained by administering HBSS, ferri-austin, or periostin protein fragment 1 to an ischemic limb animal model, and then staining the 28-day-old tissue with CD31.
FIG. 16 is a diagram showing results obtained by administering HBSS, ferri-austin, or periostin protein fragment 1 to an ischemic limb animal model, and then staining the 28-day-old tissue with? -SMA.
FIG. 17 is a graph showing blood vessel density measured after administration of HBSS, ferri-austin or ferri-austin protein fragment 1 to an ischemic limb model.
FIG. 18 is a diagram showing values of arterial density measured after administration of HBSS, ferri-austin or ferri-austin protein fragment 1 to an ischemic limb model.
FIG. 19 is a graph showing the migration of vascular endothelial progenitor cells by staining three-day-old tissues after administration of HBSS, ferri-austin, or periostin protein fragment 1 to an ischemic limb animal model.
FIG. 20 is a graph showing the results of measuring the migration of vascular endothelial progenitor cells by staining three-day-old tissues after administration of HBSS, ferri-austin, or periostin protein fragment 1 to an ischemic limb animal model.

The present invention provides a pharmaceutical composition for the prevention or treatment of an ischemic disease comprising a ferri-austin protein or a fragment thereof as an active ingredient.

The fragment of the ferri-austin protein has an amino acid sequence represented by any one of SEQ ID NOS: 1 to 5, preferably an amino acid sequence represented by SEQ ID NO: 1.

SEQ ID NO: 1 is the 95th to 234th amino acid sequence of the periostin protein (NCBI GenBank Accession No. NP006466.2), SEQ ID NO: 2 is the 230th to 368th amino acid sequence of ferriostin, SEQ ID NO: 3 is 365 to 496 The amino acid sequence of SEQ ID NO: 4, the 492 to 634th amino acid sequence of periostin and the amino acid sequence of 628 to 836th amino acid of periostin, respectively, and variants of the protein are included within the scope of the present invention.

A variant of the protein means a protein having a sequence which differs from the amino acid sequence of the ferri-austin protein or fragment thereof by one or more amino acid residues by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchange in proteins and fragments that do not globally alter the activity of the molecule is known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979).

The variant of the ferri-austin protein or fragment thereof can be prepared by natural extraction or by a recombinant method based on the synthetic sequence (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156, 1963) or DNA sequence (Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd ed., 1989).

The nucleotide encoding the fragment of the ferri-austin protein may have a nucleotide sequence represented by any one of SEQ ID NOS: 6 to 10 and includes a variant capable of functionally equivalent to a nucleotide encoding a fragment of the ferri-austin protein , But is not limited thereto.

A nucleic acid molecule that can be used as a gene encoding a ferri-austin protein or fragment thereof of the present invention is a functional equivalent of a nucleic acid molecule constituting it, for example, a deletion, substitution substitution or insertion, but includes variants capable of functionally equivalent to a ferri-austin protein or a fragment nucleic acid molecule thereof. Specifically, the gene has at least 70% homology, more preferably at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology with any one of the nucleotide sequences of SEQ ID NOS: 6 to 10 Lt; RTI ID = 0.0 > nucleotides < / RTI > "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The ferri-austin protein or fragment thereof promotes migration, adhesion and angiogenesis of vascular endothelial cells and has excellent neovascularization-promoting effect on an ischemic limb animal model. Therefore, the ferri-austin protein or fragment thereof of the present invention can be usefully used for the prophylaxis or treatment of ischemic diseases.

 The ischemic diseases include, but are not limited to, ischemic necrosis, ischemic cerebrovascular disease, ischemic renal disease, ischemic pulmonary disease, ischemic disease of limb, ischemic heart disease, stroke, cerebral infarction, myocardial infarction, ischemic heart failure and arteriosclerosis .

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. The pharmaceutical composition according to the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or other oral formulations, external preparation, suppository and sterilized injection solution, . Suitable formulations known in the art are preferably those disclosed in Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton PA.

Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations are prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, It is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspension include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For a desired effect, the composition of the present invention can be administered at a daily dose of 0.001 to 1000 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The present invention also provides a food composition for preventing or ameliorating an ischemic disease comprising a ferri-austin protein or a fragment thereof as an active ingredient.

When the composition of the present invention is used as a food additive, the composition can be added as it is or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention as defined by the appended claims. It will be obvious to you.

Example 1 Expression and Purification of Periaustin Protein Fragments Having Angiogenesis Promoting Function

The following experiments were carried out for the expression and purification of periostin protein fragments having the function of promoting angiogenesis.

More specifically, periaustin protein fragments obtained by dividing the rest of the fragments except for the EMI fragment in five fractions from the EMI fragment and four FAS I fragments into fragments 1, 2, 3, 4 and 5 (SEQ ID NO: 1 , 2, 3, 4 and 5 and the nucleotide sequences of SEQ ID NOS: 6, 7, 8, 9 and 10, respectively).

Escherichia coli expression plasmids were prepared and expressed in BL21 Escherichia coli for the expression and purification of the periostin protein fragments 1 to 5, followed by purification of endogenous toxins and endotoxin removal to purify clean proteins.

More specifically, for expression of periostin protein fragments 1 to 5, a plasmid construct in which each ferri-austin protein fragment was inserted into pET-30a (Novagen, Cat. 69909-3) vector was transformed into BL21 E. coli ). The colonies obtained by the transformation were cultured in LB broth (resistant antibiotics: kanamycin) at 30 DEG C and 150 rpm for 12 hours. The transformed E. coli was cultured for 4 hours at 25 ° C and 100 rpm in 1M IPTG (Promega, Cat. V3955) at 1/10000 (final concentration: 0.1 mM) to induce IPTG induction (Isopropyl- thio-galactoside induction. The E. coli was put into a centrifuge and precipitated and spun down at 15,000 rpm, 4 ° C and 10 minutes. After the spindown sample was removed, the lysis buffer (50 mM Tris-Cl (pH 8.0) + 100 mM NaCl + 1 mM EDTA (pH 8.0) + 1% Triton X-100 + 1 mM PMSF + DTT + tertiary sterilized water) was pipetted into a tube, and the tube was divided into a predetermined amount and sonicated (dutycycle) for 2 or 5 seconds to repeat 4 times. Min. ≪ / RTI >

To purify the expressed periostin protein fragments 1 to 5, a Ni-NTA agarose bead (Qiagen, Cat. 30210) was used and the bead volume was adjusted to be 1: 1 by making a slurry of 50% Was bound to His-tagged protein. The bound sample was incubated at 4 ° C for 2 hours using a rotor, and then precipitated and spun down at 15000 rpm, 4 ° C and 1 minute. The precipitated bead supernatant was separated using a syringe and placed in a new tube. Thereafter, the beads were washed 3 times with the elution buffer, and then the buffer was added thereto, followed by 5 to 10 times of flipping, followed by centrifugation. (20 mM Tris-Cl (pH 8.0), 500 mM NaCl, and Imidazole (10 mM / 20 mM) were added to the poly-Prep chromatography column (BIO-RAD, Catalog 731-1550) / 50mM / 100mM / 200mM / 300mM) and tertiary sterilized water) for 10 minutes, and eluting proteins attached to the beads according to the concentration of imidazole. The obtained protein was dialyzed against 1XPBS at 4 ° C for 24 hours.

To the endotoxin removal, the dialyzed proteins were mixed with 10 uL endotoxin-removed beads (Thermo scientific, Cat. 20339) made of 50% slurry and incubated at 4 ° C using a rotor. The cultured samples were spun down at 1500 rpm, 4 < 0 > C, and 1 minute, and the supernatant was taken and transferred to a new tube.

To test endotoxin release, the sample was examined for the formation of gel-clots using Pyrotell Gel-clot Formulation (Cat. G2125).

Fig. 1 shows a schematic diagram of ferri-austin fragments 1 to 5, the expression and purification process thereof is shown in Fig. 2, and finally the results of confirming ferri-austin fragments 1 to 5 through SDS-PAGE are shown in Fig.

Example 2. Verification of effect of periostin protein fragments 1 to 5 on vascular endothelial progenitor cells

Example 2-1. Selection of ferri-austin protein fragments to promote migration of vascular endothelial progenitor cells

In order to select a protein fragment of ferri-austin that promotes the migration of vascular endothelial progenitor cells, the periostin protein fragments 1 to 5 obtained in Example 1 were treated to endothelial progenitor cells, which are cells existing in human blood vessels, The movement was confirmed.

More specifically, vascular endothelial progenitor cells cultured in a medium of EGM-2 (Endothelial Cell Growth Medium-2, Clonetics Co., San Diego, USA) were inoculated into a 96-well chemotaxis chamber (Neuro Probe, Inc., Gaithersburg, Md., USA) was injected with 5,000 cells per well. The periustin protein fragments 1 to 5 were added to the lower part of the chamber with a 20 ug / ml rat-tail collagen-coated polycarbonate filter having pore therein at a concentration of 10 ug / ml and incubated at 37 캜 for 10 hours Respectively. As a positive control, vascular endothelial growth factor (VEGF) or ferriostin was treated, and the negative control group was cultured under the same conditions. After completion of the culture, each cell was stained with hematoxylin and the degree of migration of the vascular endothelial progenitor cells was measured. The results are shown in Fig.

As shown in Fig. 4, it was confirmed that the ferri-austin protein fragment 1 of the present invention has an effect of promoting migration of vascular endothelial progenitor cells superior to the other fragments 2 to 5.

In order to confirm the proper treatment concentration of the periostin protein fragment 1 having the effect of accelerating the migration of the vascular endothelial progenitor cells, a negative control without any treatment and a periostin protein fragment with a concentration of 1, 2, 5, 10 or 25 / / 1 were each treated with vascular endothelial progenitor cells, and the degree of migration of vascular endothelial progenitor cells was measured. The results are shown in Fig.

As shown in FIG. 5, it was confirmed that when the ferrier protein fragment 1 was treated at a concentration of 10 μg / ml, the migration effect was the most excellent.

Example 2-2. Selection of ferri-austin protein fragments to promote adhesion of vascular endothelial progenitor cells

In order to select a protein fragment of periostin that promotes adhesion of vascular endothelial progenitor cells, the periostein protein fragments 1 to 5 obtained in Example 1 were treated on the vascular endothelial progenitor cells and the degree of adhesion of vascular endothelial progenitor cells was measured Respectively.

More specifically, 10 ug / ml rat-tail collagen type 1 (BD Bioscience, USA), ferri-austin or the ferri-austin protein fragments 1 to 5 of the present invention were added to a 96-well plate (Neuro Probe, Inc., Gaithersburg, 5 was coated with 10 ug / ml of each, followed by blocking with 0.1% bovine serum albumin (BSA). Collagen and ferriostin were used as positive control, and none were used as negative control. 100,000 vascular endothelial progenitor cells cultured using EGM-2 medium were added to the coated wells and cultured at 37 DEG C for 1 hour. After completion of the culture, the cells were stained with hematoxylin and the degree of adhesion was measured. The results are shown in Fig.

As shown in FIG. 6, it was confirmed that the ferri-austin protein fragment 1 of the present invention had better adhesion promoting effect on vascular endothelial progenitor cells than the other fragments 2 to 5.

In order to confirm the optimum treatment concentration of the periostein protein fragment 1 having the excellent effect of promoting adhesion of the vascular endothelial cells, a negative control group treated with nothing and a positive control group treated with collagen were used, and 1, 2, 5, / Ml concentration of periostin protein fragment 1 was treated with each of the vascular endothelial progenitor cells, and then the degree of adhesion of vascular endothelial progenitor cells was measured. The results are shown in Fig.

As shown in FIG. 7, when ferrier protein fragment 1 was treated at a concentration of 10 μg / ml, it was confirmed that the adhesion promoting effect of vascular endothelial cells was most excellent.

Examples 2-3. Screening of ferri-austin protein fragments to promote angiogenesis in vascular endothelial progenitor cells

In order to select a periostin protein fragment that promotes angiogenesis of vascular endothelial cells, the periostatin protein fragments 1 to 5 obtained in Example 1 were treated on the vascular endothelial progenitor cells and the degree of promoting angiogenesis was measured.

More specifically, vascular endothelial progenitor cells cultured in EGM-2 medium containing 2% fetal bovine serum (FBS) were placed in a 96-well plate, and then 50 μl of Matrigel (BD Bioscience, USA ) To prevent air bubbles. Periyustine or the protein fragments 1 to 5 of the ferri-austin of the present invention were respectively added to a medium containing 1% fetal bovine serum and mixed. Thereafter, the cells were suspended, inoculated at 1 × 10 ⁴ cells / well, and cultured at 37 ° C. After 10 hours, the degree of angiogenesis was observed. The results are shown in Fig. 8 and Fig.

As shown in Figs. 8 and 9, it was confirmed that the ferri-austin protein fragment 1 of the present invention had better vascularization-promoting effect than the ferri-austin and ferri-austin protein fragments 2 to 5.

In order to confirm the proper treatment concentration of periostin protein fragment 1 having excellent vascularization promoting effect, a negative control group treated with nothing and a positive control group treated with 1% fetal bovine serum were used. 1, 2, 5, 10 or Periostatin protein fragment 1 at a concentration of 25 占 퐂 / ml was treated on the cultured vascular endothelial progenitor cells, and the degree of vascularization was observed. The results are shown in Fig. 10 and Fig.

As shown in FIG. 10 and FIG. 11, when the ferrier protein fragment 1 was treated at a concentration of 2 μg / ml, it was confirmed that the best promoting effect of angiogenesis was achieved.

Example 3: Verification of the therapeutic effect of the periostin protein fragment 1 on the hindered disease

Example 3-1. Identification of the neovascularization effect of the ferri-austin protein fragment 1 of the present invention in an ischemic limb animal model

Based on the results of Example 2, the following experiment was carried out in order to confirm whether or not the periostin protein fragment 1 having the neovascularization-promoting effect exhibited the same effect in the ischemic limb animal model.

More specifically, HBsAg (Hank's balanced salt solution) was used as a negative control and periostin was used as a positive control in an ischemic limb animal model. In the experimental group, periostin protein fragment 1 of the present invention was administered at a concentration of 15 ug / ml Respectively. During the first 14 days, each substance was administered to the site of ischemic injury every day, and then every other day for 14 days. On day 0, day 14, and day 28, the shape and blood flow of the mice were measured by laser Doppler and necrosis scores were measured. The results and measured values are shown in Figs. 12 to 14. Fig.

As shown in Fig. 12 and Fig. 13, it was confirmed that blood vessel regeneration was performed in the group treated with periostin or periostin protein fragment 1 at the 28th day of the experiment.

Also, as shown in Fig. 14, it was confirmed that the degree of necrosis was lower in the group treated with ferrier protein fraction 1.

Example 3-2. Identification of the effect of the ferri-austin protein fragment 1 of the present invention on the number of blood vessels and arteries in an ischemic limb animal model

Based on the results of Example 2, experiments were carried out as follows to confirm whether the ferri-austin protein fragment 1 of the present invention having neovascularization-promoting effect exhibits the same effect of increasing blood vessels and arteries in an ischemic limb animal model.

More specifically, the tissues of the experimental 28-day-old rats of Example 3-1 were detached and stained with? -Smooth muscle actin (? -SMA), which is a marker for identifying blood vessels and a marker for identifying arteries, Confocal laser scanning microscopy and blood vessel and arterial density were measured. The results are shown in Fig. 15 to Fig.

As shown in FIGS. 15 and 16, it was confirmed that CD31 and? -SMA were expressed at high levels in the group to which ferrier protein fragment 1 was administered.

In addition, as shown in Fig. 17 and Fig. 18, it was confirmed that the density of vessels and arteries was higher in the group to which ferrier protein fraction 1 was administered than in the negative control group.

Example 3-3. Identification of promoting effect of periostin protein fragment 1 of the present invention on migration of vascular endothelial progenitor cells in an ischemic limb animal model

Based on the results of Example 2, in order to confirm whether the ferri-austin protein fragment 1 of the present invention having neovascularization-promoting effect exhibits the same effect of the same vascular endothelial progenitor cells in an ischemic limb animal model, .

More specifically, CM-DiI-labeled vascular endothelial progenitor cells were injected into the tail of an ischemic limb animal model. Subsequently, HBSS was administered as a negative control and ferriostin was administered as a positive control in the area where ischemic limb disease occurred, and the ferri-austin protein fragment 1 of the present invention was injected into the experimental group. The injected date was set to 0 day, and after 3 days, the injected tissue was removed and immunostaining was performed by the method of Example 3-2 and observed with a laser microscope. The results are shown in Fig. 19 and Fig.

As shown in FIGS. 19 and 20, it was confirmed that the group treated with periostin protein fragment 1 promoted the migration of vascular endothelial progenitor cells as much as the group treated with periostein, which is a positive control group.

Hereinafter, the pharmaceutical composition of the present invention and the preparation example of the food composition will be described, but the present invention is not intended to be limited but is specifically described.

Formulation Example 1. Preparation of a pharmaceutical composition

1-1. Manufacture of Powder

20 mg of ferri-austin protein or fragment thereof

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

1-2. Manufacture of tablets

10 mg of ferri-austin protein or fragment thereof

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

1-3. Preparation of capsules

10 mg of ferri-austin protein or fragment thereof

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

1-4. Injection preparation

10 mg of ferri-austin protein or fragment thereof

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄₂ H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

1-5. Manufacture of liquid agent

20 mg of ferri-austin protein or fragment thereof

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation Example 2. Preparation of Food Composition

2-1. Manufacture of health food

Perry Austin protein or fragment thereof 100 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 [mu] g vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

2-2. Manufacture of health drinks

Perry Austin protein or fragment thereof 100 mg

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3 g

Water quantification

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 liter container, It is used in the production of the health beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

<110> Pusan National University Industry-University Cooperation Foundation <120> Compositions containing periostin or its fragment for the          prevention or treatment of ischemia <130> PNU1-158P <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 140 <212> PRT <213> Periostin domain 1 Amino acids <400> 1 Val Leu Pro Ile Asp His Val Tyr Gly Thr Leu Gly Ile Val Gly Ala   1 5 10 15 Thr Thr Gln Arg Tyr Ser Asp Ala Ser Lys Leu Arg Glu Glu Ile              20 25 30 Glu Gly Lys Gly Ser Phe Thr Tyr Phe Ala Pro Ser Asn Glu Ala Trp          35 40 45 Asp Asn Leu Asp Ser Asp Ile Arg Arg Gly Leu Glu Ser Asn Val Asn      50 55 60 Val Glu Leu Leu Asn Ala Leu His Ser His Met Ile Asn Lys Arg Met  65 70 75 80 Leu Thr Lys Asp Leu Lys Asn Gly Met Ile Ile Pro Ser Met Tyr Asn                  85 90 95 Asn Leu Gly Leu Phe Ile Asn His Tyr Pro Asn Gly Val Val Thr Val             100 105 110 Asn Cys Ala Arg Ile Ile His Gly Asn Gln Ile Ala Thr Asn Gly Val         115 120 125 Val His Val Ile Asp Arg Val Leu Thr Gln Ile Gly     130 135 140 <210> 2 <211> 139 <212> PRT <213> Periostin domain 2 Amino acids <400> 2 Leu Thr Gln Ile Gly Thr Ser Ile Gln Asp Phe Ile Glu Ala Glu Asp   1 5 10 15 Asp Leu Ser Ser Phe Arg Ala Ala Ile Thr Ser Asp Ile Leu Glu              20 25 30 Ala Leu Gly Arg Asp Gly His Phe Thr Leu Phe Ala Pro Thr Asn Glu          35 40 45 Ala Phe Glu Lys Leu Pro Arg Gly Val Leu Glu Arg Ile Met Gly Asp      50 55 60 Lys Val Ala Ser Glu Ala Leu Met Lys Tyr His Ile Leu Asn Thr Leu  65 70 75 80 Gln Cys Ser Glu Ser Ile Met Gly Gly Ala Val Phe Glu Thr Leu Glu                  85 90 95 Gly Asn Thr Ile Glu Ile Gly Cys Asp Gly Asp Ser Ile Thr Val Asn             100 105 110 Gly Ile Lys Met Val Asn Lys Lys Asp Ile Val Thr Asn Asn Gly Val         115 120 125 Ile His Leu Ile Asp Gln Val Leu Ile Pro Asp     130 135 <210> 3 <211> 132 <212> PRT <213> Periostin domain 3 Amino acids <400> 3 Leu Ile Pro Asp Ser Ala Lys Gln Val Ile Glu Leu Ala Gly Lys Gln   1 5 10 15 Gln Thr Thr Phe Thr Asp Leu Val Ala Gln Leu Gly Leu Ala Ser Ala              20 25 30 Leu Arg Pro Asp Gly Glu Tyr Thr Leu Leu Ala Pro Val Asn Asn Ala          35 40 45 Phe Ser Asp Asp Thr Leu Ser Met Asp Gln Arg Leu Leu Lys Leu Ile      50 55 60 Leu Gln Asn His Ile Leu Lys Val Lys Val Gly Leu Asn Glu Leu Tyr  65 70 75 80 Asn Gly Gln Ile Leu Glu Thr Ile Gly Gly Lys Gln Leu Arg Val Phe                  85 90 95 Val Tyr Arg Thr Ala Val Cys Ile Glu Asn Ser Cys Met Glu Lys Gly             100 105 110 Ser Lys Gln Gly Arg Asn Gly Ala Ile His Ile Phe Arg Glu Ile Ile         115 120 125 Lys Pro Ala Glu     130 <210> 4 <211> 143 <212> PRT <213> Periostin domain 4 Amino acids <400> 4 Ile Lys Pro Ala Glu Lys Ser Leu His Glu Lys Leu Lys Gln Asp Lys   1 5 10 15 Arg Phe Ser Thr Phe Leu Ser Leu Leu Glu Ala Ala Asp Leu Lys Glu              20 25 30 Leu Leu Thr Gln Pro Gly Asp Trp Thr Leu Phe Val Pro Thr Asn Asp          35 40 45 Ala Phe Lys Gly Met Thr Ser Glu Glu Lys Glu Ile Leu Ile Arg Asp      50 55 60 Lys Asn Ala Leu Gln Asn Ile Ile Leu Tyr His Leu Thr Pro Gly Val  65 70 75 80 Phe Ile Gly Lys Gly Phe Glu Pro Gly Val Thr Asn Ile Leu Lys Thr                  85 90 95 Thr Gln Gly Ser Lys Ile Phe Leu Lys Glu Val Asn Asp Thr Leu Leu             100 105 110 Val Asn Glu Leu Lys Ser Lys Glu Ser Asp Ile Met Thr Thr Asn Gly         115 120 125 Val Ile His Val Val Asp Lys Leu Leu Tyr Pro Ala Asp Thr Pro     130 135 140 <210> 5 <211> 209 <212> PRT <213> Periostin domain 5 Amino acids <400> 5 Leu Tyr Pro Ala Asp Thr Pro Val Gly Asn Asp Gln Leu Leu Glu Ile   1 5 10 15 Leu Asn Lys Leu Ile Lys Tyr Ile Gln Ile Lys Phe Val Arg Gly Ser              20 25 30 Thr Phe Lys Glu Ile Pro Val Thr Val Tyr Thr Thr Lys Ile Ile Thr          35 40 45 Lys Val Val Glu Pro Lys Ile Lys Val Ile Glu Gly Ser Leu Gln Pro      50 55 60 Ile Ile Lys Thr Glu Gly Pro Thr Leu Thr Lys Val Lys Ile Glu Gly  65 70 75 80 Glu Pro Glu Phe Arg Leu Ile Lys Glu Gly Glu Thr Ile Thr Glu Val                  85 90 95 Ile His Gly Glu Pro Ile Ile Lys Lys Tyr Thr Lys Ile Ile Asp Gly             100 105 110 Val Pro Val Glu Ile Thr Glu Lys Glu Thr Arg Glu Glu Arg Ile Ile         115 120 125 Thr Gly Pro Glu Ile Lys Tyr Thr Arg Ile Ser Thr Gly Gly Gly Glu     130 135 140 Thr Glu Glu Thr Leu Lys Lys Leu Leu Gln Glu Glu Val Thr Lys Val 145 150 155 160 Thr Lys Phe Ile Glu Gly Asp Gly His Leu Phe Glu Asp Glu Glu                 165 170 175 Ile Lys Arg Leu Leu Gln Gly Asp Thr Pro Val Arg Lys Leu Gln Ala             180 185 190 Asn Lys Lys Val Gln Gly Ser Arg Arg Arg Leu Arg Glu Gly Arg Ser         195 200 205 Gln     <210> 6 <211> 420 <212> DNA <213> Periostin domain 1 nucleotide <400> 6 gtttggccca ttgaccatgt ttatggcact ctgggcatcg tgggagccac cacaacgcag 60 cgctattctg acgcctcaaa actgagggag gagatcgagg gaaagggatc cttcacttac 120 tttgcaccga gtaatgaggc ttgggacaac ttggattctg atatccgtag aggtttggag 180 agcaacgtga atgttgaatt actgaatgct ttacatagtc acatgattaa taagagaatg 240 ttgaccaagg acttaaaaaa tggcatgatt attccttcaa tgtataacaa tttggggctt 300 ttcattaacc attatcctaa tggggttgtc actgttaatt gtgctcgaat catccatggg 360 aaccagattg caacaaatgg tgttgtccat gtcattgacc gtgtgcttac acaaattggt 420                                                                          420 <210> 7 <211> 417 <212> DNA <213> Periostin domain 2 nucleotide <400> 7 cttacacaaa ttggtacctc aattcaagac ttcattgaag cagaagatga cctttcatct 60 tttagagcag ctgccatcac atcggacata ttggaggccc ttggaagaga cggtcacttc 120 acactctttg ctcccaccaa tgaggctttt gagaaacttc cacgaggtgt cctagaaagg 180 atcatgggag acaaagtggc ttccgaagct cttatgaagt accacatctt aaatactctc 240 cagtgttctg agtctattat gggaggagca gtctttgaga cgctggaagg aaatacaatt 300 gagataggat gtgacggtga cagtataaca gtaaatggaa tcaaaatggt gaacaaaaag 360 gatattgtga caaataatgg tgtgatccat ttgattgatc aggtcctaat tcctgat 417 <210> 8 <211> 396 <212> DNA <213> Periostin domain 3 nucleotide <400> 8 ctaattcctg attctgccaa acaagttatt gagctggctg gaaaacagca aaccaccttc 60 acggatcttg tggcccaatt aggcttggca tctgctctga ggccagatgg agaatacact 120 ttgctggcac ctgtgaataa tgcattttct gatgatactc tcagcatgga tcagcgcctc 180 cttaaattaa ttctgcagaa tcacatattg aaagtaaaag ttggccttaa tgagctttac 240 aacgggcaaa tactggaaac catcggaggc aaacagctca gagtcttcgt atatcgtaca 300 gctgtctgca ttgaaaattc atgcatggag aaagggagta agcaagggag aaacggtgcg 360 attcacatat tccgcgagat catcaagcca gcagag 396 <210> 9 <211> 429 <212> DNA <213> Periostin domain 4 nucleotide <400> 9 atcaagccag cagagaaatc cctccatgaa aagttaaaac aagataagcg ctttagcacc 60 ttcctcagcc tacttgaagc tgcagacttg aaagagctcc tgacacaacc tggagactgg 120 acattatttg tgccaaccaa tgatgctttt aagggaatga ctagtgaaga aaaagaaatt 180 ctgatacggg acaaaaatgc tcttcaaaac atcattcttt atcacctgac accaggagtt 240 ttcattggaa aaggatttga acctggtgtt actaacattt taaagaccac acaaggaagc 300 aaaatctttc tgaaagaagt aaatgataca cttctggtga atgaattgaa atcaaaagaa 360 tctgacatca tgacaacaaa tggtgtaatt catgttgtag ataaactcct ctatccagca 420 gacacacct 429 <210> 10 <211> 459 <212> DNA <213> Periostin domain 5 nucleotide <400> 10 ctctatccag cagacacacc tgttggaaat gatcaactgc tggaaatact taataaatta 60 atcaaataca tccaaattaa gtttgttcgt ggtagcacct tcaaagaaat ccccgtgact 120 gtctataagc caattattaa aaaatacacc aaaatcattg atggagtgcc tgtggaaata 180 actgaaaaag agacacgaga agaacgaatc attacaggtc ctgaaataaa atacactagg 240 atttctactg gaggtggaga aacagaagaa actctgaaga aattgttaca agaagaggtc 300 accaaggtca ccaaattcat tgaaggtggt gatggtcatt tatttgaaga tgaagaaatt 360 aaaagactgc ttcagggaga cacacccgtg aggaagttgc aagccaacaa aaaagttcaa 420 ggatctagaa gacgattaag ggaaggtcgt tctcagtga 459

Claims (6)

1. A pharmaceutical composition for preventing or treating an ischemic disease comprising an effective component of a fragment of a ferri-austin protein consisting of the amino acid sequence shown in SEQ ID NO: 1. delete The method according to claim 1,
Wherein the nucleotide encoding the fragment of the ferri-austin protein is a nucleotide sequence of SEQ ID NO: 6. The method according to claim 1,
The pharmaceutical composition for preventing or treating ischemic diseases, wherein the fragment of the ferri-austin protein has the effect of promoting migration, adhesion or angiogenesis of vascular endothelial cells. The method according to claim 1,
The ischemic diseases are selected from the group consisting of ischemic necrosis, ischemic cerebrovascular disease, ischemic renal disease, ischemic pulmonary disease, ischemic disease of limb, ischemic heart disease, stroke, cerebral infarction, myocardial infarction, ischemic heart failure and occlusive atherosclerosis Wherein the composition is at least one herbicide. 1. A food composition for preventing or ameliorating an ischemic disease comprising a fragment of a ferri-austin protein consisting of an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.

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