KR20170043772A - Composition for preventing or treating erectile dysfunction comprising DKK3 - Google Patents

Abstract

The present invention relates to a composition for preventing or treating erectile dysfunction, comprising DKK3 (Dickkopf 3) protein or a polynucleotide encoding the DKK3 protein as an active ingredient. The DKK3 protein according to the present invention or the polynucleotide encoding the DKK3 protein may be useful for the treatment of excellent erectile dysfunction such as increasing the level of endothelial cell specific protein to induce regeneration of penile cavernosal endothelial cells to increase penile erection power .

Description

[0001] The present invention relates to a composition for preventing or treating erectile dysfunction, comprising DKK3 as an active ingredient,

The present invention relates to a composition for preventing or treating erectile dysfunction, comprising DKK3 (Dickkopf 3) protein or a polynucleotide encoding the DKK3 protein as an active ingredient.

Erectile dysfunction is a type of male sexual dysfunction that can not be done because male penis is not erect or erect state is not maintained. The causes of erectile dysfunction are largely classified into psychogenic causes and substrate causes. Cardiogenic erectile dysfunction is caused by excessive secretion of noradrenaline due to excessive action of sympathetic nerves due to psychological and psychological influences, increased smooth muscle tension of the penis cavernosum, and suppression of neurotransmitter secretion. Substantial erectile dysfunction, on the other hand, is classified as neurogenic, vascular, and endocrine erectile dysfunction. The vascular erectile dysfunction is caused by damage to the corpus cavernosum and vascular endothelial cells due to hyperlipidemia, diabetes, hypertension, smoking, systemic cardiovascular disease, etc., and the secretion of nitric oxide (NO) It is a disorder that does not occur.

Recently, studies on erectile dysfunction have focused more on the underlying causes, and oral PDE-5 (phosphodiesterase-5) inhibitors, including Viagra (sildenafil), have been used worldwide for its treatment. It is known that the oral drug increases the concentration of cGMP due to the inhibition of PDE-5, which is specifically distributed in the corpus cavernosum, thereby increasing the blood flow in the corpus cavernosum and inducing erection, thereby treating erectile dysfunction. However, PDE-5 inhibitors such as Viagra have been reported to have various side effects such as headache, flushing, dyspepsia, and cardiac arrest, as well as to modulate transient protein expression and related factors at the molecular level It can not be treated. Furthermore, erectile dysfunction due to diabetes has a disadvantage in that such a therapeutic effect is not well manifested and its efficacy can not be sustained for a long time even if it is effective.

Thus, there is a need for an erectile dysfunction treatment that essentially treats the abnormal normal corpus cavernosum structure in the erectile dysfunction penis, and the efficacy thereof is prolonged for a long time.

On the other hand, Dickkopf (DKK) is a protein with four isotypes (DKK 1-4). Among these, DKK 1 and 2 have been reported to be actively studied and associated with LRP 5/6 and affecting Wnt signaling (B Mao et al., Nature 417 (6889), pp. 664-667, 2002). However, the role and function of DKK3 has not been studied yet, and some studies have reported that DKK3 is expressed in vascular endothelial cells of cancer tissues and affects capillary organization (G Untergasser et al. Int J Cancer 122 (7), pp1539-1547, 2008). However, the relationship between DKK3 and penile erection, and the prevention or treatment effect of DKK3 on erectile dysfunction are unknown, and there is no study about it.

Accordingly, the inventors of the present invention conducted a study to develop a composition for preventing or treating erectile dysfunction, and found that the expression of DKK3 was decreased in STZ (streptozotocin) -induced type 1 diabetic mice and erectile tissues of diabetic patients In the animal model of type 1 diabetic erectile dysfunction, DKK3 peptide, plasmid or adenovirus induces regeneration of penile corpus cavernosum endothelial cells, thereby enhancing erectile function of penile erection. It was confirmed that DKK3 promotes tube formation and cell migration in penile corpus cavernous endothelial cells (MCECs), thus completing the present invention.

It is an object of the present invention to provide a composition for preventing or treating erectile dysfunction which comprises DKK3 as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating erectile dysfunction, comprising DKK3 protein or a polynucleotide encoding the DKK3 protein as an active ingredient.

The present invention also provides a quasi-drug composition for preventing or ameliorating erectile dysfunction, comprising a DKK3 protein or a polynucleotide encoding the DKK3 protein as an active ingredient.

The present invention also provides a food composition for preventing or improving erectile dysfunction, which comprises DKK3 protein or a polynucleotide encoding the DKK3 protein as an active ingredient.

The DKK3 protein or the polynucleotide encoding the DKK3 protein according to the present invention has an excellent erectile dysfunction treatment effect by increasing the level of endothelial cell specific protein and inducing the regeneration of the penile corpus cavernosum endothelial cell to increase the erectile power of the penis , And to prevent or treat erectile dysfunction.

FIG. 1 shows the results of immunofluorescence staining (A) and RT-PCR (B) of DKK3 expression in penile corpus callosum tissues of an erectile dysfunction mouse model.
FIG. 2 is a diagram showing the results of observation of the degree of DKK3 expression in the corpus cavernosum tissue of an erectile dysfunction patient through immunofluorescence staining.
FIG. 3 is a graph showing the effect of DKK3 peptide stimulation on in vivo angiogenesis through Matrigel Assay.
Fig. 4 is a graph showing the effect of DKK3 peptide on the promotion of tube formation of mouse penile corpus cavernosum endothelial cells (A: microscope photograph, and B: number of tubes formed).
FIG. 5 is a graph showing the effect of DKK3 peptide on the migration of endothelial cells of mouse penile corpus cavernosum (A: microscope photograph, B: number of migrating cells).
FIG. 6 is a graph showing the effect of DKK3 peptide on the expression of bFGF gene in mouse penile cavernous endothelial cells.
FIG. 7 is a graph showing the effect of DKK3 peptide on the expression of VEGF gene in mouse penile corpus cavernosal endothelial cells (A: RT-PCR B: Western blot).
FIG. 8 is a graph showing the effect of DKK3 peptide on the expression of angiopoietin 1 and angiopoietin 2 gene in mouse penile corpus cavernosal endothelial cells by RT-PCR (A: angiopoietin 1 B: angiopoietin 2).
FIG. 9 shows the results of confirming the effect of DKK3 peptide on the expression of vascular endothelial cell adhesion protein (ZO-1, Claudin-5, Occludin) in mouse penile cavernosal endothelial cells (A: RT-PCR B: ).
FIG. 10 is a graph showing the results of intracoronary cavernosal pressure (erectile force) measurement according to electrical stimulation of the penile cavernosal nerve after local injection of DKK3 peptide in the penis in the erectile function mouse model (A: measurement result of penile corpus cavernosum, B: , C: the area of the pressure curve of the cavernosal corpus cavernosum).
FIG. 11 is a graph showing the measurement results of intracoronary cavernosal pressure (erectile force) according to penile nerve electrical stimulation after local injection of DKK3 plasmid DNA in the penis using electroporation in an erectile dysfunction mouse model (A: B: highest intracoronary CaP, and C: area of CaP curve.
FIG. 12 is a graph showing the measurement results of penile sponge pressure (erectile force) according to penile nerve electrical stimulation after topical injection of DKK3 adenovirus in the penis in an erectile dysfunction mouse model.
13 shows the expression level of platelet-endothelial cell adhesion molecule-1 (Platelet-Endothelial Cell Adhesion Molucule-1), which is a specific endothelial cell-specific protein by administration of DKK3 adenovirus in the erythrocyte cavernous tissue of the erectile dysfunction mouse model (A: Immunofluorescence staining, B: PECAM-1 expression in the penis).
FIG. 14 is a graph showing the results of analysis of the expression level of ZO-1 (Zonular Occudens-1), a vascular endothelial cell-junction protein by administration of DKK3 adenovirus, in a corpus cavernosum tissue of an erectile dysfunction mouse model with a confocal microscope : Immunofluorescence staining, B: the amount of ZO-1 expression in the penis).
FIG. 15 is a graph showing confocal microscopic analysis of the expression level of claudin-5, a vascular endothelial cell-binding protein by administration of DKK3 adenovirus, in corpus cavernosal tissues of an erectile dysfunction mouse model (A: B: the amount of claudin expression in the penis).

The present invention provides a composition for preventing or treating erectile dysfunction, comprising a DKK3 protein or a polynucleotide encoding the DKK3 protein as an active ingredient.

The composition comprises a pharmaceutical composition, a quasi-drug composition or a food composition.

The term "erectile dysfunction" in the present invention means a state in which erectile function is not sufficiently maintained or maintained in the sex life, and erectile dysfunction is generally defined as a state in which such state lasts for 3 months or more. Erectile dysfunction may be caused by impairment or impairment of the corpus cavernosum endothelial cells and smooth muscle cells. Such impairment or impairment of the corpus cavernosum endothelial cells and smooth muscle cells may result from, but is not limited to, selected from hyperlipidemia (including hypercholesterolemia deficiency), diabetes, hypertension, penile nerve damage, and combinations thereof .

In the present invention, the term "corpuscle" is an erectile tissue constituting the main body of the mammalian penis or clitoris. The corpuscle is piled up with a membrane of a thick and durable connective tissue containing elastic fibers. . Within the male penis, there are two penile cavernosomes on the left and right, and one urethral cavernosum on the lower side. There are clitoral cavernosomes and urethral cavernosomes with similar structures to the penis cavernosum in women. When the cavernosum is full of venous blood, the penis becomes erect. The penile cavernosal smooth muscle and penile blood vessels are usually contracted by adrenergic sympathetic nerves, and after erectile response due to relaxation, they return to contracted state by stimulation of the adrenergic sympathetic nervous system. Although erectile tissues maintain normal contraction state, erectile tissues are relaxed by the stimulation of the nervous system, various neurotransmitters and hormones in the body and erectile tissues, and erection is started. Is maintained. After this erection reaction is completed, the erectile tissue is maintained in a contracted state.

The term "prophylactic" in the present invention refers to any action that inhibits erectile dysfunction or inhibits or delays erectile dysfunction using a composition comprising the DKK3 protein of the present invention or a polynucleotide encoding the DKK3 protein.

The term "treatment" in the present invention refers to any action that improves or benefits erectile dysfunction by using a composition comprising the DKK3 protein of the present invention or a polynucleotide encoding the DKK3 protein.

The "DKK3 (Dickkopf 3) protein" as an active ingredient in the present invention includes all DKK3 proteins derived from various mammals such as human and mouse, but is not limited thereto.

The DKK3 protein of the present invention may be composed of the amino acid sequence of SEQ ID NO: 1 or 2, and may be composed of the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 2.

Also, it can be encoded by the nucleotide sequence shown in SEQ ID NO: 3 or 4, and includes functional equivalents of the protein. The above-mentioned "functional equivalent" means an amino acid sequence having at least 70% or more, preferably 80% or more, more preferably 90% or more More preferably 95% or more, of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and has substantially the same physiological activity as the protein consisting of the amino acid sequence shown in SEQ ID NO:

In addition, the DKK3 protein of the present invention includes not only the protein having the naturally occurring amino acid sequence but also the amino acid sequence mutant thereof within the scope of the present invention. The variant of the DKK3 protein means a protein having a sequence which differs from the native amino acid sequence of the DKK3 protein by deletion, insertion, non-conservative or conservative substitution or a combination thereof. Amino acid exchanges in proteins and peptides that do not globally alter the activity of the molecule are known in the art. The DKK3 protein or variant thereof can be prepared by natural extraction or by synthesis (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156, 1963) or by DNA recombination methods (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, Second Edition, 1989).

The DKK3 protein of the present invention can be encoded by the nucleotide sequence of SEQ ID NO: 3 or 4, and variants capable of functionally equivalent to the polynucleotide are included within the scope of the present invention. Specifically, the polynucleotide encoding the DKK3 protein preferably has 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, of the nucleotide sequence of SEQ ID NO: 3 or 4, Sequence homology. "% 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 polynucleotide encoding the DKK3 protein of the present invention may be contained in a plasmid vector or an adenovirus. Preferably, DKK3 is contained in the composition of the present invention in the form of plasmid DNA. The plasmid vector of the present invention may be abm # PV172185, and the adenovirus-containing form may be abm # 190468.

The DKK3 protein or the polynucleotide encoding the DKK3 protein according to the present invention is effective for preventing, improving and treating erectile dysfunction, such as increasing the level of endothelial cell specific protein, thereby inducing regeneration of the corpus cavernosum endothelial cell, It can be used as medicines, quasi-drugs or health functional foods useful for preventing or treating erectile dysfunction.

When the composition of the present invention is formulated into a pharmaceutical composition for the purpose of preventing or treating erectile dysfunction, it may further comprise suitable carriers, excipients and diluents conventionally used in the preparation of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents that can be included in the composition 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. When the composition is formulated into a pharmaceutical composition, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which 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. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent 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 means providing the subject invention with a composition of the invention in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention will vary depending on the condition and the weight of the patient, the severity of the disease, the type of drug, the route of administration and the period of time, but for a desired effect, the composition of the present invention contains 0.001 to 1000 mg / kg ≪ / RTI > The composition may be administered once a day or divided into several doses, and the dose is not limited in any way to the scope of the present invention, and the pharmaceutical composition of the present invention may be administered to a subject by various routes . All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. However, at the time of oral administration, since the protein is digested, it is preferable to formulate the oral composition so as to coat the active agent or protect it from decomposition at the top. The pharmaceutical composition of the present invention can preferably be administered in the form of an injection.

The pharmaceutical composition of the present invention may contain one or more known active ingredients having the effect of preventing or treating erectile dysfunction together with DKK3 gene or protein.

The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for prevention and treatment of erectile dysfunction.

The composition of the present invention may be added to a quasi-drug composition for the purpose of preventing or improving erectile dysfunction.

When the composition of the present invention is used as an external composition for an external medicine, the active ingredient may be added as it is or may be used together with other quasi-drugs or quasi-drugs, and may 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 addition, the composition of the present invention can be added to a food composition for the purpose of preventing or improving erectile dysfunction, and can be preferably formulated as a health functional food.

The term " health functional food " in the present invention refers to a food having a biological control function such as prevention and improvement of disease, bio-defense, immunity, recovery after disease and aging inhibition.

The health functional food may include food-acceptable food supplementary additives, and may further include suitable carriers, excipients and diluents conventionally used in the production of health functional foods.

When the composition of the present invention is used as a food additive, the above-mentioned active ingredient can be directly added or 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, it 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.

There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

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.

Hereinafter, preferred examples of the present invention will be described in order to facilitate understanding of the present invention. However, the following experimental examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the experimental examples.

Example  1. In the corpus cavernosal tissues of diabetic mice and diabetic patients DKK3  Expression analysis of proteins and genes

Diabetes was induced by streptozotocin administration (50 mg / kg dose for 5 days) in the abdominal cavity of a 2-month old male mouse (C57BL / 6). After 8 weeks of diabetes induction, mouse penile tissues were harvested, fixed in 4% paraformaldehyde, and frozen sections were subjected to immunofluorescence staining using DKK3 antibody. In addition, DNA was extracted from the penile tissue of the same mouse, and expression of DKK3 gene was confirmed by RT-PCR. As a control group, normal mice of the same age were used. The results are shown in Fig.

As shown in Figure 1A, DKK3 expression in penile tissues was significantly reduced in diabetic mice compared to normal mice. In addition, as shown in Fig. 1B, the expression of DKK3 gene in the penile tissues of diabetic mice was markedly decreased compared to that of normal mice.

In addition, the penile tissues of normal erectile and diabetic erectile dysfunction patients were collected, and then immunofluorescent staining was performed using DKK3 and vWF (von willebrand factor) antibodies after freezing. More specifically, penile tissues were collected and fixed in a tube containing 4% paraformaldehyde at 4 ° C overnight. The penile tissue was then washed three times with PBS and sectioned to a thickness of 7 μm using a freezing section. The sections were washed three times for 5 minutes with PBS, blocked for 1 hour with antibody dilution regimen containing BSA (Invitrogen, Eugene, OR, USA), and reacted with primary antibody overnight at 4 ° C. The next day, the cells were washed 3 times with PBST for 5 minutes and reacted with the fluorescent secondary antibody for 2 hours at room temperature. After the reaction, the cells were washed 3 times with PBST for 5 minutes and fixed with a soluble mount solution. And then analyzed using a confocal microscope. The results are shown in Fig.

As shown in FIG. 2, the expression of DKK3 and vWF in the erectile tissues of patients with diabetic erectile dysfunction was significantly reduced compared to normal subjects.

Example  2. DKK3 Of peptide  In vivo or in vitro extracellular angiogenesis

2-1. In vivo Matrigel  plug experiment

The matrigel liquefied at 4 ° C was treated with DKK3 peptide (100 ng / 400 μL) and then rapidly injected 400 μL into the abdomen subcutaneously of the mice using a syringe. Five days later, the mouse was euthanized and the solidified matrigel under the abdomen was taken to assess the degree of angiogenesis. VEGF peptide (50 ng / 400 [mu] L), an angiogenic peptide known as a positive control, was used. The results are shown in Fig.

As shown in FIG. 3, the group treated with DKK3 peptide promoted angiogenesis to a level similar to that of the group treated with the positive control, VEGF.

2-2. In vitro tube formation experiments

Experiments were performed using Mouse Cavernous Endothelial Cells (MCECs) to analyze the effect of DKK3 peptide on tube formation. More specifically, 50 [mu] L of matrigel treated with DKK3 peptide was added to each well of a 96-well cell culture dish and solidified in a gel form in a 5% CO ₂ incubator at 37 ° C. 200 μL of M199 medium was added to each well, MCECs were added at a concentration of 2 × 10 ⁴ cells per well, cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours, and then observed under a microscope. Respectively. At this time, bFGF and VEGF peptide, which are known as positive control groups, were simultaneously treated, and the results are shown in FIG.

As shown in FIG. 4, in the group treated with DKK3 peptide (20 ng / mL), the tube formation was similar to that of the positive control group treated with bFGF (20 ng / mL) and VEGF .

2-3. Cell migration experiment

MCECs were cultured in 60 mm cell culture dishes at a density of about 95%, and wounded with a razor blade to a width of 2 mm. After washing with M199 medium containing no serum, M199 medium containing 1% serum, 1 mM thymidine, and DKK3 peptide (20 ng / mL) was added and cultured at 37 ° C in 5% CO ₂ Lt; / RTI > After 12 hours, the cells were washed with serum-free M199 medium and fixed with methanol. This was stained with Giemsa solution and the number of cells migrated through the optical microscope was calculated. The results are shown in Fig.

As shown in FIG. 5, in the group treated with DKK3 peptide (20 ng / mL), migration promotion promotion effect of mouse penile cavernosal endothelial cells was similar to that of bFGF and VEGF (20 ng / mL) Respectively.

These results indicate that DKK3 peptide is involved in the migration of endothelial cells of the corpus cavernosum and induces angiogenesis. There is a possibility that DKK3 may effectively work to treat erectile dysfunction induced by penile endothelial cell damage by diabetes Respectively.

Example  3. In the corpus cavernosal tissue DKK3 To peptides  by Angiogenic factor  And Seam  Protein expression analysis

MCECs were treated with DKK3 peptide for 24 hours at various concentrations, and the effects of mRNA and protein expression of angiogenic factors and segregation proteins were analyzed.

3-1. Reverse transcription Polymerase chain reaction

Trizol reagent (Invitrogen, Eugene, OR, USA) was used to extract total RNA from MCECs treated with DKK3 peptide. RT-PCR was performed using the following primers: RT PreMix (BioNEER, Daejeon, South Korea) for the synthesis of cDNA after extraction of total RNA. The primer sequences are as follows.

DKK3 : 5'-ATG CAG CGG CTC GGG GGT AT -3 '(forward), 5'-CTA AAT CTC CTC CTC TCC GC -3' (reverse), VEGF : 5'- CAG GCT GCT GTA ACG ATG AA-3 (forward), 5'-AAA TGC TTT CTC CGC TCT GA-3 '(reverse), bFGF : 5'-GGC TTC TTC CTG CGC ATC CA-3' (forward), 5'- GCT CTT AGC AGA CAT TGG AAGA-3 '(reverse), Claudin-5: 5'-GGT CAC TGG GAA CTT CCT GA-3' (forward), 5'-ATG CCA CTC ACT GCC TCT CT-3 '(reverse), occludin: 5' -TCA GGC AGC CTC GTT ACA G-3 '(forward), 5'-CCT CCT CCA GCT CAT CAC-3' (reverse), ZO -1 : 5'- ATA GCA CGG ACA GTA GAC ACA CTT C-3 (forward), 5'-GTG AGT AAG GAG GGT ATG TCT GGT T-3 '(reverse), Ang1 : 5'-AAA CAG CAA ATG GGA ACA GG- TCC ACT AA-3 '(reverse), Ang2 : 5'-CAA GGC ACT GAG AGA CAC CA-3' (forward), 5'-CTG AAC TCC CAC GGA ACA TT-3 '(reverse).

After the PCR product was obtained, it was electrophoresed using 1.0% agarose gel, and the degree of mRNA expression was analyzed. The results are shown in Figs. 6, 7A, 8 and 9A.

As shown in FIGS. 6, 7A, 8 and 9A, the angiogenic factors bFGF, Ang1, Ang2, ZO-1, claudin-5 and occludin in the DKK3 peptide- mRNA expression was significantly increased and mRNA expression of VEGF was decreased.

3-2. Western Blat

Proteins were separated from MCECs treated with DKK3 peptide, and the proteins were separated by size using SDS-PAGE and fixed on nitrocellulose membranes (Bio-Rad, Richmond, CA, USA). Membranes were blocked with PBS containing 5% non-fat milk powder and 0.05% Tween 20. The primary antibodies (VEGF, Santa Cruz Biotechnology; ZO-1, Santa Cruz Biotechnology; claudin-5, Zymed Laboratories; occludin, SantaCruz Biotechnology; -actin, Abcam) at 4 < 0 > C overnight. The cells were washed three times with PBS for 10 minutes, reacted with secondary antibody for 2 hours, and exposed to X-ray film according to the enhanced chemiluminescence protocol. The results are shown in Figs. 7B and 9B.

As shown in FIGS. 7B and 9B, the expression of ZO-1, claudin-5 and occludin proteins in the corpus cavernosum endothelial cell fusion proteins was significantly increased in the DKK3 peptide-treated group compared to the control group without any treatment , And protein expression of VEGF was decreased.

Example  4. Erectile dysfunction in animal models DKK3 Of erectile dysfunction treatment

4-1. Design of erectile dysfunctional animal models

(1) Laboratory animals and treatment

Streptozotocin (STZ, streptozotocin, 50 mg / kg) was continuously administered to abdominal cavity of 8-week-old male mouse (C57BL / 6) for 5 consecutive days to prepare a model of type 1 diabetic mouse. DKK3 peptide (R & D System, Minneapolis, Minn., USA), DKK3 plasmid (5 mg / kg) were anesthetized with ketamine (100 mg / kg) and xylazine DNA (Abm, Richmond, Canada) and DKK3 adenovirus (Abm) were divided into three independent experiments as follows.

1) Experimental design to identify the improvement of erectile potency of DKK3 peptide in diabetic model

The mice were divided into four groups: N = 4 / group 1, normal mice, group 2, STZ-induced diabetic mice + PBS twice [days -3 and 0; 20 μL] , Group 2, STZ-induced diabetic mouse + mouse DKK3 peptide (SEQ ID NO: 2) twice [days -3 and 0; 5 μg / 20 μL] ) 2 doses [days -3 and 0; 5 μg / 20 μL]). The PBS and DKK3 peptides were injected into the penile corpus caudalis twice at 3-day intervals and the erectile power was measured by the penile cavernous nerve electrical stimulation for 1 week from the last administration day.

2) Experimental design to identify the effect of electroporation on DKK3 plasmid DNA in erectile dysfunction in diabetic model

(N = 5 / group 1, normal mouse, group 2, STZ-induced diabetic mouse group + untreated group, group 3, STZ-induced diabetic mouse + bovine vector administration group) Group 5, STZ-induced diabetic mouse + DKK3-mouse plasmid DNA (abm # 20 / L); Group 4, STZ-induced diabetic mouse + DKK3- mouse plasmid DNA- (100 μg / 20 μL) in group 6, STZ-induced diabetic mice + DKK3-mouse plasmid DNA-treated group. Electrical stimulation (ECM 830, Harvard Apparatus, Holliston, MA, USA) was applied to the penis at 30 V, 40 ms, and 8 pulse intensities immediately after each empty vector or plasmid DNA was injected into the corpus cavernosum. The erectile function was measured by electrical stimulation of the penis cavernosal nerve at 2 weeks.

3) Experimental design to identify the effect of DKK3 adenovirus (Ad-DKK3) on improving erectile function in diabetic model

(STZ-induced diabetic mice + PBS-treated group [20 μL]), Group 3, STZ-induced diabetic mice (group 2, Ad-GFP treated group ^{[1x10 6 vp / 20 μL]} ; 4 group, a diabetic mice induced by STZ + Ad-DKK3 (abm # 190468) - mouse treated ^{[1x10 7 vp / 20 μL]} ; induced in five groups, STZ Group 1, diabetic mouse + Ad-DKK3-mouse group [1x10 ⁸ vp / 20 μL]; group 6, STZ-induced diabetic mouse + Ad-DKK3-mouse group [1x10 ⁹ vp / 20 μL]. Erectile function was measured by electrical stimulation of the penile cavernosal nerve at 2 weeks after Ad-GFP or Ad-DKK3-mouse administration.

4-2. Measurement of erectile force by electric stimulation

DKK3 was administered to the penile corpus cavernosum of the erectile dysfunctional animal model as in Example 4-1, and the left epidermis of the lower abdomen of each mouse was opened to measure the erectile force, and the penile cavernosal nerve located at the posterior lateral side of the prostate ) Were prepared to look good. For electrical stimulation of the penis nerve, a platinum electrode was placed on the penis nerve, and electrical stimulation was applied for about 1 minute at a strength of 5 volts and 12 hertz. When electrical stimulation is applied, the penis becomes erect, and the pressure inside the penis (internal pressure of the penis) during erection is measured using a pressure transducer (BioSpec System, USA) connected to the computer via a catheter inserted in the penis Respectively. The results are shown in Figs. 10 to 12, respectively.

ICP (Intracavernous Pressure) on the vertical axis in FIG. 10 to FIG. 12 refers to the pressure inside the penis (erosion pressure of the penis) during erection, and is generally an index indicating the erection strength. The abscissa in Figs. 10 to 12 represents the time after the electric stimulation, and the electric stimulation period for 1 minute is indicated by a black bar on the abscissa. 10 to 11 show the maximum ICP (maximal ICP), and C in Figs. 10 to 11 show the area (total ICP [area under the curve]) of the penumbra corneal pressure curve Value. 12B shows a value obtained by dividing the maximal ICP (maximal ICP) by the Mean Systolic Blood Pressure (MSBP), FIG. 12C shows the area of the ICP curve (area under the curve) ) Divided by mean systolic blood pressure (MSBP). This is to compensate for the effects of blood pressure itself because it can affect the intracranial pressure of the penis.

10, mice treated with DKK3 peptide and human DKK3 peptide showed higher erectile potency than those treated with PBS. As shown in FIG. 10, % Of the erectile function. In particular, it was confirmed that two parameters related to penile erection, namely, the maximum penile corpus cavernosal pressure and the area of the penile pressure curve show significant improvement after administration of the DKK3 peptide.

As shown in Fig. 11, the group to which DKK3 plasmid DNA was administered (10 μg of pDKK3, 40 μg of pDKK3, and 100 μg of pDKK3) showed a higher erectile function improvement effect than the non-treated group and the blank vector group, ) To about 70% of the erectile function. In particular, it was confirmed that two parameters related to penile erection, that is, the area of the maxillary cavernosal pressure and the area of the penile pressure curve, show significant improvement after administration of the DKK3 plasmid DNA.

As shown in Fig. 12, the group administered with DKK3 adenovirus (Ad-DKK3 1x10 ⁷ vp / 20 μL, 1 × ^{10 8} vp / 20 μL, 1 × ^{10 9} vp / 20 μL) And it was confirmed that the erectile potency was restored to about 90% of the normal control (control). In particular, we found that two parameters related to penile erection, the maximum penile corpus cavernosal pressure and the area of penile pressure curve, show significant improvement after administration of DKK3 adenovirus.

4-3. Expression of vascular endothelial cell-specific protein in corpus cavernosal tissue

The penis consists of cavernosal tissues that function as a penis erection and small blood vessels that supply nutrition. However, under pathological conditions such as diabetes, hypertension, nerve damage, etc., lesions are formed in the corpus cavernosum including these blood vessels and the structure of the corpuscle becomes abnormal, resulting in erectile dysfunction.

In order to analyze the expression level of platelet-endothelial cell adhesion molecule-1 (PECAM-1), which is a specific protein to vascular endothelial cells, in the penile cavernosal tissue of erectile function animal model 3) of Example 4-1, After chemical staining, quantitative changes of vascular endothelial cells were examined using a confocal microscope.

More specifically, the corpus cavernosum tissue was separated from each mouse, and then put in 4% paraformaldehyde and fixed at 4 ° C for 24 hours. The above tissues were fixed using a frozen foraging agent, and then cut into a thickness of 7 mu m in a frozen section to prepare a penile tissue section. The penile tissue sections were mounted on slides and fixed in 4% paraformaldehyde for about 5 minutes for expression analysis of PECAM-1. Fixed penis tissue sections were washed three times with wash buffer (2% FBS + 0.1% Sodium Azide in PBS) and blocked with non-specific protein blocking buffer (5% BSA in PBS) for 1 hour. Thereafter, the cells were reacted with a primary antibody (anti-PECAM-1 hamster antibody, 1: 100) at 4 ° C for 16 hours, washed three times with a washing buffer to remove remaining antibody, (TRITC-labeled anti-hamster antibody, 1: 1000) for 2 hours at room temperature. After the reaction was completed, the cells were washed twice with washing buffer to remove remaining antibody, and then incubated for 5 minutes at room temperature with DAPI (4 ', 6-diamidino-2-phenylindole; Lt; / RTI > Washed twice with wash buffer again and analyzed using a confocal microscope. The results are shown in Fig.

As shown in FIG. 13, the numbers of cavernosal and vascular endothelial cells were significantly decreased in the penile corpus cavernosum tissues of the PBS-administered group and the Ad-GFP treated group compared with the normal control group. However, Showed that the expression of vascular endothelial cell specific protein increased to normal mouse levels. It can be seen from this that regeneration of DKK3 adenovirus induces regeneration of corpus cavernosum and vascular endothelial cells reduced by diabetes and that erectile power is restored accordingly.

4-4. In the corpus cavernosal tissue Seam  Protein expression analysis

In order to analyze the expression level of the segregation protein (ZO-1, Claudin-5) in the penile corpus cavernosum of the erectile dysfunction animal model 3) of Example 4-1, a confocal microscope was used after immunohistochemical staining Quantitative changes of the joint proteins were investigated. The results are shown in Fig. 14 and Fig.

As shown in FIG. 14 and FIG. 15, the expression of the septal protein in the penile corpus cavernosum was significantly reduced in the penile corpus cavernosum tissues of the PBS-treated group and the Ad-GFP treated group compared with the normal control group, In the penile corpus callosum tissue, the expression of the joint protein increased to the normal mouse level. This demonstrates that DKK3 adenovirus recovers the normal level of expression of the reduced seam protein by diabetes and improves erectile function.

Through the above experimental results, the DKK3 protein according to the present invention or the polynucleotide encoding the DKK3 protein increased the level of endothelial cell-specific protein, thereby inducing the regeneration of the corpus cavernosum endothelial cell, thereby increasing the erectile function of the penis, And thus it can be used effectively for preventing or treating erectile dysfunction.

Hereinafter, examples of pharmaceutical compositions and food compositions according to the present invention will be described, but the present invention is not to be construed as limiting the present invention .

Formulation example  1. Preparation of pharmaceutical compositions

1-1. Preparation of capsules

DKK3 protein (gene) 10 mg

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.

1-2. Injection preparation

DKK3 protein (gene) 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

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

1-3. Liquid  Produce

DKK3 protein (gene) 20 mg

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

DKK3 protein (gene) 100 mg

Vitamin mixture quantity

Vitamin A acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

Folic acid 50 μg

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.

<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Composition for preventing or treating erectile dysfunction          comprising DKK3 <130> 1-170 p <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 350 <212> PRT <213> DKK3-human <400> 1 Met Gln Arg Leu Gly Ala Thr Leu Leu Cys Leu Leu Leu Ala Ala Ala   1 5 10 15 Val Pro Thr Ala Pro Ala Pro Ala Pro Thr Ala Thr Ser Ala Pro Val              20 25 30 Lys Pro Gly Pro Ala Leu Ser Tyr Pro Gln Glu Glu Ala Thr Leu Asn          35 40 45 Glu Met Phe Arg Glu Val Glu Glu Leu Met Glu Asp Thr Gln His Lys      50 55 60 Leu Arg Ser Ala Val Glu Glu Met Glu Ala Glu Glu Ala Ala Ala Lys  65 70 75 80 Ala Ser Ser Glu Val Asn Leu Ala Asn Leu Pro Ser Ser Tyr His Asn                  85 90 95 Glu Thr Asn Thr Asp Thr Lys Val Gly Asn Asn Thr Ile His Val His             100 105 110 Arg Glu Ile His Lys Ile Thr Asn Asn Gln Thr Gly Gln Met Val Phe         115 120 125 Ser Glu Thr Val Ile Thr Ser Val Gly Asp Glu Glu Gly Arg Arg Ser     130 135 140 His Glu Cys Ile Ile Asp Glu Asp Cys Gly Pro Ser Met Tyr Cys Gln 145 150 155 160 Phe Ala Ser Phe Gln Tyr Thr Cys Gln Pro Cys Arg Gly Gln Arg Met                 165 170 175 Leu Cys Thr Arg Asp Ser Glu Cys Cys Gly Asp Gln Leu Cys Val Trp             180 185 190 Gly His Cys Thr Lys Met Ala Thr Arg Gly Ser Asn Gly Thr Ile Cys         195 200 205 Asp Asn Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln Arg     210 215 220 Gly Leu Leu Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu Leu 225 230 235 240 Cys His Asp Pro Ala Ser Arg Leu Leu Asp Leu Ile Thr Trp Glu Leu                 245 250 255 Glu Pro Asp Gly Ala Leu Asp Arg Cys Pro Cys Ala Ser Gly Leu Leu             260 265 270 Cys Gln Pro His Ser His Ser Leu Val Tyr Val Cys Lys Pro Thr Phe         275 280 285 Val Gly Ser Arg Asp Gln Asp Gly Glu Ile Leu Leu Pro Arg Glu Val     290 295 300 Pro Asp Glu Tyr Glu Val Gly Ser Phe Met Glu Glu Val Arg Gln Glu 305 310 315 320 Leu Glu Asp Leu Glu Arg Ser Leu Thr Glu Glu Met Ala Leu Gly Glu                 325 330 335 Pro Ala Ala Ala Ala Ala Leu Leu Gly Gly Glu Glu Ile             340 345 350 <210> 2 <211> 349 <212> PRT <213> DKK3-mouse <400> 2 Met Gln Arg Leu Gly Gly Ile Leu Leu Cys Thr Leu Leu Ala Ala Ala   1 5 10 15 Val Pro Thr Ala Pro Ala Pro Ser Pro Thr Val Thr Trp Thr Pro Ala              20 25 30 Glu Pro Gly Pro Ala Leu Asn Tyr Pro Glu Glu Glu Ala Thr Leu Asn          35 40 45 Glu Met Phe Arg Glu Val Glu Glu Leu Met Glu Asp Thr Gln His Lys      50 55 60 Leu Arg Ser Ala Val Glu Glu Met Glu Ala Glu Glu Ala Ala Ala Lys  65 70 75 80 Thr Ser Ser Glu Val Asn Leu Ala Ser Leu Pro Pro Asn Tyr His Asn                  85 90 95 Glu Thr Ser Thr Glu Thr Arg Val Gly Asn Asn Thr Val His Val His             100 105 110 Gln Glu Val His Lys Ile Thr Asn Asn Gln Ser Gly Gln Val Val Phe         115 120 125 Ser Glu Thr Val Ile Thr Ser Val Gly Asp Glu Glu Gly Lys Arg Ser     130 135 140 His Glu Cys Ile Ile Asp Glu Asp Cys Gly Pro Thr Arg Tyr Cys Gln 145 150 155 160 Phe Ser Ser Phe Lys Tyr Thr Cys Gln Pro Cys Arg Asp Gln Gln Met                 165 170 175 Leu Cys Thr Arg Asp Ser Glu Cys Cys Gly Asp Gln Leu Cys Ala Trp             180 185 190 Gly His Cys Thr Gln Lys Ala Thr Lys Gly Gly Asn Gly Thr Ile Cys         195 200 205 Asp Asn Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln Arg     210 215 220 Gly Leu Leu Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu Leu 225 230 235 240 Cys His Asp Pro Thr Ser Gln Leu Leu Asp Leu Ile Thr Trp Glu Leu                 245 250 255 Glu Pro Glu Gly Ala Leu Asp Arg Cys Pro Cys Ala Ser Gly Leu Leu             260 265 270 Cys Gln Pro His Ser His Ser Leu Val Tyr Met Cys Lys Pro Ala Phe         275 280 285 Val Gly Ser His Asp His Ser Glu Glu Ser Gln Leu Pro Arg Glu Ala     290 295 300 Pro Asp Glu Tyr Glu Asp Val Gly Phe Ile Gly Glu Val Arg Gln Glu 305 310 315 320 Leu Glu Asp Leu Glu Arg Ser Leu Ala Gln Glu Met Ala Phe Glu Gly                 325 330 335 Pro Ala Pro Val Glu Ser Leu Gly Gly Glu Glu Glu Ile             340 345 <210> 3 <211> 2587 <212> DNA <213> DKK3-human <400> 3 cgcgcctctg atcgcgttcc gggacacaca ggcggcggct gcgggcgcag agcggagatg 60 cagcggcttg gggccaccct gctgtgcctg ctgctggcgg cggcggtccc cacggccccc 120 gcgcccgctc cgacggcgac ctcggctcca gtcaagcccg gcccggctct cagctacccg 180 caggaggagg ccaccctcaa tgagatgttc cgcgaggttg aggaactgat ggaggacacg 240 cagcacaaat tgcgcagcgc ggtggaagag atggaggcag aagaagctgc tgctaaagca 300 tcatcagaag tgaacctggc aaacttacct cccagctatc acaatgagac caacacagac 360 acgaaggttg gaaataatac catccatgtg caccgagaaa ttcacaagat aaccaacaac 420 cagactggac aaatggtctt ttcagagaca gttatcacat ctgtgggaga cgaagaaggc 480 agaaggagcc acgagtgcat catcgacgag gactgtgggc ccagcatgta ctgccagttt 540 gccagcttcc agtacacctg ccagccatgc cggggccaga ggatgctctg cacccgggac 600 agtgagtgct gtggagacca gctgtgtgtc tggggtcact gcaccaaaat ggccaccagg 660 ggcagcaatg ggaccatctg tgacaaccag agggactgcc agccggggct gtgctgtgcc 720 ttccagagag gcctgctgtt ccctgtgtgc acacccctgc ccgtggaggg cgagctttgc 780 catgaccccg ccagccggct tctggacctc atcacctggg agctagagcc tgatggagcc 840 ttggaccgat gcccttgtgc cagtggcctc ctctgccagc cccacagcca cagcctggtg 900 tatgtgtgca agccgacctt cgtggggagc cgtgaccaag atggggagat cctgctgccc 960 agagaggtcc ccgatgagta tgaagttggc agcttcatgg aggaggtgcg ccaggagctg 1020 gaggacctgg agaggagcct gactgaagag atggcgctga gggagcctgc ggctgccgcc 1080 gctgcactgc tgggagggga agagatttag atctggacca ggctgtgggt agatgtgcaa 1140 tagaaatagc taatttattt ccccaggtgt gtgctttagg cgtgggctga ccaggcttct 1200 tcctacatct tcttcccagt aagtttcccc tctggcttga cagcatgagg tgttgtgcat 1260 ttgttcagct cccccaggct gttctccagg cttcacagtc tggtgcttgg gagagtcagg 1320 cagggttaaa ctgcaggagc agtttgccac ccctgtccag attattggct gctttgcctc 1380 taccagttgg cagacagccg tttgttctac atggctttga taattgtttg aggggaggag 1440 atggaaacaa tgtggagtct ccctctgatt ggttttgggg aaatgtggag aagagtgccc 1500 tgctttgcaa acatcaacct ggcaaaaatg caacaaatga attttccacg cagttctttc 1560 catgggcata ggtaagctgt gccttcagct gttgcagatg aaatgttctg ttcaccctgc 1620 attacatgtg tttattcatc cagcagtgtt gctcagctcc tacctctgtg ccagggcagc 1680 attttcatat ccaagatcaa ttccctctct cagcacagcc tggggagggg gtcattgttc 1740 tcctcgtcca tcagggatct cagaggctca gagactgcaa gctgcttgcc caagtcacac 1800 agctagtgaa gaccagagca gtttcatctg gttgtgactc taagctcagt gctctctcca 1860 ctaccccaca ccagccttgg tgccaccaaa agtgctcccc aaaaggaagg agaatgggat 1920 ttttcttttg aggcatgcac atctggaatt aaggtcaaac taattctcac atccctctaa 1980 aagtaaacta ctgttaggaa cagcagtgtt ctcacagtgt ggggcagccg tccttctaat 2040 gaagacaatg atattgacac tgtccctctt tggcagttgc attagtaact ttgaaaggta 2100 tatgactgag cgtagcatac aggttaacct gcagaaacag tacttaggta attgtagggc 2160 gaggattata aatgaaattt gcaaaatcac ttagcagcaa ctgaagacaa ttatcaacca 2220 cgtggagaaa atcaaaccga gcagggctgt gtgaaacatg gttgtaatat gcgactgcga 2280 acactgaact ctacgccact ccacaaatga tgttttcagg tgtcatggac tgttgccacc 2340 atgtattcat ccagagttct taaagtttaa agttgcacat gattgtataa gcatgctttc 2400 tttgagtttt aaattatgta taaacataag ttgcatttag aaatcaagca taaatcactt 2460 caactgctct tctgtagttc ttggatttct tttccctttt gactttgaat aaatgtaaaa 2520 tcctttcagc cagaaaaagt aaaatagaaa caacctgtat taaaaatctt ccatagaaaa 2580 aaaaaaa 2587 <210> 4 <211> 3357 <212> DNA <213> DKK3-mouse <400> 4 gcacacagtg cctctccagc tgctctgtgg cagcccagct accggtcgtg accagatcca 60 gcttgcagct cagctttgtt cattcgaatt gggcggcggc cagcgcggaa caaacatgca 120 gcggctcggg ggtattttgc tgtgtacact gctggcggcg gcggtcccca ctgctcctgc 180 tccttccccg acggtcactt ggactccggc ggagccgggc ccagctctca actaccctca 240 ggaggaagct acgctcaatg agatgtttcg agaggtggag gagctgatgg aagacactca 300 gcacaaactg cgcagtgccg tggaggagat ggaggcggaa gaagcagctg ctaaaacgtc 360 ctctgaggtg aacctggcaa gcttacctcc caactatcac aatgagacca gcacggagac 420 cagggtggga aataacacag tccatgtgca ccaggaagtt cacaagataa ccaacaacca 480 gagtggacag gtggtctttt ctgagacagt cattacatct gtaggggatg aagaaggcaa 540 gaggagccat gaatgtatca ttgatgaaga ctgtgggccc accaggtact gccagttctc 600 cagcttcaag tacacctgcc agccatgccg ggaccagcag atgctatgca cccgagacag 660 tgagtgctgt ggagaccagc tgtgtgcctg gggtcactgc acccaaaagg ccaccaaagg 720 tggcaatggg accatctgtg acaaccagag ggattgccag cctggcctgt gttgtgcctt 780 ccaaagaggc ctgctgttcc ccgtgtgcac acccctgccc gtggagggag agctctgcca 840 tgaccccacc agccagctgc tggatctcat cacctgggaa ctggagcctg aaggagcttt 900 ggaccgatgc ccctgcgcca gtggcctcct atgccagcca cacagccaca gtctggtgta 960 catgtgcaag ccagccttcg tgggcagcca tgaccacagt gaggagagcc agctgcccag 1020 ggaggccccg gatgagtacg aagatgttgg cttcataggg gaagtgcgcc aggagctgga 1080 agacctggag cggagcctag cccaggagat ggcatttgag gggcctgccc ctgtggagtc 1140 actaggcgga gaggaggaga tttaggccca gacccagctg agtcactggt agatgtgcaa 1200 tagaaatggc taatttattt tcccaggagt gtccccaagt gtggaatggc cgcagctcct 1260 tcccagtagc ttttcctctg gcttgacaag gtacagtgca gtacatttct tccagccgcc 1320 ctgcttctct gacttgggaa agacaggcat ggcgggtaag ggcagcggtg agtcgtccct 1380 cgctgttgct agaaacgctg tcttgttctt catggatgga agatttgttt gaagggagag 1440 gatgggaagg ggtgaagtct gctcatgatg gatttggggg atacagggag gaggatgcct 1500 gccttgcaga cgtggacttg gcaaaatgta acctttgctt ttgtcttgcg ccgctcccat 1560 gggctgaggc agtggctaca caagagctat gctgctctgt ggcctcccac atattcatcc 1620 ctgtgtttca gctcctacct cactgtcagc acagcccttc atagccacgc cccctcttgc 1680 tcaccacagc ctaggagggg accagagggg acttctctca gagccccatg ctctctctct 1740 caaccccata ccagcctctg tgccagcgac agtccttcca aatggaggga gtgaaatcct 1800 ttggttttat tttttctcc ttcaaggcac gcctgccact aaggtcaggc tgacttgcat 1860 gtccctctaa cgttcgtagc agtgtggtgg acactgtctt ccaccgactg cttcaatacc 1920 tctgaaagcc agtgctcgga gtgcagttcg tgtaaattaa tttgcaggaa gtatacttgg 1980 ctaattgtag ggctaggatt gtgaatgaaa tttgcaaagt cgcttagcaa caatggaaag 2040 cctttctcag tcacaccgag aagtcacaac caagccaggt tgtgtagagt acagctgtga 2100 catacagaca gaagaaggct gggctggatg tcaggcctca gatgacggtt tcaggtgcca 2160 ggaactatta ccattctgta tctatccaga gttattaaaa ttgaaagttg cacacatttg 2220 tataagcatg cctttctcct gagttttaaa ttatatgtat acacaaacat gtggccctca 2280 aagatcatgc acaaaccact actctttgct aattcttgga cttttctctt tgattttcaa 2340 taaatacaaa tccccttcat gcaaaaaaat taaaacaatc tgtagtataa agagacaaaa 2400 aaattccata gaagcagatt ttccaggcat ctgcagtttc cctcttttag aatcggaatt 2460 cgttggaact ctcatccttg tctggatggg aattagcttt aacagagaaa ctacttcacc 2520 ctctcctgaa agaacaaatg gaatatatga gtcttctctt ggaggctctt tccactcaaa 2580 tgcagttctg gggctgtgct agcattgata ctgtaacaaa acggctgaag caatgaactt 2640 atatatttaa aaagttaggt taattgggtt caccatttca ggtttcagtc ctgatcccat 2700 ggggttgaaa ctaaggagag gcagcacagc gtggcaaggg aatgtggtag agtcaagctg 2760 ctccctttct ggctaacagg agagtgggca atgtgcagtc ttgtgagaat gcccaggtcc 2820 tggggggaag ggagtgccct ggacatcacc ttaaaggtgg agacttctgc agctttggtt 2880 ttagttactc ttctgggtgc tacaatcaaa cgcccaacaa gaagccacct gagggatgag 2940 ggtttatttt ggctcctggt tcaagcaggg agtccttcgt ggcaggagtg caaggttgct 3000 tcctgcagtg tggaggatca ggaagcaaag aaagagcaat gcaagactca gctttctctc 3060 tttccctgat tatttattct ggaaccccaa cccttggggt ggtgccgacc gcagtaagag 3120 tgagtgtcct ttccttagaa ccctctgaaa actcttggcc tcatagaaat gtgcagaggt 3180 gtgtcaccta aattgttcaa atccattctg ttccaagaca tgggagcgct atgtgctaag 3240 tcttccacat aagaccaccg agtacctctt aaacgcctgt aaatcgcatc tgaagatacc 3300 acagtaaaga gatgtaaaca tttaggaaaa caataaatgt aactgatgaa gtcacct 3357

Claims (7)

A pharmaceutical composition for preventing or treating erectile dysfunction, comprising DKK3 (Dickkopf 3) protein or a polynucleotide encoding said DKK3 protein as an active ingredient. The pharmaceutical composition for preventing or treating erectile dysfunction according to claim 1, wherein the DKK3 protein comprises the amino acid sequence of SEQ ID NO: 1 or 2. The pharmaceutical composition for preventing or treating impotence according to claim 2, wherein the DKK protein is composed of amino acids 22 to 350 of SEQ ID NO: 1 or amino acids 23 to 350 of SEQ ID NO: 2. The pharmaceutical composition for preventing or treating erectile dysfunction according to claim 1, wherein the polynucleotide encoding the DKK3 protein comprises the nucleotide sequence of SEQ ID NO: 3 or 4. The pharmaceutical composition for preventing or treating impotence according to claim 4, wherein the polynucleotide encoding the DKK3 protein is contained in a plasmid vector. DKK3 (Dickkopf 3) gene or protein as an active ingredient. DKK3 (Dickkopf 3) gene or protein as an active ingredient.

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