KR102176832B1 - A microparticle comprising hedgehog protein and biocompatible material and a composition for preventing or treating hair loss comprising the same - Google Patents

Abstract

The present invention relates to fine particles comprising a hedgehog protein and a biocompatible material, and a composition for preventing or treating hair loss comprising the same, and according to the present invention, the hedgehog protein can be easily delivered into the body through the skin, As a result, it is possible to provide a pharmaceutical composition for preventing or treating hair loss having an excellent effect from the fine particles, and a cosmetic composition for preventing hair loss, promoting hair growth or hair growth.

Description

Fine particles containing hedgehog protein and biocompatible material, and a composition for preventing or treating hair loss containing the same {A MICROPARTICLE COMPRISING HEDGEHOG PROTEIN AND BIOCOMPATIBLE MATERIAL AND A COMPOSITION FOR PREVENTING OR TREATING HAIR LOSS COMPRISING THE SAME}

The present invention relates to fine particles for absorbing hedgehog protein into the skin, including hedgehog protein and biocompatible material, a pharmaceutical composition for preventing or treating hair loss comprising the same, and a cosmetic composition for preventing hair loss, promoting hair growth or hair growth will be.

In the past, hair loss has been recognized as a phenomenon that occurs as aging progresses, but in recent years, it is known to occur due to a combination of genetic factors and environmental factors. Environmental factors that cause hair loss include exposure to environmental pollutants, lack of nutrition due to excessive diet, nutritional imbalance and abnormal hormone secretion due to dietary changes, stress from work and social activities, irregular lifestyle habits, and reckless use of chemicals. Etc. Due to these environmental factors, hair loss is also increasing in young men and women in their 20s and 30s, and as the demand for prevention and treatment for this increases, various studies have been attempted to overcome hair loss. As a result of the study, until now, the DHT Theory, Dihydrotestosterone Theory, Gravity Theory, and the blood supply theory are known as general hypotheses for hair loss, but the exact cause and mechanism of hair loss or hair growth are still known. Not.

Propecia and minoxidil, which are currently used alopecia relieving and hair growth promoting agents, have a problem that symptoms recur when application is stopped, and it is difficult to recover already advanced hair loss.In addition, dermatitis, hair growth in unwanted areas, and erectile dysfunction when applied for a long time And it has been reported that side effects such as decreased sexual function such as decreased libido, dizziness, headache and swelling appear. Moreover, most of the anti-hair loss formulations licensed as quasi-drugs have unclear clinical effects such as preventing hair loss or increasing the thickness of hair, so the Ministry of Food and Drug Safety is trying to manage it as a functional cosmetic through a review.

In addition to these hair loss-related agents, surgical treatment methods for preventing or treating hair loss, such as scalp reduction surgery, hair follicle transplantation surgery, scalp transplantation surgery, and injection methods, and promoting hair growth, are also used. However, it has been reported that the surgical treatment method has a risk of skin necrosis occurring at the surgical site or the formation of skin sclerosis such as scar and keloid.

As described above, the currently used hair loss-related preparations and surgical treatment methods have various problems such as insufficient effect or side effects, so more effective and safe prevention or treatment of hair loss, or prevention of hair loss, hair growth or hair growth There is a need to develop new formulations for promotion.

Meanwhile, the hedgehog (hh) protein is a signaling protein that plays a very important role in the embryonic development process. In higher organisms, the hh family consists of sonic, indian, and desert hedgehog proteins, and the sonic hedgehog (Shh) protein inhibits the patched receptor in the hedgehog signaling pathway. It plays an important role in initiating transcription of the target protein by sequentially activating the Sumo (Smoothened) membrane protein and the Gli transcription factor.

The method of applying useful substances such as hedgehog protein to the skin in order to deliver them into the body has the advantage that continuous substance delivery is possible, there is no pain, and it is easy to use. However, since the stratum corneum layer of 10 μm to 60 μm thick at the outermost part of the skin prevents the penetration of foreign substances into the body, the absorption rate in the body of the active ingredient is very low, especially if the active ingredient is hydrophilic or has a large molecular weight, the absorption rate in the body is further lowered. Therefore, in order to effectively deliver useful substances, an administration method using injection is used. However, the injection method has a drawback that it cannot be easily used at home because the injection method gives considerable pain to the subject, and injection can be performed only in a hospital or specialized skin care institution in general.

US Patent Publication No. 2011-0306546

An object of the present invention is to provide fine particles for exhibiting a desired effect by absorbing hedgehog protein into the skin.

Another object of the present invention is a safe pharmaceutical composition and cosmetic composition comprising the fine particles for preventing or treating hair loss, or for preventing hair loss, promoting hair growth or hair growth, and can be applied regardless of age or sex To provide.

According to an aspect of the present invention, a hedgehog protein provides fine particles contained in a biocompatible material.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating hair loss, comprising the fine particles.

According to another aspect of the present invention, there is provided a cosmetic composition for preventing hair loss, hair growth or promoting hair growth, including the fine particles.

According to the fine particles of the present invention, hedgehog protein can be easily delivered to the body through skin absorption, and as a result, a pharmaceutical composition for preventing or treating hair loss having excellent effects from the fine particles, and for preventing hair loss, promoting hair growth or hair growth A cosmetic composition can be provided.

1 shows the result of confirming that the Shh protein was expressed in E. coli transformed with the pET-hShh vector.
FIG. 2 shows the results of selecting mutant vectors (RanShh-6, RanShh-10, RanShh-16, and RanShh-22) whose expression rate was significantly increased than that of the Shh protein obtained from the pET-hShh vector.
Figure 3 shows the result of confirming that the ubiquitin fused form of Shh protein was expressed.
4A is a schematic diagram showing the purification process of the Shh protein in which ubiquitin is fused.
Figure 4b shows the result of purification of the Shh protein from which ubiquitin has been removed using a Ni-affinity resin.
Figure 4c shows the result of confirming the final purified Shh protein.
5 is a graph showing the results of measuring the biological activity of Shh protein.
6A to 6I are graphs showing the results of confirming that the Shh protein increases the expression of major genes during the Shh signaling process.
7 is a quantitative representation of the expression levels of hair-related genes in human skin papilloma cells by Shh treatment.
FIG. 8 is a view of a cream prepared including microparticles containing Shh protein applied to the scalp and observed after 1 month.
FIG. 9 shows the appearance of the cream prepared including microparticles containing Shh protein applied to the scalp and observed after 2.5 months and 4 months.

Hereinafter, the present invention will be described in detail.

The present invention relates to microparticles in which the hedgehog protein is contained in a biocompatible material. At this time, the microparticles are microparticles for absorbing hedgehog protein into the skin.

In one aspect of the present invention, the hedgehog protein may be derived from a mammal, preferably a human, may be a sonic hedgehog protein, an Indian hedgehog protein or a desert hedgehog protein, and the sonic hedgehog protein It is preferred, but is not limited thereto.

In one aspect of the present invention, the hedgehog protein may be a recombinant hedgehog protein derived from E. coli, and the hedgehog protein is preferably a sonic hedgehog protein, but is not limited thereto. In this case, the sonic hedgehog protein may be a full-length amino acid or fragment. When the sonic hedgehog protein is a fragment, it may include glycine, which is the 197th amino acid from glycine, which is the 25th amino acid of the amino acid sequence of the full-length sonic hedgehog protein. In addition, the sonic hedgehog protein may be in a form in which one or two amino acids are additionally bound to the N-terminus, and preferably, two isoleucine (Ile) is additionally bound to the N-terminus of the sonic hedgehog protein. It can be a form.

The sonic hedgehog protein is a concept including a protein represented by SEQ ID NO: 7 and a functional equivalent of the protein. As used herein, the term “functional equivalent” refers to at least 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more of the sequence as a result of addition, substitution, or deletion of amino acids. It has sequence homology and refers to a protein that exhibits substantially the same physiological activity as the sonic hedgehog protein.

In one aspect of the present invention, the ubiquitin protein may be bound to the N-terminus of the sonic hedgehog protein, wherein the protein represented by SEQ ID NO: 8 is fused with the protein represented by SEQ ID NO: 7 It is a form of protein. Microparticles comprising the functional equivalent of the protein represented by SEQ ID NO: 8 and compositions comprising the same are also included in the scope of the present invention.

The term “vector” is used herein to refer to a DNA fragment(s) or nucleic acid molecule that is delivered into a cell. Vectors replicate DNA and can be reproduced independently in host cells. The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence essential for expressing the coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals usable in eukaryotic cells are known.

The term "recombinant" as used herein refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid.

As used herein, the term “recombinant protein” refers to any protein expressed from a recombinant genetic material encoding an amino acid, including peptides, polypeptides, proteins, oligoproteins, and/or fusion proteins, or biologically active portions thereof (eg, It means a part that retains biological activity).

In one aspect of the present invention, the fine particles may be solid, and each surface of the fine particles may be flat, may have irregularities, and may be hollow. In addition, there may be defects such as cracking of a corner or part of a vertex of the fine particles.

In one aspect of the present invention, the biocompatible material may be any one selected from the group including polysaccharides, polyvinyl alcohol, carboxyvinyl polymers, chitosan, hyaluronic acid, cellulose polymers, and combinations thereof, and hyaluronic acid is It is preferred, but is not limited thereto.

In one aspect of the present invention, the fine particles may be in the form of a three-dimensional figure having at least one corner or vertex, and preferably in the form of a tetrahedron, a pentagram, or a cone. When the fine particles are applied on the skin and tapped or rubbed by hand, the vertices or corners of the three-dimensional figure form micropores in the keratin or remove part of the keratin. When the hedgehog protein passes through the fine pores formed in the stratum corneum or the thinned stratum corneum, the largest physical barrier among skin tissues, the hedgehog protein is relatively easy in the epidermis and dermal layers, which are rich in moisture and have relatively low cell density. Spreads. Therefore, the skin absorption of hedgehog protein is increased by the fine particles. In order for such an action to occur, the vertices or corners of the polyhedron must form a sharp angle.

In one aspect of the present invention, since the thickness of the stratum corneum of the skin is 10 μm to 60 μm and the skin is elastic, the horizontal length of one side of the fine particles is 50 μm to 500 μm in order to form micropores in the stratum corneum. It is preferred, but is not limited thereto. If the fine particles are a polyhedron such as a tetrahedron or a pentahedron, the base and height are preferably 50 μm to 500 μm, respectively, and in the case of a cone, the diameter and height of the base are preferably 50 μm to 500 μm, but are not limited thereto. .

In addition, if the fine particles continue to maintain their shape without being dissolved, there is an inconvenience that the user must wash. Moreover, if the fine particles are embedded in the stratum corneum, most of the fine pores are blocked, so that the skin absorption of the skin cosmetic material is not sufficiently improved. Therefore, in one aspect of the present invention, the fine particles are preferably easily dissolved, decomposed or swelled in body fluid or water.

In one aspect of the present invention, a depression is formed on at least one surface of the fine particle, and the depth of the depression may be within 1/2 of the height of the fine particle, but is not limited thereto.

In addition, the present invention relates to a pharmaceutical composition for preventing or treating hair loss, comprising the fine particles.

As used herein, the term "prevention" refers to any action that suppresses or delays the onset of hair loss by administration of the composition. As used herein, the term "treatment" refers to any action that improves or benefits the symptoms of hair loss already caused by administration of the composition of the present invention.

In addition, the present invention relates to a cosmetic composition for preventing hair loss, promoting hair growth or hair growth, comprising the fine particles.

In one aspect of the present invention, the composition may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions or cosmetic compositions.

In one aspect of the present invention, in the formulation of the pharmaceutical composition, it may be prepared by using excipients or diluents such as generally used pharmaceutically acceptable fillers, extenders, binders, wetting agents, disintegrants, and surfactants. In addition, anti-aggregating agents, lubricants, flavors, emulsifiers, preservatives, etc. may additionally be included, using methods well known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal. Can be formulated.

In one aspect of the present invention, the pharmaceutical composition may be formulated in a conventional pharmaceutical formulation known in the art, preferably a transdermal formulation and a skin external formulation for topical application. I can. Specifically, the pharmaceutical composition may be formulated as a cream, ointment, paste, gel, jelly, serum, aerosol spray, non-aerosol spray, foam, lotion, solution or suspension, but is not limited thereto.

In one aspect of the present invention, the cosmetic composition may be appropriately selected and blended by a person skilled in the art within a range that does not impair the object and effect of the present invention. Examples of ingredients that can be added include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, and blood circulation accelerators. , Cooling agents, restrictive agents, and purified water.

In one aspect of the present invention, the cosmetic composition may be formulated in a conventional formulation known in the art to which the present invention belongs, and hair cream, hair paste, hair tonic, hair conditioner, hair essence, hair lotion, hair Nourishing lotion, hair shampoo, hair rinse, hair treatment, hair nutrition cream, hair moisture cream, hair massage cream, hair wax, hair aerosol, hair pack, hair nutrition pack, hair soap, hair cleansing foam, hair oil, hair dryer, hair Preservatives, hair dyes, hair wave agents, hair bleaching agents, hair gels, hair glazes, hairdressers, hair lacquers, hair moisturizers, hair mousses or hair spray formulations, but are not limited thereto.

The composition according to the present invention can be applied (administered) 1 to 6 times a day to the target site when topically applied, and the daily application amount (dosage) is 0.001 mg to 1000 mg/day/weight based on the amount of hedgehog protein. kg, preferably 0.01 mg to 100 mg/day/kg of body weight, more preferably 0.1 mg to 10 mg/day/kg of body weight. Such dosage and frequency may be appropriately increased or decreased according to the patient's age, sex, and progression of hair loss.

Hereinafter, the present invention will be described in more detail by examples. However, the following examples are only illustrative of the present invention, and that various changes and modifications are possible within the scope and spirit of the present invention are obvious to those of ordinary skill in the art, and such modifications and modifications are It is natural to fall within the scope of the appended claims.

Example One. Sonic Hedgehog ( Shh ) Preparation of protein

Example 1.1.Shh CDNA gene of protein Cloning

In order to express Shh as a recombinant protein, total RNA was extracted from Huh7 cells, a cell line derived from human liver cancer, and cDNA was synthesized therefrom. Huh7, a human liver cancer-derived cell line, was cultured in 10% serum medium under 5% carbon dioxide at 37°C (1x10 ⁶ cells), and then the culture solution was removed, PBS buffer was added to the cells, washed twice, and 0.5 ml of RNA extract ( Trizol reagent, Thermo Fisher Scientific) was added directly. After the mixture to which the RNA extract was added was allowed to stand at room temperature for 10 minutes, 0.1 ml of chloroform was added and stirred for 15 seconds, followed by centrifugation at 12,000 xg for 10 minutes. Then, the separated supernatant was taken, the same volume of isopropyl alcohol was added, centrifuged at 12,000 xg for 10 minutes to remove the liquid, washed once with 75% ethanol, and the RNA was dried at room temperature. 50 μl of purified distilled water without RNAase was added, and the quantification and purity of RNA were measured using a spectrophotometer.

To synthesize cDNA, 2 μg of purified total RNA was subjected to a binding reaction with oligo dT for 5 minutes at 70°C, followed by 10X reverse transcription reaction buffer solution, 10 mM dNTP, RNAse inhibitor, and M-MLV reverse transcriptase (Enzynomics, Korea ) Was added to perform cDNA synthesis reaction at 42°C for 60 minutes. After the cDNA synthesis reaction, the reverse transcriptase was inactivated by heating at 72° C. for 5 minutes, and then RNase H was added to remove single-stranded RNA, which was used as a template for the polymerase chain reaction of the mature Shh gene. Obtaining a gene encoding a sonic hedgehog protein from a human liver cancer-derived cell line containing glycine, which is the 25th amino acid of the amino acid sequence of the full-length sonic hedgehog protein, and glycine, which is the 197th amino acid, and two additional isoleucines at the N-terminal Primer encoding the amino terminus (NdeShh) 5'-GGA TCC CAT ATG ATC ATC GGA CCG GGC AGG GGG TTC GG-3' (SEQ ID NO: 9) and primer encoding the carboxyl terminus (XShh) 5'-AAA AAA CTC GAG TTA GCC TCC CGA TTT GGC CGC CAC-3' (SEQ ID NO: 10) was synthesized, and then polymerase chain reaction was performed using the cDNA prepared above as a template.

After mixing 0.2 pmol primer (NdeShh) and 0.2 pmol primer (XShh) with dNTP 0.2 nM, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit of AccuPrime Taq DNA polymerase, polymerase The amplification reaction was performed in a chain reaction apparatus at 95°C and 40 seconds, 58°C and 30 seconds, 72°C and 40 cycles of 1 minute. After the reaction, the amplified DNA fragment of 0.55 kbp was separated by electrophoresis on a 1% agarose gel, and then inserted into pGEMT easy (Promega, USA) vector using a T4 DNA ligase, and the resulting DNA was sequenced. As a result, it was confirmed that cDNA encoding the Shh protein was obtained. The Shh gene obtained by this process was named pTA-hShh (SEQ ID NO: 1).

Example 1.2. Shh Preparation and expression of E. coli expression vector of gene

In order to express the Shh gene represented by SEQ ID NO: 1 as a recombinant protein in E. coli, the Shh gene was inserted into a pET15b expression vector having a T7 promoter, which is a protein expression vector for E. coli. First, 2 μg of the pTA-hShh gene whose base sequence was confirmed was digested with NdeI and XhoI restriction enzymes, and then subjected to electrophoresis on a 1% agarose gel to obtain a DNA fragment corresponding to 530 bp, which was then digested with NdeI and XhoI of the pET15b expression vector. It was inserted into the site. The vector prepared in this way was named pET-hShh. E. coli BL21(DE3)pLys.S strain was transformed using the plasmid pET-hShh obtained above, and selected in Luria-Bertani (LB) solid medium to which the antibiotic ampicillin was added. The single colony thus obtained was cultured in an LB liquid medium in a shaking incubator at 37° C. and the cell density reached 0.4 absorbance at 600 nm, so that the final concentration of 0.5 mM was obtained. Isopropyl-β-D-thiogalactoside, IPTG) was added, followed by further shaking culture for 4 hours.

After obtaining a part of E. coli cells by centrifugation, the cells were crushed and then subjected to SDS-polyacrylamide electrophoresis to confirm that the 19 kDa Shh protein was weakly expressed (FIG. 1).

Example 1.3. Sequence Variation for inducing an increase in Shh expression level according to a modification of Shh 5 'terminal coding region genes

In order to increase the expression of the Shh protein obtained in Example 1.2, an increase in the expression rate was induced as follows only through mutation of the base sequence without changing the amino acid sequence of the coding region at the Shh amino terminal. Primer with degenerate wobble sequence (RanNdeShh) 5'-GGA TCC CAT ATG ATH ATH GGN CCN GGN for 12 codons excluding the start codon (methionine) of the 5'end coding region of the Shh gene. AGR GGN TTY GGN AAR AGR AGR CAC CCC AAA AAG CTG ACC CCT TTA GC-3' (SEQ ID NO: 11) and primer encoding the carboxyl terminus (XShh) 5'-AAA AAA CTC GAG TTA GCC TCC CGA TTT GGC CGC CAC Polymerase chain reaction was performed using -3' (SEQ ID NO: 10) using pTA-hShh DNA as a template. After mixing 0.2 pmol primer (RanNdeShh) and 0.2 pmol primer (XShh) with dNTP 0.2 nM, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit of AccuPrime Taq DNA polymerase, polymerase chain reaction device The amplification reaction was performed at 95° C. and 40 seconds, 58° C. and 30 seconds, 72° C. and 25 cycles of 1 minute. After the reaction, the amplified 0.55 kbp DNA fragment was digested with restriction enzymes NdeI and XhoI, and then separated by electrophoresis on a 1% agarose gel. Thereafter, the plasmid pET-hRanShh was obtained by inserting into the pET15b vector digested with restriction enzymes NdeI and XhoI using T4DNA ligase.

E. coli BL21(DE3)pLys.S strain was transformed using the plasmid pET-hRanShh. Then, the colonies obtained from the Luria-Bertani (LB) solid medium to which the antibiotic ampicillin was added were cultured in a shaking incubator at 37°C in LB liquid medium, and the final concentration of 0.5 mM was reached when the cell density reached 0.4 absorbance at 600 nm. Isopropylthiogalactoside (IPTG) was added so as to be. Then, shaking culture was performed for 4 hours more. A part of E. coli cells were obtained by centrifugation, and then the cells were crushed, and then SDS-polyacrylamide electrophoresis was performed. Based on this, the mutant vectors (RanShh-6, RanShh-10, RanShh-16, and RanShh-22) having significantly increased expression rates than those obtained from conventional pET-hShh were selected (FIG. 2). The 5'terminal gene sequences of the mutants whose expression rate was significantly increased are shown in Table 1 below (SEQ ID NOs: 2, 3, 4 and 5).

Sequence number division order 2 RanShh-6 5'-ATG ATC ATT GGT CCA GGC AGA GGG TTT GGG AAG AGA AGG CACCCCAAAAAGCTGACCCCTTTA-3' 3 RanShh-10 5'-ATG ATC ATT GGT CCC GGC AGG GGA TTC GGG AAG AGA AGA CACCCCAAAAAGCTGACCCCTTTA-3' 4 RanShh-16 5'-ATG ATT ATA GGG CCG GGC AGG GGG TTT GGT AAG AGA AGG CACCCCAAAAAGCTGACCCCTTTA-3' 5 RanShh-22 5'-ATG ATA ATA GGG CCG GGG AGG GGG TTT GGG AAG AGA AGA CACCCCAAAAAGCTGACCCCTTTA-3'

Example 1.4. Ubiquitin Fused Shh Expression vector preparation and expression

In order to prepare Shh from which methionine, which is an initiation codon, is removed, an expression vector capable of expressing Shh in a fused form of ubiquitin was prepared. Primer (NdeUB) 5'-GGA TTC CAT ATG CAA CTT TTC GTC AAA ACT CTA AC-3' (SEQ ID NO: 12) and primer (T2UB) 5'-ATG ACC ACC GCG GAG TCT CAA CAC CAA- to obtain ubiquitin gene 3'(SEQ ID NO: 13) was prepared, and primer (T2RanShh-6) 5'-GGA TCC CCG CGG TGG TAT CAT TGG TCC AGG to prepare the variant RanShh-6 obtained in Example 1.3 in a ubiquitin fusion form CAG AGG G-3' (SEQ ID NO: 14) was synthesized. After mixing 0.2 pmol primer (NdeUB) and 0.2 pmol primer (T2UB) with dNTP 0.2nM, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit of AccuPrime Taq DNA polymerase, polymerase chain reaction device Gene UB was obtained by performing an amplification reaction at 95°C and 40 seconds, 58°C and 30 seconds, 72°C and 25 cycles of 1 minute.

Meanwhile, 0.2 pmol primer (T2RanShh-6) and 0.2 pmol primer (XShh) were mixed with dNTP 0.2 nM, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit of AccuPrime Taq DNA polymerase, and then polymerase. Gene T2RanShh-6 was obtained by performing an amplification reaction at 95°C and 40 seconds, 58°C and 30 seconds, 72°C and 25 cycles of 1 minute in a chain reaction apparatus. Gene UB was digested with restriction enzymes NdeI and SacII, and gene T2RanShh-6 was digested with restriction enzymes SacII and XhoI and subjected to 2% agarose gel electrophoresis to obtain DNA fragments of 210 bp and 550 bp, respectively, with restriction enzymes NdeI and XhoI. Plasmid pET-UB-hRanShh was obtained by inserting into the cut pET15b vector using T4 DNA ligase. The nucleotide sequence of the mature Shh gene in which the ubiquitin obtained by this process is fused is shown in SEQ ID NO: 6.

The single colony thus obtained was cultured in a shaking incubator at 37°C in LB liquid medium, and isopropylthiogalactoside (IPTG) was added to a final 0.5 mM concentration at the time when the cell density reached 0.4 absorbance at 600 nm, followed by 4 hours. Further shaking culture was performed. After obtaining a part of E. coli cells by centrifugation, the cells were crushed and then subjected to SDS-polyacrylamide electrophoresis to confirm that the 27 kDa ubiquitin-fused Shh protein was expressed (FIG. 3).

Example 1 . 5. Escherichia coli Derived recombination Shh Protein isolation and purification

BL21(DE3)pLys.s producing strain expressing ubiquitin-fused Shh was inoculated in LB liquid medium and cultured in a shaking incubator at 37°C. When the absorbance reached 0.4 at OD ₆₀₀ , 0.5 mM IPTG was added for 4 hours. Further shaking culture was performed to express the ubiquitin-fused Shh protein. After completion of the culture, the cells were recovered by centrifugation, and the recovered cells were washed once with PBS, and then the cells were suspended using a 20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 8.0 solution. , The suspended cells were crushed using a sonicator. The crushed cells were centrifuged using a high-speed centrifuge and the supernatant was recovered, and the recovered supernatant was filtered using a 0.45 μm filter, and then loaded onto a pre-packed nickel chromatography column to perform primary purification. After loading the crushing solution containing Shh fused with ubiquitin, use a solution of 20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, and pH 8.0 until impurities that do not bind are not detected. It was eluted using sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 8.0 solution, and the protein was eluted at a concentration of 250 mM imidazole. After measuring the amount of protein in the eluate solution recovered from 1 ^st nickel chromatography, to remove the ubiquitin protein from the ubiquitin-fused Shh protein, the first purified protein and enzyme ubiquitin carboxyl-terminal hydroxylase 1 (UBP-1) Was added in a ratio of 70:1 and reacted at 20° C. for 12 hours to specifically remove ubiquitin (FIG. 4A).

In order to remove the cleaved ubiquitin and the host-derived protein, the reaction solution was loaded onto a column prepacked with an SP cation exchange resin, followed by secondary chromatography. The reaction solution was loaded onto the column, washed with 20 mM Tris, pH 8.0 solution until unbound impurities were not detected, and then washed with 20 mM Tris, 1 M NaCl, pH 8.0 solution at a concentration of 500 mM NaCl. Shh protein from which ubiquitin was removed (SEQ ID NO: 7) was eluted (FIG. 4A). The ubiquitin fusion Shh is a Shh protein ubiquitin is removed from the SP cation exchange resin (2 ^nd SP chromatography) the ubiquitin is present not cut ubiquitin is fused to at Shh protein because it is coupled with six histidine residues at the amino terminus a Ni- affinity chromatography (3 ^rd Nickel chromatography) to separate pure water was again performed to recover the Shh protein ubiquitin is removed (Figure 4b). The recovered final Shh protein was confirmed using protein quantification and SDS-PAGE (Fig. 4c).

Example 2. Recombination Shh Preparation of water-soluble microparticles containing protein and cream containing the same

A mixed solution of 2 g of hyaluronic acid, 4 g of water, and 2,000 μg of purified Shh protein was loaded onto a plastic plate with small pores in the shape of a polyhedron with sharp corners and dried. A large amount of angular microparticles were prepared. After mixing 1 g of the prepared water-soluble microparticles with angled ends and 11 g of particles of an excipient for increasing amount such as ascorbic acid in 82 g of an oily component including olive oil, and then slowly mixing them with a stirrer for 1 hour or more. Mix evenly. Thereafter, the mixture was cooled to 30° C. to prepare a cream composition in the form of a paste in which Shh protein was entrapped and water-soluble microparticles in the form of angled ends were suspended in the oil phase component .

Comparative example 1. Recombination Shh Preparation of a composition containing water-soluble fine particles that do not contain protein

A composition in the form of a paste was prepared in the same manner as in Example 2, except that 2,000 µg of Shh protein was not mixed.

Experimental example 1. Recombination Shh Protein activity measurement test

In order to measure the biological activity of the Shh protein obtained in Example 1.5, a cell-based analysis was performed using a reporter gene. As the activity of Shh in the extracellular induces the activity of the Gli transcription factor in the cell, the nucleotide sequence 5'-GACCACCCA-3' (SEQ ID NO: 15) to which the Gli transcription factor can bind is repeated 8 times as a reporter gene. The plasmid p8XGli-Luc bound to luciferase and the plasmid pRSVb-gal, a beta-galactosidase expression vector, were transformed into NIH3T3 cells, which are mouse epithelial cells, by the lipofectamine method. Then, after 6 hours, the Shh protein was treated according to concentrations of 100 ng/ml, 200 ng/ml, 500 ng/ml, and 1,000 ng/ml. At this time, PBS was used as a control. After culturing the treated cells for 18 hours, luciferase activity was measured and analyzed. At this time, the luciferase value divided by the value obtained by measuring the activity of betagalactosidase was corrected to correct the efficiency of transformation. It was determined by the value. The degree of Shh activity was determined by calculating a fold value compared to the corrected luciferase value obtained by treating only PBS with the corrected luciferase value obtained from the cells treated with the Shh protein (FIG. 5).

Experimental example 2. Human skin Recombination in dermal papilla cells Shh According to protein processing Hedgehog Signal transduction gene expression increase test

Cell-based assays were performed as follows in order to determine whether the expression of the gene was increased by the treatment of the Shh protein obtained in Example 1.5 in human skin papilloma cells present in the hair follicle. Human skin papilloma cells were cultured in DMEM medium containing 10% FBS. ⁵ ×10 ⁵ cells were cultured in a 6-well plate, treated with Shh protein at concentrations of 100 ng/ml and 500 ng/ml after 24 hours, and cultured for an additional 24 hours. Thereafter, the culture solution was removed, and the cells were washed twice with PBS buffer solution, and 0.5 ml of RNA extract (Trizol reagent, Thermo Fisher Scientific) was directly added. After standing at room temperature for 10 minutes, 0.1 ml of chloroform was added, stirred for 15 seconds, and then centrifuged at 12,000 xg for 10 minutes. The separated supernatant was taken, the same volume of isopropyl alcohol was added, centrifuged at 12,000 xg for 10 minutes, the liquid was removed, washed once with 75% ethanol, and dried at room temperature. 50 μl of purified distilled water without RNAase was added, and the quantification and purity of RNA were measured using a spectrophotometer.

In order to synthesize cDNA, 2 μg of purified total RNA was subjected to a binding reaction with oligo dT at 70°C for 5 minutes, and then 10X reverse transcription reaction buffer, 10 mM dNTP, RNAse inhibitor, and M-MLV reverse transcriptase (Enzynomics, Korea) CDNA synthesis reaction was performed at 42° C. for 60 minutes. After the reaction, it was heated at 72°C for 5 minutes to inactivate the reverse transcriptase, and then RNase H was added to remove single-stranded RNA to obtain cDNA. Quantitative RT-PCR was performed using the primers shown in Table 2 below to determine whether the expression of genes known to be activated by Shh in the Hedgehog signaling pathway was changed. At this time, the difference in expression was corrected by quantifying the β-actin gene and the 18S gene as genes for correction. It was confirmed that the Shh protein prepared in Example 1.5 increased the expression of major genes during the Shh signaling process in a concentration-dependent manner (FIGS. 6A to 6I ).

gene Forward Reverse Gli1 5'-GGGATGATCCCACATCCTCAGTC-3'
(SEQ ID NO: 16) 5'-CTGGAGCAGCCCCCCCAGT-3'
(SEQ ID NO: 17) Gli2 5'-ACACGGGCTTTGGTCTCA-3'
(SEQ ID NO: 18) 5'-CCCTTGGGCATAGCTTCT-3'
(SEQ ID NO: 19) Cyclin D 5'-GCTCCTGTGCTGCGAAGT-3'
(SEQ ID NO: 20) 5'-TGTTCCTCGCAGACCTCCAG-3'
(SEQ ID NO: 21) Ptch1 5'-GGGTGGCACAGTCAAGAACAG-3'
(SEQ ID NO: 22) 5'-TACCCCTTGAAGTGCTCGTACA-3'
(SEQ ID NO: 23) Smo 5'-ACGAGGACGTGGAGGGCTG-3'
(SEQ ID NO: 24) 5'-CGCACGGTATCGGTAGTTCT-3'
(SEQ ID NO: 25) noggin 5'-CCATCATTTCCGAGTGCAAGTGCT-3'
(SEQ ID NO: 26) 5'-AAGCTAGGTCTCTGTAGCCCAGAA-3'
(SEQ ID NO: 27) Igfbp3 5'-TGCCCAGAAAATGAAAAAGG-3'
(SEQ ID NO: 28) 5'-GGATGACACAGCGTGAGAGA-3'
(SEQ ID NO: 29) FGFR2 5'-GATAAATACTTCCAATGCAGAAGTGCT-3'
(SEQ ID NO: 30) 5'-TGCCCTATATAATTGGAGACCTTACA-3'
(SEQ ID NO: 31) D2 5'-ACTTCCTGCTGGTCTACATTGATG-3'
(SEQ ID NO: 32) 5'-CTTCCTGGTTCTGGTGCTTCTTC-3'
(SEQ ID NO: 33)

Experimental example 3. In human skin papilloma cells Shh Comparison of gene expression according to protein treatment

In order to determine whether the expression of hair-related genes was changed by Shh protein treatment in human skin papilloma cells present in hair follicles, a cell-based assay was performed as follows. First, human skin papilloma cells were cultured in DMEM medium containing 10% FBS, and about 2×10 ⁵ human skin papilloma cells were cultured in a 6-well plate, and proteins were treated after 24 hours under the following conditions:

1) Control without protein treatment

2) Group treated with 5 ㎍/ml of Shh and cultured for 24 hours.

After the protein treatment was further cultured for about 24 hours, the culture solution was removed, and the cells were washed twice by adding a PBS buffer solution, and 0.5 ml of RNA extract (Trizol reagent, Thermo Fisher Scientific) was directly added. After standing at room temperature for 10 minutes, 0.1 ml of chloroform was added, stirred for 15 seconds, and centrifuged at 12,000 xg for 10 minutes. The separated supernatant was taken, the same volume of isopropyl alcohol was added, centrifuged at 12,000 xg for 10 minutes, and the liquid was removed, washed once with 75% ethanol, and dried at room temperature. 50 ul of purified distilled water without RNAase was added and the quantification and purity of RNA were measured using a spectrophotometer. To synthesize cDNA, 2 ug of purified total RNA was subjected to a binding reaction with oligo dT at 70°C for 5 minutes, and then 10X reverse transcription reaction buffer, 10 mM dNTP, RNAse inhibitor, and M-MLV reverse transcriptase (Enzynomics, Korea) CDNA synthesis reaction was performed at 42° C. for 60 minutes. After the reaction was heated at 72° C. for 5 minutes to inactivate the reverse transcriptase, RNase H was added to remove single-stranded RNA to obtain cDNA. Quantitative RT-PCR was performed using the primers shown in Table 3 below to determine whether the expression of genes known to be activated by Shh was changed.

primer order hVersican
forward 5'-GGG ATT GAA GAC ACA CAA GAC ACG-3' (SEQ ID NO: 34) reverse 5'-TGC TCT GGA GTT GCT ATG ACT GCC-3' (SEQ ID NO: 35) hβ-catenin
forward 5'-CCC ACT AAT GTC CAG CGT TT-3' (SEQ ID NO: 36) reverse 5'-AAC CAA GCA TTT TCA TCA CCA GG-3' (SEQ ID NO: 37)

At this time, the difference in expression was corrected by quantifying the β-actin gene and the 18S gene together as genes for correction. As shown in FIG. 7, it was found that Shh prepared in Example 1.5 increased the Versican gene and β-catenin gene.

Experimental example 4. Shh Check the efficacy of water-soluble microparticles containing protein

The cream composition prepared in Example 2 in which the water-soluble microparticles in which the Shh protein was collected was suspended was applied daily to the scalp of two subjects to confirm the hair growth effect. At this time, the subject information is as follows: 1. Subject A (Im xx, 49 years old), 2. Subject B (Team xx, 52 years old). Specifically, Subject A applied 0.2 g of cream to the scalp every day for 1 month, scrubbed for 20 to 30 seconds, and then evenly sprayed with water-containing mist 3 to 4 times, and gently massaged for 20 to 30 seconds. The water-soluble microparticles were completely dissolved by rubbing. Subject B applied 0.2 g of cream daily to the scalp in the same manner as Subject A for 4 months. The results are shown in FIGS. 8 and 9.

As shown in FIGS. 8 and 9, the effect of increasing and thickening hair in both subjects A and B was confirmed.

<110> Paean Biotechnology Inc. <120> A MICROPARTICLE COMPRISING HEDGEHOG PROTEIN AND BIOCOMPATIBLE MATERIAL AND A COMPOSITION FOR PREVENTING OR TREATING HAIR LOSS COMPRISING THE SAME <130> FPD/201810-0023 <150> KR 10-2017-0161721 <151> 2017-11-29 <160> 37 <170> KoPatentIn 3.0 <210> 1 <211> 525 <212> DNA <213> Artificial Sequence <220> <223> pTA-hShh <400> 1 atcatcggac cgggcagggg gttcgggaag aggaggcacc ccaaaaagct gaccccttta 60 gcctacaagc agtttatccc caatgtggcc gagaagaccc taggcgccag cggaaggtat 120 gaagggaaga tctccagaaa ctccgagcga tttaaggaac tcacccccaa ttacaacccc 180 gacatcatat ttaaggatga agaaaacacc ggagcggaca ggctgatgac tcagaggtgt 240 aaggacaagt tgaacgcttt ggccatctcg gtgatgaacc agtggccagg agtgaaactg 300 cgggtgaccg agggctggga cgaagatggc caccactcag aggagtctct gcactacgag 360 ggccgcgcag tggacatcac cacgtctgac cgcgaccgca gcaagtacgg catgctggcc 420 cgcctggcgg tggaggccgg cttcgactgg gtgtactacg agtccaaggc acatatccac 480 tgctcggtga aagcagagaa ctcggtggcg gccaaatcgg gaggc 525 <210> 2 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> RanShh-6 <400> 2 atgatcattg gtccaggcag agggtttggg aagagaaggc accccaaaaa gctgacccct 60 tta 63 <210> 3 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> RanShh-10 <400> 3 atgatcattg gtcccggcag gggattcggg aagagaagac accccaaaaa gctgacccct 60 tta 63 <210> 4 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> RanShh-16 <400> 4 atgattatag ggccgggcag ggggtttggt aagagaaggc accccaaaaa gctgacccct 60 tta 63 <210> 5 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> RanShh-22 <400> 5 atgataatag ggccggggag ggggtttggg aagagaagac accccaaaaa gctgacccct 60 tta 63 <210> 6 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> Ubiquitin-fused matured shh gene sequence <400> 6 atgcaacttt tcgtcaaaac tctaacaggg aagactgtaa ccctagaggt tgaatcttcc 60 gacactattg acaacgtcaa aagtaaaatt caagataaag aaggtatccc tccggatcag 120 cagagattga tttttgctgg taagcaacta gaagatggta gaaccttgtc tgactacaac 180 atccaaaagg aatctactct tcacttggtg ttgagactcc gcggtggtat cattggtcca 240 ggcagagggt ttgggaagag aaggcacccc aaaaagctga cccctttagc ctacaagcag 300 tttatcccca atgtggccga gaagacccta ggcgccagcg gaaggtatga agggaagatc 360 tccagaaact ccgagcgatt taaggaactc acccccaatt acaaccccga catcatattt 420 aaggatgaag aaaacaccgg agcggacagg ctgatgactc agaggtgtaa ggacaagttg 480 aacgctttgg ccatctcggt gatgaaccag tggccaggag tgaaactgcg ggtgaccgag 540 ggctgggacg aagatggcca ccactcagag gagtctctgc actacgaggg ccgcgcagtg 600 gacatcacca cgtctgaccg cgaccgcagc aagtacggca tgctggcccg cctggcggtg 660 gaggccggct tcgactgggt gtactacgag tccaaggcac atatccactg ctcggtgaaa 720 gcagagaact cggtggcggc caaatcggga ggctaa 756 <210> 7 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> Shh protein <400> 7 Ile Ile Gly Pro Gly Arg Gly Phe Gly Lys Arg Arg His Pro Lys Lys 1 5 10 15 Leu Thr Pro Leu Ala Tyr Lys Gln Phe Ile Pro Asn Val Ala Glu Lys 20 25 30 Thr Leu Gly Ala Ser Gly Arg Tyr Glu Gly Lys Ile Ser Arg Asn Ser 35 40 45 Glu Arg Phe Lys Glu Leu Thr Pro Asn Tyr Asn Pro Asp Ile Ile Phe 50 55 60 Lys Asp Glu Glu Asn Thr Gly Ala Asp Arg Leu Met Thr Gln Arg Cys 65 70 75 80 Lys Asp Lys Leu Asn Ala Leu Ala Ile Ser Val Met Asn Gln Trp Pro 85 90 95 Gly Val Lys Leu Arg Val Thr Glu Gly Trp Asp Glu Asp Gly His His 100 105 110 Ser Glu Glu Ser Leu His Tyr Glu Gly Arg Ala Val Asp Ile Thr Thr 115 120 125 Ser Asp Arg Asp Arg Ser Lys Tyr Gly Met Leu Ala Arg Leu Ala Val 130 135 140 Glu Ala Gly Phe Asp Trp Val Tyr Tyr Glu Ser Lys Ala His Ile His 145 150 155 160 Cys Ser Val Lys Ala Glu Asn Ser Val Ala Ala Lys Ser Gly Gly 165 170 175 <210> 8 <211> 251 <212> PRT <213> Artificial Sequence <220> <223> Ubiquitin-fused Shh protein <400> 8 Met Gln Leu Phe Val Lys Thr Leu Thr Gly Lys Thr Val Thr Leu Glu 1 5 10 15 Val Glu Ser Ser Asp Thr Ile Asp Asn Val Lys Ser Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Ile Ile Gly Pro 65 70 75 80 Gly Arg Gly Phe Gly Lys Arg Arg His Pro Lys Lys Leu Thr Pro Leu 85 90 95 Ala Tyr Lys Gln Phe Ile Pro Asn Val Ala Glu Lys Thr Leu Gly Ala 100 105 110 Ser Gly Arg Tyr Glu Gly Lys Ile Ser Arg Asn Ser Glu Arg Phe Lys 115 120 125 Glu Leu Thr Pro Asn Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu 130 135 140 Asn Thr Gly Ala Asp Arg Leu Met Thr Gln Arg Cys Lys Asp Lys Leu 145 150 155 160 Asn Ala Leu Ala Ile Ser Val Met Asn Gln Trp Pro Gly Val Lys Leu 165 170 175 Arg Val Thr Glu Gly Trp Asp Glu Asp Gly His His Ser Glu Glu Ser 180 185 190 Leu His Tyr Glu Gly Arg Ala Val Asp Ile Thr Thr Ser Asp Arg Asp 195 200 205 Arg Ser Lys Tyr Gly Met Leu Ala Arg Leu Ala Val Glu Ala Gly Phe 210 215 220 Asp Trp Val Tyr Tyr Glu Ser Lys Ala His Ile His Cys Ser Val Lys 225 230 235 240 Ala Glu Asn Ser Val Ala Ala Lys Ser Gly Gly 245 250 <210> 9 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> NdeShh primer <400> 9 ggatcccata tgatcatcgg accgggcagg gggttcgg 38 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> XShh primer <400> 10 aaaaaactcg agttagcctc ccgatttggc cgccac 36 <210> 11 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> RanNdeShh primer <400> 11 ggatcccata tgathathgg nccnggnagr ggnttyggna aragragrca ccccaaaaag 60 ctgacccctt tagc 74 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> NdeUB primer <400> 12 ggattccata tgcaactttt cgtcaaaact ctaac 35 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> T2UB primer <400> 13 atgaccaccg cggagtctca acaccaa 27 <210> 14 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> T2RanShh-6 primer <400> 14 ggatccccgc ggtggtatca ttggtccagg cagaggg 37 <210> 15 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequences which can be binded by Gli transcription factor <400> 15 gaccaccca 9 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer (Gli 1, forward) <400> 16 gggatgatcc cacatcctca gtc 23 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer (Gli 1, reverse) <400> 17 ctggagcagc ccccccagt 19 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer(Gli 2, forward) <400> 18 acacgggctt tggtctca 18 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer (Gli 2, reverse) <400> 19 cccttgggca tagcttct 18 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer (cyclin D, forward) <400> 20 gctcctgtgc tgcgaagt 18 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer (cyclin D, reverse) <400> 21 tgttcctcgc agacctccag 20 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer(Ptch1, forward) <400> 22 gggtggcaca gtcaagaaca g 21 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer(Ptch1, reverse) <400> 23 taccccttga agtgctcgta ca 22 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer(Smo, forward) <400> 24 acgaggacgt ggagggctg 19 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer (Smo, reverse) <400> 25 cgcacggtat cggtagttct 20 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer (noggin, forward) <400> 26 ccatcatttc cgagtgcaag tgct 24 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer (noggin, reverse) <400> 27 aagctaggtc tctgtagccc agaa 24 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer(Igfbp3, forward) <400> 28 tgcccagaaa atgaaaaagg 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer(Igfbp3, reverse) <400> 29 ggatgacaca gcgtgagaga 20 <210> 30 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer(FGFR2, forward) <400> 30 gataaatact tccaatgcag aagtgct 27 <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer (FGFR2, reverse) <400> 31 tgccctatat aattggagac cttaca 26 <210> 32 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer(D2, forward) <400> 32 acttcctgct ggtctacatt gatg 24 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer(D2, reverse) <400> 33 cttcctggtt ctggtgcttc ttc 23 <210> 34 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> hVersican forward primer <400> 34 gggattgaag acacacaaga cacg 24 <210> 35 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> hVersican reverse primer <400> 35 tgctctggag ttgctatgac tgcc 24 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> h beta-catenin forward primer <400> 36 cccactaatg tccagcgttt 20 <210> 37 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> h beta-catenin reverse primer <400> 37 aaccaagcat tttcatcacc agg 23

Claims (13)

The hedgehog protein includes microparticles contained in a biocompatible material,
The biocompatible material is selected from the group consisting of polysaccharides, polyvinyl alcohol, carboxyvinyl polymers, chitosan, hyaluronic acid, cellulose polymers, and combinations thereof,
The fine particles are dissolved in body fluid or water, and have at least one vertex in the form of a three-dimensional figure, and the horizontal length of one side is 50 μm to 500 μm,
External pharmaceutical composition for preventing or treating hair loss. The method of claim 1,
The hedgehog protein is characterized in that the sonic hedgehog protein, pharmaceutical composition. delete delete The method of claim 1,
The fine particles are characterized in that the tetrahedral, pentahedral or conical shape, pharmaceutical composition. The method of claim 1,
A pharmaceutical composition, characterized in that a depression is formed on at least one surface of the fine particle, and the depth of the depression is within 1/2 of the height of the fine particle. delete delete delete delete The method of claim 1,
The pharmaceutical composition is a cream, ointment, paste, gel, jelly, serum (serum), aerosol spray, non-aerosol spray, a pharmaceutical composition, characterized in that selected from the formulation of a foam, lotion, solution or suspension. The hedgehog protein includes microparticles contained in a biocompatible material,
The biocompatible material is selected from the group consisting of polysaccharides, polyvinyl alcohol, carboxyvinyl polymers, chitosan, hyaluronic acid, cellulose polymers, and combinations thereof,
The fine particles are dissolved in body fluid or water, and have at least one vertex in the form of a three-dimensional figure, and the horizontal length of one side is 50 μm to 500 μm,
A cosmetic composition for preventing hair loss, promoting hair growth or hair growth. The method of claim 12,
The cosmetic composition is hair cream, hair paste, hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair rinse, hair treatment, hair nutrition cream, hair moisture cream, hair massage cream, hair wax , Hair aerosol, hair pack, hair nutrition pack, hair soap, hair cleansing foam, hair oil, hair dryer, hair preservation agent, hair dye, hair wave agent, hair bleach, hair gel, hair glaze, hairdresser, hair lacquer, hair A cosmetic composition, characterized in that it is selected from the formulation of a moisturizer, hair mousse or hair spray.

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