Hair follicle repair protein composition and method for making same and use thereof

Abstract

The hair follicle repair protein composition is prepared by adding 20 U / mL to 35 U / mL of either one or a combination of nuclease and ultranuclease to a cell protein extract, enzymatically decomposing the extract for 15 to 40 minutes at 37°C±1°C, and isolating and purifying the enzymatic decomposition solution. The obtained cell protein extract and cell protein composition have a repairing effect on damaged hair follicle cells, efficiently repair damaged hair follicles, and significantly improve the activity of hair follicles. They also have the advantages of being highly pure, stable, safe, and effective, and effectively solving the problems that viable cells require refrigeration and that their activity is limited to the cell viability time.

Description

[Technical field]

[0001] The present invention relates to the field of biomedical technology, specifically to hair follicle repair protein compositions and methods for making and using same. [Background technology]

[0002] A large amount of hair is an important indicator of body health and hair health. Hair follicle health is the basis of hair health. The key to maintaining hair follicle health is to activate the follicular stromal cells in the scalp system. Hair follicles undergo a cyclic growth cycle of anagen, catagen, and telogen. The cyclic changes of hair follicles are closely related to the cyclic division, proliferation, differentiation, etc. of follicular stromal cells. Highly active follicular stromal cells continuously release large amounts of follicular stromal cell growth factors to promote the benign cycle of hair follicle cells to repair themselves, grow, divide, and grow hair.

[0003] Modern people, influenced by busy lifestyles, work stress, environmental pollution and other factors, suffer from hair breakage, premature aging, severe hair loss, hair regeneration disorder and other problems, which seriously affect personal image and even lead to psychological disorders. According to survey data from the National Health Commission of China, there are more than 260 million people suffering from hair loss in China, with male hair loss accounting for approximately 63%, mainly androgen-induced hair loss. The trend of hair loss occurring at a younger age has become evident, with 60% of people experiencing hair loss symptoms around the age of 25, and the proportion of people suffering from hair loss after the 90s (the Chinese term for young people born between 1990 and 2000) is as high as 84%, with severe hair loss as high as 46.7%.

[0004] Hair diseases include physiological or pathological symptoms such as hypotrichosis, hypertrichosis, abnormal hair distribution, abnormal hair density, abnormal hair diameter, abnormal hair pigmentation, abnormal hair shaft, etc. Hair loss includes physiological hair loss and pathological hair loss. Physiological hair loss includes natural hair loss, seasonal hair loss, infantile hair loss, senile hair loss, and postpartum hair loss. Pathological hair loss includes non-scarring hair loss and cicatricial hair loss. Non-scarring hair loss includes androgenic alopecia (ratio is about 95%), alopecia areata (ratio is about 4%), syphilitic alopecia, trichotillomania, telogen effluvium, anagen effluvium, traction alopecia, physical alopecia, alopecia folliculitis, perifolliculitis capitis abscedens et suffodiens, congenital hypotrichosis, etc. When the pathogenic factor of non-scarring hair loss is removed, hair will grow again. Cicatricial alopecia is caused by the permanent destruction of hair follicles, resulting in permanent hair loss, and causes include trauma, infection, inflammatory skin diseases (e.g., discoid lupus erythematosus, hairy lichen planus, etc.), scalp lichen planus, scalp discoid lupus erythematosus, radioactive alopecia, female fibrous alopecia, scalp burns, etc.

[0005] The amount of hair in patients with pathological hair loss is obviously reduced due to abnormal hair loss and excessive shedding. Common causes of hair loss include excess male hormones, heredity, excessive stress, pregnancy, diseases, drugs, injuries, etc. Diseases such as autoimmune diseases, thyroid diseases, lupus erythematosus, diabetes, iron deficiency, eating disorders, and anemia cause changes in the body's hormone levels, which exacerbate hair loss. Antitumor drugs, anticoagulants, antihypertensive drugs, contraceptives, burns, various injuries, X-ray radiation, etc. can cause temporary hair loss.

[0006] Seborrheic alopecia (androgenic alopecia, AGA) is a hair disease whose main symptoms are excessive oil leakage from the scalp, causing hair to become gradually thinner, softer, and less abundant, and may be accompanied by scalp itching, dandruff, and itching. It is a progressive hair follicle disorder that involves genetic factors and is dependent on the action of male hormones. The total number of AGA patients in China exceeds 100 million, and the prevalence rate is higher in men (20.2%) than in women (5.1%).

[0007] Cells are the basic unit of life activity and the basis of the health of living organisms. When the redox balance of living organisms is destroyed, it will cause the interruption of redox signaling and control, induce oxidative stress disorders, and induce various diseases. Research has already shown that factors such as the number and activity of hair follicle stem cells and the regeneration ability of hair follicles are directly related to hair loss. The decrease in the number and activity of hair follicle stem cells weakens the differentiation ability of hair follicle cells, affecting the hair regeneration cycle, which causes hair follicles to be blocked and permanent hair loss to occur. The main methods for treating hair loss include hair washing care, drug treatment, laser hair growth, hair transplantation, etc. Hair washing care mainly stimulates the scalp with topical products such as hair growth liquid or hair growth agent to promote blood circulation in the scalp, promote blood supply to the head, nutrition of the head, and hair growth, but has the disadvantages of poor hair growth and hair loss prevention, and is ineffective against damaged hair follicles and dormant hair follicles. Drug treatments include oral medications (e.g. finasteride, spironolactone, VB2, VB6, tanshinone capsules, glycyrrhizin combination tablets, white peony sugar capsules, etc.) and topical medications (e.g. minoxidil solution, halcinonide solution, selenium sulfide, etc.), but they have shortcomings such as poor hair loss treatment effects, significant side effects, and drug dependency. Laser hair growth uses low-energy laser irradiation with a wavelength of 670 nm to enhance cell activity, promote blood microcirculation, increase blood supply and nutrition to the head, promote the absorption of nutrients in hair, improve the activity of hair matrix cells and hair follicle stem cells, accelerate protein synthesis, and activate hair follicles, but has shortcomings such as slow hair growth and high cost. Hair transplantation requires surgery, in which hair follicles from the patient's back and both sides of the head are selected as the hair source, the harvested hair follicles are separated into single or multiple hair follicles, and then the single or multiple hair follicles are transplanted to the bald area that needs to be transplanted through precise microsurgery, so that the newly transplanted single or multiple hair follicles survive and grow in the new hair transplantation area, and further restore the local hair distribution and distribution density.Although hair transplantation technology can solve the problem of completely blocked hair follicles, it is limited by the limited transplantable sites and hair follicle resources, and hair transplantation surgery is painful, has the risk of infection, the survival rate of newly transplanted hair follicles is not guaranteed, the problem of repairing damaged hair follicles cannot be solved, scars remain at the transplantation surgery site, which affects the aesthetic appearance, and is expensive, etc. Therefore, in clinical practice, there is an urgent need for a safe and effective hair growth composition with better hair growth and hair loss prevention effects, reliable treatment effects, and high efficacy.

[0008] When cells and microorganisms are subjected to stress induction by external stimuli and exogenous stress substances (including cold, heat, acid, alkali, electric current, radiation, chemical substances, etc.), the cells and microorganisms are induced to produce stress proteins through a stress response. Literature 1 (New limonophyllines AC from the stem of Atalantia monophylla and cytotoxicity against cholangiocarcinoma and HepG2 cell lines, Arch. Pharm. Res. (2018) 41:431-437) discloses that compounds 1-16 prepared by extraction from a Rutaceae plant (Atalantia monophylla) have activity to inhibit the proliferation of tumor cells, etc.

[0009] Mesenchymal stem cells (MSCs) have the ability to self-renew and differentiate in multiple directions. They are widely distributed in bone marrow, fat, synovium, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amniotic membrane, peripheral blood, muscle, urine and other tissues. They have characteristics such as wide supply source, no need for matching, low infection rate, strong differentiation ability, strong proliferation ability and easy collection. They can produce activating factors such as stem cell factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), interferon (IFN) and other activating factors. They are involved in the regulation of processes such as cell proliferation, apoptosis, cell differentiation, antiviral and immune maturation, and can be used for immune regulation, tissue repair and treatment of diseases such as acute lung injury, severe pneumonia and acute respiratory distress syndrome. However, MSCs products require refrigeration during production, storage, transportation, and use, and their cell activity can only be maintained for 12 hours or less, limiting their therapeutic use. Therefore, there is a need to develop a safe and effective skin repair drug to meet clinical needs. Summary of the Invention [Problem to be solved by the invention]

[0010] It is an object of the present invention to provide a hair follicle repair protein composition. [Means for solving the problem]

[0011] A hair follicle repair protein composition, the preparation of which comprises: (1) adding 20 U / mL to 35 U / mL of any one or a combination of nuclease and ultranuclease to the cell protein extract of the present invention, and subjecting the resulting mixture to enzymatic degradation at 37°C±1°C for 15 to 40 minutes to prepare an enzymatic degradation solution; and (2) preparing the enzymatic hydrolysis solution produced in step (1) at 5 to 15 mg / ml with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride at 2°C to 8°C, passing the solution through a chromatography column at an elution flow rate of 0.1 to 1 ml / min, and monitoring and collecting the eluted portion at an ultraviolet wavelength of 280 nm.

[0012] In a preferred technical solution of the present invention, the preparation of the protein extract comprises: Density is 5.0×10 6 pieces / mL~1.0×10 7 Step S-1: placing mesenchymal passaged cells (cells / mL) in a medium containing 40-50% DMEM / F12, 40-50% RPMI1640, 0.1-2% bovine serum protein (BSA), 1-15ug / mL epidermal growth factor (EGF), 1-15ug / mL fibroblast growth factor (FGF), 1-15ug / mL insulin transferrin, 0.01-0.1% amino acid compound (18AA), and 2-10μmol / L of any one or combination of compounds 1-16, and then culturing the medium for 10-60min at 37.0℃±0.5℃ and 5%±1.0% CO2, followed by separating, washing, and collecting the cells; [ka] [ka] The collected cells were 5.0 x 10 6 pieces / mL~5.0×10 7 Step S-2 of dispersing the cells in a solvent selected from physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer, TBST buffer, and Tris buffer to a density of 10 ... and step S-3 of separating the cell lysate prepared in step S-2 and then filtering the resulting separation solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0013] In a preferred technical proposal of the present invention, the medium in step S-1 contains 42-45% DMEM / F12, 42-45% RPMI1640, 0.5-1.5% bovine serum albumin (BSA), 5-10 ug / mL epidermal growth factor (EGF), 5-10 ug / mL fibroblast growth factor (FGF), 5-10 ug / mL insulin transferrin, 0.02-0.05% amino acid compound (18AA), and 3-8 μmol / L of a stress substance.

[0014] In a preferred technical solution of the present invention, the medium in step S-1 contains 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10 ug / mL epidermal growth factor (EGF), 10 ug / mL fibroblast growth factor (FGF), 10 ug / mL insulin transferrin, 0.05% amino acid compound (18AA), and 4 to 6 μmol / L of a stress substance.

[0015] In the preferred technical solution of the present invention, the mesenchymal passage cell density in step S-1 is 6.0 × 10 6 ~2.0×10 7 5.0×10 6 ~1.0×10 7 pieces / mL.

[0016] In a preferred technical solution of the present invention, the mesenchymal passaged cells in step S-1 are cultured in the medium for 15 to 50 minutes, preferably 20 to 40 minutes.

[0017] In a preferred technical solution of the present invention, the solvent for washing the cells in step S-1 is any one or combination selected from physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer, TBST buffer, and Tris buffer, and the number of washing the cells is 2 to 5 times, preferably 3 to 4 times.

[0018] In a preferred technical solution of the present invention, the separation described in step S-1 is any one selected from centrifugation and filtration or a combination thereof, wherein the centrifugation conditions are 1000-2000 rpm*3-15 min, preferably 1200 rpm-1500 rpm*5-10 min.

[0019] In a preferred technical solution of the present invention, the ultrasonic conditions in step S-2 are 2°C to 8°C, 25 kHz, and 360 W, and ultrasonic treatment is performed for 1 to 5 minutes with an interval of 1 second for 3 seconds.

[0020] In a preferred technical solution of the present invention, the separation described in step S-3 is any one or combination selected from 2000-8000 rpm*10-30 min centrifugation, multi-stage centrifugation, and multi-stage filtration, preferably 3000-7000 rpm*15-25 min.

[0021] In a preferred technical solution of the present invention, the multi-stage centrifugation in step S-3 is performed in the order of 3000-4000 rpm*3-5 min, 5000-6000 rpm*3-5 min, and 7000 rpm*5-8 min.

[0022] In a preferred technical solution of the present invention, the pore size of the filtration membrane of the multi-stage filtration is any one selected from 80 um, 50 um, 30 um, 10 um, and 5 um.

[0023] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 is enzymatically digested using either nuclease or ultranuclease, and then separated and purified.

[0024] In a preferred technical solution of the present invention, the nuclease is selected from the group consisting of RNA nuclease, DNA nuclease, or a combination thereof.

[0025] In a preferred technical solution of the present invention, the molecular weight of the hair follicle repair protein composition is 20 kDa-300 kDa, preferably 50 kDa-200 kDa.

[0026] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 or the protein composition prepared in step (2) is frozen and stored, preferably at -40°C to -20°C.

[0027] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 or the protein composition prepared in step (2) is added with a freeze-drying protection agent selected from any one or combination of mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, glucan, tricaprylin (HES), polyethylene glycol, ethylene vinylidene, phosphate, acetate, citrate, sorbitol, and starch, and then freeze-dried to prepare a freeze-dried cell protein extract preparation or a freeze-dried protein composition preparation.

[0028] In a preferred technical solution of the present invention, the lyophilization protection agent is contained in the lyophilized preparation of the cell protein extract or protein composition in a mass % range of 0.5 to 8%, preferably 1 to 5%.

[0029] In a preferred technical solution of the present invention, a protein stabilizer is optionally added to the cellular protein extract prepared in step S-3 or the protein composition prepared in step (2), which is any one selected from albumin, zinc salts, and aluminum salts.

[0030] In a preferred technical solution of the present invention, the lyophilized preparation of the cell protein extract or the lyophilized preparation of the protein composition has a pH of 6 to 8, preferably 7 to 7.5.

[0031] In a preferred technical solution of the present invention, the protein composition in the hair follicle repair protein composition is as shown in FIG.

[0032] In a preferred technical solution of the present invention, the freeze-dried preparation is reconstituted immediately before use with an isotonic solution selected from physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer, or a combination thereof, and then used by one or a combination of smear, rolling needle, microneedle, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupuncture injection, and lumbar puncture.

[0033] In a preferred technical solution of the present invention, the culture of the primary mesenchymal stem cells is a culture method in the art.

[0034] In a preferred technical solution of the present invention, the mesenchymal stem cell culture is performed by culturing 5.0×10 primary mesenchymal stem cells. 5 ~5.0×10 6 The method includes the steps of adding the cells to a subculture medium so as to give an initial density of 10 cells / ml, culturing the medium at 37.0°C±0.5°C and 5%±1.0% CO2 for 10 to 15 days, and after observing the yellowing of the subculture medium every 2 to 3 days, replacing half of the subculture medium, wherein the subculture medium is a DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 ug / ml streptomycin.

[0035] In a preferred technical solution of the present invention, the primary mesenchymal stem cells are cultured by: After cleaning and disinfecting the umbilical cord, the tissue was dissected and the Wharton's jelly layer tissue was removed and cut into 3 mm pieces. 3 Step A is to cut the tissue into small pieces, centrifuge and wash the tissue pieces to collect the tissue pieces, place them in DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100ug / ml penicillin, and 100ug / ml streptomycin, and then culture them under conditions of 37.0℃±0.5℃ and 5%±1.0% CO2, changing half of the medium every 2 to 3 days, and culturing until cells emerge from the tissue pieces; and step B, shaking the mixture to collect the lower layer cells and washing them with PBS, adding 0.25% trypsin to digest for 2-3 minutes, adding an equal volume of trypsin stop solution to stop the digestion, gently blowing with a pipette, centrifuging at 1200-1500 rpm / min* for 5-8 minutes, and then collecting and obtaining the cells.

[0036] The object of the present invention is to provide a method for producing a cellular protein extract having a hair follicle repair effect, the method comprising: Density is 5.0×10 6 pieces / mL~1.0×10 7 Step S-1: placing mesenchymal passaged cells (cells / mL) in a medium containing 40-50% DMEM / F12, 40-50% RPMI1640, 0.1-2% bovine serum protein (BSA), 1-15ug / mL epidermal growth factor (EGF), 1-15ug / mL fibroblast growth factor (FGF), 1-15ug / mL insulin transferrin, 0.01-0.1% amino acid compound (18AA), and 2-10μmol / L of any one or combination of compounds 1-16, and then culturing the medium at 37.0℃±0.5℃ and 5%±1.0% CO2 for 20-40 minutes, followed by separating, washing, and collecting the cells; The collected cells were 5.0 x 10 6 pieces / mL~5.0×10 7 Step S-2 of dispersing the cells in a solvent selected from physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer, TBST buffer, and Tris buffer to a density of 10 ... and step S-3 of separating the cell lysate prepared in step S-2 and then filtering the resulting separation solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0037] In a preferred technical proposal of the present invention, the medium in step S-1 contains 42-45% DMEM / F12, 42-45% RPMI1640, 0.5-1.5% bovine serum albumin (BSA), 5-10 ug / mL epidermal growth factor (EGF), 5-10 ug / mL fibroblast growth factor (FGF), 5-10 ug / mL insulin transferrin, 0.02-0.05% amino acid compound (18AA), and 3-8 μmol / L of a stress substance.

[0038] In a preferred technical solution of the present invention, the medium in step S-1 contains 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10 ug / mL epidermal growth factor (EGF), 10 ug / mL fibroblast growth factor (FGF), 10 ug / mL insulin transferrin, 0.05% amino acid compound (18AA), and 4 to 6 μmol / L of a stress substance.

[0039] In a preferred technical solution of the present invention, the mesenchymal passage cell density in step S-1 is 6.0×10 6 ~2.0×10 7 5.0×10 6 ~1.0×10 7 pieces / mL.

[0040] In a preferred technical solution of the present invention, the mesenchymal passaged cells in step S-1 are cultured in the medium for 15 to 50 minutes, preferably 20 to 40 minutes.

[0041] In a preferred technical solution of the present invention, the solvent for washing the cells in step S-1 is any one or combination selected from physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer, TBST buffer, and Tris buffer, and the number of washing the cells is 2 to 5 times, preferably 3 to 4 times.

[0042] In a preferred technical solution of the present invention, the separation described in step S-1 is any one selected from centrifugation and filtration or a combination thereof, wherein the centrifugation conditions are 1000-2000 rpm*3-15 min, preferably 1200 rpm-1500 rpm*5-10 min.

[0043] In a preferred technical solution of the present invention, the ultrasonic conditions in step S-2 are 2°C to 8°C, 25 kHz, and 360 W, and ultrasonic treatment is performed for 1 to 5 minutes with an interval of 1 second for 3 seconds.

[0044] In a preferred technical solution of the present invention, the separation described in step S-3 is any one or combination selected from 2000-8000 rpm*10-30 min centrifugation, multi-stage centrifugation, and multi-stage filtration, preferably 3000-7000 rpm*15-25 min.

[0045] In a preferred technical solution of the present invention, the multi-stage centrifugation in step S-3 is performed in the order of 3000-4000 rpm*3-5 min, 5000-6000 rpm*3-5 min, and 7000 rpm*5-8 min.

[0046] In a preferred technical solution of the present invention, the pore size of the filtration membrane of the multi-stage filtration is any one selected from 80 um, 50 um, 30 um, 10 um, and 5 um.

[0047] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 is frozen and stored, preferably at -40°C to -20°C.

[0048] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 is enzymatically digested using either nuclease or ultranuclease, and then separated and purified.

[0049] In a preferred technical solution of the present invention, the culture of the primary mesenchymal stem cells is a culture method in the art.

[0050] In a preferred technical solution of the present invention, the mesenchymal stem cell culture is performed by culturing 5.0×10 primary mesenchymal stem cells. 5 ~5.0×10 6 The method includes the steps of adding the cells to a subculture medium so as to achieve a density of 1000 cells / ml, culturing the medium at 37.0°C±0.5°C and 5%±1.0% CO2 for 10 to 15 days, and after observing the subculture medium for yellowing every 2 to 3 days, replacing half of the subculture medium, wherein the subculture medium is a DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 ug / ml streptomycin.

[0051] In a preferred technical solution of the present invention, the primary mesenchymal stem cells are cultured by: After cleaning and disinfecting the umbilical cord, the tissue was dissected and the Wharton's jelly layer tissue was removed and cut into 3 mm pieces. 3 Step A is to cut the tissue into small pieces, centrifuge, wash, collect the tissue pieces, add them to DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100ug / ml penicillin, and 100ug / ml streptomycin, and then culture them under conditions of 37.0℃±0.5℃ and 5%±1.0% CO2, changing half of the medium every 2 to 3 days, and culturing until cells emerge from the tissue pieces; and step B, shaking the mixture to collect the lower layer cells and washing them with PBS, adding 0.25% trypsin to digest for 2-3 minutes, adding an equal volume of trypsin stop solution to stop the digestion, gently blowing with a pipette, centrifuging at 1200-1500 rpm / min* for 5-8 minutes, and then collecting and obtaining the cells.

[0052] An object of the present invention is to provide a method for producing a hair follicle repair protein composition, the method comprising: (1) adding 20 U / mL to 35 U / mL of any one or a combination of nuclease and ultranuclease to the cell protein extract of the present invention, and subjecting the resulting mixture to enzymatic degradation at 37°C±1°C for 15 to 40 minutes to prepare an enzymatic degradation solution; and (2) preparing the enzymatic hydrolysis solution produced in step (1) at 5 to 15 mg / ml with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride at 2°C to 8°C, passing the solution through a chromatography column at an elution flow rate of 0.1 to 1 ml / min, and monitoring and collecting the eluted portion at an ultraviolet wavelength of 280 nm.

[0053] In a preferred technical solution of the present invention, the nuclease is selected from the group consisting of RNA nuclease, DNA nuclease, or a combination thereof.

[0054] In a preferred technical solution of the present invention, the molecular weight of the hair follicle repair protein composition is 20 kDa-300 kDa, preferably 50 kDa-200 kDa.

[0055] In a preferred technical solution of the present invention, the protein composition prepared in step (2) is frozen and stored, preferably at -40°C to -20°C.

[0056] In a preferred technical solution of the present invention, the protein composition prepared in step (2) is added with a freeze-drying protection agent, which is any one or combination of selected from mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, glucan, tricaprylin (HES), polyethylene glycol, ethylene vinylidene, phosphate, acetate, citrate, sorbitol, and starch, and then freeze-dried to obtain the protein composition.

[0057] In a preferred technical solution of the present invention, the freeze-dried preparation contains a freeze-drying protection agent in an amount of 0.5 to 8%, preferably 1 to 5%, by mass.

[0058] In a preferred technical solution of the present invention, a protein stabilizer is optionally added to the protein composition prepared in step (2), wherein the protein stabilizer is any one selected from albumin, zinc salt, and aluminum salt.

[0059] In a preferred technical solution of the present invention, the pH of the lyophilized protein composition preparation is 6-8, preferably 7-7.5.

[0060] In a preferred technical solution of the present invention, the protein composition in the hair follicle repair protein composition is as shown in FIG.

[0061] In a preferred technical solution of the present invention, the freeze-dried preparation is reconstituted immediately before use with an isotonic solution selected from physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer, or a combination thereof, and then used by one or a combination of smear, rolling needle, microneedle, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupuncture injection, and lumbar puncture.

[0062] Another object of the present invention is to provide a hair follicle repair composition comprising any one or combination of the cellular protein extract or hair follicle repair protein composition having the hair follicle repair effect of the present invention and a pharma- ceutically acceptable carrier.

[0063] The amount or type of the pharma- ceutically acceptable carrier of the present invention is determined by factors such as the physicochemical properties and content of the active ingredient in the composition, the type of formulation, the dissolution and bioavailability of the formulation, and the like.

[0064] The compositions of the present invention may be in any dosage form known in the art and may be prepared using formulation techniques known in the art.

[0065] In a preferred technical solution of the present invention, the composition is any one selected from the group consisting of freeze-dried agents, gelling agents, nasal sprays, ointments, creams, emulsions, liquid dressings, injections, and suppositories.

[0066] In a preferred technical solution of the present invention, the administration method of the composition is selected from the following: smearing, rolling needle, microneedle, massage, or a combination thereof.

[0067] Another object of the present invention is to provide the use of a cell protein extract, hair follicle repair protein composition or composition thereof having the hair follicle repair effect described in the present invention in the preparation of a cell repair or hair follicle repair product.

[0068] In a preferred technical solution of the present invention, the product is any one selected from hair growth products, products for preventing and treating hair diseases, products for preventing and treating pathological hair loss, products for delaying physiological hair loss, and products for improving scalp environment, wherein the hair disease is any one selected from thin hair, excessive hair, distribution abnormality, abnormal hair density, abnormal hair diameter, abnormal hair pigmentation, and hair shaft, or a combination thereof, the pathological hair loss is any one selected from androgenic alopecia, alopecia areata, syphilitic alopecia, trichotillomania, physical alopecia, alopecia alopecia, perifolliculitis capitis abscedens et suffodiens, and congenital hypotrichosis, and any one selected from scalp lichen planus, hair lichen planus, scalp discoid lupus erythematosus, radioactive alopecia, female fibrous alopecia, and scalp burn, and the physiological hair loss is any one selected from natural alopecia, seasonal alopecia, infantile alopecia, senile alopecia, and postpartum alopecia. Another object of the present invention is to provide a use of the hair growth product of the present invention in an administration regimen for hair growth treatment and hair loss prevention, in which a subject is administered one dose every 2 to 4 weeks, for a course of three doses, and the hair loss site is punctured with either a microneedle or a rolling needle, and then the product is administered locally in a smearing manner and massaged until completely absorbed.

[0069] In a preferred technical solution of the present invention, the treatment area is locally anesthetized prior to treatment.

[0070] Another object of the present invention is to provide a use of compounds 1-16 in stress-inducing stem cells to produce functional proteins with repair effects.

[0071] In a preferred technical solution of the present invention, the repair is any one of cell repair, hair follicle repair, joint repair, and nerve repair.

[0072] Unless otherwise stated, where the invention relates to percentages of liquids to liquids, said percentages are volume / volume percentages, where the invention relates to percentages of liquids to solids, said percentages are volume / weight percentages, where the invention relates to percentages of solids to liquids, said percentages are weight / volume percentages, and the remainder are weight / weight percentages.

[0073] Unless otherwise specified, the identification of the mesenchymal stem cells (MSCs) of the present invention refers to "Standards for the culture and quality control of umbilical cord mesenchymal stromal cells for neurorestorative clinical application." Effect of the Invention

[0074] Compared with the conventional technology, the present invention has the following advantageous effects:

[0075] 1. In the present invention, a medium containing a stress substance that induces stress in mesenchymal stem cells to produce functional proteins having the effect of cell repair and hair follicle repair is scientifically screened, and contains various growth factors and proteins that have the function of regulating cell growth and differentiation, cell repair, cell nutrition, etc., and the obtained cell protein extract, cell protein composition, or composition thereof has a repair effect on cells and damaged hair follicle cells. After puncturing the hair loss site with either a microneedle or a rolling needle, the hair follicle repair protein composition of the present invention is locally and percutaneously introduced to the surface of the scalp and the root of the hair follicle. The present invention has the advantages of forming an effective administration route that directly reaches the scalp, efficiently repairing damaged hair follicles and significantly improving the activity of hair follicles, and being highly pure, stable, safe and effective, and effectively resolving the problem that viable cells require refrigeration and that their activity is limited by the cell viability time. The present invention provides a clinically useful treatment method that promotes the repair of damaged hair follicles and the production of collagen protein, promotes hair regeneration, and significantly improves accompanying symptoms such as scalp itching, excessive oil loss, and dandruff, as well as patient compliance with medication, and has a reliable therapeutic effect, a high absorption rate, is not irritating to the scalp, and is safe and effective, and provides a simple and rapid treatment method.

[0076] 2. The preparation method of the present invention has the advantages of being simple in operation, environmentally friendly, cost-effective, and suitable for industrial production. [Brief description of the drawings]

[0077] [Figure 1] Electrophoretic separation results of the hair follicle repair protein composition of the present invention. [Diagram 2] 1 is a high performance liquid chromatography detection result of the hair follicle repair protein composition of the present invention. [Diagram 3] 1 is a study of the repair effect of the cellular protein extract of the present invention on oxidatively damaged skin. [Figure 4] 1 shows the results of a test group and a model group in a study of the hair follicle repair effect of the hair follicle repair protein composition of the present invention on an AGA mouse model. [Diagram 5]1 shows the results of H&E staining of the test group and model group in a study of the hair follicle repair effect of the hair follicle repair protein composition of the present invention on an AGA mouse model (scale bar: 200 μm). [Figure 6] Quantification of HF in mice in the test group and model group in a study of the hair follicle repair effect of the hair follicle repair protein composition of the present invention on an AGA mouse model, where *** indicates p<0.005. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0078] The present invention will be further explained and described in detail with reference to the following specific examples, but the scope of protection of the present invention is not limited thereto.

[0079] 1. Cultivation of primary mesenchymal stem cells Primary mesenchymal stem cell culture After cleaning and disinfecting the umbilical cord, the tissue was dissected and the Wharton's jelly layer tissue was removed and cut into 3 mm pieces. 3 Step 1) Cut the tissue into small pieces, centrifuge, wash, collect the tissue pieces, put them into a culture flask, add DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100ug / ml penicillin, and 100ug / ml streptomycin, then culture it under 37℃ and 5% CO2 conditions to promote adhesion to the wall, and after observing the yellowing of the medium every 2-3 days, replace half of the medium and culture for 10-12 days until cells are seen emerging from the edge of the tissue pieces; 2) gently shaking the plate to remove the tissue fragments, and then collecting the tissue fragments and the lower layer cells, and then reattaching and culturing the collected tissue fragments; The collected lower layer cells are washed with PBS, and then an appropriate amount of 0.25% trypsin is added to digest the cells for 2 to 3 minutes. The digestion is stopped by adding an equal volume of trypsin stop solution. The bottom of the flask is gently blown with a pipette, and the cells are centrifuged at 1500 rpm / min for 5 minutes. Then, the cells are collected and obtained.

[0080] 2. Subculture of primary mesenchymal stem cells Subculture of primary mesenchymal stem cells: 1.0×10 primary mesenchymal stem cells 5 ~6.0×10 5 Add the cells to DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin to an initial density of cells / ml, then culture it at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10 to 15 days. After observing the yellowing of the medium every 2 to 3 days, replace half of the medium.

[0081] 3. For the preparation of compounds 1 to 16, refer to Reference 1 (New limonophyllines AC from the stem of Atalantia monophylla and cytotoxicity against cholangiocarcinoma and HepG2 cell lines, Arch. Pharm. Res. (2018) 41:431-437).

[0082] (Example 1) Preparation of a cell protein extract having a hair follicle repair effect according to the present invention The method for producing a cell protein extract having a hair follicle repair effect of the present invention comprises the steps of: Mesenchymal cell lineages: 5.0 x 10 6 Step (1) of adding the cells to a medium containing 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum protein (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% amino acid compound (18AA) and 5μmol / L compound 16 so as to achieve a density of 100μmol / mL cells; then culturing the medium at 37℃ and 5%CO2 for 30min, centrifuging the medium at 1200rpm*5min, washing the medium with PBS three times, and then collecting the cells; 8 × 10 cells collected in step (1) 6 (2) dispersing the cells in physiological saline to a density of 1000 / mL, and sonicating for 2 min at 2-8 °C, 25 kHz, and 360 W with an interval of 1 s for 3 s to prepare a cell lysate; and (3) centrifuging the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filtering the resulting centrifuged solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0083] Example 2: Preparation of hair follicle repair protein composition of the present invention The hair follicle repair protein composition of the present invention is prepared by: (1) adding 25 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 1, and carrying out enzymatic digestion at 37° C. for 30 minutes to prepare an enzymatic digestion solution; and (2) preparing the enzymatic hydrolysis solution prepared in step (1) at 10 mg / ml with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride under conditions of 2°C to 8°C, passing the solution through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 x 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 x 250 mm) in that order, and monitoring and collecting the elution site at an elution flow rate of 0.1 to 1 ml / min and an ultraviolet wavelength of 280 nm.

[0084] Example 3: Preparation of a freeze-dried preparation of the hair follicle repair protein composition of the present invention The required amount of mannitol is added to the cell protein composition prepared in Example 2, stirred and mixed uniformly, and then lyophilized. The resulting lyophilized preparation contains 2% mannitol (m / m).

[0085] (Example 4) Preparation of cell protein extract having hair follicle repair effect of the present invention The method for producing a cell protein extract having a hair follicle repair effect of the present invention comprises the steps of: Mesenchymal cell lineages: 8.0 x 10 6Step (1) of adding the cells to a medium containing 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum protein (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% amino acid compound (18AA) and 5μmol / L compound 13 so as to achieve a density of 100μmol / mL cells; then culturing the medium at 37℃ and 5% CO2 for 30min, centrifuging the medium at 1200rpm*5min, washing the medium with PBS three times, and then collecting the cells; The cells collected in step (1) were diluted to 1.0 × 10 7 (2) dispersing the cells in physiological saline to a density of 1000 / mL, and sonicating for 2 min at 2-8 °C, 25 kHz, and 360 W with an interval of 1 s for 3 s to prepare a cell lysate; and (3) centrifuging the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filtering the resulting centrifuged solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0086] Example 5: Preparation of hair follicle repair protein composition of the present invention The hair follicle repair protein composition of the present invention is prepared by: (1) adding 20 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 4, and carrying out enzymatic digestion at 37° C. for 30 minutes to prepare an enzymatic digestion solution; and (2) preparing the enzymatic hydrolysis solution prepared in step (1) at 10 mg / ml with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride under conditions of 2°C to 8°C, passing the solution through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 x 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 x 250 mm) in that order, and monitoring and collecting the elution site at an elution flow rate of 0.1 to 1 ml / min and an ultraviolet wavelength of 280 nm.

[0087] Example 6: Preparation of a freeze-dried preparation of the hair follicle repair protein composition of the present invention Sorbitol was added to the cell protein composition prepared in Example 5, stirred and mixed uniformly, and then lyophilized. The resulting lyophilized preparation contained 5% sorbitol (m / m).

[0088] (Example 7) Preparation of cell protein extract having hair follicle repair effect of the present invention The method for producing a cell protein extract having a hair follicle repair effect of the present invention comprises the steps of: Mesenchymal cell lineages 1.0 x 10 7 Step (1) of adding the cells to a medium containing 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% amino acid compound (18AA) and 8μmol / L compound 14 to a density of 100μmol / mL, culturing the medium at 37℃ and 5% CO2 for 30min, centrifuging the medium at 1200rpm*5min, washing the medium with PBS three times, and then collecting the cells; 5.0 × 10 cells collected in step (1) 7 (2) dispersing the cells in physiological saline to a density of 1000 / mL, and sonicating for 2 min at 2-8 °C, 25 kHz, and 360 W with an interval of 1 s for 3 s to prepare a cell lysate; and (3) centrifuging the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filtering the resulting centrifuged solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0089] Example 8: Preparation of hair follicle repair protein composition of the present invention The hair follicle repair protein composition of the present invention is prepared by: (1) adding 30 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 7, and carrying out enzymatic digestion at 37° C. for 30 minutes to prepare an enzymatic digestion solution; and (2) preparing the enzymatic hydrolysis solution prepared in step (1) at 10 mg / ml with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride under conditions of 2°C to 8°C, passing the solution through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 x 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 x 250 mm) in that order, and monitoring and collecting the elution site at an elution flow rate of 0.1 to 1 ml / min and an ultraviolet wavelength of 280 nm.

[0090] Example 9: Preparation of a freeze-dried preparation of the hair follicle repair protein composition of the present invention Mannitol was added to the cell protein composition prepared in Example 8, stirred and mixed uniformly, and then lyophilized, so that the resulting lyophilized preparation contained 5% mannitol (m / m).

[0091] (Example 10) Preparation of cell protein extract having hair follicle repair effect according to the present invention The method for producing a cell protein extract having a hair follicle repair effect of the present invention comprises the steps of: Mesenchymal cell lineages: 5.0 x 10 6 Step (1) of adding the cells to a medium containing 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum protein (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% amino acid compound (18AA) and 2μmol / L compound 15 so as to achieve a density of 100μg / mL cells; then culturing the medium at 37℃ and 5% CO2 for 30min, centrifuging the medium at 1200rpm*5min, washing the medium with PBS three times, and then collecting the cells; The cells collected in step (1) were diluted to 8.0 × 10 6(2) dispersing the cells in physiological saline to a density of 1000 / mL, and sonicating for 2 min at 2-8 °C, 25 kHz, and 360 W with an interval of 1 s for 3 s to prepare a cell lysate; and (3) centrifuging the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filtering the resulting centrifuged solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0092] Example 11: Preparation of hair follicle repair protein composition of the present invention The hair follicle repair protein composition of the present invention is prepared by: (1) adding 30 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 10, and carrying out enzymatic digestion at 37° C. for 30 minutes to prepare an enzymatic digestion solution; and (2) preparing the enzymatic hydrolysis solution prepared in step (1) at 10 mg / ml with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride under conditions of 2°C to 8°C, passing the solution through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 x 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 x 250 mm) in that order, and monitoring and collecting the elution site at an elution flow rate of 0.1 to 1 ml / min and an ultraviolet wavelength of 280 nm.

[0093] (Example 12) Preparation of a freeze-dried preparation of the hair follicle repair protein composition of the present invention Dextran was added to the cell protein composition prepared in Example 11, stirred and mixed uniformly, and then freeze-dried to obtain a freeze-dried preparation containing 1% dextran (m / m).

[0094] (Example 13) Preparation of freeze-dried preparation of cell protein extract of the present invention The cell protein extract prepared in Example 1 was added with a required amount of mannitol, stirred and mixed uniformly, and then lyophilized to obtain a lyophilized preparation containing 2% mannitol (m / m).

[0095] (Example 14) Detection of molecular weight distribution of hair follicle repair protein composition of the present invention The molecular weight distribution of the nerve repair protein composition of the present invention was detected using standard molecular weight polyacrylamide gel electrophoresis; Step 1: Select a glass plate with a thickness of 1.5 mm, place it horizontally, and apply the prepared gel solutions (15% lower gel, 4% separating gel) on the glass plate in sequence as shown in Table 1, and insert the comb vertically into the lower gel; [Table 1] After thawing the marker (20KDa-245KDa) frozen at -40℃, the freeze-dried powder of the marker and the hair follicle repair protein composition prepared in Examples 3, 6, 9, and 12 are mixed with PBS to prepare a test sample of 10ug / ul, and 20ul of the test sample is added to the loading well, and then electrophoresis is performed under 60-80V conditions until a relatively clear band appears, and the voltage is adjusted to 100-120V until the marker separation is completed, and the PAGE gel is placed in Coomassie Brilliant Blue staining solution, and when a clear band appears on the gel, the staining is stopped, and after washing with pure water, it is destained with 10% acetic acid solution, and destaining is stopped when the gel becomes transparent. The results are shown in Figure 1, where band A is Example 3, band B is Example 6, band C is Example 9, and band D is Example 12.

[0096] Example 15: High performance liquid chromatography detection of hair follicle repair protein composition of the present invention The lyophilized powder of the hair follicle repair protein composition of Example 3 is dissolved in deionized water to make it into a test solution of 10 mg / ml. Chromatography column: SHIMSEN Ankylo (300mm*4.6mm.D., 3um; P / N: 380-01215-05) Shimadzu Mobile phase: 50 mmol / L phosphate buffer (pH=6.8) containing 300 mmol / L sodium chloride, flow rate is 0.3 mL / min, injection volume is 10 ul, column temperature is 25° C., detection wavelength is 280 nm, isocratic elution, 30 min collection. The results are shown in FIG. 2.

[0097] (Test Example 1) Study of the oxidative damage repair effect of the cell protein extract of the present invention having hair follicle repair effect 3D Skin Model: EpiKutis (R) , commercially available and purchased from EpiKutis. SLS working solution: 0.0080 g of SLS was weighed out and dissolved in 2 mL of PBS solution, filtered through a 0.22 μm filter to prepare a 0.4% SLS mother solution. 0.5 mL of the SLS mother solution was aspirated and 0.5 mL of PBS was added to prepare a 0.2% SLS working solution. WY14643 working solution: 10 mg of WY14643 (PPARα stimulant) was weighed and dissolved in 1 mL of DMSO to prepare a 30 mM WY14643 mother solution. Then, 10 μL of WY14643 mother solution (30 mM) was added to 6 mL of model culture medium to prepare a 50 μM WY14643 working solution. Test sample: The freeze-dried powder of the cell protein extract prepared in Example 13 was added to and dissolved in physiological saline to obtain a 1% solution of the freeze-dried cell protein extract preparation. Model culture medium: DMEM basal medium. 0.9 mL of model culture medium was added to a 6-well plate, and the 3D skin model was transferred to the 6-well plate and labeled with the test number. Blank control (BC): No treatment was applied to the skin model, which was then incubated in a CO2 incubator (37°C, 5% CO2) for 24 h. Negative control (NC): 25 μL of 0.2% SLS working solution was added to the surface of the skin model, which was then placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 h. Positive control (PC): 25 μL of 0.2% SLS working solution was added to the surface of the skin model, which was then placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 h. After that, 25 μL of 50 μM WY14643 working solution was added, and then the model was incubated in a CO2 incubator (37°C, 5% CO2) for 24 h.

[0098] Test group: 25 μL of 0.2% SLS working solution was added to the surface of the skin model, which was then placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 h. Then, 25 μL of 1% cell protein extract solution was added, and the model was then incubated in a CO2 incubator (37°C, 5% CO2) for 24 h. After 24 hours of incubation, the skin model was washed with PBS solution to remove any remaining liquid inside or outside the skin model. After 24 hours of fixation with 4% paraformaldehyde, the model was removed by circular excision, stained with H&E, and then photographed under a microscope for observation. The results are shown in Figure 3. The cell protein extract of the present invention significantly repairs oxidatively damaged skin.

[0099] (Test Example 2) Consideration of the hair growth effect of the hair follicle repair protein composition of the present invention Eleven male C57BL / 6J mice weighing 20-25g were selected and randomly divided into a model group and a drug group, with five mice in the model group and six mice in the drug group. A testosterone solution was applied topically to the test animals every day to establish an AGA mouse model. First, the back skin of the test mice was depilated with animal scissors and depilatory cream, and then 0.5% testosterone ethanol solution (w / v) (prepared by dissolving an appropriate amount of testosterone in 50% ethanol solution (v / v)) was applied at 0.1 mL / cm. 2The hair follicle repair protein composition of Example 3 of the present invention was smeared twice a day on the depilated skin of the test mice to give a dose of 2 mg / time / mouse from the 8th to 24th days after depilation. The hair growth status of the mice was observed on the 0th, 8th, 16th, and 24th days after depilation. The mice were killed on the 25th day after administration to the test animals, and the back skin of the mice was taken and HE stained (scale bar: 200 μm) to observe the hair growth and hair follicle regeneration status of the depilated skin, and three fields were randomly selected to calculate the number of HF. The results are shown in Figures 4 to 6. The hair loss area of ​​the mice in the model group remains pink, and there is almost no new hair growth and hair follicle regeneration, more than half of the HFs are in the telogen phase, and the miniaturized HFs are completely located in the dermis, with incomplete hair tissue and skin morphology.The hair loss area of ​​the mice in the test group shows obvious new hair growth and significantly more regenerated hair follicles, with enlarged bulbs and inner root sheaths located in the subcutaneous tissue, and a large proportion of HFs have already entered the anagen phase, and by the 33rd day after hair loss, dense hair grows on the back skin of the mice, with complete hair tissue and skin morphology, promoting hair follicle growth, maintaining skin morphology, and maintaining the physiological function of the skin.The hair follicle repair protein composition of the present invention significantly promotes the repair of damaged hair follicles and hair regeneration, and activates HF stem cells (p<0.05).

[0100] The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various modifications or variations based on the present invention, and any such modifications or variations should fall within the scope of protection of the claims of the present invention as long as they do not deviate from the spirit of the present invention.

Claims

1. A hair follicle repair protein composition, the preparation of which comprises: (1) adding 20 U / mL to 35 U / mL of either one of nuclease or ultranuclease or a combination thereof to a cell protein extract, and subjecting the resulting mixture to enzymatic degradation at 37°C ± 1°C for 15 to 40 minutes to prepare an enzymatic degradation solution; and (2) preparing the enzymatic decomposition solution prepared in step (1) at 5 to 15 mg / ml with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride at 2°C to 8°C, passing the solution through a chromatography column at an elution flow rate of 0.1 to 1 ml / min, and monitoring and collecting the eluted portion at an ultraviolet wavelength of 280 nm. Hair follicle repair protein composition.

2. The preparation of the cell protein extract comprises: Density is 5.0 x 10 ６ pieces / mL ~ 1.0×10 ７ The mesenchymal passaged cells (cells / mL) were placed in a medium containing 40-50% DMEM / F12, 40-50% RPMI1640, 0.1-2% bovine serum protein (BSA), 1-15 ug / mL epidermal growth factor (EGF), 1-15 ug / mL fibroblast growth factor (FGF), 1-15 ug / mL insulin transferrin, 0.01-0.1% amino acid compound (18AA), and 2-10 μmol / L of any one or combination of compounds 1-16, and then incubated at 37.0° C.±0.5° C., 5%±1.0% CO ２ Step S-1: culturing the cells for 10 to 60 minutes under the above conditions, and then isolating, washing, and collecting the cells; 【Chemistry 1】 【Chemistry 2】 【Chemistry 3】 The collected cells were 5.0 × 10 ６ pieces / mL ~ 5.0×10 ７ Step S-2 of dispersing the cells in a solvent selected from physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer to a density of 10 ... Step S-3: separating the cell lysate prepared in step S-2, and then filtering the resulting separation solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate. The protein composition of claim 1.

3. The molecular weight of the hair follicle repair protein composition is 20 kDa to 300 kDa, preferably 50 kDa to 200 kDa; A protein composition according to any one of claims 1 to 2.

4. The mesenchymal passaged cells of step S-1 are cultured in a medium for 15 to 50 minutes, preferably 20 to 40 minutes; A protein composition according to any one of claims 1 to 3.

5. The ultrasonic conditions for step S-2 are 2°C to 8°C, 25 kHz, and 360 W, and ultrasonic treatment is performed for 1 to 5 minutes with an interval of 1 second for 3 seconds. A protein composition according to any one of claims 1 to 4.

6. Add a lyoprotectant selected from mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, glucan, tricaprylin (HES), polyethylene glycol, ethylene vinylidene, phosphate, acetate, citrate, sorbitol, and starch, or a combination thereof, to the protein composition prepared in step (2), and lyophilize to prepare a lyophilized preparation of the protein composition. A protein composition according to any one of claims 1 to 5.

7. The freeze-dried preparation contains a freeze-drying protection agent in an amount of 0.5 to 8%, preferably 1 to 5%, by mass. A protein composition according to any one of claims 1 to 6.

8. The lyophilized preparation of the protein composition has a pH of 6 to 8, preferably a pH of 7 to 7.

5. A protein composition according to any one of claims 1 to 7.

9. A hair follicle repair composition comprising the hair follicle repair protein composition of any one of claims 1 to 7 and a pharma- ceutically acceptable carrier.

10. 10. Use of a hair follicle repair protein composition according to any one of claims 1 to 7 or a composition according to claim 9 in the preparation of a cell repair or hair follicle repair product.

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