CN104622904A - Skin stem cell active component and application of active component - Google Patents

Abstract

The invention discloses a skin stem cell active component. The active component is secreted by skin stem cells, exists in supernatant generated by culturing skin stem cells and is expressed in a form of DIk1+/Scal-. The skin stem cell active component is applied to treatment on alopecia and can be used for activating the stem cells of the body, accelerating growth and proliferation of the hairs; the hair growing effects of the active component in the normal model and the pathological model are superior to those of commercially-available minoxidil or finasteride; and the skin stem cell active component is relatively high in safety.

Description

A kind of skin stem cell active ingredient and its application

technical field

The invention belongs to the field of biotechnology, and in particular relates to an active ingredient of skin stem cells and a method for obtaining the active ingredient of skin stem cells. The skin stem cell active ingredient obtained by the invention can be applied to the treatment of hair loss.

Background technique

Alopecia refers to hair pathological loss and causes hair thinning or forms alopecia plaque, serious can cause hair to all fall off, and pathogenic factor is unclear, is the commonly encountered diseases of department of dermatology, frequently-occurring disease. In the west, the male incidence rate is about 30% at the age of 30, 40% at the age of 40, and 50% at the age of 50, and at least 80% of whites at the age of 70 suffer from hair loss. In China, the prevalence of hair loss is about 30.29%, and 25% of men aged 25 to 55 have androgenetic alopecia. With the increase of social pressure, hair loss is developing towards a younger age, and the number of patients is increasing year by year. Although hair loss does not endanger human life, it obviously affects the appearance and brings great mental pressure and psychological burden to patients, especially for children in the growing period.

In the past 30 years, the U.S. Food and Drug Administration has only approved two drugs for the treatment of hair loss, minoxidil and finasteride, and they have been the first-line drugs since they were approved, and they have a good hair growth effect on most patients. However, in the long-term application of the two drugs, some shortcomings have gradually been exposed, such as ineffectiveness for a small number of patients, slow onset of effect, and easy recurrence after drug withdrawal. What bothers patients even more is the side effects of the two drugs. Minoxidil has a high incidence of skin allergies, which can cause side effects such as rapid heartbeat and increased blood pressure in severe cases. There are also reports of non-specific allergic reactions with unclear causality, such as wheals, allergic rhinitis, facial swelling, allergic shortness of breath, Headache, neuritis and other adverse reactions. Finasteride is only suitable for male patients in the treatment of androgenetic alopecia. Long-term use of common sexual dysfunction (impotence, loss of libido, ejaculation disorder), breast discomfort (mammary gland enlargement, breast pain) and rash, and others include Allergic reactions such as itching, urticaria and swelling of the face and lips, and adverse reactions such as testicular pain. Some patients have adopted expensive hair follicle transplantation when the effect of drug treatment is not satisfactory. However, for patients with thinning hair, hair follicle transplantation is difficult to perform, and complications such as infection and epidermal cysts are prone to occur, and the appearance is often unnatural. .

Stem cells are a kind of cell population with self-renewal, high proliferation and multi-directional differentiation potential, and are the "ancestor cells" and "universal cells" that form various tissues and organs of the human body. Stem cells can produce and secrete a large number of bioactive factors, which can effectively regulate body cell signal transduction, activate human stem cells, and then physiologically repair or replace damaged, diseased and aging cells in the body. For example, stem cells can produce stem cell growth factor (SCF), nerve growth factor (NGF), stromal cell-derived growth factor (SDF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin Like growth factor (IGF), epidermal growth factor (EGF), interleukin-6 and interleukin-7 (IL-6 and IL-7), megakaryocyte colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), interference These cytokines can promote cell proliferation, differentiation, anti-apoptosis and other functions; stem cells can also produce natural immune proteins, such as IgG, IgA, IgM, IgD, IgE, etc., which can resist or repair themselves and the outside world Factors that cause damage to body cells.

Using the bioactive factors secreted by stem cells to activate the stem cells of the body, and then through the stem cells to physiologically repair or replace the damaged, diseased and aging cells of the body, so as to treat the problem of hair loss, it has broad application prospects in the fields of disease prevention and health care and beauty.

Contents of the invention

The purpose of the present invention is to provide an active ingredient of skin stem cells and a method for obtaining the active ingredient of skin stem cells.

An active ingredient of skin stem cells, the active ingredient is secreted by skin stem cells, exists in the supernatant produced by culturing skin stem cells, is expressed in the form of Dlk1 ⁺ /Sca1 ^?, and is obtained by the following method: according to the combination of skin tissue and enzyme solution The volume ratio is 1:3, add the chopped skin tissue into the collagenase III enzyme solution with a concentration of 1.0 mg/ml dissolved in DMEM containing double antibody and place it for 2 hours, filter the cells with a 200-mesh filter, collect the filtrate, and use An equal amount of DMEM containing 10wt% FBS neutralized the activity of the enzyme; the remaining tissue pieces were digested with 0.25wt% trypsin/EDTA at 37°C, and the cells were filtered through a 200-mesh filter to collect the filtrate; Bead-labeled Dlk antibody and Sca1 antibody were used to screen out Dlk1 ⁺ /Sca1 ^? skin stem cells by magnetic bead screening.

Wherein, the shredded skin tissue is washed with PBS and then cut into a skin tissue with a size of 1 mm×1 mm×1 mm.

The purity of Dlk1 ⁺ /Sca1 ^? skin stem cells after screening was identified by flow cytometry. The results showed that the purity of extracted Dlk1 ⁺ /Sca1 ^? skin stem cells could reach 92.0%.

The present invention also provides a method for cultivating the above-mentioned active ingredients of skin stem cells, including:

1) Inoculate the sorted skin stem cells into a culture bottle, add 5ml of serum-free medium for skin stem cells, and culture in an environment with humidity ≥ 95%, temperature 37°C, and CO ₂ volume concentration 5%, and change every 2 days liquid;

2) After the cells are 80% confluent, they are digested and passaged with 0.25wt% trypsin and 0.02wt% EDTA, and the cells produced by the passage are transferred into a new culture flask;

3) Repeat the above culture and amplification process, and adjust the optimal cell number according to the needs during the culture process.

Wherein, the serum-free special medium for skin stem cells is DMEM containing 50ug/ml bovine pituitary extract and 5ng/ml hEGF, pH=7.0-7.4.

Collect the above cultured skin stem cell supernatant, centrifuge at 1000r/min for 5min, discard the cell pellet, transfer the supernatant into a 50ml centrifuge tube, centrifuge at 9000r/min for 15min, discard the liquid, and resuspend the sediment at the bottom of the centrifuge tube with 1ml PBS , that is, the active ingredients of skin stem cells in the supernatant of skin stem cells. Transfer the collected PBS containing the active ingredients of skin stem cells into a 1.5ml EP tube and store at -20°C.

The skin stem cell active ingredient obtained above in the present invention can be used as an active ingredient of a medicine for treating hair loss, and a remarkable effect has been obtained through tests.

The invention maintains the high proliferative ability and stable shape of skin stem cells through in vitro culture and expansion, and collects the supernatant of skin stem cells during the period when skin stem cells have high proliferative ability and stable shape, centrifuges at low speed to discard the cells, and separates them at low temperature. Purification and other modern biotechnology methods have obtained high-purity active ingredients of skin stem cells.

The biologically active factors or substances of the present invention can be developed into products with cell biologically active functions, which can activate body stem cells after acting on the human body, thereby promoting the growth and proliferation of hair. The hair growth effect of the present invention is better than that of the commercially available medicine minoxidil or finasteride under normal model and pathological model, which shows that the hair growth effect of the present invention is remarkable. The safety test shows that the present invention does not show skin irritation and allergy, and has relatively high safety. The present invention uses regenerative medicine technology to complete skin cosmetic repair, and has the advantages of simple and safe operation method, easy acceptance by patients, time-saving, fast and significant curative effect, so as to achieve the purpose of promoting hair proliferation and eliminating the troubles of people with thinning hair.

Description of drawings

Fig. 1 is a diagram of flow cytometry results of Dlk1 ⁺ /Sca1 ^? skin stem cells of the present invention.

Fig. 2 is a diagram of the growth status of Dlk1 ⁺ /Sca1 ^? skin stem cells of the present invention.

Detailed ways

The present invention is further described by way of specific examples below, but the following examples do not constitute any limitation to the present invention. On the premise of not departing from the technical solutions of the present invention, any modifications or changes made to the present invention that are easily realized by those skilled in the art will fall within the scope of the claims of the present invention.

Example 1: Isolation of skin stem cells.

1. Clean the skin tissue with PBS, put it into a 5ml Ep tube, cut it into a size of 1mm×1mm×1mm with ophthalmic scissors, add 3-4ml collagenase III enzyme solution, this enzyme solution contains 1.0mg/ml collagenase III , and dissolved in DMEM containing double antibody. The mixture of the above enzyme solution and cells was left for 2h.

2. Filter the cells with a 200-mesh filter into a round-bottomed polystyrene tube, collect the filtrate, and neutralize the enzyme activity with an equal amount of DMEM containing 10% FBS. The remaining tissue pieces were digested with 0.25% trypsin/0.02% EDTA at 37°C for 10 min. Filter the cells with a 200-mesh filter into a round-bottomed polystyrene tube, combine the two filtrates, and count the cells.

3. From the above cells, Dlk1 ⁺ cells were screened out by magnetic bead screening method using magnetic bead-labeled Dlk antibody (SIGMA company). The specific steps are counting the collected cells with a counting plate, controlling the number of cells between 10 ⁷ and 10 ⁸ , and then operating according to the instructions of the sorting kit.

4. From the sorted Dlk1 ⁺ cells, Dlk1 ⁺ /Sca1 ^? cells were screened out by magnetic bead screening method using magnetic bead-labeled Sca1 antibody (SIGMA company). The specific steps are counting the collected cells with a counting plate, controlling the number of cells between 10 ⁷ and 10 ⁸ , and then operating according to the instructions of the sorting kit.

5. The purity of Dlk1 ⁺ /Sca1 ^? skin stem cells after screening was identified by flow cytometry. The results showed that the purity of extracted Dlk1 ⁺ /Sca1 ^? skin stem cells could reach 92.0% (Figure 1).

Example 2: Culture of skin stem cells.

1. The sorted cells were inoculated on a 25mm culture flask, and a serum-free medium for skin stem cells was added. The medium was composed of DMEM containing 50ug/ml bovine pituitary extract and 5ng/ml hEGF, pH=7.0-7.4. The medium was changed every 2 days, and the culture environment was kept at a humidity ≥ 95%, a temperature of 37°C, and a CO ₂ volume concentration of 5%.

2. After the cells are 80% confluent, they can be passaged and digested with 0.25% trypsin and 0.02% EDTA.

3. This culture and expansion process was repeated many times to obtain a large number of epidermal stem cells (see Figure 2 for the growth status of the cells).

Example 3: Acquisition of skin stem cell active substances.

1. The skin stem cell supernatant was collected, centrifuged at 1000r/min for 5min, and the cell pellet was discarded.

2. Transfer the collected supernatant into a 50ml centrifuge tube, use a low-temperature high-speed centrifuge to control the temperature, and collect the skin stem cell active substances in the skin stem cell supernatant by centrifugation at a centrifugation speed of 9000r/min and a centrifugation time of 15min.

3. Discard the liquid in the 50ml centrifuge tube after centrifugation, resuspend the sediment at the bottom of the centrifuge tube with 1ml PBS solution, transfer the PBS containing active substances of skin stem cells into a 1.5ml EP tube, and store at -20°C.

Application example 1: The pharmacodynamic experiment of the skin stem cell active substance obtained in the above-mentioned embodiment of the present invention.

1.1 The effect of the skin stem cell active substance of the present invention on depilatory C57BL/6 mice.

Male C57BL/6 mice, 6 weeks old, in telogen (pink skin). They were adapted to feeding for one week, depilated with rosin paraffin (1:1) on the back, and randomly divided into 6 groups, 10 in each group. The blank group was not given any test substance; the excipient group was given 70% ethanol, each dosage 0.1ml/piece; the skin stem cell active substance low-dose group, the skin stem cell active substance middle-dose group, and the skin stem cell active substance high-dose group group, each dose of 0.05mg/, 0.1mg/ and 0.2mg/ only; minoxidil tincture group, given commercially available minoxidil tincture, each dose of 0.1ml/ .

The mice in each group were administered once a day in the morning and evening, and an appropriate amount was evenly applied to the depilated area, and the medication was continued until the skin entered the telogen (observation end point). Record the transition time of each mouse from resting phase to growth phase and observe the end point time. At the end of the observation time, the mice were killed by dislocation of the neck, and the new hair of the same part on the back was taken and weighed. Statistical analysis of the transition time, observation end time and gross weight difference of the mice of each test substance, the results are shown in Table 1.

The transition time is the time from the telogen phase to the anagen phase, and the shorter the time, the stronger the induction effect and the stronger the ability to promote the transformation of hair to the anagen phase. As can be seen from Table 1, the skin stem cell active substance of the present invention is compared with the minoxidil tincture group, and the transformation time is significantly shortened (P<0.05).

The observation end point time is closely related to the hair growth phase time, the longer the time is, the longer the hair growth time is, and the stronger the growth-promoting effect is. Table 1 shows that all medication groups are compared with the auxiliary material group and the blank group, and the observation endpoint time is significantly longer than the auxiliary material group and the blank group (P<0.05); There is a tendency to increase, but there is no significant difference (P<0.05); the effect of the low-dose group and the middle-dose group is basically the same as that of the minoxidil tincture group (P>0.05), and the time of the high-dose group is longer than that of the minoxidil tincture group ( P<0.05).

Gross weight index weighs hair growth quality situation, and table 1 shows, blank group and auxiliary material group have no significant difference in the test, and all medication groups compare with auxiliary material group, and observation gross weight is all significantly higher than auxiliary material group/blank group (P<0.05); The present invention The hair weight of the skin stem cell active substance high dose group is higher than the minoxidil tincture group, and the effect of the low dose group and the middle dose group is no different from the minoxidil tincture group (P>0.05); each dosage group of the skin stem cell active substance of the present invention Gross weight showed an increasing trend (P<0.05).

1.2 The effect of the skin stem cell active substance of the present invention on androgenetic alopecia model mice.

Six-week-old male C57BL/6 mice, weighing 18g to 22g, were randomly divided into 5 groups, 10 mice in each group. The sodium chloride solution group was given 0.9% sodium chloride solution, and the dosage was 0.1ml/one each time; the skin stem cell active substance low-dose group, the skin stem cell active substance middle dose group and the skin stem cell active substance high-dose group, each The administration doses were respectively 0.05mg/, 0.1mg/ and 0.2mg/; the finasteride group was given finasteride tablet water suspension.

The mice in each group were intramuscularly injected with 0.1ml Tritonate Propionate Injection, once a day, to create a model of androgenetic alopecia. In the finasteride tablet group, the finasteride suspension was given by intragastric administration according to the dose of 0.17 mg/kg finasteride, once a day; for the other groups, each sample was evenly applied to the back of the mice, and the drug was administered in the morning and evening every day One time, continuous administration for 12 weeks. At the end of the observation time, the mice were killed by dislocation of the neck, and the skin and hair at the medication site were clipped and weighed. The dehaired skin of mice was cut into pieces with scissors, added to PBS and homogenized at a high speed with a tissue homogenizer, the homogenate was centrifuged at 3000rpm for 20min, the supernatant was collected, and the content of dihydrotritonin (DHT) was determined according to the kit instructions. See Table 2 for the results of the gross weight determination of the mice at the end point of observation.

As can be seen from Table 2, after the mouse was given the skin stem cell active substance of the present invention, the DHT content in the skin decreased significantly, the hair weight was high, and the hair loss was better prevented (P<0.05). The skin stem cell active substance high dose group of the present invention reduces skin DHT and increases the gross weight effect and is better than the positive drug finasteride group (P<0.05); the skin stem cell active substance group of the present invention increases with dosage, and the gross weight is significantly enhanced (P<0.05). 0.05), the DHT content in the skin decreases with increasing dose; the skin stem cell active substance low and middle dose group of the present invention has an effect close to that of the positive drug finasteride group (P>0.05).

1.3 The effect of the skin stem cell active substance of the present invention on cyclophosphamide hair loss model mice.

Male C57BL/6 mice, 6 to 8 weeks old, in telogen (pink skin). They were adapted to feeding for one week, depilated with rosin paraffin (1:1), and randomly divided into 5 groups, 10 in each group. Adjuvant group, give 70% ethanol, each dose 0.1ml/; skin stem cell active substance low dose group, skin stem cell active substance middle dose group, skin stem cell active substance high dose group, each administration dose is 0.05mg / only, 0.1mg/ and 0.2mg/ only; cyclosporine group, given 0.5% cyclosporine 0.15ml / only.

After depilation in mice, the hair follicles in the depilation area transitioned from the quiescent phase to the anagen phase, and when the hair in the depilated area was close to growing Two days before the period, take an appropriate amount of each test substance and apply it evenly on the depilatory area, administer once a day in the morning and evening, and continue to use the medicine until the skin enters the resting period (observation end point). Mice in each group grew Cyclophosphamide was injected intraperitoneally at a dose of 150 mg/kg once during the period. At the end of the observation period, the mice were killed by dislocation of the neck, and the new hair of the same part on the back with an area of 2.5cm×2.5cm was weighed. The results are shown in Table 3.

Giving cyclophosphamide to mice when their hair follicles were in the anagen phase prevented hair growth and resulted in a significant decrease in gross weight. As can be seen from Table 3, mice are given skin stem cell active substance of the present invention and cyclosporine, all can improve the amount of new hair, and have better anti-hair loss effect (P<0.05); Skin stem cell active substance of the present invention increases gross weight with dosage The effect of the high-dose group was better than that of the cyclosporine group (P<0.05).

1.4 The effect of the skin stem cell active substance of the present invention on patients with seborrheic alopecia.

Dermatology outpatients, aged 16-60 years. Hair loss caused by radiotherapy and chemotherapy or those with a history of drug contact allergy were excluded. A total of 78 patients with seborrheic alopecia were selected and divided into treatment group and control group to ensure that the sex, age and course of disease of the two groups of patients with seborrheic alopecia were balanced. The treatment group used the skin stem cell active substance of the present invention externally, rubbed the affected part with a soft brush or cotton wool dipped in the medicine, and spread the medicine solution all over the affected part, 3 times/day. Patients in the control group were treated with 3% minoxidil shampoo. During the treatment period, the two groups of patients washed their hair with warm water once/3 days, and the affected area was protected from wind and cold stimulation, and no other hair growth therapy was used during this period. 60 days as a course of treatment, 3 consecutive courses.

Criteria for judging the curative effect: markedly effective: hair loss is significantly reduced or returned to normal, dandruff and itching disappear, new hairs grow in the affected area or the original vellus-like hair becomes thicker, black, hard, and long, and the appearance is obviously improved; effective: hair loss, dandruff Reduced, relieved itching, new hair growth in the affected area, and improved appearance; invalid: alopecia, dandruff slightly reduced or no change, itching was slightly lighter or the same as before, no new hair growth, and the appearance did not improve significantly.

The higher the curative effect of seborrheic alopecia patients, the earlier the onset of days, indicating that the treatment is more effective. As can be seen from Table 4, the curative effect of the seborrheic alopecia patients using the skin stem cell active substance group of the present invention is significantly higher than that of the matched group 3% minoxidil (P<0.05). Simultaneously, table 5 also illustrates that the onset days of the seborrheic alopecia patients using the skin stem cell active substance group of the present invention are significantly earlier than the onset days of the matched group 3% minoxidil (P<0.05).

1.5 The effect of the skin stem cell active substance of the present invention on patients with alopecia areata.

Dermatology outpatients, aged 16-60 years. Hair loss caused by radiotherapy and chemotherapy or those with a history of drug contact allergy were excluded. Select 72 patients with alopecia areata and divide them into a treatment group and a control group to ensure that the gender, age, and course of the disease of the two groups are balanced. The treatment group used the skin stem cell active substance of the present invention externally, rubbed the affected part with a soft brush or cotton wool dipped in the medicine, and spread the medicine solution all over the affected part, 3 times/day. Patients in the control group were treated with 3% minoxidil shampoo. During the treatment period, the two groups of patients washed their hair with warm water once/3 days, and the affected area was protected from wind and cold stimulation, and no other hair growth therapy was used during this period. 60 days as a course of treatment, 3 consecutive courses.

Criteria for judging curative effect: cure: there is terminal hair growth in the hair loss area, the appearance returns to normal, the hair around the hair loss area is not loose, and the new hair no longer falls off; marked effect: there is generally vellus hair growth in the hair loss area, and the area covered by terminal hair exceeds 1/2 of the hair loss area , the hair around the hair loss area is not loose, and the new hair does not fall off; effective: the hair loss area generally has vellus hair growth, there is no terminal hair or the terminal hair does not reach 1/2 of the hair loss area, and the hair loss is significantly reduced; invalid: there is no or only hair loss in the hair loss area A small amount of vellus hair grows and the hair continues to fall out.

The higher the curative effect of patients with alopecia areata, the earlier the onset of days, indicating that the treatment is more effective. As can be seen from Table 6, the curative effect of the alopecia areata patients using the skin stem cell active substance group of the present invention is significantly higher than that of the matched group 3% minoxidil (P<0.05). Simultaneously, table 7 also illustrates that the onset days of the alopecia areata patients using the skin stem cell active substance group of the present invention are significantly earlier than the onset days of the control group 3% minoxidil (P<0.05).

In this application example, three kinds of models are used for research to comprehensively evaluate the pharmacological effect of the skin stem cell active substance of the present invention. 1) Depilate the normal C57BL/6 mice with rosin paraffin wax, induce their hair to enter a new growth cycle, and investigate the effect of the skin stem cell active substance of the present invention on its growth. The test results show that the skin stem cell active substance of the present invention can shorten the transition from the quiescent phase to the anagen phase, prolong the anagen phase, and thus effectively increase the weight of new hair. 2) In order to investigate the effect under the pathological model, the androgen depilation model and the cyclophosphamide depilation model were established, and the skin stem cell active substance of the present invention showed the effects of inhibiting depilation and promoting hair growth under two kinds of pathological models; the skin of the present invention The stem cell active substance is generally dose-dependent, and with the increase of the dose, the drug effect is enhanced, and the effect is better than the existing drugs for treating hair loss. 3) The case of being applied to patients with seborrheic alopecia and alopecia areata shows that the skin stem cell active substance of the present invention is still significant in curative effect when applied to human body.

Application Example 2: Safety evaluation of the skin stem cell active substance of the present invention.

The safety of the skin stem cell active substance of the present invention is studied with reference to the national drug safety evaluation method, the irritation to the skin is investigated by rabbits, and the allergic reaction to the skin is investigated by guinea pigs.

2.1 The stimulating effect of the skin stem cell active substance of the present invention on the skin.

2.1.1 Single-dose skin irritation test.

10 male New Zealand white rabbits, weighing 2.0kg to 3.0kg, 24 hours before the experiment, select symmetrical parts on both sides of the middle of the spine, carefully remove the hair with hair scissors, each side is 10cm×10cm, there should be no erythema, edema and damage in the depilated part.

Adopt the self-contrast method of the left and right sides of the same body, apply 0.5 mg of the skin stem cell active substance of the present invention to the left side of each rabbit's back, and 0.5 ml of 0.9% sodium chloride solution to the right side of the hair removal area as a contrast. Cover with gauze and fix with adhesive tape; after 4 hours, wash with warm water to remove the residual smear, and observe the local reaction at 1 hour, 24 hours, 48 hours and 72 hours after removing the drug. The result shows that each group of animals has no irritation symptoms such as erythema and edema at the test site, and the general state, behavior and signs of each animal in the experiment are normal, indicating that the skin stem cell active substance of the present invention is non-irritating to the skin.

2.1.2 Multiple administration skin irritation test.

10 male New Zealand white rabbits, weighing 2.0kg to 3.0kg, 24 hours before the experiment, select symmetrical parts on both sides of the middle of the spine, carefully remove the hair with hair scissors, each side is 10cm×10cm, there should be no erythema, edema and damage in the depilated part.

Adopt the self-contrast method of the left and right sides of the same body, apply 0.5 mg of the skin stem cell active substance of the present invention to the left side of the hair removal area of each rabbit back, and 0.5 ml of 0.9% sodium chloride solution to the right side of the hair removal area as a contrast, disinfect gauze after administration Cover and fix with adhesive tape; 2 times a day, once in the morning and once in the evening, with the same administration time each time, wash with warm water after 4 hours to remove the residual smear, and apply continuously for 4 weeks. On the 7th day, the 14th day and the 21st day, 1 hour after each drug removal, before each administration and 1 hour, 24 hours, 48 hours and 72 hours after the last drug removal, the local reactions were observed respectively. The results show that each group of animals has no irritation symptoms such as erythema and edema at the test site, and the general state, behavior and signs of each animal in the experiment are normal, indicating that the skin stem cell active substance of the present invention has no irritation to the skin after repeated administration.

2.2 The allergic reaction of the skin stem cell active substance of the present invention to the skin.

Guinea pigs, weighing 250g to 300g, were randomly divided into 0.9% sodium chloride solution group, skin stem cell active substance group and 2,4-dinitrochlorobenzene positive control group, 6 in each group; 24 hours before the experiment, the middle part of the spine was selected For symmetrical parts on both sides, carefully remove the hair with hair scissors, 3cm×3cm on each side, and there should be no erythema, edema and damage at the depilated part.

Sensitization contact: On the 0th day, the 7th day and the 14th day, apply 0.9% sodium chloride solution, 0.5mg/guinea skin stem cell active substance of the present invention and 1% 2,4 - Dinitrochlorobenzene 0.5ml/piece, covered with sterile gauze after administration, fixed with adhesive tape, and contacted for 6 hours each time.

Excitation exposure: 14 days after the last administration, evenly smear 0.9% sodium chloride solution, 0.5mg/guinea skin stem cell active substance of the present invention and 0.1% 2,4-dinitrochlorobenzene 0.5% on the depilated area on the right side of the back of the guinea pig ml/only, challenged, removed the test substance after 6h, observed immediately, and observed the skin allergic reaction again at 24h, 48h, and 72h.

The results show that the 0.9% sodium chloride solution group and the skin stem cell active substance group of the present invention have no skin allergies such as edema and erythema on the guinea pig skin, and no systemic allergies such as guinea pig asthma, unsteady standing or shock. That is, there is no sensitization effect. Moderate erythema or edema appeared on the skin of the guinea pigs in the positive control 2,4-dinitrochlorobenzene group, and the sensitization rate was 100%. It is proved that the skin stem cell active substance of the present invention does not show skin irritation and allergy, and has high safety.

Claims (6)

1. An active ingredient of skin stem cells, said active ingredient is secreted by skin stem cells, exists in the supernatant produced by culturing skin stem cells, is expressed in the form of Dlk1 ⁺ / ^Sca1 , and is obtained by the following method: according to skin tissue and enzyme The volume ratio of the solution is 1:3, the chopped skin tissue is added to the collagenase III enzyme solution with a concentration of 1.0 mg/ml dissolved in DMEM containing double antibodies and placed for 2 hours, the cells are filtered through a 200-mesh filter, and the filtrate is collected , use an equal amount of DMEM containing 10wt% FBS to neutralize the activity of the enzyme; the remaining tissue pieces are digested with 0.25wt% trypsin/EDTA at 37°C, filter the cells with a 200-mesh filter, and collect the filtrate; combine the two filtrates, respectively Dlk1 ⁺ /Sca1 ^? skin stem cells were screened out by magnetic bead screening method using magnetic bead-labeled Dlk antibody and Sca1 antibody. 2. The skin stem cell active ingredient according to claim 1, wherein the shredded skin tissue is washed with PBS and then cut into skin tissue with a size of 1mm×1mm×1mm. 3. The culture method of the skin stem cell active ingredient that claim 1 obtains, comprising: 1) Inoculate the sorted skin stem cells into a culture bottle, add 5ml of serum-free medium for skin stem cells, and culture in an environment with humidity ≥ 95%, temperature 37°C, and CO ₂ volume concentration 5%, and change every 2 days liquid; 2) After the cells are 80% confluent, they are digested and passaged with 0.25wt% trypsin and 0.02wt% EDTA, and the cells produced by the passage are transferred into a new culture flask; 3), repeat the above culture and amplification process, and adjust the optimal cell number according to the needs during the culture process; Wherein, the serum-free special medium for skin stem cells is DMEM containing 50ug/ml bovine pituitary extract and 5ng/ml hEGF, pH=7.0-7.4. 4. the application of the skin stem cell active ingredient that claim 1 obtains as the medicine for the treatment of alopecia. 5. the application of the skin stem cell active ingredient that claim 1 obtains as the medicine for the treatment of seborrheic alopecia. 6. the application of the skin stem cell active ingredient that claim 1 obtains as the medicine for the treatment of alopecia areata.