Preparation method and application of umbilical cord extract for improving and repairing hair follicle miniaturization

Abstract

The present invention discloses a preparation method and application of an umbilical cord extract for improving and repairing hair follicle miniaturization. The preparation of the umbilical cord extract is as follows: the animal umbilical cord is washed and cut into pieces, digested with trypsin, added with serum-free mesenchymal stem cell culture medium, dispersed tissue by blowing, filtered, the filtered tissue fluid is incubated for 10 to 12 h after low-energy red light irradiation, an equal volume of serum-free mesenchymal stem cell culture medium is added, and the incubation is continued for 48 to 72 h, an equal volume of serum-free mesenchymal stem cell culture medium is added, and mir‑135b is added at the same time, and the tissue fluid is processed for 48 to 72 h, and the tissue fluid is taken out, and freeze-thawed, ultrasonically treated, filtered, and the filtrate is diluted with sterile water to obtain. The umbilical cord extract of the present invention can significantly improve and repair miniaturized hair follicles, promote the hair diameter to return to normal, and can be used as a new type of raw material for preparing cosmetics and medicines with the effects of improving and repairing hair follicle miniaturization, promoting hair growth or preventing hair loss.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of an umbilical cord extract for improving and repairing hair follicle miniaturization. Background Art

[0002] In mature individuals, hair follicles generally cannot regenerate, but instead present a cyclical cycle, including the growth phase, regression phase, and resting phase. During the growth phase, hair papilla cells secrete cytokines, hair follicle stem cells begin to differentiate asymmetrically, and daughter cells migrate to the hair bulb to differentiate into matrix cells. The matrix cells rapidly proliferate and differentiate into the hair follicle structure, hair shaft, and inner root sheath; during the regression phase: the matrix cells in the lower part of the hair follicle and a small part of the hair papilla cells begin to apoptosis, the hair papilla moves up and the cells are in a tight state, and the secretory function of the hair papilla cells is inhibited; during the resting phase: the hair papilla reaches the bottom of the follicle bulge, the hair shaft stops growing, and the hair falls off. In the cyclic growth of hair follicles, the structure of the upper 1 / 3 remains stable, and the morphology of the lower 2 / 3 of the hair follicles undergoes a cyclic cycle. In a normal adult scalp, the growth phase of hair follicles usually lasts for more than 2 years, the regression phase is 2 weeks, and the resting phase is about 2-3 months. The characteristics of hair follicle miniaturization caused by various reasons are: on the one hand, the hair bulb becomes smaller, and on the other hand, the growth period of the hair follicle is shortened. These changes cause the hair to change from terminal hair to vellus hair.

[0003] The hair bulb is a tissue structure formed by the fusion and expansion of the lower end of the epithelial sheath of the hair root and the hair follicle. The main cells are the matrix cells, which proliferate and differentiate into the epithelial sheath of the hair root and the hair follicle, thereby maintaining the size of the hair bulb and the duration of the growth phase during the hair follicle cycle, thereby maintaining the normal shape of the hair.

[0004] Animal umbilical cord is an animal-derived cosmetic raw material that is easy to obtain and has relatively low immunogenicity. The biologically active substances obtained from animal raw materials vary greatly due to the different extraction processes, extraction methods and treatment methods. The present invention provides a method for extracting animal umbilical cords. The extracted biologically active substances have the effect of improving and repairing the miniaturization of hair follicles, can repair and improve the miniaturized hair follicles to restore their normal size and shape, and thus improve the shape of the hair. It can be used as a cosmetic, raw material or medicine to improve and repair the miniaturization of hair follicles. Summary of the invention

[0005] The first object of the present invention is to provide a preparation method of an umbilical cord extract for improving and repairing hair follicle miniaturization, comprising the following steps: washing and chopping a fresh animal umbilical cord, digesting it with trypsin, adding a serum-free mesenchymal stem cell culture medium, blowing and dispersing the tissue, and then filtering it, irradiating the filtered tissue fluid with low-energy red light and incubating it for 10 to 12 hours, adding an equal volume of serum-free mesenchymal stem cell culture medium, continuing to incubate for 48 to 72 hours, adding an equal volume of serum-free mesenchymal stem cell culture medium, and adding mir-135b at the same time, treating it for 48 to 72 hours, taking out the tissue fluid, performing freeze-thaw treatment, then ultrasonically treating it, filtering it, and diluting the filtrate with sterile water to obtain an umbilical cord extract.

[0006] Preferably, the low-energy red light irradiation is irradiation with 620 nm low-energy red light for 10-15 min with an energy density of 0.5-1 J / cm 2 .

[0007] Preferably, the dosage of mir-135b is calculated based on a final concentration of 1 nmol / L.

[0008] Preferably, the incubation is carried out in a 37°C incubator.

[0009] Preferably, the freeze-thaw treatment is specifically as follows: quick freezing at -80°C for 1 h, and thawing in warm water at 37°C; quick freezing at -80°C for 1 h, and thawing in warm water at 37°C.

[0010] Preferably, the ultrasonic treatment is specifically: ultrasonic treatment at 4° C. for 4-10 min, with an operating frequency of 80 kHz.

[0011] Preferably, the first filtration is performed using a 100-300 μm filter, and the second filtration is performed using a 10-40 μm filter.

[0012] Preferably, the trypsin digestion treatment is specifically: using 0.25% trypsin at 4°C for 8-12 h.

[0013] Preferably, the dilution with sterile water is specifically diluted to a concentration of 10% by mass.

[0014] The second object of the present invention is to provide an umbilical cord extract prepared by the above preparation method.

[0015] The third object of the present invention is to provide the use of the umbilical cord extract in preparing products for improving and repairing hair follicle miniaturization.

[0016] The fourth object of the present invention is to provide the use of the umbilical cord extract in the preparation of products for promoting hair growth or preventing hair loss.

[0017] Preferably, the products include cosmetics and medicines.

[0018] A fifth object of the present invention is to provide a product for improving and repairing hair follicle miniaturization, comprising the above-mentioned umbilical cord extract as an active ingredient.

[0019] The present invention has discovered a method for processing the umbilical cord, and the extract obtained can significantly repair the miniaturized hair follicles, promote the growth of hair follicles to normal levels, and thicken the hair. The extract can be used as a new type of raw material for preparing cosmetics or medicines with the effects of improving and repairing the miniaturization of hair follicles, promoting hair growth or preventing hair loss, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Umbilical cord extract improves and repairs hair follicle miniaturization.

[0021] Figure 2 The statistical results of the average diameter of hair bulbs in each group, *p<0.05, ***p<0.001. DETAILED DESCRIPTION

[0022] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0023] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0024] Example 1

[0025] Preparation of umbilical cord extract:

[0026] In the clean bench, take fresh pig umbilical cord and wash it in PBS containing penicillin (100 U / mL) and streptomycin (0.1 mg / mL) for 3 times, 5 min each time; then use sharp scissors to cut the tissue into 1 mm 2 About 100 μm, wash twice with calcium-free and magnesium-free PBS, 5 min each time; then add an equal volume of 0.25% trypsin and shake at 4°C for 12 h. Add an equal volume of serum-free mesenchymal stem cell culture medium (serum-free culture medium produced by Ecosai Biotechnology (Taicang) Co., Ltd., with a catalog number of ME000-N023), blow and disperse the tissue for 2 h, and filter the tissue liquid with a 100 μm filter; the filtered tissue liquid is placed on an open plate and irradiated with 620 nm low-energy red light for 10 min, with an energy density of 0.5-1 J / cm 2Then, the tissue fluid was placed in a 37°C incubator with 5% CO2 for 12 h, an equal volume of serum-free mesenchymal stem cell culture medium was added, and the incubation continued for 72 h. An equal volume of serum-free mesenchymal stem cell culture medium was added, and mir-135b (final concentration 1 nmol / L) was added for treatment for 72 h. The tissue fluid was taken out, bottled, 10 mL / bottle, and placed at -80°C for 3 h until it became solid; thawed in warm water at 37°C; frozen at -80°C for 3 h until it became solid, and thawed in warm water at 37°C; then ultrasonically treated at 4°C, 500W ultrasonic cell disruptor, working frequency was 80 kHz, duration was 4 min; the obtained tissue fluid was filtered using a 40 μm filter; then the filtered tissue fluid was diluted with sterile water to a concentration of 10% by mass, and the obtained liquid was the umbilical cord extract, which was stored at -80°C.

[0027] Example 2

[0028] Umbilical cord extract improves miniaturized hair follicles and promotes hair to return to normal:

[0029] The skin of 6-8 week old C57BL / 6 mice is pink and the hair follicles are in the resting phase. First, DHT (dihydrotestosterone, 1 mg / 100 μL / day) was injected intraperitoneally for 4 consecutive days, and then the back of the mice was depilated. After depilation, the color of the back of the mice was recorded and marked as the 0th day of treatment. Then, from 1st to 22nd day, DHT was injected intraperitoneally for 2 consecutive days and one day was stopped to induce the miniaturization of hair follicles in mice. On the 22nd day of treatment, the hair and skin of the back of the mice were taken, the fat tissue was removed, and the morphology of the hair follicle bulb and the hair in the skin tissue were observed under a stereo microscope.

[0030] Grouping and treatment: 1: Normal control group, normal mice were not treated in any way; 2. Model control group: After the androgenic alopecia model in mice was induced by DHT, no other treatment was performed; 3: RL-UCE group, mice were intraperitoneally injected with DHT to induce hair follicle miniaturization, and umbilical cord extract irradiated with 620 nm low-energy red light (prepared according to the preparation method of Example 1 of ZL202311368155.6) was applied to the back every day from the 12th day to the 22nd day, 200 μl / mouse; 4: mir-135b group, mice were intraperitoneally injected with DHT to induce hair follicle miniaturization, and mir-135b was applied to the back every day from the 12th day to the 22nd day, 0.1 nmol / day; 5. RL-UCE+mir-135b group: mice were intraperitoneally injected with DHT to induce hair follicle miniaturization, and a mixture was applied to the back every day from the 12th day to the 22nd day: 0.1 nmol of mir-135b was added to the 620 nm low-energy red light. 6: RL-mir-UCE group, mice were intraperitoneally injected with DHT to induce hair follicle miniaturization, and 620 nm low-energy red light was applied to the back every day from the 12th day to the 22nd day, and then mir-135b was added to continue incubation to obtain the umbilical cord extract (prepared in Example 1 of the present invention), 200 μl / mouse. There were 3 mice in each treatment group.

[0031] Results Figure 1 and Figure 2 .

[0032] Figure 1 and Figure 2 The results showed that on the 22nd day of treatment, the bulbs of the hair follicles in the back skin of mice in the normal control group were clearly visible, the bulbs were obviously funnel-shaped, the maximum diameter of the bulbs was larger, and the hair was thicker; in the model control group, RL-UCE group, mir-135b group and RL-UCE+mir-135b group, after DHT induction, the bulbs of the hair follicles in the back skin of mice were only partially visible, and although a small part of the bulbs were funnel-shaped, the maximum diameter of the bulbs was smaller, and the hair was thinner; in the RL-Mir-UCE group, although DHT was induced, the bulbs of the hair follicles in the skin were still clearly visible, the bulbs were obviously funnel-shaped, the maximum diameter of the bulbs was larger, and the hair was thicker, similar to the normal control group. The results show that compared with the use of umbilical cord extract or mir-135b alone irradiated with 620 nm low-energy red light, or a simple mixture of the two, the extract obtained by first irradiating the umbilical cord tissue fluid with 620 nm low-energy red light and then adding mir-135b for continued incubation has a better effect in improving and repairing hair follicle miniaturization.

[0033] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing an umbilical cord extract for improving and repairing hair follicle miniaturization, characterized in that: The following steps are involved: The fresh pig umbilical cord was washed and cut into pieces, digested with trypsin, added with serum-free mesenchymal stem cell culture medium, dispersed by blowing, and then filtered. The filtered tissue fluid was irradiated with 620 nm low-energy red light for 10-15 min with an energy density of 0.5-1 J / cm 2 Then, incubate in a 37°C incubator for 10-12 h, add an equal volume of serum-free mesenchymal stem cell culture medium, continue incubation for 48-72 h, add an equal volume of serum-free mesenchymal stem cell culture medium, and add mir-135b with a final concentration of 1 nmol / L, treat for 48-72 h, take out the tissue fluid, perform freeze-thaw treatment, then perform ultrasonic treatment, filter, and dilute the filtrate with sterile water to obtain the umbilical cord extract; The freeze-thaw treatment is specifically as follows: quick freezing at -80°C for 1 hour, thawing in warm water at 37°C; quick freezing at -80°C for 1 hour, thawing in warm water at 37°C; the ultrasonic treatment is specifically as follows: ultrasonic treatment at 4°C for 4-10 minutes, with an operating frequency of 80 kHz.

2. The umbilical cord extract prepared by the preparation method according to claim 1.

3. Use of the umbilical cord extract according to claim 2 in the preparation of products for improving and repairing hair follicle miniaturization.

4. Use of the umbilical cord extract according to claim 2 in the preparation of products for promoting hair growth or preventing hair loss.

5. The use according to claim 3 or 4, characterized in that: The products include cosmetics and medicines.

6. A product for improving and repairing hair follicle miniaturization, characterized in that: Contains the umbilical cord extract according to claim 2 as an active ingredient.

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