CN116077623A

CN116077623A - Use of LGALS1 protein and functional peptide fragments thereof for promoting hair regeneration

Info

Publication number: CN116077623A
Application number: CN202310027436.9A
Authority: CN
Inventors: 薛慧玲
Original assignee: Shenyang Agricultural University
Current assignee: Shenyang Agricultural University
Priority date: 2023-01-09
Filing date: 2023-01-09
Publication date: 2023-05-09

Abstract

The invention discloses an application of LGALS1 protein and a functional peptide fragment thereof in promoting hair regeneration, belonging to the technical field of regenerative medicine. The invention discovers that an experimental group injected with LGALS1 protein enters the hair follicle regeneration process earlier than a control group, and the number of regenerated hair follicles of the LGALS1 protein injection group is obviously more than that of the control group, so that the LGALS1 and the functional peptide thereof can effectively promote hair regeneration, and further the application of the LGALS1 protein and the functional peptide thereof in preparing medicaments for promoting hair regeneration is protected. The invention provides a new thought for the hair regeneration of the hirsute animals and a new potential treatment means for the clinical pets with related diseases such as hair loss caused by diseases or physical injuries.

Description

Use of LGALS1 protein and functional peptide fragments thereof for promoting hair regeneration

Technical Field

The invention belongs to the technical field of regenerative medicine, and particularly relates to application of LGALS1 protein and a functional peptide thereof in promoting hair regeneration, and a specific application method of the LGALS1 protein and the functional peptide thereof in promoting hair regeneration is utilized.

Background

Hair follicles are a tiny and complex skin accessory that can grow and anchor hair. Hair follicles play a vital role in skin homeostasis, thermoregulation and sensory processes. Hair follicles have the characteristic of periodic regeneration, from the morphogenic and rapid growth phase (anagen phase) of the follicle to the catagen phase of apoptotic drive (catagen phase), followed by a relative resting phase (telogen phase) and a return to anagen phase. The hair follicle tissue contains a plurality of stem cells related to self-renewal, differentiation and regulation of hair growth, and maintains the homeostasis of the skin, while the Hair Follicle Stem Cells (HFSCs) located at Long Tubu (Bulge) are not isolated from the hair follicle tissue.

Hair follicle stem cells are a group of undifferentiated cells with multipotency, and have strong self-renewal capacity and proliferation capacity. During the cyclic regeneration of hair, the hair follicle stem cells can be continually activated to form new hair follicles. During this process, the activated hair follicle stem cells continue to proliferate and differentiate, thereby forming the various cell types required for mature hair follicles. The hair follicle stem cells continuously migrate downwards to form an Outer Root Sheath (ORS) and wrap hair Matrix cells (Matrix) differentiated from the hair follicle stem cells, and the hair Matrix cells continue to differentiate to generate hair follicle structures such as Inner Root Sheath (IRS) and Hair Shaft (HS). In the case of skin lesions, hair follicle stem cells proliferate rapidly and migrate upward, participating in the formation of new epithelium during wound healing and regeneration of hair follicles in the wound.

Regeneration of complex tissue or organ structures of mammals is not common compared to other animals. When the human embryo and neonate skin is damaged, the skin has the capability of complete regeneration and can heal in a scar-free manner, but the capability gradually disappears after birth, and the auxiliary structures such as skin, hair follicles and the like can be replaced by fibrotic scars. However, dann et al in 1941 demonstrated that de novo hair follicle regeneration occurred in the wound center of adult mice (Dann et al, 1941). It was found that the skin of the back of the mice was wound and then epithelialized, i.e. wound healing was followed by gradual appearance of the hair matrix, formation of hair follicle structures, hair shaft production, and eventually new intact hair follicles and hair growth, a process of wound-induced hair neogenesis (WIHN). Lineage tracking analysis shows that WIHN-induced hair neogenesis is similar to embryonal follicle development, both morphological and molecular, but the number of new follicles is generally small and the hair is sparse.

Galectin-1 (Lgals 1) belongs to the galectin family, has affinity for β -galactoside, and is widely present in various tissues of humans and animals, and is involved in regulating immune response, cell growth, cell migration, lineage differentiation, tissue development, tissue regeneration, and other functions (Fan et al, 2018). LGALS1 has been reported to promote the hyperproliferative of fibroblasts. However, there is currently no report on the involvement of LGALS1 in promoting hair-regrowth function.

At present, the main method for repairing and improving the skin injury condition clinically is to replace damaged skin by tissue engineering skin, but even the skin tissue obtained by the forefront tissue engineering technology does not have accessory organs such as hair follicles and the like, and the functions, the characteristics and the appearance of the skin tissue are far from those of natural skin. Moreover, the most intuitive treatment method at present is generally hair transplantation, but the hair transplantation cost is high and the requirements are strict, and the success rate of hair transplantation at skin parts such as burns, scalds and the like can be greatly reduced. The final goal of regenerative medicine is to restore the function and structure of the original tissue, thus exploring the issue of hair regeneration, which can provide a basis for developing new methods for treating hair loss.

Disclosure of Invention

In view of this, it is an object of the present invention to provide the use of LGALS1 proteins and functional peptide fragments thereof for promoting hair regeneration.

The inventor researches and discovers that the experimental group injected with the LGALS1 protein enters the hair follicle regeneration process earlier than the control group, and further experimental researches and discovers that the number of regenerated hair follicles of the LGALS1 protein injection group is significantly more than that of the control group, so that the LGALS1 can effectively promote hair regeneration.

The invention aims at realizing the following steps:

the present invention provides the use of LGALS1 proteins and functional peptide fragments thereof for promoting hair regrowth.

In another aspect, the invention provides the use of LGALS1 protein and functional peptide fragments thereof in the manufacture of a medicament for promoting hair regrowth.

Further, the medicament is prepared into a pharmaceutically acceptable dosage form.

Further, the dosage forms include drops, injection preparations, gel preparations and solid preparations.

Further, the preparation is an injection preparation.

Further, the injection preparation comprises an effective amount of LGALS1 protein and a functional peptide fragment thereof and pharmaceutically acceptable auxiliary materials.

Further, the pharmaceutically acceptable auxiliary materials comprise a filling agent, a pH regulator, a suspending agent, an antioxidant, a bacteriostatic agent, an emulsifying agent and a drug carrier without toxic or side effect.

Further, the pharmaceutically acceptable auxiliary materials comprise collagen, hyaluronic acid and chitosan.

Further, the medicament is prepared in a single dose form, and the single dose medicament contains 0.01-100 mu g LGALS1 protein and functional peptide fragments thereof.

Further, the concentration of LGALS1 protein and its functional peptide fragment in the single dose drug is 0.1-100 mug/ml.

Compared with the prior art, the invention has the following beneficial effects:

1. the application of the LGALS1 protein and the functional peptide thereof in promoting hair regeneration provides a new thought for human or hair regeneration of animals with hair, provides a new potential treatment means for related diseases such as hair loss and the like caused by diseases or physical injuries of clinical animals, and provides a new strategy for improving the hair quality and the hair yield of economic animals with hair.

2. The invention has the advantages of simple operation, easy control, low price and easy clinical practice.

Drawings

In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described.

FIG. 1 shows the results of H & E staining of paraffin sections of LGALS1 protein group injected on day 15 after wound in 53-day old mice according to the present invention, wherein FIG. 1A is a 50X enlarged picture, and FIGS. 1B and 1C are 100X enlarged pictures;

FIG. 2 shows the H & E staining results of the PBS control group injected on day 15 after the trauma of the 53-day old mice of the present invention;

FIG. 3 shows alkaline phosphatase staining results on day 28 after wounding of 21-day old mice according to the invention, wherein FIGS. 3A-3C show LGALS1 protein-injected groups and FIGS. 3D-3F show PBS-injected control groups;

FIG. 4 shows alkaline phosphatase staining results on day 28 after wounding of 53 day old mice according to the invention, wherein FIGS. 4A-4C show LGALS1 protein-injected groups and FIGS. 4D-4F show PBS-injected control groups;

FIG. 5 shows the results of regenerative hair follicle count for the LGALS1 protein group of the present invention and the control group.

Detailed Description

The following detailed description of the invention is provided in connection with examples, but the implementation of the invention is not limited thereto, and it is obvious that the examples described below are only some examples of the invention, and that it is within the scope of protection of the invention to those skilled in the art to obtain other similar examples without inventive faculty.

The specific techniques or conditions not identified in the examples of this application may be carried out according to techniques described in the literature in this field or according to the specifications of the product, and the reagents or instruments used, not identified to the manufacturer, are conventional products available commercially.

The main reagents used in the examples: diethyl ether was purchased from Hongsheng fine chemical company, inc. of ordinary city; PBS, hematoxylin staining, eosin staining, neutral gum caplets were purchased from bioengineering (Shanghai) limited; BCIP/NBT alkaline phosphatase chromogenic kit is purchased from Shanghai Biyun biotechnology Co., ltd; LGALS1 protein was purchased from offshore protein technologies Inc.; pancreatin substitutes were purchased from sameimers technology (china) limited.

The experimental animals used in the examples were 6 healthy 21-day-old C57BL/6J mice for post-traumatic hair follicle regeneration number detection; of the 53 day-old C57BL/6J mice, 6 were used for the number of post-traumatic hair follicle regeneration detection and 6 were used for the post-traumatic hair follicle regeneration progress detection.

Example 1

LGALS1 promotes hair regeneration process experiments, mainly comprising the following steps:

1. the mice of 53 days old are anesthetized in a sealed box by diethyl ether, and the anesthetized mice are in an unoccupied state;

2. shaving the skin on the back of the anesthetized mice by using an electric shaver to remove the hair around the predicted wound;

3. wiping and sterilizing the wound area with alcohol, and taking sterilized scissors and forceps to cut off the whole skin of the wound area, wherein the wound area is 1.5cm multiplied by 1.5cm;

4. observing and recording wound healing conditions every day after the wound;

5. on day 7 post-traumatic mice were randomized into two groups, 0.1ml LGALS1 protein (1. Mu.g/ml) and equal amounts of PBS were injected subcutaneously at the site of scab, and injection was stopped after 7 consecutive days (day 14 post-traumatic);

6. on the 15 th day after the wound, the scar part after the wound healing is taken, paraffin section H & E staining is carried out, and the regeneration condition of the scar skin hair is observed.

The specific procedure for H & E staining of paraffin sections was as follows:

(1) The scar tissue paraffin section on day 15 post-traumatic is placed in xylene and incubated on a shaker for 10 minutes at room temperature, this step is repeated 2 times;

(2) The liquid was discarded and incubated with 100%, 95%, 85%, 75%, 50%, 25% ethanol on a shaker for 2 minutes at room temperature;

(3) Discard liquid, use ddH ₂ O is washed on a shaker for 2 minutes at room temperature, and this step is repeated 2 times;

(4) Hematoxylin staining: paraffin sections were stained with hematoxylin for 3 minutes, rinsed with running water in immunohistochemical cups for 1 minute, and this step was repeated 2 times;

(5) Dropwise adding hydrochloric acid ethanol (1% hydrochloric acid solution, and mixing with 70% ethanol and concentrated hydrochloric acid according to a certain proportion) onto the washed paraffin slice for leaching;

(6) Carefully rinsing with running water in an immunohistochemical cup for 1 minute, and repeating the step for 2 times;

(7) Paraffin sections were placed in ddH ₂ In O, shake on a shaker for 5 minutes at room temperature;

(8) Eosin staining: dripping with eosin staining solution for 15 seconds, washing with 95% ethanol for 3 times, and washing for 15 minutes each time;

(9) Slicing the washed paraffin, placing the slices in a 100% ethanol solution, and incubating on a shaking table for 5 minutes;

(10) Paraffin sections were removed from the ethanol solution, placed in xylene and incubated on a shaker for 10 minutes at room temperature, and this step was repeated 2 times;

(11) The sections were removed and, when the xylene solution on them was not completely dried, neutral gum was added dropwise and the slide was quickly blocked.

On day 15 after the injury, the scar part after the wound healing is taken, paraffin section H & E staining is carried out to observe the regeneration condition of scar skin hair, the H & E staining results of the LGALS1 protein group and the PBS control group are shown in fig. 1 and 2, it can be seen that the experiment group (fig. 1) for injecting the LGALS1 protein obviously observes that the epidermis part is uneven, the dermis direction is concave, and the PBS control group (fig. 2) does not observe the trend of hair follicle regeneration, so that the LGALS1 protein group injected after the injury enters the hair follicle regeneration process earlier than the control group.

Example 2

The LGALS1 protein promotes hair regeneration number experiment, mainly comprising the following steps:

1. the mice with the age of 21 days and 53 days are anesthetized in a sealed box by diethyl ether, and the anesthetized mice are in an unoccupied state;

3. wiping and sterilizing the wound area with alcohol, and removing the whole skin of the wound area with sterilized scissors and forceps, wherein the back wound of a 21-day-old mouse is 1.0cm multiplied by 1.0cm wound, and the back wound of a 53-day-old mouse is 1.5cm multiplied by 1.5cm wound;

4. observing and recording wound healing conditions every day after the wound;

5. on day 7 post-wound, the proteome and control were subcutaneously injected under the scab site with 0.1ml LGALS1 protein (1. Mu.g/ml) and equal amounts of PBS, respectively, and stopped after 7 consecutive days (day 14 post-wound);

6. on the 28 th day after the wound, the scar part of the wound mice after healing is taken, alkaline phosphatase staining is carried out after connective tissues are removed, and the number of regenerated hair follicles is counted.

The specific procedure for alkaline phosphatase staining is as follows:

(1) Taking the whole skin of the scar region on the back of the mice on the 28 th day after the trauma;

(2) True epidermis separation: the removed mouse skin is spread in a culture dish, and 2mg/ml pancreatin substitute solution is incubated overnight at 4 ℃;

(3) The epidermis was removed under a dissecting microscope and washed 3 times for 1 minute with PBS;

(4) Fixing: the rest dermis tissues are spread in a culture dish, soaked in 4% paraformaldehyde solution for 10 minutes, washed 3 times with PBS for 3 minutes each time;

(5) Dyeing: incubation of BCIP/NBT working solution (BCIP volume: NBT volume=1:2) for 30 min at room temperature, running water rinse to terminate development, 3 times of rinsing for 3 min each;

(6) Counting: photographs were taken under a dissecting microscope and regenerative hair follicle counts were performed.

The results of the number of regenerated hair follicles in the LGALS1 protein group and the PBS control group are shown in table 1 and fig. 3 to 5.

TABLE 1 LGALS1 protein group and PBS control group comparison of regenerated follicle count

As can be seen from table 1 and fig. 3 to 5, the LGALS1 protein was injected to grow more regenerated hair follicles in the scar region after the injury of the mice, and the difference was very remarkable compared with the control group, the number of regenerated hair follicles of the LGALS1 protein group was about 50 on average at the 28 th day after the injury, and the control group was only 9 on average, so that the LGALS1 protein had the effect of promoting hair regeneration.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

Use of lgals1 protein for promoting hair regrowth.
Use of lgals1 protein and a functional peptide fragment thereof for the preparation of a medicament for promoting hair regrowth.
3. The use according to claim 2, wherein the medicament is prepared in a pharmaceutically acceptable dosage form.
4. The use according to claim 3, wherein said dosage forms include drops, injectable formulations, gel formulations and solid formulations.
5. The use according to claim 4, wherein the dosage form is an injectable formulation.
6. The use according to claim 5, wherein the injectable formulation comprises an effective amount of LGALS1 protein and functional peptide fragments thereof and pharmaceutically acceptable excipients.
7. The use according to claim 6, wherein the pharmaceutically acceptable excipients comprise fillers, pH adjusters, suspending agents, antioxidants, bacteriostats, emulsifiers and drug carriers without toxic side effects.
8. The use according to claim 6, wherein the pharmaceutically acceptable excipients comprise collagen, hyaluronic acid and chitosan.
9. The use according to claim 8, wherein the medicament is prepared in a single dose form comprising 0.01-100 μg of LGALS1 protein and functional peptide fragments thereof.
10. The use according to claim 9, wherein the concentration of LGALS1 protein and its functional peptide fragment in the single dose of medicament is 0.1-100 μg/ml.