阅读说明：本技术 Vegf-b在维持毛囊细胞抗损伤能力上的应用 (Application of VEGF-B in maintaining anti-injury capacity of hair follicle cells ) 是由 李旭日 李万红 刘熠 于 2021-07-08 设计创作，主要内容包括：本发明提供VEGF-B在维持毛囊细胞抗损伤能力上的应用。VEGF-B具有增强毛囊细胞抵抗基因组损伤物质伤害的能力,在外源基因组损伤物质处理的情况下,通过保护毛囊细胞,能够起到促进毛发生长、防止毛发脱落的作用效果。(The invention provides the application of VEGF-B in maintaining the anti-injury capacity of hair follicle cells. VEGF-B has the function of enhancing the ability of hair follicle cells to resist the damage of genome damaging substances, and can play the role of promoting hair growth and preventing hair loss by protecting hair follicle cells under the condition of treating exogenous genome damaging substances.)

Use of VEGF-B to maintain the ability of hair follicle cells to resist damage.

2. A method of increasing the damage resistance of hair follicle cells, comprising: comprising the step of administering exogenous VEGF-B, and/or the step of activating the VEGF-B signaling pathway.

3. A formulation for enhancing the ability of hair follicle cells to resist damage, characterized by: the active component comprises at least one of vascular endothelial growth factor VEGF-B, VEGF-B signal channel activator.

4. A shampoo for preventing and treating alopecia in the growing period is characterized in that: the agent for enhancing the anti-damage ability of hair follicle cells according to claim 3 is included in the active ingredient thereof.

5. The medicine for preventing and treating the chemotherapy-induced alopecia is characterized in that: the agent for enhancing the anti-damage ability of hair follicle cells according to claim 3 is included in the active ingredient thereof.

6. An antitumor agent characterized by: the agent for enhancing the anti-damage ability of hair follicle cells according to claim 3 is included in the active ingredient thereof.

Technical Field

The invention belongs to the technical field of growth factor application, and particularly relates to application of VEGF-B in maintaining the anti-injury capacity of hair follicle cells.

Background

Alopecia is a common disease of dermatology, and as social competition is increased, social stress such as working stress, mental stress, hormone imbalance and other social stress and psychophysiological factors cause or increase the occurrence of alopecia, so that the mental health and the life quality of patients are obviously affected. Alopecia can be divided into reversible non-scarring alopecia and irreversible scarring alopecia. Irreversible cicatricial alopecia whereas non-cicatricial alopecia, due to hair follicle damage or loss of hair follicle pores, can promote the regeneration of the shed hair or reverse the condition of hair follicle damage and mild atrophy after specific treatment. Non-ecchymotic alopecia can be classified into androgenetic alopecia, alopecia areata, alopecia caused by head moss, stationary phase alopecia, anagen phase alopecia, etc.

Anagen alopecia is an acute and severe lesion that affects most hair follicles in the anagen phase and can cause diffuse hair loss of 80-90%. Anagen alopecia, also commonly referred to as chemotherapy alopecia, typically occurs during tumor chemotherapy due to cytotoxicity inhibition of hair matrix cell division caused by genome damage induced by antimetabolites, alkylating agents, and mitotic inhibitors, causing hair thinning and shedding. Chemotherapy alopecia is one of the most disturbing long-term side effects in cancer treatment. It is estimated that 65% of patients receiving traditional chemotherapy will experience alopecia. Common antitumor drugs such as antimetabolites, alkylating agents, mitotic inhibitors and the like can kill tumor cells by damaging or interfering the genome of the cells. However, because the drugs have an injury effect on normal cells, under the condition of large drug dosage or poor targeting, the genome of the normal cells can be injured, and the normal physiological function of the cells is affected. Cytotoxicity caused by antitumor agents can inhibit division of hair matrix cells, leading to hair loss and inhibition of hair regrowth. Although most anagen hair loss is reversible, it sometimes results in permanent hair loss and can have devastating effects on the patient's mind, negatively impacting the individual's appearance, physical appearance and self-esteem. For some patients, very severe emotional trauma may result, which may result in discontinuation or rejection of beneficial treatment. In this case, intervention and treatment for anagen alopecia is of significant importance and necessity. However, no special method and medicine for treating anagen alopecia exists in clinic at present.

The existing treatment for alopecia mainly aims at various causes causing alopecia to carry out intervention treatment. Androgenetic alopecia is currently treated with topical milodel solutions, in addition finasteride is used in male androgenetic alopecia patients and spironolactone is used in female androgenetic alopecia patients. The intralesional glucocorticoid injection is used for treating alopecia areata; oral antifungal drugs, such as terbinafine, itraconazole, fluconazole or griseofulvin, are used for systemic treatment to treat alopecia areata caused by trichophyton infection. At present, the clinical treatment for anagen alopecia is mainly to cool the scalp, but the effect is not ideal. For alopecia in the growing period, particularly alopecia caused by antineoplastic drug treatment, no effective treatment method or drug is available at present.

VEGF-B, one of the vascular endothelial growth factor family members, was discovered in 1996, and the function and mechanism of VEGF-B has not been completely understood to date, as compared to VEGF-A, which has been extensively studied. VEGF-B is abundantly expressed in most cells and organs, especially in the heart, adipose tissue, skeletal muscle and blood vessels. VEGF-B has been shown to have protective effects on a variety of cells by participating in cellular antioxidant and regulating cellular metabolic processes.

Disclosure of Invention

The invention aims to provide application of VEGF-B in maintaining the anti-injury capacity of hair follicle cells so as to play an effective role in preventing and treating physiological alopecia caused by hair follicle cell injury.

According to one aspect of the invention, there is provided the use of VEGF-B to maintain the ability of hair follicle cells to resist damage.

According to one aspect of the present invention, there is provided a method of increasing the damage resistance of hair follicle cells: comprising the step of administering exogenous VEGF-B, and/or the step of activating the VEGF-B signaling pathway.

According to one aspect of the present invention, there is provided a formulation for enhancing the ability of hair follicle cells to resist damage: the active component comprises at least one of vascular endothelial growth factor VEGF-B, VEGF-B signal channel activator.

According to one aspect of the present invention, there is provided a shampoo for preventing and treating anagen alopecia: the active ingredients of the preparation comprise the preparation for enhancing the anti-injury capability of hair follicle cells.

According to one aspect of the present invention, there is provided a medicament for the prevention and treatment of chemotherapy-induced alopecia: the active ingredients of the preparation comprise the preparation for enhancing the anti-injury capability of hair follicle cells.

According to one aspect of the present invention, there is provided an antitumor agent: the active ingredients of the preparation comprise the preparation for enhancing the anti-injury capability of hair follicle cells.

Hair follicle cells, one of the skin cells, are the basis for hair to remain young, grow and regenerate. VEGF-B has the function of enhancing the ability of hair follicle cells to resist the damage of genome damaging substances, and can play the role of promoting hair growth and preventing hair loss by protecting hair follicle cells under the condition of treating exogenous genome damaging substances. During the course of anti-tumor therapy, patients with tumors often have clinical manifestations of hair loss due to the damage to the genome of hair follicle cells caused by anti-tumor therapy. The preparation for enhancing the anti-injury capability of hair follicle cells provided by the invention is used for participating in the preparation of anti-tumor medicaments, and is beneficial to preventing and relieving alopecia caused by tumor treatment.

Drawings

FIG. 1 is a schematic view showing the result of tissue section staining of the skin of a Vegf-b knockout mouse and a littermate wild type mouse in example 1, in which Co represents a collagen layer, Ad represents a fat layer, and SM represents a skeletal muscle layer;

FIG. 2 is a graph showing statistics of relative densities of collagen in a dermal layer of a skin tissue section corresponding to the graph provided in FIG. 1;

FIG. 3 is a fat layer relative thickness statistic corresponding to the skin tissue section provided in FIG. 1;

FIG. 4 is a graph of the relative thickness statistics of the collagen layer of the skin tissue sections provided in correspondence with FIG. 1;

FIG. 5 is a skeletal muscle layer relative thickness statistic corresponding to the skin tissue section provided in FIG. 1;

FIG. 6 is a photograph showing immunofluorescence staining of skin tissue after applying a cell genome damaging agent 2, 2-dimethylolbutyric acid (DMBA) to a Vegf-b knockout mouse and a littermate wild-type mouse for 24 hours in example 1;

FIG. 7 is a statistical plot of the number of epidermal gamma H2AX positive cells for the skin tissue provided in correspondence with FIG. 6;

FIG. 8 is a statistical graph of the expression levels of Vegf-b in various cells of skin tissues of young and old rats;

FIG. 9 shows hair states of 27-month-old Vegf-b knockout mice and littermate wild-type mice thereof in example 2;

FIG. 10 is a statistical graph corresponding to the degree of graying of hair in the mice shown in FIG. 8;

FIG. 11 is a graph showing the results of experiments in example 2 in which mouse hair was knocked out to study the effect of VEGF-B on mouse hair regrowth;

FIG. 12 is a statistical chart of hair regrowth in the mouse hair-shaved area corresponding to the mouse shown in FIG. 10.

In the diagrams of the above figures, WT means a wild type mouse, Vegf-b^-/-It is a Vegf-b knockout mouse.

Detailed description of the preferred embodiments

In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.

Example 1

The dorsal skin tissues of 6-month-old Vegf-b knockout mice and littermate wild-type mice were stained by H & E section, pictures were taken by microscope, the thickness of the epidermal, dermal and adipose layers of the skin was measured and the structural change at the dermal-epidermal junction was observed. As shown in fig. 1, the results of skin tissue section staining showed that the dermal-epidermal junction was flattened and the surface area of the junction was reduced, increasing the fragility of the skin and decreasing the transfer of nutrients between the dermis and epidermis, compared to the skin of a wild type mouse; atrophy of dermis layer and damage of reticular structure; the fat layer was thinned, showing a pronounced skin aging phenotype. The structural characterization statistical results of the skin tissue sections are shown in FIGS. 2-5.

The method comprises the following steps of taking a Vegf-b gene knockout mouse and a wild mouse in the same nest, removing back hairs of the Vegf-b gene knockout mouse, uniformly coating cell genome damage substances DMBA for 24 hours, and taking skin to be frozen sections. Immunofluorescent staining was performed with the genomic lesion-specific marker antibody γ H2 AX. Counting the number of gamma H2AX positive cells, evaluating the damage degree of a skin cell genome, and evaluating the protective effect of VEGF-B on skin cells. The staining results are shown in fig. 6, under the stimulation of genome damage drugs, gamma H2AX signals in the skin of Vegf-b knockout mice are obviously stronger compared with wild type mice in the same fossa, which indicates that the genome damage of skin cells of the Vegf-b knockout mice is more serious, and the statistical result of the number content of the corresponding epidermal layer gamma H2AX positive cells is shown in fig. 7, and the statistical result has statistical significance.

Hair follicle cells, one of the skin cells, are the basis for hair to remain young, grow and regenerate. The expression level of Vegf-B in various cells of rat skin tissues of different ages was determined by using a single cell sequencing technique, and as a result, as shown in FIG. 8, Vegf-B expression in hair follicle cells of old rats was significantly lower than that in hair follicle cells of young rats, indicating that VEGF-B has a close correlation with the youthful state and hair growth ability of hair follicle cells. In fig. 8, the meanings of the respective symbols are as follows:

fib, Fibroblast (fibroplast); EC, Endothelial cells (Endothelial cells); SMC, Smooth muscle cell (Smooth muscle cell); epi, Epithelial cells (Epithelial cells); spi, spine cells; HFC, Hair follicle cells (Hair follicle cells); mit1, mitochondrial cells (Mitotic cells); CC, Channel cells; BC1, Krt15+ Basal cells (Krt15+ Basal cells); BC2, Krt14+ Basal cells (Krt14+ Basal cells); NKT, Natural killer T cell (Natural killer T cell)

Example 2

Fig. 9 and 10 show the hair state of 27-month-old Vegf-b knockout mice and littermate wild-type mice, and it was observed that the Vegf-b knockout mice became thin and lusterless in hair, changed in color to grayish white, exhibited a remarkable aging hair quality, and the condition was more remarkable in male mice, except that the hair loss was more severe and the hair was sparse. The experimental results show that Vegf-b helps to maintain the young state of the hair.

About 2cm of Vegf-b knockout mice at 6, 12, 18, and 24 months old and wild-type mice in the same litter were shaved on the backs of the mice²The hair growth rate was evaluated by measuring the area of the uncovered part of the hair 30 days after shaving, calculating the hair re-coverage. After 30 days of hair removal, as shown in FIGS. 11 and 12, the hair re-coverage of the hairshaved area of the littermate wild-type mice was close to 100%, while the hair shaved area of the Vegf-b knockout mice still had about 20% of the hair-deleted area, revealing that the deletion of the Vegf-b gene had an inhibitory effect on hair growth.

Example 3

(1) Test one: effect of VEGF-B recombinant proteins on Hair growth Rate

Wild type mice of 12 months of age were randomly divided into two groups, one group was subcutaneously implanted with an osmotic pump containing physiological saline as a control group, and the other group was subcutaneously implanted with an osmotic pump containing VEGF-B recombinant protein as a treatment group, at an output rate of 0.15. mu.l/hr. Two groups of mice were shaved about 2cm of their backs²The area of the hair uncovered portion of the skin was measured 10 days, 15 days, and 20 days after the hair was removed, and the hair re-coverage rate was calculated to evaluate the hair growth rate. The loss of VEGF-B function has an inhibitory effect on hair growth, suggesting that VEGF-B has a correlation with hair growth. It is presumed that when VEGF-B recombinant protein is applied to the skin, it has a promoting effect on hair growth, as demonstrated by the fact that the rate of hair re-coverage in the mice in the proteome is higher than that in the normal saline group.

(2) And (2) test II: prevention and treatment effect of VEGF-B recombinant protein on hair loss caused by antitumor drugs

Selecting 6-month-old wild-type mice, randomly dividing the mice into five groups, respectively numbering a blank group, a control group, a VEGF-B group, an AS101 inhibitor group and a minoxidil group, implanting osmotic pumps filled with physiological saline under the skin of the blank group mice and the control group mice, implanting osmotic pumps filled with VEGF-B recombinant proteins under the skin of the VEGF-B group mice, implanting osmotic pumps filled with AS101 inhibitors (CAS: 106566-58-9) under the skin of the AS101 inhibitor group mice, implanting osmotic pumps filled with minoxidil under the skin of the minoxidil group mice, wherein the output speed of the osmotic pumps is 0.15 mu l/hr. The control group mice, the VEGF-B group mice, the AS101 inhibitor group mice and the minoxidil group mice are subjected to single intraperitoneal injection of a freshly prepared alkylating agent anti-tumor drug cyclophosphamide at a dose of 120mg/kg, and the blank group mice are injected with the same dose of physiological saline. Hair loss and regrowth were observed at 1 week, 2 weeks, 3 weeks, and 4 weeks after injection, respectively. Cyclophosphamide is used as a typical alkylating agent antineoplastic drug and can cause serious anagen-phase alopecia, and a large amount of clinical data can predict that the mice in a control group can rapidly and massively shed hairs after being injected with cyclophosphamide, and the hairs grow slowly and are difficult to recover to a pre-treatment state; the AS101 inhibitor group and the minoxidil group have no genome damage resistance effect, so that the serious hair shedding phenomenon can occur, but the mice of the AS101 inhibitor group and the minoxidil group can have faster hair regrowth compared with the control group; VEGF-B has the function of protecting skin cells due to the function of resisting genome damage, can reduce cytotoxicity caused by genome damage and maintain normal physiological functions of cells, and is shown as remarkably improving the condition of hair loss, and meanwhile, VEGF-B has the function of promoting hair growth and can quickly regrow part of lost hair. Therefore, VEGF-B has significant superiority in preventing and treating alopecia caused by antitumor drugs, unlike AS101 inhibitors and minoxidil.

Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the present invention.