Application of PBX1 in promotion of hair growth

Abstract

The invention relates to an application of PBX1 or TAT-PBX1 as an active ingredient in promoting hair growth, such as an application in preparing a medicine for promoting hair growth and an application in preparing a medical instrument for promoting hair growth. Through verification, the umbilical cord mesenchymal stem cells loaded or overexpressed with the TAT-PBX1 fusion protein or culture supernatant or the TAT-PBX1 fusion protein have the capability of remarkably promoting hair regeneration.

Description

Technical Field

[0001] The present invention relates to an application of a biological substance, and in particular to an application of PBX1 in promoting hair growth, and an application of the biological substance as an active ingredient in preparing a drug or medical device for promoting hair growth. Background Art

[0002] Hair follicles are one of the appendages of human and mammal skin, originating from the interaction between the epidermis and mesenchyme during embryonic development. Human and mammal hair follicles continuously undergo a growth phase dominated by cell proliferation, a regression phase dominated by cell apoptosis, and a resting phase in which cells are in a relatively resting state, which repeats over and over again to form a hair follicle cycle. The hair follicle cycle accompanies humans throughout their lives, and any factors that cause imbalance in the homeostasis of the hair follicle cycle will lead to hair loss.

[0003] In practice, the treatment of androgenic alopecia mainly adopts drugs such as minoxidil and finasteride. Minoxidil was originally used to treat hypertension. Later studies found that it can also reasonably control the hair follicle cycle and is used to treat hair loss. Taking finasteride may break the balance between estrogen and androgen, leading to adverse effects including decreased sexual function. Treatments for alopecia areata include glucocorticoid drugs, immunosuppressant drugs, and photoelectric therapy, which also have many adverse reactions. Not only are the costs high, but the recurrence rate is high after stopping the drug.

[0004] Studies have found that hair follicles contain a variety of stem cells, including keratin stem cells, melanin stem cells, mesenchymal stem cells, and neural crest stem cells. These stem cells form an interactive stem cell network in time and space, which not only participates in hair occurrence, but also participates in hair growth and hair follicle cycle. They work together to maintain the homeostasis of hair follicles and enable hair regeneration. Promoting the transition of hair follicles from the resting phase to the growth phase, prolonging the growth phase of hair follicles, and shortening the resting phase of hair follicles are the keys to hair regeneration and hair growth.

[0005] It is known that the common cause of various types of hair loss is the apoptosis and dysfunction of hair follicle stem cells caused by excessive hormone secretion, immune disorders and inflammatory responses. The fundamental solution to hair loss is to inhibit the apoptosis of hair follicle stem cells and maintain hair follicle homeostasis and the normal operation of the hair follicle cycle. Although there have been reports that mesenchymal stem cells can promote hair growth, there are still large individual differences in practice. One of the reasons is that mesenchymal stem cells are only one of the four types of hair follicle stem cells mentioned above, and hair regeneration requires the synergistic effect of these four stem cells to have the potential to regenerate hair.

[0006] Therefore, regulating the synergistic effects of the four stem cells involved in hair growth and hair follicle cycle is the key to treating hair loss. Summary of the invention

[0007] One object of the present invention is to provide a use of PBX1 as an active ingredient in the preparation of a drug for promoting hair growth.

[0008] Another object of the present invention is to provide a use of PBX1 as an active ingredient in the preparation of a medical device for promoting hair growth.

[0009] PBX1 is a transcription factor that promotes the proliferation of hair follicle mesenchymal stem cells, but there is still a lack of evidence to show whether cells overexpressing PBX1 (such as mesenchymal stem cells) have the ability to promote hair growth, and whether their hair growth-promoting effect is better than that of mesenchymal stem cells.

[0010] Cell penetrating peptides (CPPs) have been used to deliver a variety of molecules, especially large molecular weight active proteins from the extracellular to the intracellular delivery, such as protamine, TAT peptide or R9, among which transcription transactivator (TAT) is a peptide derived from human immunodeficiency virus and is one of the most commonly used cell penetrating peptides. Many studies have shown that when TAT binds to cells, it can enhance the cell's absorption of proteins.

[0011] The PBX1 provided by the present invention exists in the culture medium of cells (such as mesenchymal stem cells, neural crest stem cells, melanin stem cells and fibroblasts). In order to facilitate membrane permeation, PBX1 usually also includes a membrane-penetrating peptide, such as TAT.

[0012] The TAT-PBX1 overexpressing cells or culture supernatant provided by the present invention are cells infected with lentivirus to overexpress TAT-PBX1 protein, and the TAT transmembrane peptide enables the PBX1 protein contained in the cell culture supernatant to enter the cells.

[0013] The TAT-PBX1 fusion protein provided by the present invention is an exogenous protein expressed by Escherichia coli, yeast or CHO cells. Because the TAT cell-penetrating peptide enhances the absorption of proteins by mesenchymal stem cells, PBX1 can pass through the cell membrane and enter the cell nucleus, and experimental results show that the prokaryotically expressed TAT-PBX1 still has transcriptional activity after entering the cell nucleus.

[0014] The TAT-PBX1 fusion protein provided by the present invention is packaged with lentivirus, adenovirus, adeno-associated virus and retrovirus, and then transduced into cells (such as mesenchymal stem cells, neural crest stem cells, melanocyte stem cells and fibroblasts).

[0015] Whether it is androgenic alopecia (AGA), alopecia areata (AA) and telogen effluvium (TE) and other diseases that cause hair loss, they are essentially caused by excessive hormone secretion, immune disorders and inflammatory responses that lead to apoptosis and dysfunction of hair follicle stem cells. The PBX1 and TAT-PBX1 of the present invention increase the number of hair follicles in the dermis, increase the diameter of the hair bulb and increase the length of the hair shaft, maintain the homeostasis of the hair follicles and promote the rapid entry of the hair follicles into the adult stage, thereby achieving the effect of promoting hair growth, thereby solving the essential problem that causes hair loss, and are suitable for all diseases of hair loss. This is used as an active ingredient to prepare a drug that promotes hair growth, or a medical device (such as a microneedle) carrying such an active ingredient is used to treat androgenic alopecia (AGA), alopecia areata (AA) and telogen effluvium (TE) and other diseases.

[0016] The PBX1 or TAT-PBX1 prepared by the present invention is used as an active ingredient to promote hair growth. The PBX1 or TAT-PBX1 is mixed with other auxiliary materials to prepare a medicine (preparation) for promoting hair growth, which is used in preventing hair loss, protecting hair and growing hair.

[0017] These pharmaceutical excipients can be those conventionally used in various preparations, such as but not limited to isotonic agents, buffers, flavoring agents, excipients, fillers, adhesives, disintegrants and lubricants, etc.; they can also be selected for use in order to adapt to the substance, such as emulsifiers, solubilizers, antibacterial agents, analgesics and antioxidants, etc. Such excipients can effectively improve the stability and solubility of the compounds contained in the composition or change the release rate and absorption rate of the compounds, thereby improving the metabolism of various compounds in the organism, and further enhancing the administration effect of the composition.

[0018] In aqueous solution injections, excipients generally include isotonic agents and buffers, as well as necessary emulsifiers (such as Tweeen-80, Pluronic and Poloxamer), solubilizers and antibacterial agents, etc. In addition, other pharmaceutically acceptable excipients are also included, such as antioxidants, pH regulators and analgesics.

[0019] The excipients used to prepare liquid preparations generally include solvents, water, oils (such as fatty acids), emulsifiers, and necessary preservatives.

[0020] Various adjuvants and the culture medium supernatant of the present invention are made into dosage forms that are conducive to drug delivery, such as but not limited to aqueous solution injection, powder injection, powder, patch, suppository, emulsion, cream, gel, aerosol, spray, powder aerosol, sustained release agent and controlled release agent, etc. In addition, adjuvants used to achieve specific drug delivery purposes or methods, such as sustained release drug delivery, controlled release drug delivery and pulse drug delivery, etc., such as but not limited to gelatin, albumin, chitosan, polyether and polyester polymer materials, such as but not limited to polyethylene glycol, polyurethane, polycarbonate and copolymers thereof, etc. The so-called "conducive to drug delivery" mainly manifests itself in but not limited to improving therapeutic effects, improving bioavailability, reducing toxic side effects and improving patient compliance, etc.

[0021] It is also common to combine drugs and medical devices to make medicated medical devices, such as dressings containing PBX1 or TAT-PBX1 of the present invention. PBX1 or TAT-PBX1 of the present invention is also loaded or coated on materials as an active ingredient to make medical devices that promote hair growth. Common scaffold materials include PLA, PLGA, GelMA and metals. It can also be mixed with biocompatible degradable materials to make microneedles and microneedle arrays thereof, or loaded into metal microneedles to make microneedle chips. When the microneedle penetrates the skin, PBX1 or TAT-PBX1 is released in the epithelial tissue to regulate the hair follicle cycle and promote hair growth.

[0022] Stem cells (such as mesenchymal stem cells) are also used as a PBX1 carrier to directly promote hair growth. Stem cells overexpressing PBX1 or TAT-PBX1 promote hair growth and are used in preventing hair loss, hair care and hair growth, as well as in the treatment of androgenic alopecia (AGA), alopecia areata (AA) and telogen effluvium (TE). BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 TAT-PBX1 plasmid map

[0024] Figure 2 This is the bright field observation result of the control group and stem cells overexpressing TAT-PBX1;

[0025] Figure 3 Western Blot images of the control group and TAT-PBX1 overexpression;

[0026] Figure 4 This is a Western Blot image of TAT-PBX1 protein in the culture supernatant of umbilical cord mesenchymal stem cells overexpressing TAT-PBX1;

[0027] Figure 5This is a general observation diagram showing that the culture supernatant of stem cells overexpressing TAT-PBX1 significantly promotes hair regeneration in mice;

[0028] Figure 6 The results of HE staining of hair follicles of mice in the control group and the TAT-PBX1 overexpression stem cell culture supernatant group;

[0029] Figure 7 The results of the hair follicle count in the control group and the TAT-PBX1 overexpression stem cell culture supernatant group;

[0030] Figure 8 The results of hair bulb diameter of mice in the control group and the group with overexpression of TAT-PBX1 stem cell culture supernatant;

[0031] Fig. 9 This is a general observation diagram showing that TAT-PBX1 fusion protein significantly promotes hair regeneration in mice;

[0032] Fig.10 The results of HE staining of hair follicles of mice in the control group and TAT-PBX1 fusion protein group;

[0033] Fig.11 The results of the number of hair follicles per visual field in the control group and TAT-PBX1 fusion protein group mice;

[0034] Fig.12 This is the result graph of hair rod length of mice in the control group and TAT-PBX1 fusion protein group. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0036] The various test methods used in the following examples of the present invention are specifically described as follows:

[0037] 1) Preparation of TAT-PBX1 fusion protein

[0038] Preparation of TAT-PBX1 fusion protein: The coding gene of PBX1 was amplified by PCR and cloned by TA.

[0039] The PCR product was connected to the cloning vector pT-EASY, and then the correctly connected white clones were picked by blue-white screening. After bacterial liquid PCR, restriction digestion identification and gene sequencing identification, the PBX1 coding gene fragment in the positive clone was inserted into the prokaryotic expression vector pTAT-HA (purchased from Addgene plasmid #35612) by restriction digestion and T4 connection, and then transferred into the prokaryotic expression strain Rosetta (DE3) (purchased from Tiangen Biochemical Technology Co., Ltd.), and the positive transformants were obtained by ampicillin resistance screening. Activate overnight at 37°C in the expression medium, expand the culture, and collect the precipitate by centrifugation; the precipitate was placed in the lysis solution for ultrasonic disruption and dissolution, centrifuged at 12000g×30min, and the supernatant was collected. After the supernatant was filtered with a 0.45μm filter, it was purified by nickel column affinity chromatography, and then renatured by pulse dilution. The renatured solution was dialyzed and ultrafiltered to obtain a high concentration of TAT-PBX1 fusion protein.

[0040] Purification of TAT-PBX1 fusion protein includes the following steps: 2+ -NTA-agarose column, equilibrated with binding buffer (1-200mM Tris-HCl, 1-200mM imidazole, 1-500mM NaCl, 1-8M urea, pH9.5), the bacterial supernatant was loaded onto the column, the column was rinsed with washing buffer (1-200mM Tris-HCl, 1-509mM imidazole, 500mM NaCl, 8M urea, pH9.5), and then the TAT-PBX1 fusion protein bound to the column was eluted with elution buffer (1-100mM Tris-HCl, 1-500mM imidazole, 1-500mM sodium chloride, 1-8M urea, pH9.5).

[0041] The refolding of TAT-PBX1 fusion protein includes the following steps: the eluted sample is refolded in a refolding buffer containing 1-20 mM Tris, 1-20 mM glycine, 1-10 mM EDTA, 0.1-0.5 M arginine, pH 9.5 at a dilution ratio of 1:1-20. After the refolding is completed, the supernatant is introduced into a dialysis bag and dialyzed overnight at 4°C with 1-10 mM phosphate buffer (pH 5.4). After this, the denatured protein is concentrated using a 30-kDa ultrafiltration tube (purchased from Millipore, USA). The concentration of the fusion protein is determined using a BCA protein detection kit.

[0042] 2) Preparation of umbilical cord mesenchymal stem cells overexpressing TAT-PBX1

[0043] Packaging of empty vector TAT (Vector) into lentiviral particles: When the density of 293T cells (kindly donated by the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) reaches 70% to 75%, prepare for transfection. First, add 500 μL serum-free culture medium (Opti-MEM), 1 to 20 μg of TAT plasmid (constructed in this laboratory), 3 to 15 μg of packaging plasmid PAX2 (purchased from Addgene), and 1 to 5 μg of PMD2G (purchased from Addgene) into a 1.5 mL EP tube; at the same time, add 1 to 50 μL of LipoFiter TM Add transfection reagent (Thermo Fisher) to 500 μL of Opti-MEM, and incubate both at room temperature for 5 to 100 minutes. Add Opti-MEM containing plasmid to Opti-MEM containing lipofectamine 2000 (Thermo fisher), incubate at room temperature for 15 to 20 minutes, then add to 293T and culture at 37°C and 5% CO2. After 6 to 8 hours of culture, change to DMEM containing 10% FBS. After continuing to culture for 36 hours, collect the DMEM supernatant containing virus particles and centrifuge at 3000 r / min for 5 to 10 minutes. Collect the supernatant, filter it through a 0.45 μm filter, and set aside.

[0044] Packaging TAT-PBX1 into lentiviral particles: When the density of 293T cells reaches 70% to 75%, prepare for transfection. First, add 500uL serum-free culture medium (Opti-MEM), 1 to 30μg of TAT-PBX1 plasmid (constructed in this laboratory), 3 to 10μg of packaging plasmid PAX2 (purchased from Addgene), and 1 to 5μg of PMD2G (purchased from Addgene) into a 1.5mL EP tube; at the same time, add 1 to 30μL lipofectamine 2000 into 500μL Opti-MEM, and both are incubated at room temperature for 5 to 10min. After incubating the Opti-MEM containing plasmid and Opti-MEM containing lipofectamine 2000 at room temperature for 15 to 20min, add it dropwise to 293T (kindly donated by the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), and culture it at 37°C and 5% CO2 for 6 to 8h, and then change it to DMEM containing 10% FBS. Continue culturing for 36 hours, collect the DMEM supernatant containing virus particles, centrifuge at 3000r / min for 5-10min, collect the supernatant, filter it through a 0.45μm filter and set aside.

[0045] 3) Preparation of umbilical cord mesenchymal stem cells loaded with TAT-PBX1 protein

[0046] Umbilical cord mesenchymal stem cells were cultured with DMEM medium. When the cell density reached (70% to 75%), TAT-PBX1 fusion protein was added to the medium to a final concentration of 1ng / ml to 100μg / ml. After continuing to culture the cells for 1 minute to 120 hours, the TAT-PBX1 fusion protein penetrated the cell membrane and entered the cell nucleus, promoting cell growth.

[0047] Umbilical cord mesenchymal stem cells were cultured with DMEM medium. When the cell density reached (70% to 75%), the umbilical cord mesenchymal stem cell medium was discarded and 7.5 μL polybrene, 7.5 uL bFGF and virus supernatant mixture were added. After 24 hours, the transduction was completed and umbilical cord mesenchymal stem cells overexpressing TAT-PBX1 were successfully established. The TAT-PBX1 protein penetrated the cell membrane and was secreted into the cell culture supernatant.

[0048] 4) Preparation of culture supernatant of umbilical cord mesenchymal stem cells overexpressing or loading TAT-PBX1 protein

[0049] Umbilical cord mesenchymal stem cells overexpressing or loaded with TAT-PBX1 protein were cultured with DMEM medium. When the cells reached 70% to 75% density, they were replaced with phenol red-free and serum-free DMEM medium. After 24 hours of continuous culture, the cell culture supernatant was collected and the concentration was quantitatively determined using BCA protein. The culture supernatant was freeze-dried in a vacuum freezer, sealed, and stored at -80°C for later use.

[0050] 5) Adenovirus packaging of TAT-PBX1

[0051] When 293A cells (Wuhan Punosai Life Science Co., Ltd.) reach 50% to 70% confluence, prepare for transfection, and recombinant TAT-PBX1 into the AdMAX system (Hanheng Bio), which consists of pHBAd series shuttle plasmids and adenovirus backbone vector pBHGlox (delta) E13Cre (Hanheng Bio) dual vectors. Replace with fresh culture medium 6 hours after transfection. Before collecting the virus, observe whether the virus plaques are formed. In order to limit the spread of the virus and allow the plaques to form better, low-melting point agarose is usually added to the culture medium. Small plaques can generally be seen under a microscope on the 10th to 21st day after transfection. After the plaques are formed, pick up the plaques together with the agarose and place them in 1ml fresh culture medium overnight. Usually 3-6 plaques are picked, and then the titers are compared, and the plaque with the highest titer is used for subsequent experiments. On the second day, the virus in the culture medium is added to the fresh 293A cell culture medium for a small amount of virus amplification. When the cells show plaques again, the cells and supernatant are collected, and the virus is collected by repeated freezing and thawing three times. This virus is the P1 virus, and the P1 adenovirus is used to infect 293A cells. The infection is carried out for three generations in a row, and the adenovirus is amplified in large quantities to the P4 generation. After the plaques are formed, the virus is collected and purified and concentrated in vitro. Umbilical cord mesenchymal stem cells are cultured with DMEM medium. When the cell density reaches 70% to 75%, 5 μl of adenovirus is added to each 1 ml of culture medium. After 72 hours, the transduction is completed, and the successfully transduced TAT-PBX1 umbilical cord mesenchymal stem cells are selected using puromycin, and verified by immunoprotein blotting.

[0052] 6) Adeno-associated virus packaging of TAT-PBX1

[0053] When the 293A cells reach 50-70% confluence, prepare for transfection. Change to serum-free medium within 2 hours before transfection. Preheat OptiMEM, LipoFiter in a 37°C water bath. TMTransfection reagent (Thermo Fisher) can be used after returning to room temperature. Both need to be shaken before use. TAT-PBX1 was recombined into pAAV-IRES-PURO vector (Addgene). The TAT-PBX1 plasmid of AAV vector was transfected into 293A cells. Fresh complete medium was replaced after 6 hours. After 72 hours, the cells containing AAV particles were gently scraped and collected in a 15mL centrifuge tube, centrifuged at 150rpm / min for 3min, and the supernatant was removed and the cell pellet was retained. The cell pellet was washed once with PBS and resuspended with 300μL PBS. The centrifuge tube containing the cell suspension was repeatedly frozen and thawed in liquid nitrogen and 37℃ water bath for 3 times. Centrifuged at 4℃, 3,000rpm / min for 5min, and the lysed supernatant containing AAV particles was filtered through a 0.45μM filter and collected. To remove the residual plasmid DNA and cell genome in the crude virus extract, 0.1 μL Benonase enzyme (Sigma) was added to 1 mL of the virus solution and incubated in a 37°C water bath for 1 h. After that, centrifuge at 4°C, 600 rpm / min for 10 min and take the supernatant. The virus was then purified and concentrated. Umbilical cord mesenchymal stem cells were cultured with DMEM medium. When the cell density reached (70% to 75%), 0.5 ml of adeno-associated virus was added per 1 ml. After 72 h, transduction was completed. Puromycin was used to screen for successfully transduced TAT-PBX1 umbilical cord mesenchymal stem cells, which were verified by immunoblotting.

[0054] 7) Hair growth promotion experiment

[0055] 7-week-old SPF male C57BL / 6 mice were randomly divided into 5 groups: blank control group, solvent control group, umbilical cord mesenchymal stem cell culture supernatant group, empty umbilical cord mesenchymal stem cell culture supernatant group, and umbilical cord mesenchymal stem cell culture supernatant group overexpressing TAT-PBX1. The mice were kept at a constant temperature of 22°C and constant humidity, and the environment was kept quiet. The hair on the back of the mice was removed, and the dorsal side of the mice was depilated using a melted paraffin / rosin mixture (1:1), exposing about 4 cm × 2 cm of skin to ensure that all hair follicles in the dorsal skin of the depilated mice were in the same hair follicle cycle.

[0056] First, dissolve the lyophilized powder of umbilical cord mesenchymal stem cell culture supernatant overexpressing TAT-PBX1 or umbilical cord mesenchymal stem cell supernatant loaded with TAT-PBX1 protein, and then appropriately dilute the solvent, umbilical cord mesenchymal stem cell culture supernatant, empty umbilical cord mesenchymal stem cell culture supernatant, and umbilical cord mesenchymal stem cell culture supernatant overexpressing TAT-PBX1, and inject it into the subcutaneous part of the mouse back hair removal area. Inject twice, and observe the hair growth of the mice 14 days later and take pictures.

[0057] The mice were killed by cervical dislocation, and the hair on the back was shaved with electric clippers and razors. The skin tissue under the depilated area of ​​the mice was taken, and the subcutaneous fat layer was removed, and paraffin sections were made. After HE staining, the growth of hair follicles was observed under a microscope.

[0058] 8) Statistical analysis

[0059] All data are expressed as mean ± standard deviation, and statistical analysis and statistical graph generation were performed using GraphPad Prism 8 software. When comparing between groups, if the data conformed to normal distribution, one-way ANOVA test was used, and the least significant difference test (LSI D) was used for multiple comparisons between groups; if the data variance was unequal, the Kruskal-Wallis test was used, and the test level was a-0.05. P<0.05 was considered statistically significant, and P<0.01 was considered statistically significant.

[0060] Example 1 Preparation and verification of stem cells overexpressing TAT-PBX1

[0061] Umbilical cord mesenchymal stem cells were cultured in DMEM medium. When the cells reached a density of 70%-75%, they were subcultured at a ratio of 1:3. Then, lentivirus (packaged in this laboratory) was infected. This patent uses lentivirus as a vector for introducing TAT-PBX1 target plasmid as an example: blank control, empty vector (Jikai Gene), PBX1 (Jikai Gene), TAT (purchased from Addgene), TAT-PBX1 (constructed by the laboratory) ( Figure 1 ). Packaging plasmids: psPAX2-gag / pol / tat / rev and pMD2.G-VSVG (purchased from Addgene). PBX1 or TAT-PBX1 was packaged into lentiviral particles in 293T cells (kindly donated by the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) and then transduced into umbilical cord mesenchymal stem cells. PBX1 transduction was observed under a fluorescence microscope 72 hours after transduction. Puromycin was used to select umbilical cord mesenchymal stem cells that overexpressed TAT-PBX1 ( Figure 2 ) was validated using Western blot ( Figure 3 ).

[0062] Example 2 Preparation and verification of umbilical cord mesenchymal stem cell culture supernatant loaded with TAT-PBX1 protein

[0063] Umbilical cord mesenchymal stem cells loaded with TAT-PBX1 protein were cultured with DMEM medium. When the cells reached a density of 70%-75%, they were replaced with phenol red-free and serum-free DMEM medium. After culturing for 24 hours, the cell culture supernatant was collected and the concentration was quantitatively determined using BCA protein. The culture supernatant was freeze-dried in a vacuum freezer, sealed, and stored at -80°C for later use. The expression of TAT-PBX1 protein in the cell culture supernatant was detected by immunoblotting ( Figure 4 ).

[0064] Example 3 Verification of the effect of culture supernatant of umbilical cord mesenchymal stem cells overexpressing TAT-PBX1 on promoting hair growth

[0065] Compared with the solvent group, the supernatant of umbilical cord mesenchymal stem cells cultured, the supernatant of empty umbilical cord mesenchymal stem cells cultured, and the supernatant of umbilical cord mesenchymal stem cells overexpressing TAT-PBX1 all significantly promoted the regeneration of hair in mice. Moreover, compared with the supernatant of umbilical cord mesenchymal stem cells cultured and the supernatant of empty umbilical cord mesenchymal stem cells cultured, the supernatant of umbilical cord mesenchymal stem cells overexpressing TAT-PBX1 was able to more significantly promote the regeneration of hair in mice ( Figure 5 HE staining showed that the hair follicles in the solvent group were still in the resting phase, but the hair follicles in the skin of mice overexpressing TAT-PBX1 umbilical cord mesenchymal stem cell culture supernatant entered the growth phase ( Figure 6 Compared with the solvent group, the number of skin hair follicles in the supernatant of umbilical cord mesenchymal stem cells overexpressing TAT-PBX1 was significantly increased ( Figure 7 ) and the diameter of the hair bulb increased significantly ( Figure 8 ).

[0066] Example 4: Verification of TAT-PBX1 fusion protein promoting hair growth

[0067] Compared with the control group, the hair of mice injected subcutaneously with TAT-PBX1 fusion protein entered the growth phase faster ( Fig. 9 ).

[0068] Analyze the HE staining results (see Fig.10 ) It can be seen that compared with the solvent group, the number of hair follicles per unit area in the TAT-PBX1 group of mice increased ( Fig.11 ), and the increase in hair shaft length ( Fig.12 ). This indicates that the hair follicles in the skin of mice in the TAT-PBX1 fusion protein group quickly entered the growth phase from the resting phase, causing an increase in the number of hair follicles and the length of hair rods in the mice.

[0069] It can be seen that overexpression of TAT-PBX1 umbilical cord mesenchymal culture supernatant or TAT-PBX1 fusion protein has the ability to significantly promote hair regeneration.

Claims

1. A use of PBX1 or TAT-PBX1 as an active ingredient in the preparation of a drug or medical device for promoting hair growth.

2. The use according to claim 1, characterized in that The medical device is a microneedle or a microneedle array.

3. The use according to claim 1, characterized in that The PBX1 or TAT-PBX1 is packaged by lentivirus, adenovirus, adeno-associated virus or retrovirus and then transduced into cells.

4. The use according to claim 1, characterized in that The PBX1 or TAT-PBX1 is contained in cells, or is expressed by cells and then secreted outside the cells.

5. The use according to claim 4, characterized in that The cells are mesenchymal stem cells, neural crest stem cells, melanin stem cells and fibroblasts.

6. The use according to claim 4, characterized in that The cells are carriers, and PBX1 or TAT-PBX1 is loaded into the cells.

7. The use according to claim 4, characterized in that The cells overexpress PBX1 or TAT-PBX1.

8. The use according to claim 4, characterized in that Used to stimulate the hair follicle cycle.

9. Use of a fusion protein in the preparation of a drug for promoting hair growth, characterized in that The fusion protein is TAT-PBX1 fusion protein.

10. The use according to claim 9, characterized in that The fusion protein is expressed by Escherichia coli or yeast or CHO cells.

Images