Application of DBC1 gene in prevention or treatment of alopecia diseases
By expressing the DBC1 gene in Treg cells, the targeting and side effects of existing treatments for hair loss diseases have been addressed, enabling the proliferation of hair follicle stem cells and hair regeneration, thus providing a new therapeutic and diagnostic target.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI SONGJIANG DISTRICT CENTRAL HOSPITAL
- Filing Date
- 2026-01-21
- Publication Date
- 2026-04-17
AI Technical Summary
Current technologies lack highly targeted therapies with controllable side effects for treating hair loss, and the interaction mechanism between skin Treg cells and hair follicle stem cells is unclear, resulting in limited treatment effects or skin inflammation.
By utilizing the normal or overexpression of the DBC1 gene in Treg cells, the number or functional homeostasis of Treg cells can be maintained, promoting the proliferation of hair follicle stem cells, supporting the normal growth and structural integrity of hair follicles, driving hair regeneration, and avoiding the blind activation of the immune system by targeting the DBC1 pathway.
It provides a more targeted treatment strategy with a lower risk of side effects, clarifies the role of DBC1-Treg-hair follicle stem cells, provides a new specific target for the diagnosis and treatment of hair loss diseases, and avoids adverse reactions such as skin inflammation.
Smart Images
Figure CN121868520A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to the application of the DBC1 gene in the prevention or treatment of hair loss diseases. Background Technology
[0002] Alopecia is a group of diseases characterized by abnormal or excessive hair loss from the scalp or other parts of the body, resulting in a significant reduction in hair density and localized or widespread baldness. Due to the easily diagnosed but difficult-to-treat nature of alopecia, there is an urgent need to develop innovative targeted therapies.
[0003] Hair originates from hair follicle stem cells (HFSCs) in the skin. The cells surrounding these stem cells, along with the extracellular matrix, form the HFSC microenvironment, which influences their biological behavior, such as proliferation, differentiation, and apoptosis, through complex mechanisms. Given the crucial role of HFSCs in hair regeneration, in-depth research into the mechanisms by which the HFSC microenvironment regulates hair regeneration is essential for finding new treatments for hair loss.
[0004] To target the hair follicle stem cell microenvironment, existing technologies attempt to promote hair regeneration through immunomodulation. Among these, the strategy of increasing the number of skin Treg cells using low-dose IL-2 was once considered a potential approach. IL-2 can activate Treg cells and promote their proliferation, theoretically enhancing their positive regulatory effect on hair follicle stem cells. However, preclinical and clinical data show that this approach has limited efficacy in treating alopecia areata and other hair loss diseases, and may even fail to effectively improve the condition. Fundamentally, existing technologies suffer from the following key shortcomings: 1. The core molecular mechanism by which skin Treg cells promote hair regeneration is not yet clear. The interaction between Treg cells and hair follicle stem cells involves multi-factor and multi-pathway cross-regulation, and the key molecular targets that play a decisive role have not yet been revealed, resulting in a lack of precise guidance for therapy development. 2. While increasing the number of Treg cells, non-specific immunomodulators such as IL-2 may activate other immune cell subsets, disrupting local immune balance and even triggering skin inflammation, thus exacerbating hair follicle damage.
[0005] Therefore, the core issue in this field is how to identify the key molecular mechanisms by which Treg cells regulate the proliferation of hair follicle stem cells, and on this basis, develop new therapies with strong targeting and controllable side effects. Thus, finding new mechanisms by which Treg cells primarily regulate the proliferation of hair follicle stem cells is of great significance for the development of novel Treg therapies for hair loss.
[0006] Therefore, we propose the application of the DBC1 gene in the prevention or treatment of hair loss diseases. Summary of the Invention
[0007] The purpose of this invention is to provide the application of the DBC1 gene in the prevention or treatment of hair loss diseases, and to use the DBC1 gene as the main target for future Treg cell therapy to treat hair loss diseases, providing new ideas for the prevention and treatment of hair loss diseases.
[0008] The DBC1 gene is used in the preparation of medicines or health products for the prevention or treatment of hair loss, and the nucleotide sequence of the DBC1 gene is shown in SEQ ID NO.1.
[0009] The application involves preventing or treating hair loss by promoting the normal or overexpression of the DBC1 gene in Treg cells.
[0010] Any of the following applications of the DBC1 gene with a nucleotide sequence as shown in SEQ ID NO.1:
[0011] 1) To maintain the number or functional homeostasis of Treg cells, or to prepare drugs or health products that maintain the number or functional homeostasis of Treg cells;
[0012] 2) Maintaining or increasing the number of hair follicle stem cells, or preparing medicines or health products that maintain or increase the number of hair follicle stem cells;
[0013] 3) To support the normal growth and structural integrity of hair follicles, or to prepare medicines or health products that support the normal growth and structural integrity of hair follicles.
[0014] 4) To drive hair regeneration, or to prepare drugs or health products that drive hair regeneration.
[0015] 5) Maintain the stability of the immune microenvironment surrounding hair follicle stem cells, or prepare drugs or health products that maintain the stability of the immune microenvironment surrounding hair follicle stem cells.
[0016] This invention has at least the following beneficial effects:
[0017] This invention reveals the key role of the DBC1 gene in hair regeneration: for the first time, through gene knockout animal models (systemic knockout and specific knockout), it has been confirmed that DBC1 is an essential gene for promoting hair regeneration, extending the function of DBC1 from other known fields to the fields of skin biology and hair regeneration.
[0018] This invention clarifies a new axis of action: the DBC1-Treg-hair follicle stem cell. The DBC1 gene plays a role in regulatory T cells (Tregs), thereby promoting the proliferation of hair follicle stem cells and ultimately driving hair regeneration. This discovery connects immune regulation and stem cell biology, providing a completely new perspective for understanding hair regeneration.
[0019] This invention provides a novel and specific target for the diagnosis and treatment of hair loss: based on the above mechanism, DBC1 itself, its expression products, or its activity in Treg cells can all serve as new targets for the development of diagnostic reagents or therapeutic drugs.
[0020] This invention proposes a treatment strategy with stronger targeting and lower risk of side effects: compared with existing technologies (such as low-dose IL-2), it directly targets the DBC1 pathway in Treg cells, which helps to avoid adverse reactions such as skin inflammation that may be caused by blindly activating the immune system, and achieves the beneficial effect of "targeting Treg cells without causing widespread skin inflammation and dysregulation". Attached Figure Description
[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0022] Figure 1 Example 1 showed that hair regrowth was slowed in Dbc1 knockout mice.
[0023] Figure 2 Example 2 illustrates the slowed hair regeneration in Treg cell-specific knockout Dbc1 mice;
[0024] Figure 3 Example 3 illustrates how Treg cell-specific knockout of Dbc1 resulted in reduced proliferation of mouse hair follicle stem cells. Detailed Implementation
[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0026] The main objective of this invention is to provide the application of the DBC1 gene in the prevention or treatment of hair loss diseases.
[0027] The nucleotide sequence of the DBC1 gene can be found in GenBank:NC_000008.11. This application also provides the nucleotide sequence of the human DBC1 gene, as shown in SEQ ID NO.1 below.
[0028] SEQ ID NO.1:
[0029]
[0030] The following are several examples, mainly used to disclose relevant research and validation of DBC1 in the diagnosis or treatment of hair loss diseases.
[0031] Example 1: This example is used to construct a mouse model with the Dbc1 gene knocked out throughout the body (Dbc1 KO) and compare it with wild-type mice (WT) to observe the differences in the hair regeneration process.
[0032] The experimental method is as follows:
[0033] 1. Constructing and evaluating a hair regeneration model.
[0034] 1.1 Hair removal on the back was performed using a razor and a commercially available depilatory cream (Nair). The patient was first anesthetized with isoflurane, and the longer hairs on the back were shaved off. The depilatory cream was then applied evenly to the shaved skin for 35 seconds, followed by thorough cleaning with sterile water and 75% alcohol to establish an artificially induced hair growth cycle model.
[0035] 1.2 Calculation of hair regeneration ratio: The area of the skin that was de-haired on day 0 is the baseline value. The hair growth area detected subsequently is divided by the baseline value to obtain the hair regeneration ratio.
[0036] 2. Flow cytometry detection of mouse skin Treg cells
[0037] 2.1 Six- to eight-week-old female C57BL / 6 mice were selected, and they were euthanized by cervical dislocation after anesthesia with 5% chloral hydrate. The skin on the back of the mice was taken, and the subcutaneous fat was gently scraped off with a scalpel. The mice were then washed twice with PBS buffer (containing 1% penicillin / streptomycin solution).
[0038] 2.2 Mince the skin tissue and prepare a digestion solution using 1640 basal culture medium (containing 2 mg / ml type IV collagenase (Gibco, 17104-019), 0.5 mg / ml hyaluronidase (Sigma & Aldrich, H3506), and 0.1 mg / ml DNase I (Roche, 10104159001)). Digest at 37°C and 200 rpm for 45 minutes. After grinding and filtration through a sieve, obtain a cell suspension. Centrifuge at 500 g and 4°C for 5 minutes, and discard the supernatant. Resuspend the cell pellet in 4 ml of 30% Percoll (cytiva, 17089101) and slowly load it onto 2 ml of 70% Percoll. Centrifuge at 800 g and 26°C for 20 minutes, with the acceleration and deceleration rates set to 0. After centrifugation, take the cells in the middle cloudy turbid layer, wash once with 5 times the volume of PBS buffer, centrifuge at 500 g, 4 ℃ for 5 min, discard the supernatant and resuspend in PBS or 1640 medium containing serum for subsequent experiments.
[0039] 2.3 Flow cytometry staining: Add Treg cell labeling antibodies, 0.5 μL of each antibody, incubate at 4℃ for 20 min, and detect Treg (CD4+ CD25+ Foxp3+).
[0040] 3. HE staining of mouse skin
[0041] 3.1 Take the back skin of the above-mentioned hair regeneration model mice or normal mice, and fix it with 4% paraformaldehyde, dehydrate it, decalcify it, and embed it in paraffin.
[0042] 3.2 Cut the paraffin sections into 4-6 μm sections using a microtome.
[0043] 3.3 The sections were stained with HE dye for 5 min.
[0044] 3.4 Images were obtained using a conventional optical microscope, and the length of hair follicles was quantified using ImageJ to evaluate the morphology and number of hair follicles in mice.
[0045] 4. The experimental results are as follows (please refer to the relevant documents). Figure 1 ):
[0046] 4.1 Data on hair regrowth rate indicate that Dbc1 deficiency leads to delayed regrowth:
[0047] Figure 1 Parts (a) and (b) visually demonstrate the quantitative results of hair regrowth rate. Figure 1 (a) shows the appearance comparison of hair regeneration on the back, and it can be observed that the hair coverage of Dbc1 KO mice is significantly sparse. Figure 1 The statistical curve in (b) shows that from day 6-9 after hair removal, the hair regeneration rate in the Dbc1 KO group was significantly lower than that in the wild group, and the difference increased over time. Therefore, we can conclude that the delayed regeneration indicates that the deletion of the Dbc1 gene directly inhibits the hair regeneration rate, conversely proving that its normal expression has a promoting effect.
[0048] 4.2 Morphological evidence of hair follicles supports impaired regenerative function
[0049] Figure 1 (c) and (d) further validated the regenerative impairment at the tissue level by HE staining. Figure 1 In the HE staining diagram in (c), the hair follicles in the Dbc1 KO group are shorter and sparser. Figure 1 Statistical data in (d) show that the hair follicle length in the Dbc1 KO group was significantly shortened. Therefore, since the hair follicle is the functional unit of hair regeneration, its morphological degradation directly indicates that Dbc1 deficiency not only affects the regeneration rate but also damages the structure and function of the hair follicle.
[0050] 4.3Dbc1 affects regeneration by regulating the proportion of Treg cells.
[0051] Figure 1 Flow cytometry results in (e) and (f) revealed a significantly reduced proportion of Treg cells in the skin of Dbc1 KO mice. Given that Treg cells are known to promote hair follicle stem cell activation, Dbc1 deficiency leads to a reduction in Treg cells and indirectly inhibits stem cell function. This demonstrates that Dbc1 can maintain the proportion of Treg cells in the skin, thereby promoting hair follicle stem cell proliferation and ultimately hair regeneration.
[0052] Example 2: This example mainly uses a mouse model with Treg cell-specific knockout of Dbc1 (Foxp3creDbc1fl / fl) to evaluate the hair regeneration process by comparing it with normal wild-type mice (control group).
[0053] The experimental method is the same as in Example 1; please refer to the experimental results. Figure 2 .
[0054] Figure 2 (a) and (b) primarily validate hair regeneration, through Figure 2 (a) It can be visually observed that mice with Treg-specific Dbc1 knockout (Foxp3) cre Dbc1 fl / fl The hair regeneration on the back was significantly sparse and uneven, while the control group had dense hair coverage. This indicates that the absence of Dbc1 in Treg cells directly inhibits regeneration efficiency. Figure 2 (b) Regeneration rate statistics: Quantitative curves showed that from day 6-9 after hair removal, the hair regeneration rate in the knockout group was significantly lower than that in the control group, and the difference persisted until the end of the observation period. Therefore, we can conclude that the delayed regeneration is not a systemic effect, but specifically stems from the loss of function of Dbc1 in Treg cells, proving that Treg cells rely on DBC1 to drive the hair regeneration process.
[0055] Figure 2 (c) HE staining results: The hair follicles in the control group were intact and orderly arranged, while the hair follicles in the knockout group were significantly shortened and sparsely distributed. This indicates that Dbc1 deficiency leads to disordered hair follicle growth cycle and prevents them from entering the anagen phase normally. Figure 2 (d) It can be seen that the quantitative results of hair follicle number and length both show a significant decrease in the knockout group. Therefore, the hair follicle is the functional unit of hair growth, and its morphological degeneration directly reflects the impairment of growth potential. Combined with the Treg-specific knockout background, it can be seen that DBC1 maintains hair follicle structure and proliferation capacity through Treg cells.
[0056] In Example 1, compared with the systemic knockout in Example 1, the Treg-specific knockout in Example 2 reproduced a similar slowed regeneration phenomenon, excluding interference from other cell types and confirming that the effect of DBC1 is concentrated in Treg cells. This further demonstrates that normal expression of DBC1 in Treg cells regulates the hair follicle stem cell microenvironment, thereby promoting stem cell proliferation and differentiation, accelerating hair follicle growth, and ultimately promoting hair regeneration.
[0057] Example 3 primarily verifies whether DBC1 indirectly regulates the proliferation of hair follicle stem cells through Treg cells. A mouse model with DBC1 specifically knocked out by Treg cells was used, compared with normal wild-type mice (control group), focusing on detecting the proportion and proliferation status of hair follicle stem cells. The specific experimental methods are as follows:
[0058] 1. Flow cytometry detection of hair follicle stem cells
[0059] 1.1 Preparation of Epidermal Single-Cell Suspension: Six- to eight-week-old female C57BL / 6 mice were selected, euthanized by cervical dislocation after anesthesia with 5% chloral hydrate, and the skin from the back of the mice was collected. Subcutaneous fat was gently scraped off with a scalpel, and the mice were washed twice with PBS buffer (containing 1% penicillin / streptomycin solution). The skin was then floated face down on 0.25% trypsin digestion solution and digested at 4°C for 1 hour, followed by digestion at 37°C for 1 hour; or digested overnight at 4°C. The epidermal layer was separated with forceps and washed with EDTA to disrupt intercellular junctions. Digestion was terminated with DMEM culture medium containing 10% fetal bovine serum, and the cells were filtered through a 40 μm filter to obtain the epidermal single-cell suspension.
[0060] 1.2 Flow cytometry staining of hair follicle stem cells: 250g of the above single-cell suspension was centrifuged at 4℃ for 10 minutes, resuspended with PBS, and stained with CD45, CD34, EpiCam, ITGA6, and Sca-1 mouse flow cytometry antibodies. The staining was carried out at 4℃ for 20 minutes, and the cells were washed once with PBS before flow cytometry detection.
[0061] Immunofluorescence of hair follicle stem cells
[0062] Paraffin sections were baked, dewaxed, and hydrated, then rinsed twice with running water. The antigen retrieval solution and sections were boiled four times in a microwave oven for 6 minutes each time, with the antigen retrieval solution added as needed during this process to prevent drying. The sections were then allowed to cool naturally with sodium citrate antigen retrieval solution. After rinsing with PBS, the liquid was blotted dry with filter paper. The tissue was circled with an immunohistochemical pen, and 10% goat serum was added. The sections were incubated at 37°C for 1 hour. The blocking solution was blotted dry, and α-CD34 immunofluorescence primary antibody working solution was added. The sections were incubated overnight at 4°C. The next day, after rinsing three times with PBS, immunofluorescence secondary antibody working solution (prepared according to the instructions, protected from light) was added, and the sections were incubated at 37°C in the dark for 1 hour. The secondary antibody was discarded, and the sections were rinsed three times with PBS. The sections were then incubated at room temperature in the dark for DAPI and Ki67 staining. After rinsing, the sections were blotted dry, and a fluorescent anti-quenching agent was added to cover the tissue. Cover sections were then prepared. Images were acquired under a fluorescence microscope to evaluate the number and proliferation of mouse hair follicle stem cells.
[0063] 2. Experimental Results (See attached document) Figure 3 )
[0064] 2.1 The decrease in the proportion of hair follicle stem cells indicates that DBC1 maintains the stem cell pool through Treg. Figure 3 Parts (a) through (c) demonstrate the changes in the number of hair follicle stem cells using flow cytometry. Figure 3 (a) It clarifies the identification pathways for hair follicle stem cells, ensuring the accuracy of the test. Figure 3 (b) Visually demonstrates Foxp3 cre Dbc1 fl / fl The number of hair follicle stem cells in the group was significantly reduced. Figure 3 (c) Statistical results confirmed that the proportion of hair follicle stem cells in the knockout group was significantly lower than that in the control group. Therefore, we can conclude that normal expression of DBC1 in Treg cells is a necessary condition for maintaining the number of stem cells.
[0065] 2.2 Decreased proliferation activity confirms that the DBC1-Treg axis drives stem cell activation. Figure 3 Parts (d) to (f) were further revealed to have impaired proliferative function by immunofluorescence and flow cytometry. Figure 3 (d) Immunofluorescence staining of skin sections showed that the Ki67 positive signal in the hair follicle area of the knockout group was significantly weaker than that in the control group, and the colocalization of stem cell markers was reduced, indicating that the stem cell proliferation activity was reduced. Figure 3 (e) shows that the expression level of Ki67 in the hair follicle stem cell population was significantly downregulated in the knockout group. Figure 3(f) Confirmed that the average fluorescence intensity of Ki67 in the knockout group was significantly reduced, directly reflecting a slowed proliferation rate. This indicates that Ki67 is a key protein in the cell cycle, and its reduced expression suggests that after Dbc1 deficiency, Treg cells cannot effectively transmit proliferation signals to hair follicle stem cells, thus hindering the transition of stem cells from a resting state to an activated state. This explains the reason for the slow hair follicle growth in Example 2—insufficient stem cell proliferation, unable to support hair follicle regeneration.
[0066] Therefore, in conjunction with Example 2, which showed that Dbc1 deficiency in Treg cells leads to slowed hair regeneration, this example further reveals that this deficiency directly reduces hair follicle stem cell proliferation, demonstrating that Treg cells are a key mediator of DBC1 action. This further illustrates that DBC1 expression within Treg cells can regulate paracrine or contact-dependent signaling in Treg cells, thereby activating hair follicle stem cell proliferation, increasing stem cell numbers, and ultimately promoting hair follicle growth and hair regeneration.
[0067] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.
Claims
1. The application of the DBC1 gene in the preparation of medicines or health products for the prevention or treatment of hair loss, characterized in that, The nucleotide sequence of the DBC1 gene is shown in SEQ ID NO.
1.
2. The application according to claim 1, characterized in that, The application involves preventing or treating hair loss by promoting the normal or overexpression of the DBC1 gene in Treg cells.
3. Any of the following applications of the DBC1 gene with a nucleotide sequence as shown in SEQ ID NO.1: 1) To maintain the number or functional homeostasis of Treg cells, or to prepare drugs or health products that maintain the number or functional homeostasis of Treg cells; 2) Maintaining or increasing the number of hair follicle stem cells, or preparing medicines or health products that maintain or increase the number of hair follicle stem cells; 3) To support the normal growth and structural integrity of hair follicles, or to prepare medicines or health products that support the normal growth and structural integrity of hair follicles. 4) To drive hair regeneration, or to prepare drugs or health products that drive hair regeneration; 5) Maintain the stability of the immune microenvironment surrounding hair follicle stem cells, or prepare drugs or health products that maintain the stability of the immune microenvironment surrounding hair follicle stem cells.