Application of teterocyte culture cultured under low-oxygen condition in treatment of wound difficult to heal

Abstract

The invention discloses application of a teterocyte culture cultured under a low-oxygen condition in treatment of a wound difficult to heal, and belongs to the technical field of skin wound repair. It is found that the teterocyte cultured under the low-oxygen condition and / or the cell supernatant of the teterocyte can obviously accelerate healing of a wound difficult to heal, meanwhile, wound surface inflammation infiltration is inhibited, wound surface hair follicle growth is promoted, and the effect of the teterocyte and / or the cell supernatant is better than that of a conventional growth factor spray (EGF + bFGF) and a conventional cell therapeutic agent (MSCs supernatant). Therefore, the teterocyte cultured under the low-oxygen condition and / or the cell supernatant thereof can be prepared into an external preparation for the skin for treating the wound difficult to heal, and a new thought is provided for wound surface treatment and infection control of the clinical wound difficult to heal.

Description

Technical Field

[0001] The invention belongs to the technical field of skin wound repair, and specifically relates to an application of a teratocyte culture cultured under hypoxic conditions in the treatment of difficult-to-heal wounds. Background Art

[0002] Chronic and difficult-to-heal wounds on the body surface refer to wounds caused by various reasons that have not healed after more than one month of treatment and have no healing trend. They often occur in patients with severe chronic diseases and acute injuries such as diabetes, trauma, varicose veins, vascular sclerosis, paraplegia, long-term bed rest, etc. They have complex pathogenesis, difficult treatment, long treatment cycle, and high cost, which seriously affect the physical and mental health and quality of life of the people.

[0003] Difficult-to-heal wounds not only affect physical health, but may also bring psychological and economic burdens, and require timely treatment and management. Difficult-to-heal wounds may bring the following hazards: 1. Increased risk of infection: Wounds that do not heal for a long time are prone to breed bacteria and cause infection, and face the possibility of the spread of infection, leading to serious complications such as sepsis; 2. Chronic pain: Wounds that do not heal for a long time may cause chronic pain and affect the quality of life; 3. Limited functions: When the wound is located in a joint or active part, it may limit activity and affect function; 4. Psychological impact: Long-term non-healing wounds may cause a series of emotional problems such as anxiety and depression, and even cause psychological problems; 5. Economic burden: Long-term treatment and care increase economic pressure; 6. Risk of complications: Long-term non-healing wounds may cause necrosis of surrounding tissues. If the infection spreads to the bones, it may cause osteomyelitis; 7. Decreased quality of life: Wounds affect daily activities and reduce the quality of life; 8. Potential diseases: Difficult-to-heal wounds may be manifestations of chronic diseases such as diabetes and vascular diseases; 9. Scars and deformities: Long-term non-healing wounds may leave obvious scars and even cause deformities; 10. Risk of amputation: In extreme cases, difficult-to-heal wounds may cause serious infection and further induce the risk of amputation. In short, difficult-to-heal wounds not only damage local tissues, but may also cause systemic complications, seriously affecting the patient's quality of life and prognosis. Early intervention, comprehensive treatment and multidisciplinary cooperation are the key to reducing its harm.

[0004] The treatment of non-healing wounds requires the formulation of personalized treatment plans based on specific causes and conditions. The common treatment methods are as follows: 1. Wound surface treatment: Remove necrotic tissue and foreign bodies, reduce the risk of infection, and use appropriate dressings (such as hydrogels, foam dressings, etc.) to keep the wound surface moist and promote healing; 2. Infection control: Use antibiotics locally or systemically to control infection, change dressings regularly, keep the wound surface clean, and prevent infection; 3. Improve blood circulation: Improve local blood supply through drug or surgical vascular treatment, or promote blood circulation through physical therapy such as ultrasound and hyperbaric oxygen; 4. Nutritional support: A high-protein diet is required to help promote tissue repair and supplement necessary vitamins and minerals such as vitamin C and zinc to accelerate healing; 5. Chronic disease management: For example, patients with diabetes need to strictly control their blood sugar, and patients with vascular diseases need to treat vascular diseases to improve blood circulation; 6. Biological treatment: Such as epidermal growth factor (EGF) to promote cell proliferation, or stem cell treatment to promote tissue regeneration; 7. Surgical treatment: Skin grafting is required for large-area wounds, and flap surgery is performed to repair deep tissue defects; 8. Negative pressure wound therapy (NPWT) uses negative pressure suction to promote wound healing and reduce infection; 9. Psychological support: Psychological treatment of patients may be necessary to help them cope with the stress of long-term treatment; 10. Comprehensive management: Multidisciplinary collaboration: Combine surgery, endocrinology, nutrition, etc. to develop a comprehensive treatment plan. In short, the treatment of non-healing wounds needs to comprehensively consider various factors such as wound surface treatment, infection control, and nutritional support, and personalized treatment is the key.

[0005] Telocytes (TCs) are a type of cells that express CD34, c-kit, and Vimentin, and have a supporting and regulatory effect on the growth of immune cell interstitial stem cells. They can provide a reticular structure support for fibroblasts, secrete a variety of growth factors to promote cell growth, and have tissue repair and immune regulation functions. Lung TCs are widely distributed in the alveolar interstitium, small blood vessels, and smooth muscle layers of small airways, express vascular endothelial growth factor (VEGF), and are involved in the formation of blood vessels and the air-blood barrier. TCs can secrete more repair-related cytokines such as EGF, VEGF, GM-CSF, HGF, FGF, and inflammation-regulating related factors such as IGF-1 and TGF-β

[10] . Lung TCs express vascular endothelial growth factor (VEGF) and are involved in the formation of blood vessels and the air-blood barrier. When the conditioned medium of human lung TCs is co-cultured with human lung microvascular endothelial cells after LPS injury, VEGF and epidermal growth factor (EGF) produced by TCs can significantly promote the regeneration of human lung microvessels.

[0006] Telocytes are present in almost all human tissues and organs. However, due to their extremely low content in each tissue, it is difficult to obtain cells, and it is also difficult to expand them. Most current technologies focus on isolating TC cells from mouse organ tissues for research, or isolating TCs from human lung tissues and then immortalizing them. Moreover, a large number of TCs cells for research can only be amplified by culturing with fetal bovine serum. The currently established TCs cell lines have problems such as slow amplification speed, easy aging, and loss of surface markers after dozens of passages. At the same time, the concentration of paracrine factors is also very limited, which is not conducive to clinical applications. Summary of the Invention

[0007] In view of this, after establishing a seed bank with the extracted primary lung telocytes (TCs), the present invention cultures them under low oxygen (O 2 concentration of 5%) conditions, applies the TCs cell suspension or cell supernatant to the surface of LPS-induced non-healing wounds, and at the same time compares them with TCs cells cultured under normal conditions. The results show that TCs cultured under low oxygen and / or their cell supernatant can significantly accelerate wound healing. At the same time, it is confirmed on the cytological model that the function of TCs cultured under low oxygen and / or their cell supernatant in accelerating cell scratch healing is superior to the conventional growth factor spray (EGF + bFGF) on the market and the supernatant of MSCs commonly used in cell therapy.

[0008] Based on the above experimental findings, the present invention provides the use of a telocyte culture obtained under low oxygen conditions in the preparation of a drug for treating non-healing wounds. The telocyte culture includes cells and / or cell supernatant. Preferably, the telocyte culture is cell supernatant. The second object of the present invention is to provide a skin topical preparation for non-healing wounds.

[0009] The present invention includes the following technical solutions:

[0010] In the first aspect of the present invention, the present invention provides the application of a telocyte culture in at least one of the following:

[0011] a1) Application in the preparation of a drug for promoting the healing of non-healing wounds;

[0012] a2) Application in the preparation of a drug for inhibiting inflammatory infiltration of non-healing wounds;

[0013] a3) Application in the preparation of a drug for promoting hair follicle growth.

[0014] The telocyte culture is selected from telocytes and / or telocyte supernatant obtained by culturing under low oxygen conditions.

[0015] The drug has at least one of the following effects:

[0016] b1) Promote cell healing;

[0017] b2) Promote cell proliferation;

[0018] b3) Promote cell migration;

[0019] b4) Promote the secretion of VEGF in skin tissue;

[0020] b5) Inhibit the release of inflammatory cytokines IL-17, IL-6, IL-1β and TNFα in skin tissue.

[0021] The cells are cells that form skin tissue, including but not limited to fibroblasts, keratinocytes, and vascular endothelial cells.

[0022] In some embodiments of the present invention, the hypoxic condition is selected from a cell culture environment with an O 2 concentration lower than 21%.

[0023] Preferably, the hypoxic condition is selected from a cell culture environment with an O 2 concentration of 5-10%.

[0024] More preferably, the hypoxic condition is selected from a cell culture environment with an O 2 concentration of 5%.

[0025] In a specific embodiment of the present invention, the telocyte culture condition is an O 2 concentration of 5%, a CO 2 concentration of 5%, and a cell culture environment at a temperature of 37°C.

[0026] In the most preferred embodiment of the present invention, the telocyte culture is selected from the telocyte supernatant obtained by culturing under hypoxic conditions.

[0027] Unless otherwise specified, the telocyte culture obtained by culturing under the hypoxic conditions described in the present invention only limits the O 2 concentration in the culture conditions, and other culture conditions are all conventional operations for those skilled in the art. And those skilled in the art can obtain the optimal culture conditions other than the O 2 concentration through a limited number of experiments, including but not limited to temperature, CO 2 concentration, and humidity. For the telocyte culture method, reference can be made to the content disclosed in the patent document CN 117757731 A (A hypoxic culture of telocytes for vascular repair and its preparation method).

[0028] The terms "wound", "injured area", and "lesion surface" in the present invention have the same meaning and can be used interchangeably in the present invention, and refer to skin damage caused by rupture of the skin and its surrounding tissues.

[0029] The "difficult-to-heal wound" described in the present invention is a chronic wound on the body surface, which refers to a wound that has not healed after more than one month of treatment for various reasons and shows no tendency to heal.

[0030] In a second aspect of the present invention, the present invention provides a topical skin preparation for difficult-to-heal wounds, characterized in that the topical skin preparation comprises an effective dose of the telocyte culture described in the first aspect of the present invention.

[0031] Preferably, the telocyte culture is selected from the telocyte supernatant obtained by culturing under hypoxic conditions.

[0032] In a specific embodiment of the present invention, the topical skin preparation is a dressing, and the dressing comprises an effective dose of the telocyte culture described in the first aspect of the present invention.

[0033] Furthermore, the dressing further comprises a pharmaceutically acceptable excipient.

[0034] The pharmaceutically acceptable excipients include any solvents, excipients, diluents, film-forming agents, etc. that can form a dressing. In a preferred embodiment of the present invention, the pharmaceutically acceptable excipients are selected from polyethylene glycol and glycerol.

[0035] In a specific embodiment of the present invention, the application method of the dressing is to spray or smear the dressing on the surface and / or around the skin wound.

[0036] The present invention applies a culture system with a low oxygen content to TCs cells and finds that it can not only rapidly increase the amplification rate of TCs cells but also greatly enhance their paracrine and vascular adsorption functions, delaying the phenomenon of cell senescence during multiple passages. It is a method to achieve rapid and efficient amplification of TCs without senescence and maintain stable phenotypes without gene editing and transformation means, laying a solid foundation for the safer and more efficient clinical application of TCs.

[0037] Furthermore, the present invention applies TCs cells or TCs cell supernatant obtained by culturing under hypoxic conditions to the surface of LPS-induced difficult-to-heal wounds and finds that TCs cells or TCs cell supernatant have the effect of promoting wound healing, can inhibit inflammatory infiltration on the wound surface, accelerate wound repair, and promote hair follicle growth on the wound surface. Based on this, the present invention creatively proposes to prepare TCs cells or TCs cell supernatant obtained by culturing under hypoxic conditions into a topical skin preparation for treating difficult-to-heal wounds. The technical solution provided by the present invention provides new ideas for the wound surface treatment and infection control of difficult-to-heal wounds, has high clinical application value, and has high clinical transformation potential. Description of the Drawings

[0038] Figure 1 Statistical chart of the wound healing situation and healing rate of the experimental group.

[0039] Figure 2 HE staining results of the skin tissue of the experimental group.

[0040] Figure 3 Statistical charts of the expressions of VEGF, IL-17, IL-6, IL-1β, and TNFα in the skin tissue of the experimental group.

[0041] Figure 4 Results of the fibroblast scratch assay and cell proliferation curve.

[0042] Figure 5 Results of the keratinocyte scratch assay and cell proliferation curve.

[0043] Figure 6 Results of the tube formation assay, cell migration assay, and scratch assay of vascular endothelial cells. Detailed implementation manners

[0044] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only partial embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0045] The method for obtaining TCs cells cultured under 5% hypoxic conditions and cell supernatant used in the specific embodiments of the present invention is as described in the patent document CN 117757731 A.

[0046] Example 1 Therapeutic ability of hypoxic-cultured TCs on in vivo non-healing wounds

[0047] 1.1 Establishment of a non-healing wound model in mice

[0048] Mice were anesthetized intraperitoneally with 0.3% pentobarbital at 50 ml / kg (about 0.2 - 0.3 ml in volume). After anesthesia, each mouse was modeled. First, use a modeling sheet to shave the back hair of the mouse at a convenient part, preferably a part that the mouse cannot scratch. Push the back hair in a large circle (about it is okay), and it can be pushed with a clipper or removed with hair removal cream. After hair removal, make a mark with a marker pen in a small circle, and cut off the epidermis and dermis according to the mark.

[0049] 1.2 Experimental design and procedures

[0050] Thirty-six mice were randomly divided into 6 groups, with 6 mice in each group, namely the normal wound group (Normal group), the non-healing wound group (Control group), the non-healing wound + TCs treatment group (TCs group), the non-healing wound + TCs cultured under hypoxia treatment group (lo-TCs group), the non-healing wound + TCs supernatant treatment group (sTCs group), and the non-healing wound + TCs supernatant cultured under hypoxia treatment group (slo-TCs group). The non-healing wound models were established for all mice according to the method provided in 1.1 above.

[0051] The Normal group had ordinary wounds, without LPS induction or drug treatment. The remaining experimental groups were induced with non-healing wounds by applying LPS every other day. The specific method was as follows: The pre-prepared LPS was applied to the wound, 0.1 ml for each mouse. The Control group had non-healing wounds without drug treatment; the TCs group was directly applied with conventional telocytes, with a dose of 1E6 / time; the lo-TCs group was directly applied with telocytes cultured under 5% hypoxia, with a dose of 1E6 / time. The sTCs group and the slo-TCs group represented the application of the supernatants of the corresponding cells, which were applied once every other day. The drug application method was: After the LPS was slightly absorbed, 0.1 ml of the corresponding drug was applied to each mouse.

[0052] 1.3 Statistical methods for experimental results

[0053] On the 0th, 2nd, 4th, and 8th days of the experiment, the wound healing conditions of the mice were photographed and recorded, and the wound healing rate was statistically analyzed. The wound healing rate was the percentage of the wound healing area. The initially modeled wound was simply regarded as a circle for calculation. The healing rate = (initial area - daily area) / initial area × 100%. On the 8th day, tissues were taken for HE staining, and the secretion of VEGF and inflammatory factors (IL-17, IL-6, IL-1β, TNFα) in the skin tissue grinding fluid of each experimental group was detected.

[0054] 1.4 Experimental results

[0055] The wound healing conditions and healing rate results of each experimental group were as Figure 1 shown. It can be seen from the figure that on the 8th day of the experiment, the wounds of the mice in the Normal group had healed, with the highest healing rate; followed by the hypoxia TCs treatment group and the hypoxia TCs supernatant treatment group, which had no significant difference from the Normal group, but were significantly higher than other treatment groups, such as the TCs treatment group and the TCs supernatant treatment group.

[0056] The tissue HE staining structure was as Figure 2As shown, it can be seen that the Normal wound has healed completely, without a fat layer, and hair follicles have not grown yet; the Control wound does not have a complete layer and is still in a fractured stage, the wound in the TCs treatment group has basically healed, but the inflammatory infiltration is very severe, without a fat layer; the wound in the TCs supernatant treatment group has healed well, with a complete layer and a small amount of inflammatory infiltration; the skin layer in the hypoxic TCs treatment group is complete, with a small number of hair follicles growing; the wound in the hypoxic TCs supernatant treatment group has healed completely, with a complete layer and a large number of hair follicles growing.

[0057] The statistical results of the expressions of VEGF, IL-17, IL-6, IL-1β, and TNFα in the skin tissues of each experimental group are as Figure 3 shown. Regarding the expression level of VEGF, the hypoxic TCs supernatant treatment group has the most, followed by the TCs supernatant treatment group, and then the hypoxic TCs treatment group. Regarding the expression levels of inflammatory factors, the hypoxic TCs supernatant treatment group has the least expression, with a statistical difference from the Control group, followed by the TCs supernatant treatment group.

[0058] Based on the above experimental results, it can be seen that compared with each therapeutic drug, treating difficult-to-heal wounds with hypoxic TCs supernatant has the best effect, followed by TCs supernatant and TCs cultured under hypoxia, and then TCs.

[0059] Example 2 Fibroblast Wound Healing Experiment

[0060] 2.1 Scratch Assay

[0061] The specific operation steps are as follows:

[0062] S1. Cell culture: Culture cells in an appropriate culture dish until they reach about 80 - 90% confluence;

[0063] S2. Scratch treatment: Use a sterile pipette tip or a scratch tool to make a scratch on the cell monolayer to simulate a wound, and gently wash the culture dish to remove floating cells;

[0064] S3. Add culture medium: Add the cell supernatants of each experimental group and the blank control culture medium;

[0065] S4. Observation and photography: Observe and take an immediate image (0 hours) of the scratch using a microscope, and place the culture dish back into the incubator;

[0066] S5. Regular observation: Take out the culture dish at 48 hours, observe and take an image of cell migration;

[0067] S6. Measurement and analysis: Use image analysis software (such as ImageJ) to measure the width of the scratch or the number of migrated cells at different time points, calculate the cell migration rate, and perform statistical analysis.

[0068] 2.2 Cell Proliferation RTCA Experiment

[0069] The specific operation steps are as follows:

[0070] S1. Digest the cells with about 80 - 90% confluence and seed them in the RTCA well plate, with 1E4 cells per well;

[0071] S2. After the bottom of the well plate is covered by adherent cells, the resistance changes, and the instrument detection will convert it into a numerical value of the cell number;

[0072] S3. After 72 hours, read the values of each experimental group for statistics.

[0073] 2.3 Experimental Results

[0074] The microscopic pictures of the cells in the scratch assay are as shown in Figure 4 a. Among them, Control is the blank medium, TCs is the supernatant of telocytes, 5% TCs is the supernatant of telocytes cultured under hypoxia, MSCs is the supernatant of umbilical cord mesenchymal stem cells, and EGF + bFGF is a common growth factor spray on the market. Figure 4 b is the statistical chart of the migration and coverage area in the middle of the cell scratch. The cell growth curve in the cell proliferation RTCA experiment is as shown in Figure 4 c. As can be seen from the results shown in Figure 4 a, Figure 4 b and Figure 4 c, the supernatant of TCs cultured under 5% hypoxia conditions significantly promotes cell scratch healing, and the effect is better than that of the TCs supernatant and the MSCs supernatant; meanwhile, the supernatant of TCs cultured under 5% hypoxia conditions has an obvious promoting effect on the growth of fibroblasts.

[0075] Example 3 Keratinocyte Wound Healing Experiment

[0076] Perform the scratch assay and the cell proliferation RTCA experiment. The operation steps are the same as those in 2.1 and 2.2 of Example 2. The experimental results are as shown in Figure 5 The microscopic pictures of the cells in the scratch assay are as shown in Figure 5 a. Among them, Control is the blank medium, TCs is the supernatant of telocytes, 5% TCs is the supernatant of telocytes cultured under hypoxia, MSCs is the supernatant of umbilical cord mesenchymal stem cells, and EGF + bFGF is a common growth factor spray on the market. Figure 5 b is the statistical chart of the migration and coverage area in the middle of the cell scratch. The cell growth curve in the cell proliferation RTCA experiment is as shown in Figure 5 c. As can be seen from the results shown in Figure 5 a, Figure 5 b and Figure 5As can be seen from the results shown in c, the supernatant of TCs cultured under 5% hypoxia significantly promoted cell scratch healing, with a better effect than the supernatant of TCs and the supernatant of MSCs; meanwhile, the supernatant of TCs cultured under 5% hypoxia had a significant promoting effect on the growth of keratinocytes.

[0077] Example 4 Vascular Endothelial Cell Wound Healing Experiment

[0078] 4.1 Tube Formation Assay

[0079] The specific operation steps are as follows:

[0080] S1. Resuscitate umbilical cord vascular endothelial cells and inoculate them into a 12-well plate that has been surface-treated for tissue culture; Step 5.2, prepare Matrigel.

[0081] S2. When the cell confluence reaches 70%-80%, add the Matrigel that has been placed on ice and melted overnight at 4°C to the wells by vortex oscillation to ensure that the gel solution is in a uniform state.

[0082] S3. Digest the cells with trypsin and observe under the microscope at the same time. When the cells become round, stop digestion in time. The number of cells inoculated on the Matrigel is 30,000 cells / well.

[0083] S4. Collect the cell supernatant after 5 days of culture, and add the supernatant of telocytes cultured under normal oxygen conditions, the supernatant of telocytes cultured under 5% hypoxia, and blank medium as controls respectively. Observe the tube formation situation every 4 hours and record the tube formation situation after 72 hours.

[0084] 4.2 Migration Assay

[0085] The specific operation steps are as follows:

[0086] S1. Prepare Transwell inserts: Place Transwell inserts in a 24-well plate. Add an appropriate amount of serum-free medium to the upper chamber and add the supernatant of each experimental group to the lower chamber.

[0087] S2. Treat the cells: Wash the cultured cells with sterile PBS, digest the cells with trypsin and count them, and adjust the cell suspension to an appropriate concentration (e.g., 1×10 5 cells / mL);

[0088] S3. Inoculate the cells: Add an appropriate amount of cell suspension to the upper chamber, handle it carefully to avoid the formation of air bubbles, and place the Transwell plate in an incubator at 37°C and 5% CO 2 for 24 - 48 hours;

[0089] S4. Fixation and staining: After the incubation is completed, wash the upper chamber with sterile PBS to remove non-migrated cells. Fix the cells migrated in the lower chamber with a fixative (such as methanol or 4% paraformaldehyde), and then wash and stain with PBS (such as 0.1% crystal violet staining or DAPI staining);

[0090] S5. Observation and counting: Observe the migrated cells on the lower chamber membrane using a microscope, count the number of migrated cells in multiple fields of view, and calculate the average value.

[0091] 4.3 Scratch assay, the operation steps are the same as those in 2.1 of Example 2.

[0092] 4.4 Experimental results

[0093] Control is the blank medium, TCs represents the supernatant of telocytes cultured under conventional conditions, 5% TCs represents the supernatant of telocytes cultured under hypoxic conditions, MSCs is the supernatant of umbilical cord mesenchymal stem cells, and EGF + bFGF is a common growth factor spray on the market.

[0094] The results of the tube formation assay are as Figure 6 shown in a. The more cell branches are formed, the better the tube formation effect. From the cell photos and statistical results, it can be seen that the supernatant of TCs cultured under 5% hypoxic conditions promotes tube formation best. The results of the cell migration assay are as Figure 6 shown in b. The upper layer is the blank medium, and the lower layer corresponds to each experimental group. Stain to see the number of cells migrated to the lower layer. From the statistical results, it can be seen that the number of cells migrated to the lower layer treated with the supernatant of TCs cultured under 5% hypoxic conditions is the largest. The results of the scratch assay are as Figure 6 shown in c. From the statistical results, it can be seen that the supernatant of TCs cultured under 5% hypoxic conditions can significantly promote cell scratch healing, showing a significant difference compared with the control group and being superior to other experimental groups.

[0095] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of the Teluo cell culture in at least one of the following: a1) Use in the preparation of a medicament for promoting the healing of difficult-to-heal wounds; a2) Use in the preparation of a drug for inhibiting inflammatory infiltration of difficult-to-heal wounds; a3) Use in the preparation of a drug for promoting hair follicle growth; The telogen cell culture is selected from telogen cells and / or telogen cell supernatant obtained by culturing under hypoxic conditions.

2. The use according to claim 1, characterized in that: The drug has at least one of the following effects: b1) Promote cell healing; b2) Promote cell proliferation; b3) Promote cell migration; b4) Promote VEGF secretion in skin tissue; b5) Inhibit the release of inflammatory cytokines IL-17, IL-6, IL-1β and TNFα in skin tissue.

3. The use according to claim 1, characterized in that: The hypoxic condition is selected from a cell culture environment in which the O2 concentration is lower than 21%.

4. The use according to claim 3, characterized in that: The hypoxic condition is selected from a cell culture environment with an O2 concentration of 5-10%.

5. The use according to claim 4, characterized in that: The hypoxic condition is selected from a cell culture environment with an O2 concentration of 5%.

6. The use according to claim 1, characterized in that: The Teluo cell culture is selected from the Teluo cell supernatant obtained by culturing under hypoxic conditions.

7. A skin external preparation for treating difficult-to-heal wounds, characterized in that: The skin external preparation comprises an effective dose of the Telu cell culture described in claim 1.

8. The external skin preparation according to claim 7, characterized in that The skin external preparation is a dressing, which includes an effective dose of the teratocyte culture described in claim 1.

9. The external skin preparation according to claim 8, characterized in that The dressing further comprises a pharmaceutically acceptable excipient.

10. The external skin preparation according to any one of claims 8 or 9, characterized in that The dressing is applied by spraying or applying the dressing on the surface and / or surrounding of the skin wound.

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