Exosome derived from endometrial mesenchymal stem cells and application of exosome in preparation of medicine for treating diabetic wounds

Abstract

The invention provides an endometrial mesenchymal stem cell-derived exosome and application thereof in preparation of a medicine for treating diabetic wounds, and belongs to the technical field of biological medicines. The endometrial mesenchymal stem cell-derived exosome provided by the invention can effectively promote HUVEC tube forming ability, promote wound angiogenesis of diabetic model mice, increase collagen deposition and reduce wound area, finally achieves the purpose of promoting healing of diabetic wounds, and solves the problem of limited treatment effect of exosomes in the prior art. The endometrial mesenchymal stem cell-derived exosome provided by the invention can be used as a medicine for promoting angiogenesis and wound healing to be applied to diabetes wound treatment, and also provides a new strategy for treatment of other chronic wounds including burn.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to exosomes derived from endometrial mesenchymal stem cells and their application in the preparation of drugs for treating diabetic wounds. Background Art

[0002] Diabetes is a group of metabolic syndromes characterized by hyperglycemia. It is a highly prevalent disease worldwide. In 2021, there were approximately 529 million diabetic patients globally, and the overall prevalence in people aged 65 and above exceeded 20%. Among diabetic patients, about 20% have refractory wounds, which can lead to diabetic foot and even amputation, and even threaten the lives of patients, causing great psychological, physical, and economic burdens to the patients.

[0003] Normal wound healing undergoes four gradually occurring and overlapping processes: the hemostasis phase, the inflammation phase, the proliferation phase, and the tissue remodeling phase. Diabetes is induced by multiple factors such as poor long-term blood glucose control, local trauma, and infection, resulting in a delayed inflammation phase and inhibited proliferation of the wound surface, making chronic wounds difficult to heal. Among them, abnormal angiogenesis during the proliferation phase is an important reason for the difficult healing of diabetic wounds. Sustained hyperglycemia causes abnormalities in endothelial metabolism and function, inhibits the activity of endothelial cells, reduces the formation of new capillaries, and ultimately leads to slow wound healing. Therefore, improving the vascular state of the wound surface tissue and accelerating the repair of damaged tissues are of great significance for the healing of diabetic wounds.

[0004] For diabetic refractory wounds, clinical treatments such as blood glucose lowering, infection control, and new dressings such as recombinant human epidermal growth factor gel are used, but each has its own limitations and it is difficult to obtain an ideal treatment effect.

[0005] Chinese Patent CN119548528A discloses a method for promoting diabetic fracture healing with bone marrow mesenchymal stem cell exosomes, including the following steps: isolating MSCs from the bone marrow of diabetic patients, culturing and extracting exosomes; then quantitatively analyzing the extracted exosomes, and then injecting the exosomes into the fracture site by intravenous injection, local injection, or minimally invasive injection, and continuously treating. The miRNA or mRNA in the exosomes promotes the proliferation and differentiation of osteoblasts by targeting cells, and enhances angiogenesis, thereby accelerating fracture healing. The miRNA or mRNA in the exosomes promotes the proliferation and differentiation of osteoblasts, enhances their ability to synthesize bone matrix, thereby accelerating the healing of fractures. At the same time, the exosomes regulate vascular endothelial cells to promote angiogenesis, providing the necessary blood supply for fracture healing, thereby effectively improving the repair ability of the fracture site.

[0006] However, the above-mentioned existing exosomes have limited therapeutic effects on refractory wounds. Summary of the Invention

[0007] The object of the present invention is to provide exosomes derived from endometrial mesenchymal stem cells and their application in the preparation of drugs for treating diabetic wounds, which have a good effect on promoting wound angiogenesis and have excellent treatment prospects.

[0008] In order to achieve the above object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides the application of exosomes derived from endometrial mesenchymal stem cells in the preparation of drugs for chronic wound repair.

[0010] Preferably, the chronic wounds include diabetic wounds, venous ulcers, arterial ulcers, pressure ulcers, post-traumatic chronic ulcers, postoperative infectious wounds, radiation dermatitis ulcers, immune-related ulcers, lymphedema-related ulcers, malignant tumor-related ulcers or vasculitis ulcers.

[0011] In the present invention, the diabetic wound refers to an open wound on the foot or lower limb skin and deep tissues caused by long-term hyperglycemia leading to increased risks of microvascular lesions, neuropathy and infection, and is difficult to heal by conventional treatment.

[0012] In the present invention, the venous ulcer refers to a chronic wound caused by insufficient function of the lower limb venous valves or deep vein thrombosis, resulting in blood reflux disorder, long-term congestion of the calf skin, malnutrition and finally ulceration.

[0013] In the present invention, the arterial ulcer refers to a necrotic ulcer of the skin of the toes, heel or anterior tibia caused by peripheral arteriosclerosis obliterans or thromboembolism, leading to ischemia and hypoxia at the distal end of the limb.

[0014] In the present invention, the pressure ulcer refers to a deep skin and subcutaneous tissue injury formed by long-term compression of the body's bony prominences, resulting in local tissue ischemia and necrosis, and is common in the sacrococcygeal region and heel of bedridden patients.

[0015] In the present invention, the post-traumatic chronic ulcer refers to an open injury that has not healed for more than 4 weeks due to improper treatment of a burn, trauma or surgical incision, resulting in continuous infection, inflammation or insufficient blood supply to the wound surface.

[0016] In the present invention, the postoperative infectious ulcer refers to a complex wound caused by secondary bacterial or fungal infection or fat liquefaction at the surgical site, resulting in redness, suppuration and delayed healing of the incision.

[0017] In the present invention, the radiation dermatitis ulcer refers to a chronic erosive wound surface caused by ionizing radiation damage to the skin and subcutaneous tissues after tumor radiotherapy, resulting in DNA breakage, microcirculation disorder and epidermal necrosis.

[0018] In the present invention, the immune-related ulcer refers to a lesion in which the skin and mucous membranes repeatedly ulcerate and do not heal due to autoimmune diseases such as systemic lupus erythematosus, vasculitis or Behcet's disease;

[0019] In the present invention, the lymphedema-related ulcer refers to a chronic exudative ulcer formed after secondary infection due to lymphedema, which causes limb swelling, hyperkeratosis and increased brittleness of the skin;

[0020] In the present invention, the malignant tumor-related ulcer refers to a refractory skin ulcer caused by the infiltration and destruction of normal structures by primary or metastatic skin cancer tissues, or the side effects of chemotherapy and targeted therapy;

[0021] In the present invention, the vasculitis ulcer refers to a painful deep ulcer formed after local tissue ischemia and necrosis caused by the inflammatory necrosis of the small blood vessel wall, leading to lumen occlusion;

[0022] Preferably, the endometrial mesenchymal stem cells are human endometrial mesenchymal stem cells.

[0023] Preferably, the exosomes derived from endometrial mesenchymal stem cells are obtained by a preparation method including the following steps:

[0024] The exosomes derived from endometrial mesenchymal stem cells are extracted from the culture supernatant of endometrial mesenchymal stem cells by differential centrifugation.

[0025] The present invention also provides the application of exosomes derived from endometrial mesenchymal stem cells in the preparation of drugs for treating or alleviating diabetic wounds.

[0026] The present invention also provides the application of a medical dressing in the preparation of drugs for treating or alleviating diabetic wounds, wherein the medical dressing contains exosomes derived from endometrial mesenchymal stem cells.

[0027] In the present invention, the treatment of diabetic wounds refers to an intervention process in which diabetic wounds achieve complete healing by promoting physiological closure of the wound surface and reconstruction of the skin barrier, including accelerating granulation tissue formation, epidermal regeneration and controlling the hyperglycemic-related pathological microenvironment.

[0028] In the present invention, the alleviation of diabetic wounds refers to reducing the degree of deterioration or clinical symptoms of diabetic wounds by inhibiting infection, reducing inflammatory reactions or improving local microcirculation, including reducing exudation, shrinking the ulcer area and creating conditions for subsequent healing.

[0029] Preferably, the medical dressing includes a matrix material, and the matrix material is selected from one or more of chitosan, alginate, hyaluronic acid, polylactic acid, polycaprolactone or gelatin.

[0030] Preferably, the medical dressing contains an active ingredient in combination with exosomes derived from endometrial mesenchymal stem cells, and the active ingredient is selected from one or more of hypoglycemic drugs: insulin, metformin sustained-release microparticles, pirfenidone, interferon, lidocaine or ibuprofen.

[0031] Preferably, the medical dressing contains auxiliary components, and the auxiliary components include one or more of silver nanoparticles, gentamicin, epidermal growth factor, fibroblast growth factor, curcumin or pirfenidone.

[0032] The present invention also provides an application of a compound preparation in the preparation of a drug for treating or alleviating diabetic wounds, and the compound preparation contains exosomes derived from endometrial mesenchymal stem cells.

[0033] Advantages of the present invention:

[0034] The exosomes derived from endometrial mesenchymal stem cells provided by the present invention can effectively promote the tube formation ability of HUVEC, promote wound angiogenesis in diabetic model mice, increase collagen deposition, reduce the wound area, and ultimately achieve the purpose of promoting the healing of diabetic wounds. The exosomes derived from endometrial mesenchymal stem cells provided by the present invention can be used as drugs for promoting angiogenesis and wound healing in the treatment of diabetic wounds, and also provide a new strategy for the treatment of other chronic wounds including burns. Description of the drawings

[0035] Figure 1 It is a result diagram of Exos uptake;

[0036] Figure 2 It is a result diagram showing the ability of Exos to promote angiogenesis in the Matrigel matrix gel tube formation experiment;

[0037] Figure 3 It is a result diagram showing the wound healing situation in the wound appearance images of each group of mice;

[0038] Figure 4 It is a result diagram showing the wound width of mice by HE staining;

[0039] Figure 5 It is a result diagram showing wound collagen deposition by Masson staining;

[0040] Figure 6 It is a result diagram showing wound angiogenesis by CD31 immunohistochemistry. Detailed implementation manners

[0041] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0042] Example 1

[0043] Extraction of exosomes from hEMSCs (human endometrial mesenchymal stem cells) (hEMSC-Exos, exosomes derived from human endometrial mesenchymal stem cells):

[0044] 1) Resuscitate human endometrial mesenchymal stem cells (hEMSCs), and select passages P3 - P6 with good status for exosome extraction. When hEMSCs are cultured to 60% - 70% confluence with DMEM / F12 complete medium, discard the DMEM / F12 complete medium, and wash twice with PBS to completely remove residual medium and cell debris. Replace it with serum-free mesenchymal stem cell medium and culture for 48 h, then collect the supernatant.

[0045] 2) Extract exosomes from the collected supernatant using differential ultracentrifugation. First, pre-cool the centrifuge to 4°C in advance. Centrifuge at low speed to remove cell debris, macromolecules, etc., and centrifuge at high speed to concentrate and collect exosomes. The centrifugation sequence is 300 rcf for 10 min, 2000 rcf for 20 min, 10000 rcf for 30 min (filter out large vesicles using a 0.22 μm filter membrane), 100000 rcf for 70 min × 2. After ultra-high speed centrifugation, discard the supernatant, and use a pipette to aspirate and discard the remaining liquid at the bottom of the tube. Gently pipette the tube wall with 50 μl PBS to collect exosomes, and store them in a -80°C refrigerator.

[0046] Use hAMSC-Exos (exosomes derived from human adipose mesenchymal stem cells) as a control. The extraction method is the same as that of exosomes derived from human endometrial mesenchymal stem cells above, and human adipose mesenchymal stem cells are used as the initial cells.

[0047] Detection of exosome uptake ability.

[0048] 1) Seed HUVECs at 8×10 4 cells / well in a dish with a diameter of 2.0 cm.

[0049] 2) Label hEMSC-Exos / hAMSC-Exos (1 mg / ml, 100 μl) with Dil (working solution concentration of 5 μg / ml) for 1 h, ultracentrifuge to remove free Dil dye solution, and resuspend with 50 μl PBS.

[0050] 3) Add hEMSC-Exos / hAMSC-Exos to HUVECs and incubate in an incubator for 12 h.

[0051] 4) Wash the cells 3 times with PBS.

[0052] 5) Fix with 4% paraformaldehyde for 10 min and wash 3 times with PBS.

[0053] 6) Stain with DAPI for 10 min and wash 3 times with PBS.

[0054] 7) Observe and take pictures under a confocal microscope.

[0055] Figure 1 It is shown that the two kinds of exosomes are taken up by HUVECs and internalized into the cells, where they can further exert biological effects.

[0056] Detection of the tube formation ability of hEMSC-Exos / hAMSC-Exos on HUVECs

[0057] 1) Preparation before the experiment: Place Matrigel in a 4°C refrigerator overnight to melt it into a liquid; pre-cool 96-well plates and 200 μl pipette tips at -20°C in advance.

[0058] 2) Use pre-cooled pipette tips to aspirate 100 μl of Matrigel and spread it on pre-cooled 96-well plates (on ice). After spreading, place the 96-well plates in an incubator at 37°C and 5% CO2 for 45 min to solidify the gel.

[0059] 3) During the gelation process, digest HUVECs, count them, and prepare cell suspensions.

[0060] 4) Seed HUVECs at 3×10 4 cells / well on 96-well plates coated with Matrigel gel and incubate them in an incubator at 37°C and 5% CO2.

[0061] 5) After incubating for 3 h, observe the formation of tubules under an inverted microscope and take pictures.

[0062] Figure 2 It is shown that hEMSC-Exos have stronger angiogenesis-promoting ability than hAMSC-Exos.

[0063] Example 2

[0064] Preparation of a diabetic mouse model.

[0065] Use the streptozotocin (STZ) method, which is widely recognized and most commonly used at home and abroad, to induce a diabetic mouse model.

[0066] 1) Purchase C57 mice and fast them for 12 h one day before induction.

[0067] 2) Mark C57 mice with ear tags, weigh them, and measure their blood glucose.

[0068] 3) Prepare an STZ working solution with sodium citrate buffer and inject the STZ working solution (50 mg / kg) intraperitoneally into the mice. Inject an equal amount of sodium citrate buffer into the control group.

[0069] 4) Inject the same dose of STZ working solution continuously for 5 days.

[0070] 5) Weigh the mice weekly and monitor their blood glucose. If the blood glucose concentration is ≥ 16.7 mM after 4 weeks, the establishment of the diabetic mouse model is considered successful.

[0071] Evaluation of the wound healing of mice in each group.

[0072] Preparation of a full-thickness skin wound model in mice

[0073] 1) Disinfect the operating table in the operating room with ultraviolet light, and sterilize all surgical instruments by high-temperature and high-pressure treatment.

[0074] 2) After the mice are anesthetized by inhaling isoflurane, remove the hair on the back with depilatory cream.

[0075] 3) Disinfect the skin with iodophor and alcohol in sequence, and cut a circular wound with a diameter of 1 cm on the back.

[0076] 4) Inject PBS, hEMSC-Exos / hAMSC-Exos (200 μg, in 100 ml PBS) subcutaneously around the wound.

[0077] 5) Observe the wound healing and take pictures at 0, 3, 7, 10, and 14 days respectively.

[0078] 6) Use ImageJ software to calculate the wound area, and calculate the wound healing rate according to the following formula:

[0079] Healing rate = (initial area - current area) / (initial area) × 100%.

[0080] Figure 3 It shows that hEMSC-Exos has a good effect on promoting the healing of diabetic wounds. The wound healing speed of diabetic mice is slower than that of non-diabetic mice. After treatment with hEMSC-Exos / hAMSC-Exos, the wound healing speed of diabetic mice is accelerated, and the effect of hEMSC-Exos is better than that of hAMSC-Exos.

[0081] Detection of the wound width of mice in each group.

[0082] After taking pictures and recording on the 14th day, cut the skin of the mice. Fix it with 4% paraformaldehyde solution, dehydrate, embed, and section. Perform HE staining on the paraffin sections.

[0083] 1) Dewaxing and hydration: 10 min each in xylene 1 and 2 → 3 min each in absolute ethanol 1 and 2, 3 min in 95% ethanol, 3 min in 85% ethanol, 3 min in 75% ethanol; soak in tap water for 1 min.

[0084] 2) Stain with hematoxylin for 5 min; rinse with tap water.

[0085] 3) Differentiation: Differentiate with hydrochloric acid alcohol for 2 s; rinse with tap water.

[0086] 4) Blueing: Blue with ammonia water for 10 s.

[0087] 5) Stain with eosin for 1 min; rinse with tap water.

[0088] 6) Immerse in 75% ethanol, 85% ethanol, and 95% ethanol for 30 s each → immerse in absolute ethanol for 2 min → immerse in xylene 1 and 2 for 1 min each.

[0089] 7) Mount with neutral balsam.

[0090] 8) Take microscope photos and analyze the wound width with ImageJ software.

[0091] Figure 4 It shows that the wound width of diabetic mice treated with hEMSC-Exos / hAMSC-Exos is smaller, and the hEMSC-Exos treatment group is smaller than the hAMSC-Exos treatment group.

[0092] Detection of wound collagen deposition in each group of mice.

[0093] Perform Masson staining on paraffin sections.

[0094] 1) Baking the section: At 62 °C for 1 h.

[0095] 2) Dewaxing and hydration: Immerse in xylene 1 and 2 for 10 min each → immerse in absolute ethanol 1 and 2 for 3 min each, 95% ethanol for 3 min, 85% ethanol for 3 min, 75% ethanol for 3 min, and distilled water for 1 min.

[0096] 3) Immerse in Weigert's working solution for 4 min, rinse with running water for 5 min, and rinse with distilled water 3 times, 5 s each time.

[0097] 4) Drop Masson composite staining solution (solution A) to cover the tissue, incubate for 12 min, and rinse slightly with distilled water.

[0098] 5) Drop phosphomolybdic acid solution (solution B) to cover the tissue, incubate for 5 min and then pour it off, and drain.

[0099] 6) Drop aniline blue solution (solution C) to cover the tissue, incubate for 3 min, and rinse with distilled water.

[0100] 7) Drop acetic acid differentiation solution (solution D) to cover the tissue, incubate for 1 min and then pour it off.

[0101] 8) Dehydration: Immerse in 85% ethanol, 95% ethanol, absolute ethanol 1 and 2, xylene 1 and 2 for 30 s each.

[0102] 9) Mount the section with neutral balsam.

[0103] 10) Microscopic photography and analysis of collagen deposition levels using ImageJ software.

[0104] Figure 5 It was shown that after treatment with hEMSC-Exos / hAMSC-Exos, the collagen deposition in the wounds of diabetic mice was denser and arranged more regularly and orderly, indicating that the wound healing was more mature. Moreover, the effect of hEMSC-Exos on promoting collagen deposition was superior to that of hAMSC-Exos.

[0105] Detection of angiogenesis in the wounds of mice in each group.

[0106] Paraffin sections were stained by CD31 immunohistochemistry.

[0107] 1) Baking the sections: at 62 °C for 4 h.

[0108] 2) Deparaffinization and hydration: 10 min each in xylene 1 and 2 → 3 min each in absolute ethanol 1 and 2, 3 min in 95% ethanol, 3 min in 85% ethanol, 3 min in 75% ethanol, 1 min in distilled water, 2 min in PBS.

[0109] 3) Treat with 3% H2O2 for 10 min and wash twice with PBS, 3 min each time.

[0110] 4) Antigen retrieval of the sections was performed using EDTA antigen retrieval solution (pH 9.0) in a pressure cooker, and then washed twice with PBS, 3 min each time.

[0111] 5) Block the sections with ready-to-use goat serum for 1 h.

[0112] 6) Drop CD31 primary antibody (1:2000) above the tissue, incubate overnight at 4 °C. The next day, incubate at room temperature for 1 h. Wash three times with PBS, 3 min each time.

[0113] 7) Drop enzyme-labeled goat anti-mouse / rabbit Ig polymer (PV-6000) above the tissue, incubate at 37 °C for 30 min. Wash three times with PBS, 3 min each time.

[0114] 8) Incubate with DAB for 7 min.

[0115] 9) Rinse with tap water to terminate the color development.

[0116] 10) Stain with hematoxylin for 5 s and terminate in tap water.

[0117] 11) Dehydration and clearing: 2 min each in 85% ethanol, 95% ethanol, 100% ethanol 1, 2, xylene 1, 2.

[0118] 12) Mount the sections with neutral balsam.

[0119] Figure 6 Demonstrate the ability to enhance neovascularization after treatment with hEMSC-Exos / hAMSC-Exos. In this invention, neovascularization in wounds was evaluated by immunohistochemical staining of the vascular endothelial cell marker CD31. The results showed that the number of new blood vessels in the hEMSC-Exos group was significantly higher than that in the hAMSC-Exos group.

[0120] As can be seen from the above examples, in this invention, hEMSC-Exos was obtained from the culture supernatant of human endometrial mesenchymal stem cells by differential centrifugation, and added to the HUVEC medium. The ability to promote angiogenesis was observed by the Matrigel tube formation assay; it was resuspended with PBS to prepare an exosome preparation, which was injected around the wounds of diabetic model mice. The appearance of the mouse wounds was photographed to observe the wound healing situation, HE staining was used to observe the width of the mouse wounds, Masson staining was used to observe the degree of collagen deposition, and CD31 immunohistochemistry was used to observe angiogenesis in the wounds. The results showed that hEMSC-Exos could effectively promote the tube formation ability of HUVECs, promote angiogenesis in the wounds of diabetic model mice, increase collagen deposition, and reduce the wound area, ultimately achieving the purpose of promoting the healing of diabetic wounds. The exosomes derived from human endometrial mesenchymal stem cells provided by this invention can be used as drugs to promote angiogenesis and wound healing in the treatment of diabetic wounds, and also provide a new strategy for the treatment of other chronic wounds including burns.

[0121] The above description is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of exosomes derived from endometrial mesenchymal stem cells in the preparation of drugs for chronic wound repair.

2. The application according to claim 1, characterized in that The chronic wounds include diabetic wounds, venous ulcers, arterial ulcers, pressure ulcers, post-traumatic chronic ulcers, postoperative infectious wounds, radiation dermatitis ulcers, immune-related ulcers, lymphedema-related ulcers, malignant tumor-related ulcers or vasculitis ulcers.

3. The application according to claim 1, wherein The endometrial mesenchymal stem cells are human endometrial mesenchymal stem cells.

4. The application according to claim 1, characterized in that The exosomes derived from endometrial mesenchymal stem cells are obtained by a preparation method comprising the following steps: The exosomes derived from endometrial mesenchymal stem cells are extracted from the culture supernatant of endometrial mesenchymal stem cells by differential centrifugation.

5. Use of exosomes derived from endometrial mesenchymal stem cells in the preparation of drugs for treating or alleviating diabetic wounds.

6. Use of a medical dressing in the preparation of a medicament for treating or alleviating diabetic wounds, characterized in that, The medical dressing contains exosomes derived from endometrial mesenchymal stem cells.

7. The application according to claim 6, wherein The medical dressing includes a matrix material selected from one or more of chitosan, alginate, hyaluronic acid, polylactic acid, polycaprolactone or gelatin.

8. The application according to claim 6, characterized in that The medical dressing contains an active ingredient combined with the exosomes derived from endometrial mesenchymal stem cells, and the active ingredient is selected from one or more of insulin, metformin sustained-release microparticles, pirfenidone, interferon, lidocaine or ibuprofen.

9. The application according to claim 6, wherein The medical dressing contains an auxiliary ingredient, and the auxiliary ingredient includes one or more of silver nanoparticles, gentamicin, epidermal growth factor, fibroblast growth factor, curcumin or pirfenidone.

10. Use of a compound preparation in the manufacture of a medicament for treating or alleviating diabetic wounds, characterized in that, The compound preparation contains exosomes derived from endometrial mesenchymal stem cells.

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