Preparation methods of regulatory T cell vesicles, composite hydrogels and their applications

Abstract

The present invention discloses a preparation method of regulatory T cell vesicles, a composite hydrogel and its application, wherein the regulatory T cell vesicle composite hydrogel comprises regulatory T cell vesicles and hydrogel, wherein the mass volume ratio of the regulatory T cell vesicles to the hydrogel is 0.5-5 g / L, and the preparation method of the regulatory T cell vesicles comprises: extracting regulatory T cells from umbilical cord blood, amplifying the extracted regulatory T cells, and obtaining amplified umbilical cord blood regulatory culture fluid; taking the supernatant of the amplified umbilical cord blood regulatory culture fluid, performing at least one centrifugation, and collecting the supernatant after centrifugation; taking the obtained supernatant, performing at least one centrifugation again, collecting the precipitate, and obtaining regulatory T cell vesicles. The regulatory T cell vesicle composite hydrogel provided by the present invention can significantly promote wound healing, and has a good therapeutic effect on the treatment of diabetic wound healing.

Description

Technical Field

[0001] The present invention relates to the technical field of cell therapy, and in particular to a preparation method of regulatory T cell vesicles, a composite hydrogel and applications thereof. Background Art

[0002] Diabetic wound injury is a serious complication of diabetes, mainly caused by diabetic peripheral neuropathy and peripheral vascular disease. The most common manifestation is diabetic foot ulcer (DFU), which requires surgical amputation or even death in the most serious cases. Diabetic foot has a high hospitalization rate, long hospitalization time, and high cost, but generally has a poor prognosis and a high mortality and disability rate.

[0003] Currently, antiplatelet and anticoagulant, anti-inflammatory, and hypoxia-improving treatment measures for improving diabetic peripheral neuropathy and vascular disease are all helpful for DFU healing to a certain extent, but how to more effectively promote wound healing under the premise of severe vascular disease needs to be solved urgently. Summary of the invention

[0004] The present invention provides a preparation method of regulatory T cell vesicles, a composite hydrogel and applications thereof, in order to solve the technical problem of how to more effectively promote the healing of diabetic wounds under the premise of severe vascular lesions.

[0005] According to one aspect of the present invention, a regulatory T cell vesicle composite hydrogel is provided, comprising regulatory T cell vesicles and hydrogel, wherein the mass volume ratio of the regulatory T cell vesicles to the hydrogel is 0.5 to 5 g / L.

[0006] Furthermore, the hydrogel is a medical hydrogel.

[0007] According to another aspect of the present invention, a method for preparing regulatory T cell vesicles is provided, comprising the following steps:

[0008] (1) extracting regulatory T cells from umbilical cord blood, amplifying the extracted regulatory T cells, and obtaining an amplified umbilical cord blood regulatory culture medium;

[0009] (2) taking the supernatant of the expanded cord blood regulated culture medium, performing at least one centrifugation treatment, and collecting the supernatant after centrifugation;

[0010] (3) Taking the supernatant obtained in step (2), performing at least one centrifugation treatment, collecting the precipitate, and obtaining regulatory T cell vesicles.

[0011] Further, the at least one centrifugation treatment in step (2) comprises:

[0012] Taking the supernatant of the amplified cord blood regulated culture medium, performing a first centrifugation treatment, removing the precipitate, and collecting the supernatant;

[0013] The supernatant obtained from the first centrifugation was taken and subjected to a second centrifugation to remove apoptotic bodies and cell debris, and the supernatant was obtained again.

[0014] Furthermore, the conditions of the first centrifugal treatment are: temperature 0-10°C, centrifugal force 200-500g, and time 10-20min.

[0015] Furthermore, the conditions of the second centrifugal treatment are: temperature 4-25°C, centrifugal force 500-10000g, and time 10-30min.

[0016] Further, the at least one centrifugation treatment in step (3) comprises:

[0017] Taking the supernatant obtained from the last centrifugation in step (2), performing an Nth centrifugation, removing the supernatant, and obtaining a regulatory T cell vesicle precipitate;

[0018] The cord blood regulatory T cell vesicle precipitate is taken and centrifuged for the N+1th time, and the supernatant is removed to obtain the regulatory T cell vesicle, wherein N is an integer greater than or equal to 3.

[0019] Furthermore, the conditions of the Nth centrifugal treatment and the N+1th centrifugal treatment are: temperature of 4 to 25° C., centrifugal force of 3000 to 20000 g, and time of 10 to 60 min.

[0020] According to yet another aspect of the present invention, there is also provided a use of the above-mentioned regulatory T cell vesicle composite hydrogel in promoting wound healing.

[0021] Furthermore, the wound surface includes a common wound surface or a diabetic wound surface.

[0022] The present invention has the following beneficial effects:

[0023] Diabetic wounds are difficult to treat due to their complex nature and limited understanding of their pathogenic mechanisms. Among them, it is clear that the disordered inflammatory environment of the wound and the persistence of inflammation are one of the important reasons for the delayed healing of diabetic wounds. Large vesicles derived from umbilical cord blood regulatory T cells have similar effects to stem cells: anti-inflammatory, immunomodulatory, and promotion of tissue regeneration. They have a strong ability to promote healing and can chemotaxis to the site of inflammation. The direct application of extracellular vesicles has limitations: short half-life and high clearance rate. Hydrogels have good biocompatibility, porosity, and unique temperature sensitivity. Cell vesicles loaded on hydrogels can achieve continuous and stable release to prevent the vesicles from being cleared prematurely. The regulatory T cell vesicle composite hydrogel provided by the present invention can significantly promote wound healing, and can more effectively promote the healing of diabetic wounds under the premise of severe vascular lesions.

[0024] The preparation method of regulatory T cell vesicles provided by the present invention first extracts regulatory T cells from umbilical cord blood, amplifies them, and obtains regulatory T cell vesicles after a series of centrifugation treatments. The vesicles prepared by this method are immune cell vesicles, have a high concentration per unit volume, and a short preparation cycle.

[0025] The regulatory T cell vesicles prepared by the preparation method of the regulatory T cell vesicles provided by the present invention have obvious effects on inhibiting the proliferation ability of single core cells of diabetic patients, and can also promote the migration ability of vascular endothelial cells.

[0026] In addition to the above-described purposes, features and advantages, the present invention has other purposes, features and advantages. The present invention will be further described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0028] Figure 1 It is a flow cytometry-based inhibitory function profile of cord blood regulatory T cells according to a preferred embodiment of the present invention;

[0029] Figure 2 It is a graph of the ability of regulatory T cell vesicles to promote the migration of vascular endothelial cells according to a preferred embodiment of the present invention;

[0030] Figure 3 This is a comparison diagram of the regulatory T cell vesicle (Treated group) composite hydrogel promoting diabetic wound healing according to the preferred embodiment of the present invention;

[0031] Figure 4 This is a comparison chart of the regulatory T cell vesicle (Treated group) composite hydrogel in the preferred embodiment of the present invention promoting the healing of common wounds. DETAILED DESCRIPTION

[0032] In order to make the invention purpose, technical scheme and beneficial technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. It should be understood that the embodiments described in this specification are only for explaining the present invention, not for limiting the present invention.

[0033] For simplicity, only some numerical ranges are explicitly disclosed herein. However, any lower limit can be combined with any upper limit to form an unambiguous range; and any lower limit can be combined with other lower limits to form an unambiguous range, and any upper limit can be combined with any other upper limit to form an unambiguous range. In addition, although not explicitly stated, each point or single value between the range endpoints is included in the range. Thus, each point or single value can be combined with any other point or single value as its own lower limit or upper limit or with other lower limits or upper limits to form an unambiguous range.

[0034] In the description of this article, it should be noted that, unless otherwise specified, “above” and “below” are inclusive of the number, and the “multiple” in “one or more” means two or more, and the “multiple” in “one or more” means two or more.

[0035] Regulatory T cells (Treg) are a group of immune cell subsets with immune regulatory functions. They have the ability to inhibit the proliferation of autoimmune cells (CD4+CD25-T) and mainly play an inhibitory role in the feedback regulation of the immune system. T cells, Treg cells after in vitro expansion have good function and low immunogenicity, and Treg cells secrete a large number of cell vesicles during the expansion process. The multiple functions of Treg cell vesicles as biological agents make them the most potential therapeutic means to promote the recovery of damaged wounds, especially the repair of blood vessels and tissues in diabetic ulcers.

[0036] An embodiment of the first aspect of the present invention provides a regulatory T cell vesicle composite hydrogel, comprising regulatory T cell vesicles and hydrogel, wherein the mass volume ratio of the regulatory T cell vesicles to the hydrogel is 0.5 to 5 g / L.

[0037] In some embodiments, the mass-to-volume ratio of the regulatory T cell vesicles to the hydrogel is: 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5, etc. The mass-to-volume ratio of the regulatory T cell vesicles to the hydrogel can also be any combination range of the above values.

[0038] It should be noted that the mass volume ratio of the above-mentioned regulatory T cell vesicles to the hydrogel is: 0.5-5 g / L. When the hydrogel is in a liquid state, the concentration of the regulatory T cell vesicles is correspondingly 0.5-5 g / L.

[0039] Large vesicles derived from umbilical cord blood regulatory T cells have similar effects to stem cells: anti-inflammatory, immunomodulatory, promoting tissue regeneration, with a strong ability to promote healing, and can chemotaxis to the site of inflammation. The direct application of extracellular vesicles has limitations: short half-life and high clearance rate. The hydrogel has good biocompatibility, porosity, and unique temperature sensitivity. The cell vesicles loaded on the hydrogel can achieve continuous and stable release and prevent the vesicles from being cleared prematurely. The regulatory T cell vesicle composite hydrogel provided by the present invention can significantly promote wound healing, and can more effectively promote diabetic wound healing under the premise of severe vascular lesions.

[0040] In an embodiment of the present invention, the hydrogel is a medical hydrogel.

[0041] In some embodiments, the hydrogel may be Pluronic F127, with the molecular formula: (C3H6O·C2H4O)x, where x is a positive integer. The hydrogel is a triblock polymer of polyoxyethylene polypropylene, and its hydrogel solution has a unique temperature-sensitive property, which can be transformed from a liquid state to a porous three-dimensional gel state at a certain concentration and temperature. Because of its medical injectability and good biocompatibility, it has been approved by the US FDA for use in humans. For example, the hydrogel of Merck Sigma, CAS No.: 9003-11-6.

[0042] The hydrogel solution has good biocompatibility, porosity, and unique temperature sensitivity. It can transform from a liquid state to a porous three-dimensional gel state at a certain concentration and temperature. After the cell vesicles are loaded on the hydrogel, they can be released continuously and stably, preventing the vesicles from being cleared prematurely.

[0043] An embodiment of the second aspect of the present invention provides a method for preparing regulatory T cell vesicles, comprising the following steps:

[0044] (1) extracting regulatory T cells from umbilical cord blood, amplifying the extracted regulatory T cells, and obtaining an amplified umbilical cord blood regulatory culture medium;

[0045] (2) taking the supernatant of the expanded cord blood regulated culture medium, performing at least one centrifugation treatment, and collecting the supernatant after centrifugation;

[0046] (3) Taking the supernatant obtained in step (2), performing at least one centrifugation treatment, collecting the precipitate, and obtaining regulatory T cell vesicles.

[0047] In an embodiment of the present invention, extracting regulatory T cells from cord blood in step (1) comprises the following steps:

[0048] 1. Disinfect the umbilical cord blood bag and prepare umbilical cord blood autologous plasma: transfer the umbilical cord blood in the umbilical cord blood bag to a 50mL centrifuge tube for centrifugation: centrifugation conditions: 20℃, 3000rpm, 10min, +8, -9. Transfer the supernatant to a new 50mL centrifuge tube for a second centrifugation: centrifugation conditions: 20℃, 4000rpm, 10min, +8, -9, absorb the yellow clarified plasma, inactivate it in a 56℃ water bath for 30 minutes, and place it in the refrigerator for use. Resuspend the umbilical cord blood with a volume of physiological saline equal to the volume of the aspirated plasma;

[0049] 2. Slowly add the cord blood resuspended in saline to the lymphocyte separation solution (cord blood volume: lymphocyte separation solution volume = 1:1) and centrifuge under the following conditions: 20°C, *400rcf-*1610rcf, 25min, +0, -0. After centrifugation, separate the cord blood into layers and use a pipette to absorb the peripheral blood mononuclear cell (PBMC) layer. Move slowly and gently, and absorb as little lymphocyte fluid as possible.

[0050] 3. Continue to centrifuge the PBMC layer, centrifugation conditions: 20℃, *580rcf, 10min, +8, -9. Take 5ml-10ml 10× red blood cell lysis buffer and 45ml sterile water to prepare red blood cell lysis solution. After the centrifugation is completed, remove the supernatant, add red blood cell lysis solution for 2 to 10 minutes, and then add 0.9% saline to 35ml to stop red blood cell lysis. Centrifuge and remove the supernatant, centrifugation conditions: 20℃, *290rcf, 10min, +8, -9. Repeat the steps of adding 0.9% saline to the volume, centrifuging, and removing the supernatant 2 to 3 times;

[0051] 4. After centrifugation, remove the supernatant, flick the cells, add 0.9% saline, and take 10μL for counting. Add 0.9% saline to the remaining volume to 40mL and centrifuge again. Centrifugation conditions: 20℃, *290rcf, 10min, +8, -9. The counting steps are as follows: put the cell solution into an EP tube, take 90μL of trypan blue and add it to the EP tube to mix. Take 10μL on the counting plate, quickly operate and count: (sum of the four corners / 4)×10 5 Cells / mL*dilution multiple, cells on the line are recorded but not on the bottom, cells on the left are recorded but not on the right;

[0052] 5. After centrifugation, remove the supernatant and resuspend the cells to 5×107cell / mL with Easy Buffer. The liquid volume after resuspension is 0.5mL-6mL;

[0053] 6. Transfer the resuspended cell suspension to a sterile tube that matches the magnetic pole;

[0054] 7. Add CD25 positive selection antibody in an amount equal to the resuspension volume × 50uL and mix well;

[0055] 8. Incubate for 5 minutes;

[0056] 9. Vortex the releasable spheres for more than 30 seconds until the magnetic bead clumps disappear;

[0057] 10. Add releasable spheres: the amount added is the resuspension volume × 30uL, mix well;

[0058] 11. Add CD4+T cell enrichment antibody mixture (T cell Enrichment Cocktail): the amount added is the resuspension volume × 50uL, and mix well;

[0059] 12. Incubate for 5 minutes;

[0060] 13. Add Easy Buffer to make up to 10 mL and mix gently up and down 2-3 times;

[0061] 14. Place the sterile tube on the magnetic pole without the cover;

[0062] 15. Incubate for 10 min.

[0063] 16. Prepare a centrifuge tube to collect CD25- cells, and pour the liquid in the tube into the tube together with the magnetic pole and sterile tube;

[0064] 17. Remove the sterile tube from the magnetic pole, add 10 mL of Easy Buffer to it, mix gently 2-3 times, place it on the magnetic pole, and incubate for 5 minutes;

[0065] 18. Repeat steps 16-17 twice;

[0066] 19. Add Easy Buffer to the initial resuspension volume, making sure to flush all cells off the tube wall;

[0067] 20. Add magnetic bead removal buffer (Release Buffer): the amount added is the resuspension volume × 100uL, and mix vigorously for more than 5 times;

[0068] 21. Add CD127high Depletion Cocktail: add the resuspension volume × 50uL and mix well;

[0069] 22. Incubate for 5 minutes;

[0070] 23. Add Easy Buffer to make up to 10 mL and mix up and down 2-3 times;

[0071] 24. Place the sterile tube on the magnetic pole and incubate for 5 minutes;

[0072] 25. Prepare centrifuge tubes to collect regulatory T cells. Pour the liquid into the prepared centrifuge tubes together with the magnetic pole and sterile tube to obtain CD4+CD25+CD127- regulatory T cells.

[0073] The above process of obtaining CD4+CD25+CD127- regulatory T cells can be performed using EasySep TM Human CD4+CD25+CD127- regulatory T cell (Treg) isolation kit, brand: STEMCELL Technologies, catalog number: 18063.

[0074] In an embodiment of the present invention, the process of amplifying the extracted regulatory T cells in step (1) to obtain the amplified cord blood regulatory culture medium is as follows:

[0075] 1. The regulatory T cells were seeded in a 48-well plate at 2-8×105 / mL. On day 0, 0.5 ml of culture medium containing magnetic beads (number of magnetic beads: number of cells = 1:1) was added to each well (the formula of the culture medium is shown in Table 1). The culture medium was incubated at 37°C and 5% CO. 2 Concentration culture.

[0076] Table 1 Formula for preparing 50 ml of cord blood regulatory T cell expansion medium

[0077]

[0078] 2. On the 1st to 2nd day (24h-48h) after seeding, add 0.5mL-1mL of expansion medium to each well of cells, and add 100-400U / mL of rhIL-2. Incubate at 37℃, 5% CO 2 Cultivated at concentration.

[0079] 3. 3-4 days after seeding, first subculture: remove 0.5 mL of expansion medium, mix the remaining expansion medium with cells, subculture 2 wells per well, add expansion medium with 100-400 U / mL rhIL-2 to 1 mL, and incubate at 37°C, 5% CO 2 After adding, each well has 1 mL of expansion medium, and the amount of rhIL-2 added is based on the content of 1 mL of expansion medium. The amount of rhIL-2 added in the remaining steps is the same as this step.

[0080] 4. On the 5th to 6th day after seeding, the second subculture: aspirate 0.5 ml of the expansion medium, mix the remaining expansion medium with the cells, subculture each well to 2 wells, and add expansion medium with 100-400 U / mL rhIL-2 to 1 mL, and incubate at 37°C, 5% CO 2 Cultivated at concentration.

[0081] 5. 7-10 days after seeding, the third passage: first remove the anti-CD3CD28 magnetic beads in the culture dish, then add the washed new anti-CD3CD28 magnetic beads (according to the number of magnetic beads: regulatory T cells = 1:1), and incubate at 37°C, 5% CO 2 Cultivated at concentration.

[0082] 6. On the 10th-11th day after seeding, the fourth subculture: aspirate 0.5 mL of the expansion medium, mix the remaining expansion medium with the cells, subculture each well to 2 wells, and add expansion medium with 100-400 U / mL of rhIL-2 to 1 ml, and incubate at 37°C, 5% CO 2 Cultivated at concentration.

[0083] 7. 11-12 days after seeding, 5th subculture: aspirate 0.5 ml of expansion medium, mix the remaining expansion medium with cells, subculture 2 wells per well, add expansion medium with 100-400 U / mL rhIL-2 to 1 mL, and incubate at 37°C, 5% CO 2 Cultivated at concentration.

[0084] 8. On the 12th to 13th day after seeding, the 6th subculture: remove 0.5 mL of the expansion medium, mix the remaining expansion medium with the cells, subculture each well to 2 wells, and add expansion medium with 100-400 U / mL of rhIL-2 to 1 ml, and incubate at 37°C, 5% CO 2 Cultivated at concentration.

[0085] 9. On the 13th-14th day after seeding, the 7th subculture: remove 0.5 mL of the expansion medium, mix the remaining expansion medium with the cells, subculture each well to 2 wells, and add expansion medium with 100-400 U / mL of rhIL-2 to 1 mL, and incubate at 37°C, 5% CO 2 Cultivated at concentration.

[0086] 10. Expand according to the above method until the cells stop growing, which takes about 20-40 days.

[0087] According to the embodiments of the present invention, the number of cord blood regulatory T cells after amplification is significantly increased, and the amplified cord blood regulatory T cells can significantly inhibit the proliferation ability of aggressive T cells.

[0088] In an embodiment of the present invention, in step (2), the supernatant of the regulated culture medium of the cord blood after amplification is taken and centrifuged at least once, and the supernatant after centrifugation is collected. The number of centrifugation can be 1 time, 2 times, 3 times or more. The purpose of step (2) is to remove the precipitate, apoptotic bodies and debris in the culture medium and retain the supernatant. When the number of centrifugation is multiple times, the yield of vesicles can be increased.

[0089] In an embodiment of the present invention, the at least one centrifugation treatment in step (2) comprises:

[0090] The supernatant of the expanded cord blood regulated culture medium is taken and centrifuged for the first time to remove the precipitate and collect the supernatant; the supernatant obtained from the first centrifugation is taken and centrifuged for the second time to remove apoptotic bodies and cell debris and obtain the supernatant again.

[0091] According to the embodiments of the present application, impurities (residual cells, cell debris and apoptotic bodies) are removed through the above two centrifugations to obtain highly purified vesicles.

[0092] In an embodiment of the present invention, the conditions of the first centrifugal treatment are: temperature 0-10° C., centrifugal force 200-500 g, and time 10-20 min.

[0093] In an embodiment of the present invention, the conditions of the second centrifugation treatment are: temperature 4-25° C., centrifugal force 500-10000 g, time 10-30 min. Purified large vesicles can be obtained through the second centrifugation treatment.

[0094] In an embodiment of the present invention, in step (3), the supernatant obtained in step (2) is centrifuged at least once, and the precipitate is collected to obtain regulatory T cell vesicles, wherein the number of centrifugation can be 1 time, 2 times, 3 times or more. Multiple centrifugation can increase the yield of large vesicles.

[0095] In an embodiment of the present invention, the at least one centrifugation treatment in step (3) comprises:

[0096] The supernatant obtained from the last centrifugation in step (2) is taken and centrifuged for the Nth time, and the supernatant is removed to obtain a regulatory T cell vesicle precipitate; the cord blood regulatory T cell vesicle precipitate is taken and centrifuged for the N+1th time, and the supernatant is removed to obtain a regulatory T cell vesicle, wherein N is an integer greater than or equal to 3, such as N is 3, 4, 5 or other positive integers greater than 5. The yield of large vesicles can be increased by the above multiple centrifugations.

[0097] In the embodiment of the present invention, the conditions of the Nth centrifugation treatment and the N+1th centrifugation treatment are: temperature of 4-25° C., centrifugal force of 3000-20000 g, and time of 10-60 min. The above centrifugation conditions can increase the yield of large vesicles.

[0098] An embodiment of the third aspect of the present invention provides an application of the above-mentioned regulatory T cell vesicle composite hydrogel in promoting wound healing.

[0099] In some embodiments, the above-mentioned cord blood regulatory T cell vesicles are mixed evenly with the medical hydrogel at a concentration of 0.5-5 g / L and then transplanted to the wound surface once a day for 4 consecutive times.

[0100] The hydrogel solution has good biocompatibility, porosity, and unique temperature sensitivity. It can transform from a liquid state to a porous three-dimensional gel state at a certain concentration and temperature. After the cell vesicles are loaded on the hydrogel, they can be released continuously and stably, preventing the vesicles from being cleared prematurely.

[0101] In an embodiment of the present invention, the wound surface includes a common wound surface or a diabetic wound surface.

[0102] A wound is a destruction of the continuity and integrity of normal skin, as well as the loss and defect of skin tissue, caused by external injury factors. Common injury factors include mechanical trauma, surgery, heat, electric current, chemicals, low temperature, spontaneous infection, and tissue damage caused by endogenous factors such as local blood supply disorders. Wounds are divided into acute wounds and chronic wounds according to the length of wound healing time. Different wounds have different phenotypic characteristics, which leads to different outcomes of wound healing. Ulcers formed by radiation damage to skin and soft tissue are usually divided into two categories: one is that the skin and soft tissue are irradiated with large doses of radiation, which produces local gangrenous dermatitis, and acute ulcers are formed after the necrotic tissue detaches.

[0103] The characteristics of diabetic wounds (the most common are diabetic foot wounds) can generally be divided into three degrees: Mild, the patient has ulcers, and the ulcer area is generally less than two centimeters, and the skin infection is limited to the epidermal tissue. During this period, debridement should be performed, and strengthening of anti-inflammatory treatment can achieve better results. Moderate, the wound has ulcers, and the ulcer tissue around the ulcer is generally larger than two centimeters. The skin infection can reach the subcutaneous tissue, and there may be abscesses in the deep part, accompanied by swollen lymph nodes. During this period, debridement should be performed, and deep abscesses should be drained. In the severe stage, the infection is relatively rapid, huge hard nodes appear in the foot tissue, the color is black, and gangrene occurs. The patient will have severe symptoms of poisoning. At this stage, the main treatment is foot resection or even amputation.

[0104] Example

[0105] The following examples describe the disclosure of the present invention in more detail, and these examples are intended for illustrative purposes only, as various modifications and variations within the scope of the disclosure of the present invention will be apparent to those skilled in the art. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are by weight, and all reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further processing, and the instruments used in the examples are commercially available.

[0106] This embodiment provides a method for preparing regulatory T cell vesicles, comprising the following steps:

[0107] (1) extracting regulatory T cells from umbilical cord blood, amplifying the extracted regulatory T cells until the cells stop growing, and obtaining an amplified umbilical cord blood regulatory culture medium;

[0108] (2) Collect the supernatant of the regulated culture of cord blood, centrifuge at 200-500×g for 10-20 minutes at 4°C, remove the precipitate, and collect the supernatant.

[0109] (3) Centrifuge at 500-10,000 × g for 10-30 min at 4°C to remove apoptotic bodies and cell debris.

[0110] (4) Centrifuge at 3,000 to 20,000 × g for 10 to 60 min at 4 to 25°C and remove the supernatant.

[0111] (5) Resuspend the cord blood Treg vesicle pellet at the bottom of the tube with PBS, centrifuge at 3000-20000×g for 10-60 min at 4-25°C, and remove the supernatant.

[0112] (6) Resuspend the vesicles in PBS and use immediately or freeze at -80°C for later use.

[0113] The prepared regulatory T cell vesicles were subjected to the following experiments:

[0114] 1. Experiment on the inhibition of proliferation of mononuclear cells (PBMC) from diabetic patients by regulatory T cell vesicles.

[0115] 1. Collect mononuclear cells (PBMC) from patients with autoimmune diabetes and take 1×10 6 cells.

[0116] 2. Resuspend cells (1×10 6 cells).

[0117] 3. Add 1 μl of cell trace stock solution (CellTrace TM CFSE Cell Proliferation Kit (CellTrace TM CFSE Cell Proliferation Kit), catalog number: C34554; Thermofisher Scientific. This kit contains ten disposable bottles of CellTrace TMCFSE (Component A), CFSE (Carboxyfluorescein diacetate succinimidyl ester) and 1 bottle of DMSO (Component B), DMSO (dimethyl sulfoxide), cell trace stock solution refers to the mixed solution prepared according to the kit instructions for CFSE staining of cells: 18ulDMSO + 1 bottle of CellTrace TM CFSE.

[0118] Incubate at 37°C in the dark for 20 min.

[0119] 5. Add 5 mL of washing solution (serum-free culture medium + 10% FBS (fetal bovine serum)).

[0120] 6. Incubate at 37°C in dark for 5 min.

[0121] 7. Centrifuge at 20°C, *290rcf, 10min, +8, -9.

[0122] 8. Resuspend the cells in 37°C pre-warmed culture medium (serum-free culture medium + 10% FBS).

[0123] 9. Take one well of regulatory T cells (Treg cells), remove the magnetic beads, count, and centrifuge at 20°C, *290rcf, 10min, +8, -9.

[0124] Resuspend 10.1×106 Treg cells in culture medium (serum-free culture medium + 10% FBS) pre-warmed at 37°C.

[0125] 11. Different concentrations of regulatory T cell vesicles (0 g / L, 0-0.1 g / L, 0.1-5 g / L) were co-cultured with mononuclear cells (PBMC) in 96-well plates (200 μl per well, plus 3 μl magnetic beads). PBMC cells treated with carboxyfluorescein diacetate succinimidyl ester (CFSE) alone were set as the negative control group, and the PBMC cell group treated with CFSE (magnetic bead stimulation) was set as the positive control group.

[0126] 12. Flow cytometry to detect the inhibitory function of cord blood regulatory T cells, such as Figure 1 As shown, it is a schematic diagram that different concentrations (Group 1 and Group 2) of cord blood regulatory T cell vesicles inhibit the proliferation of CFSE-labeled PBMCs, and the group without vesicle treatment is set as the control group (Control). Figure 1 The right side shows the graphical analysis of the results of the three groups. The results show that the cord blood regulatory T cell vesicles have the ability to inhibit the proliferation of inflammatory cells.

[0127] 2. Experiment on the ability of regulatory T cell vesicles to promote the migration of vascular endothelial cells.

[0128] 1. Culture plate streaking: streak evenly across the bottom of a 6-well culture dish, three lines per well, for subsequent photography and positioning;

[0129] 2. Cell plating: Add about 5×10 5 cells;

[0130] 3. Cell streaking: When the cells grow and fuse to 80%, use a sterile pipette to evenly streak three lines on the cell surface.

[0131] Longitudinal lines, perpendicular to the lines on the back of the tablet, 3 scratches each on the left, middle, and right;

[0132] 4. Wash cells: After the scratching is completed, wash the cells 3 times with 1× PBS (phosphate buffered saline) to wash away the floating cells so that the scratching

[0133] The gaps were clearly visible, and fresh serum-free medium was then replaced;

[0134] 5. Treat with regulatory T cell vesicles of different concentrations (0 g / L, 0-0.1 g / L, 0.1-5 g / L), cell culture and observation: place the cells at 37°C, 5% CO 2 Then, the culture dish was taken out at 0h and 24h after incubation, and the cells were observed and photographed under a microscope.

[0135] 6. Result analysis: Image J software was used to calculate the mean width of cell scratches. The results showed that compared with the control group, 0-0.1g / L (group 1) and 0.1-5g / L (group 2) regulatory T cell vesicles promoted the migration of vascular endothelial cells. Figure 2 As shown, Figure 2 The left side shows the schematic diagram of the ability of cord blood regulatory T cell vesicles of different concentrations (Group 1 and Group 2) to inhibit the migration of vascular endothelial cells. The group without vesicle treatment was set as the control group (Control). Figure 1 The right side shows the graphical analysis of the results of the three groups. The results show that the cord blood regulatory T cell vesicles have the ability to inhibit the migration of vascular endothelial cells.

[0136] 3. Experiment on promoting diabetic wound healing using regulatory T cell vesicle composite hydrogel.

[0137] 1. Preparation of diabetic mouse wound model: C57 mice were selected for the experiment and randomly divided into a control group (hydrogel-treated group without regulatory T cell vesicles) and an experimental group (composite hydrogel-treated group with regulatory T cell vesicles). STZ 40 mg / kg was injected intraperitoneally for 5 consecutive days. After the last injection, the model was considered successful when random blood glucose > 16.7 mmol / L. The wound of mice was treated according to the following steps: 1% pentobarbital solution was prepared; mice were anesthetized by intraperitoneal injection (40 mg / kg), and the back hair was removed in a clean bench after anesthesia; the wound mark was disinfected with iodine, and a 1.0×1.0 cm wound was cut on the back of the mouse with ophthalmic scissors. 2 Full-thickness skin defect wound;

[0138] 2. The cord blood regulatory T cell vesicles prepared in this example were mixed with the hydrogel at a concentration of 2.5 g / L and then transplanted into the wound surface of mice, 1.0×1.0 cm 2 75 μL of mixed hydrogel was applied to the wound surface each time; the control group was transplanted with an equal amount of hydrogel without umbilical cord blood regulatory T cell vesicles; the transplantation was performed once a day for 4 consecutive times.

[0139] 3. The mice were photographed before surgery and on the 5th and 7th days after surgery to observe the wound healing. The results showed that compared with the control group (control group), the regulatory T cell vesicle (Treated group) composite hydrogel could significantly promote wound healing, such as Figure 3 shown.

[0140] 4. Experiment on promoting the healing of common wounds by using regulatory T cell vesicle composite hydrogel.

[0141] 1. Preparation of mouse wound model: C57 mice were selected for the experiment and randomly divided into a control group (hydrogel treatment group without regulatory T cell vesicles) and an experimental group (composite hydrogel treatment group with regulatory T cell vesicles). The wound of mice was treated according to the following steps: 1% pentobarbital solution was prepared; mice were anesthetized by intraperitoneal injection (40 mg / kg), and the back hair was removed in a clean bench after anesthesia; the wound marking area was disinfected with iodine, and a 1.0×1.0 cm wound was cut on the back of the mouse with ophthalmic scissors. 2 Full-thickness skin defect wound;

[0142] 2. The cord blood regulatory T cell vesicles prepared in this example were mixed with the hydrogel at a concentration of 2.5 g / L and then transplanted into the wound surface of mice, 1.0×1.0 cm 2 75 μL of mixed hydrogel was applied to the wound surface each time; the control group was transplanted with an equal amount of hydrogel without umbilical cord blood regulatory T cell vesicles; the transplantation was performed once a day for 4 consecutive times.

[0143] 3. The wound healing of mice was observed before and 7 days after surgery. The results showed that compared with the control group (control group), the regulatory T cell vesicle (Treated group) composite hydrogel could significantly promote the proliferation of wound epithelial cells and fibrous tissue, such as Figure 4 As shown (HE staining is the hematoxylin-eosin staining method. The cytoplasm is stained with eosin to different shades of pink to pink. Collagen fibers are light pink, elastic fibers are bright pink, and proteinaceous fluid is pink. Masson staining is the most classic method for connective tissue staining. After Masson staining, muscle fibers appear red and collagen fibers appear blue. It is mainly used to distinguish collagen fibers from muscle fibers.)

[0144] Although the present invention has been described with reference to preferred embodiments, various modifications may be made thereto and parts thereof may be replaced by equivalents without departing from the scope of the present invention. In particular, the various technical features mentioned in the various embodiments may be combined in any manner as long as there is no structural conflict. The present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. Use of a regulatory T cell vesicle composite hydrogel in the preparation of a drug for treating diabetic wounds, characterized in that: The regulatory T cell vesicle composite hydrogel comprises regulatory T cell vesicles and hydrogel, and the mass volume ratio of the regulatory T cell vesicles to the hydrogel is: 0.5-5 g / L; The method for preparing the regulatory T cell vesicles comprises the following steps: (1) extracting regulatory T cells from umbilical cord blood, amplifying the extracted regulatory T cells, and obtaining amplified umbilical cord blood regulatory culture medium; (2) taking the supernatant of the expanded cord blood regulated culture medium, performing at least one centrifugation treatment, and collecting the supernatant after centrifugation; (3) Take the supernatant obtained in step (2), centrifuge it again at least once, collect the precipitate, and obtain regulatory T cell vesicles.

2. The use of the regulatory T cell vesicle composite hydrogel according to claim 1 in the preparation of a drug for treating diabetic wounds, characterized in that: The hydrogel is a medical hydrogel.

3. The use of the regulatory T cell vesicle composite hydrogel according to claim 1 in the preparation of a drug for treating diabetic wounds, characterized in that: The at least one centrifugation treatment in step (2) comprises: Taking the supernatant of the amplified cord blood regulated culture medium, performing a first centrifugation treatment, removing the precipitate, and collecting the supernatant; The supernatant obtained from the first centrifugation was taken and subjected to a second centrifugation to remove apoptotic bodies and cell debris, and the supernatant was obtained again.

4. The use of the regulatory T cell vesicle composite hydrogel according to claim 3 in the preparation of a drug for treating diabetic wounds, characterized in that: The conditions of the first centrifugation treatment are: temperature 0-10°C, centrifugal force 200-500g, and time 10-20min.

5. The use of the regulatory T cell vesicle composite hydrogel according to claim 3 in the preparation of a drug for treating diabetic wounds, characterized in that: The conditions of the second centrifugation treatment are: temperature 4-25°C, centrifugal force 500-10000g, and time 10-30min.

6. Use of the regulatory T cell vesicle composite hydrogel according to claim 1 in the preparation of a drug for treating diabetic wounds, characterized in that: The at least one centrifugation treatment in step (3) comprises: Taking the supernatant obtained from the last centrifugation in step (2), performing an Nth centrifugation, removing the supernatant, and obtaining a regulatory T cell vesicle precipitate; The cord blood regulatory T cell vesicle precipitate is taken and centrifuged for the N+1th time, and the supernatant is removed to obtain the regulatory T cell vesicle, wherein N is an integer greater than or equal to 3.

7. Use of the regulatory T cell vesicle composite hydrogel according to claim 6 in the preparation of a drug for treating diabetic wounds, characterized in that: The conditions for the Nth centrifugation treatment and the N+1th centrifugation treatment are: temperature of 4~25°C, centrifugal force of 3000~20000g, and time of 10~60min.

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