Preparation method of decidua natural killer cells, composite hydrogel and application thereof

Abstract

The invention discloses a preparation method of decidua natural killer cells, composite hydrogel and application thereof, and the preparation method of the decidua natural killer cells comprises the following steps: S1, blood cell suspension preparation, S2, albuginea layer cell collection, S3, CD3-cell sorting, S4, CD3-CD56 + cell sorting, and S5, multiplication culture to obtain the required decidua natural killer cells. In addition, the invention further provides decidua natural killer cell composite hydrogel which comprises decidua natural killer cells and hydrogel, and 6 * 10 < 5 >-1 * 10 < 6 > decidua natural killer cells are added into each 1ml of hydrogel; the surface marker of the decidua natural killer cells is CD3-CD56 < + > CD16-CD49a < + >. According to the preparation method of the decidua natural killer cells, the composite hydrogel and the application of the composite hydrogel, the problems that at present, infectious diabetic foot ulcer is difficult to control, and the treatment effect is poor are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of cell therapy, in particular to a preparation method of decidual natural killer cells, a composite hydrogel and their applications. Background Art

[0002] Diabetic foot is a serious complication of diabetes patients caused by long-term hyperglycemia leading to lower limb nerve damage and vascular lesions. Its symptoms include foot ulcers, infections, osteomyelitis, bone and joint destruction, and gangrene. Diabetic wounds are typical complex and chronic non-healing wounds, most of which occur on the feet, forming diabetic foot ulcers (DFU). Once a diabetic foot ulcer is formed, it is prone to infection and then develops into an infected diabetic foot ulcer (IDFU). Infected diabetic foot ulcer is a public health problem and the main cause of non-traumatic amputation.

[0003] According to clinical studies, extensive bacterial infections can lead to diabetic foot ulcers, among which multi-drug resistant facultative anaerobic bacterial infections are the main cause of IDFU. Due to the high drug resistance of aerobic and facultative anaerobic bacteria to antibiotics, the infection is difficult to control, the treatment difficulty increases, and a large proportion of patients have multi-drug resistant infections. The existing main treatment method is to use antibiotics, but due to the high drug resistance of bacteria, multiple antibiotics are ineffective and the treatment effect is poor. There is an urgent need for a method for anti-infection and repair of diabetic wounds. Summary of the Invention

[0004] Aiming at the above defects, the purpose of the present invention is to provide a preparation method of decidual natural killer cells, a composite hydrogel and their applications, so as to solve the problems of difficult control and poor treatment effect of current infected diabetic foot ulcers.

[0005] To achieve this purpose, the present invention adopts the following technical solutions:

[0006] A preparation method of decidual natural killer cells, comprising the following steps:

[0007] S1. Preparation of blood cell suspension: Collect decidual tissues of normal early pregnancy in humans, extract blood cells therefrom, and resuspend them with physiological saline to obtain a blood cell suspension;

[0008] S2. Collection of buffy coat cells: Add the blood cell suspension to a centrifuge tube containing an equal volume of lymphocyte separation medium. After the first centrifugation, take the buffy coat cells, add phosphate buffer solution and mix well, take a sample for counting, and perform the second centrifugation to obtain a known number of buffy coat cells;

[0009] S3. Sort CD3 - cells: At 80 - 90 μl per 1×107 cells, resuspend the buffy coat cells with pre-cooled sorting buffer, add 20 - 30 μl of CD3 magnetic beads per 1 × 10 7 cells, mix well, incubate at 3 - 5 °C for 20 min. After incubation, add three times the volume of sorting buffer, mix well and centrifuge, discard the supernatant, and resuspend with sorting buffer to obtain the sample solution labeled with CD3 magnetic beads; negatively sort to obtain the cell suspension of CD3 - , take samples for counting, centrifuge to obtain CD3 cells with a known number - ;

[0010] S4. Sort CD3 - CD56 + cells: at 80 - 90 μl per 1 × 10 7 cells, resuspend the CD3 - cells with pre-cooled sorting buffer, add 20 - 30 μl of CD56 magnetic beads per 1 × 10 7 cells, mix well, incubate at 3 - 5 °C for 20 min. After incubation, add three times the volume of sorting buffer, mix well and centrifuge, discard the supernatant, and resuspend with sorting buffer to obtain the sample solution labeled with CD56 magnetic beads; positively sort to obtain the cell suspension of decidual natural killer cells of CD3 - CD56 + ;

[0011] S5. Amplification and culture: Mix the cell suspension of CD3 - CD56 + with the complete medium for NK cells, take samples for counting and perform flow cytometry; centrifuge, discard the supernatant, resuspend with the complete medium for NK cells, and the density of the resuspended cells is 1 × 10 6 - 2 × 10 6 cells / ml; inoculate into a T75 culture flask, culture in a carbon dioxide incubator at 37 °C with saturated humidity and 5% CO2 concentration, take samples for counting every two days, and adjust the cell density to 1 × 10 6 - 2 × 10 6 cells / ml according to the counting results, and obtain the required decidual natural killer cells after 14 days of culture.

[0012] Preferably, in step S1, extracting blood cells from the decidual tissue of normal early pregnancy in humans includes the following steps:

[0013] ① Transfer the decidual tissue to a sterilized container, rinse it with physiological saline containing 1% double antibody to remove the residual blood components on the outer surface of the decidual tissue, and remove the blood clots in the decidual tissue with forceps without teeth without damaging the structure of the decidual tissue;

[0014] ② After draining the liquid remaining on the surface of the decidual tissue, cut the tissue into pieces, add anticoagulant buffer to soak and rinse, recover the liquid flowing out during washing, and obtain the first blood component; add anticoagulant buffer again to soak and rinse the decidual tissue, pipette to suspend the tissue, filter with a 200-mesh sieve to remove tissue blocks and blood clots, and recover the washed liquid to obtain the second blood component.

[0015] ③ Centrifuge the first blood component and the second blood component at 750 - 850 g for 10 min, with a rise of 9 and a fall of 6. After centrifugation, remove the supernatant, and the obtained cell pellet is blood cells.

[0016] Preferably, in step S2, the conditions for the first centrifugation are centrifugation at 800 g for 20 min, with the centrifuge rising at 7 and falling at 0; the conditions for the second centrifugation are centrifugation at 800 g for 10 min, with the centrifuge rising at 9 and falling at 9.

[0017] Preferably, in steps S3 and S4, the conditions for centrifugation are centrifugation at 300 g for 10 min, with the centrifuge rising at 9 and falling at 9.

[0018] Preferably, the decidual natural killer cells are preserved in a cryopreservation protective solution at a temperature of -196 °C.

[0019] A decidual natural killer cell composite hydrogel, which comprises decidual natural killer cells and a hydrogel, and 6×10 5 -1×10 6 decidual natural killer cells are added to each 1 ml of the hydrogel;

[0020] The surface markers of the decidual natural killer cells are CD3 - CD56 + CD16 - CD49a + .

[0021] Preferably, the matrix material of the hydrogel is sodium alginate.

[0022] An application of a decidual natural killer cell composite hydrogel in the preparation of a drug for treating infectious diabetic foot ulcers. The decidual natural killer cell composite hydrogel comprises decidual natural killer cells and a hydrogel, and 6×10 5 -1×10 6 decidual natural killer cells are added to each 1 ml of the hydrogel;

[0023] The surface markers of the decidual natural killer cells are CD3 - CD56 + CD16 - CD49a + .

[0024] Preferably, the decidual natural killer cell composite hydrogel needs to be placed in a container and stored sealed at 4°C.

[0025] The technical solution provided by the present invention may include the following beneficial effects:

[0026] Mix decidual natural killer cells with hydrogel, and add 6×10 5 -1×10 6 decidual natural killer cells into each 1 ml of hydrogel. Apply the obtained decidual natural killer cell composite hydrogel to the wound surface of infectious diabetic foot ulcer. Decidual natural killer cells can secrete the antibacterial peptide granulysin to selectively eliminate the multi-drug resistant facultative anaerobic bacteria causing IDFU, achieving the purpose of anti-infection and repair. Brief Description of the Drawings

[0027] Figure 1 It is a diagram of the phenotypic detection results of the decidual natural killer cells of the present invention.

[0028] Figure 2 It is a diagram of the phenotypic detection results of the decidual natural killer cells of the present invention.

[0029] Figure 3 It is a colony statistical chart of Example Group 1 of the present invention.

[0030] Figure 4 It is a colony statistical chart of Example Group 2 of the present invention.

[0031] Figure 5 It is a colony statistical chart of Example Group 3 of the present invention.

[0032] Figure 6 It is a graph of the change in the healing rate of Example Group 4 of the present invention. Detailed Embodiments

[0033] The technical solution of the present invention will be further described below through specific embodiments.

[0034] For ease of understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0035] For those without specific technical or conditions indicated in the embodiments, the techniques or conditions described in the literature in this field or according to the product instructions are followed. For reagents or instruments without the manufacturer indicated, they are all conventional products that can be obtained through commercial purchase.

[0036] The preparation method of decidual natural killer cells includes the following steps:

[0037] S1. Preparation of blood cell suspension: Collect decidual tissues of normal early pregnancy in humans, extract blood cells from them, and resuspend with physiological saline to obtain a blood cell suspension;

[0038] S2. Collection of cells in the buffy coat layer: Add the blood cell suspension to a centrifuge tube containing an equal volume of lymphocyte separation medium. After the first centrifugation, take the cells in the buffy coat layer, add phosphate buffer solution and mix well, take a sample for counting, and perform the second centrifugation to obtain a known number of cells in the buffy coat layer;

[0039] S3. Sorting of CD3 - cells: At a ratio of 80 - 90 μl per 1×10 7 cells, resuspend the cells in the buffy coat layer with pre-cooled sorting buffer, add 20 - 30 μl of CD3 magnetic beads per 1×10 7 cells, mix well, incubate at 3 - 5 °C for 20 min. After incubation, add 3 times the volume of sorting buffer, mix well and centrifuge, discard the supernatant, and resuspend with sorting buffer to obtain a sample solution labeled with CD3 magnetic beads; Perform negative sorting to obtain a cell suspension of CD3 - cells, take a sample for counting, centrifuge to obtain a known number of CD3 - cells;

[0040] S4. Sorting of CD3 - CD56 + cells: At a ratio of 80 - 90 μl per 1×10 7 cells, resuspend the CD3 - cells with pre-cooled sorting buffer, add 20 - 30 μl of CD56 magnetic beads per 1×10 7 cells, mix well, incubate at 3 - 5 °C for 20 min. After incubation, add 3 times the volume of sorting buffer, mix well and centrifuge, discard the supernatant, and resuspend with sorting buffer to obtain a sample solution labeled with CD56 magnetic beads; Perform positive sorting to obtain a cell suspension of decidual natural killer cells of CD3 - CD56 + ;

[0041] S5. Amplification and culture: Mix the cell suspension of CD3 - CD56 + with NK cell complete medium, take a sample for counting and perform flow cytometry detection; Centrifuge, discard the supernatant, resuspend with NK cell complete medium, and the density of the resuspended cells is 1×10 6 - 2×10 6 cells / ml; Inoculate into a T75 culture flask and culture in a carbon dioxide incubator at 37 °C with saturated humidity and 5% CO2 concentration. Take a sample for counting every two days, and adjust the cell density to 1×10 6 - 2×10 6cells / ml, and the desired decidual natural killer cells are obtained after 14 days of culture.

[0042] Currently, diabetic foot ulcer has become one of the main causes of non-traumatic lower limb amputation globally. Once a diabetic foot ulcer forms, it is prone to infection and then develops into an infected diabetic foot ulcer (IDFU). The high drug resistance of aerobic and facultative anaerobic bacteria causing the infection to antibiotics makes the treatment effect of antibiotics poor. Decidual natural killer cells can secrete the antimicrobial peptide granulysin, which can resist infection and facilitate wound repair.

[0043] Collect decidual tissues from normal early pregnancy in humans, that is, from women with normal implantation and development of the embryo in the uterine cavity from 6 to 8 weeks of pregnancy who plan to undergo elective termination of pregnancy. In the early stage of normal pregnancy, decidual natural killer cells account for 50%-90% of decidual lymphocytes and are one of the most numerous immune cells in decidual tissues. Abnormal early pregnancy is manifested as poor development of the gestational sac, abnormal morphology of the germ or fetus, absence of primitive cardiac tube pulsation, or pregnancy occurring outside the uterine cavity. Under abnormal early pregnancy, the cell composition and function of decidual tissues may change, affecting the subsequent extraction of decidual natural killer cells.

[0044] In step S1, blood cells are extracted. At this time, the blood cells contain decidual natural killer cells to obtain a blood cell suspension. In step S2, the blood cell suspension is separated using an equal volume of lymphocyte separation medium, and the buffy coat cells are obtained by centrifugation. The buffy coat cells include lymphocytes and monocytes. Among them, lymphocytes include T cells, B cells, and dNK cells, etc. Through the sorting in steps S3 and S4, according to CD3 - CD56 + natural killer cells are sorted out. Through the above steps, it is ensured that the obtained cells are decidual natural killer cells, which is beneficial to the subsequent preparation of decidual natural killer cell composite hydrogel for use on diabetic foot wounds.

[0045] In step S5, the decidual natural killer cells are amplified and cultured, which is beneficial to the subsequent preparation of the composite hydrogel. Among them, samples are taken and counted every two days, and according to the counting results, NK complete medium is added to adjust the cell density to 1×10 6 -2×10 6 cells / ml. At this cell density, it is beneficial for decidual natural killer cells to proliferate and expand to obtain more decidual natural killer cells. And the cell suspension of CD3 - CD56 + is mixed with NK cell complete medium, samples are taken and counted, and flow cytometry is performed to facilitate subsequent cell sampling according to the counting results to determine the proportion of decidual natural killer cells in the starting cells.

[0046] According to the above operations, the decidual natural killer cells obtained can be mainly of the CD56 + CD16 - phenotype, with low cytotoxicity and strong ability to secrete cytokines, which is beneficial to the secretion of antimicrobial peptide granulysin, selectively clears the multi-drug resistant facultative anaerobic bacteria causing IDFU, and achieves the purpose of anti-infection and repair of infectious diabetic foot ulcers, solving the problems of difficult control and poor treatment effect of current infectious diabetic foot ulcers.

[0047] Preferably, in step S1, extracting blood cells from the decidual tissue of normal early pregnancy in humans includes the following steps:

[0048] ① Transfer the decidual tissue to a sterilized container, rinse it with physiological saline containing 1% double antibiotics to remove the blood components remaining on the outer surface of the decidual tissue, and remove the blood clots in the decidual tissue with forceps without teeth on the premise of not destroying the structure of the decidual tissue;

[0049] ② After draining the liquid remaining on the surface of the decidual tissue, cut the tissue into pieces, add anticoagulant buffer to soak and rinse, recover the liquid flowing out during washing to obtain the first blood component; add anticoagulant buffer again to soak and rinse the decidual tissue, pipette to suspend the tissue, filter it through a 200-mesh sieve to remove tissue blocks and blood clots, and recover the washed liquid to obtain the second blood component;

[0050] ③ Centrifuge the first blood component and the second blood component at 750 - 850 g for 10 min, with an acceleration of 9 and a deceleration of 6. After centrifugation is completed, remove the supernatant, and the obtained cell pellet is the blood cells.

[0051] Specifically, there is a risk of bacterial contamination during the collection of decidual tissue. In step ①, rinsing the decidual tissue with physiological saline containing 1% double antibiotics can inhibit the growth of bacteria and prevent bacterial contamination. Among them, the double antibiotics refer to a mixture of penicillin and streptomycin, with a concentration of 100 U / ml of penicillin and 100 μg / ml of streptomycin.

[0052] In addition, through step ②, two recoveries are carried out to fully wash and recover the blood components in the decidual tissue, ensuring that the blood cells in the tissue can be effectively collected, which is beneficial to obtaining more decidual natural killer cells in subsequent sorting. In step ③, centrifuge at 750 - 850 g for 10 min, with an acceleration of 9 and a deceleration of 6, collect the blood cells in the blood components, and remove the plasma and the anticoagulant buffer added during the washing process.

[0053] It should be noted that the acceleration and deceleration of the centrifuge refer to the process in which the rotational speed of the centrifuge gradually increases from a stationary state to the working rotational speed or gradually decreases from the working rotational speed to the stationary state during the start-up and stop processes. Accelerating at N and decelerating at M means that the acceleration speed is N rpm / min and the deceleration speed is M rpm / min.

[0054] Preferably, in step S2, the conditions for the first centrifugation are centrifugation at 800 g for 20 min, with the centrifuge accelerating at 7 and decelerating at 0; the conditions for the second centrifugation are centrifugation at 800 g for 10 min, with the centrifuge accelerating at 9 and decelerating at 9.

[0055] Specifically, the first centrifugation belongs to density gradient centrifugation to ensure that blood cells can be stratified to obtain the desired buffy coat cells. The purpose of the second centrifugation is to precipitate the buffy coat cells and remove the phosphate buffer. By adding phosphate buffer and mixing evenly for sampling and counting, the number of buffy coat cells in the cell pellet after centrifugation can be determined, which is convenient for determining the dosage of the sorting buffer in subsequent sorting.

[0056] Preferably, in steps S3 and S4, the conditions for centrifugation are centrifugation at 300 g for 10 min, with the centrifuge accelerating at 9 and decelerating at 9.

[0057] In steps S3 and S4, the purpose of the centrifugation operation is to remove unbound magnetic beads and for sampling and counting. The centrifugal force is limited to 300 g to reduce the impact of excessive shear force during centrifugation on the cell viability decline, ensure the activity of decidual natural killer cells therein, and reduce the impact of the activity of decidual natural killer cells on the treatment effect when preparing the composite hydrogel subsequently.

[0058] Preferably, the decidual natural killer cells are preserved in a cryopreservation protective solution at a temperature of -196 °C.

[0059] Specifically, when mixing the decidual natural killer cells with the hydrogel, the decidual natural killer cells used can be fresh decidual natural killer cells obtained after 14 days of culture or cryopreserved and thawed decidual natural killer cells. To ensure the activity of the cells after thawing, the decidual natural killer cells need to be preserved in a cryopreservation protective solution at a temperature of -196 °C. This extremely low temperature can effectively maintain the activity and function of the cells, ensuring that the cells can still maintain their biological characteristics after thawing. During the cryopreservation process, using the cryopreservation protective solution can reduce the formation of ice crystals and protect the cells from cryogenic damage.

[0060] It should be noted that when these decidual natural killer cells are needed, they are taken out from the low-temperature environment, quickly thawed, and resuscitated and cultured under appropriate culture conditions.

[0061] A decidual natural killer cell composite hydrogel, the decidual natural killer cell composite hydrogel comprises decidual natural killer cells and a hydrogel, and 6×10 5 -1×10 6 decidual natural killer cells are added to each 1 ml of the hydrogel;

[0062] The surface marker of the decidual natural killer cells is CD3- CD56 + CD16 - CD49a + 。

[0063] Specifically, the decidual natural killer cell composite hydrogel includes decidual natural killer cells and a hydrogel. The hydrogel is formed by chemical or physical cross-linking of one or more hydrophilic high molecular polymers to form a three-dimensional cross-linked network structure, which can absorb a large amount of water and swell without dissolving. It can serve as a carrier for decidual natural killer cells, does not affect the action of decidual natural killer cells on the wound surface, and is similar to biological tissues, which is beneficial to wound healing. Add 6×10 5 -1×10 6 decidual natural killer cells per 1 ml of the hydrogel to ensure its effective biological function on the wound surface. If the concentration is too low, the treatment effect is not obvious, while if the concentration is too high, it will cause excessive immune responses or other adverse effects. Decidual natural killer cells are living cells with biological activity and can continuously secrete antibacterial peptide granulysin to play a role. Antibacterial peptide granulysin belongs to protein polypeptide substances and there is a problem of degradation in vitro. Using the hydrogel in combination with decidual natural killer cells for treatment can ensure that decidual natural killer cells can secrete antibacterial peptide granulysin to act on the wound surface and avoid the degradation of antibacterial peptide granulysin in vitro, which may not guarantee the treatment effect. Moreover, the antibacterial peptide granulysin used to exert the antibacterial function is derived from decidual natural killer cells and is naturally synthesized by the human body, with the characteristics of no cytotoxicity and specific bactericidal activity.

[0064] In addition, the surface markers of the decidual natural killer cells are CD3 - CD56 + CD16 - CD49a + , CD3 is a specific marker on the surface of T cells. As natural killer cells, dNK cells do not express CD3, so their surface markers are CD3 - , which distinguishes decidual natural killer cells from T cells; CD56 is one of the specific markers of natural killer cells, and CD16 is another marker of natural killer cells, mainly related to antibody-dependent cytotoxicity. Decidual natural killer cells mainly perform immune regulation and tissue repair functions in the decidual tissue rather than antibody-dependent cytotoxicity. Therefore, the CD16 expression of decidual natural killer cells is relatively low, which is CD16 - ; Decidual natural killer cells are mainly of the CD56 + CD16 - phenotype, with low cytotoxicity and strong ability to secrete cytokines, which is beneficial to the secretion of antibacterial peptide granulysin. CD3 - CD56 +CD16 - CD49a + The surface markers of ensure that the resulting cells are human decidual natural killer cells, which can secrete the antibacterial peptide granulysin and are required for the present invention.

[0065] Preferably, the matrix material of the hydrogel is sodium alginate.

[0066] Specifically, sodium alginate is a natural polysaccharide extracted from brown algae such as kelp or sargassum, with high water absorption and water-locking properties, relatively mild gelation conditions, low toxicity and degradability. The COOH in its polymer chain - and Ca 2+ Under mild conditions, a hydrogel with stable structure can be obtained, ensuring that the decidual natural killer cells bound therein can effectively act on the infectious wound surface and reduce the constraints on cell therapy.

[0067] Use of a decidual natural killer cell composite hydrogel in the preparation of a drug for treating infectious diabetic foot ulcers, the decidual natural killer cell composite hydrogel comprising decidual natural killer cells and a hydrogel, with 6×10 5 -1×10 6 decidual natural killer cells added to each 1 ml of the hydrogel;

[0068] The surface markers of the decidual natural killer cells are CD3 - CD56 + CD16 - CD49a + .

[0069] Infectious diabetic foot ulcer is a public health problem and one of the main causes of non-traumatic amputation. Among them, multi-drug resistant facultative anaerobic bacterial infection is the main cause of IDFU. Common pathogenic bacteria include Staphylococcus aureus, Streptococcus, and Pseudomonas aeruginosa. The high drug resistance of pathogenic bacteria makes the treatment of IDFU more difficult. Hyperglycemia is conducive to the growth and reproduction of bacteria, making the infection more difficult to control. The drug resistance of bacteria renders multiple antibiotic treatments ineffective and the treatment effect is poor. To improve the treatment effect, the present invention proposes the application of decidual natural killer cell composite hydrogel in the preparation of drugs for treating infectious diabetic foot ulcers. Natural killer cells can kill a variety of tumor cells and pathogen-infected cells without prior sensitization. In the first three months of pregnancy in women, decidual natural killer (dNK) cells are the immune cells with the largest proportion in the decidual microenvironment, playing multiple roles such as resisting microbial infection, maintaining immune tolerance, and promoting embryonic growth and development. Existing studies have shown that in the case of Listeria infection, human decidual natural killer cells can control the proliferation of Listeria. dNK cells can express a large amount of antibacterial peptide granulysin (GNLY) and selectively transfer it into infected trophoblast cells through special nanotube structures, selectively killing Listeria intracellularly without killing trophoblast cells. Correspondingly, the decidual natural killer cells in the decidual natural killer cell composite hydrogel can selectively eliminate multi-drug resistant facultative anaerobic bacteria causing IDFU, prevent further infection of the wound surface, maintain the activity of cells on the wound surface, enable cells to perform their functions more efficiently, and promote wound repair. The application of decidual natural killer cells reduces the use of antibiotics and the impact of high bacterial drug resistance on treatment. While protecting the cells of the wound surface itself, it prevents further infection by pathogenic bacteria, solving the problem that the current infectious diabetic foot ulcer is difficult to control and the treatment effect is poor.

[0070] Preferably, the decidual natural killer cell composite hydrogel needs to be placed in a container and stored sealed at 4°C.

[0071] The decidual natural killer cells in the decidual natural killer cell composite hydrogel are live cells. When in use, they can continuously secrete antibacterial peptide granulysin to play a role. Storing sealed at 4°C ensures that it is not contaminated by the outside world and ensures the activity of the cells.

[0072] The technical solution of the present invention will be further described below through specific embodiments.

[0073] (1) Preparation of hydrogel dressing:

[0074] The formula of the hydrogel is: deionized water, sodium alginate, glycerol, tartaric acid, ethylenediaminetetraacetic acid, polyvinyl alcohol - 124, aluminum glycinate, partially neutralized sodium polyacrylate, tetra-arm polyethylene glycol - epoxide.

[0075] Preparation method:

[0076] (1) Add 20 ml of deionized water to a beaker, heat it to 60 °C, then add 0.5 g of polyvinyl alcohol - 124 and stir for 30 minutes. Raise the temperature to 80 °C and continue stirring for 1 hour until the particles are completely dissolved, obtaining an aqueous solution of polyvinyl alcohol - 124, marked as solution A for later use.

[0077] (2) Weigh 5 g of glycerol, 0.02 g of glycine aluminum, 0.01 g of ethylenediaminetetraacetic acid, and 0.5 g of partially neutralized sodium polyacrylate, and add them to the beaker in sequence. Stir evenly at room temperature and mark it as solution B.

[0078] (3) Weigh 0.02 g of tartaric acid and 0.5 g of tetra - arm polyethylene glycol - epoxide, add 22 g of deionized water and stir for 15 minutes to dissolve and mix evenly, marked as solution C.

[0079] (4) Mix solution A and solution C, add 1.5 g of sodium alginate, and stir at room temperature for at least 1 hour until there are no solid particles. Add solution B to the 20 mL PVA aqueous solution prepared above and mix, and at the same time add SA and stir continuously at room temperature until there is no powder in the mixture (stir for at least 1 h). This mixture is the aqueous phase mixture. Finally, mix and stir the oil - phase mixture and the aqueous - phase mixture at room temperature for 30 min to obtain the hydrogel.

[0080] (II) Preparation of decidual natural killer cells:

[0081] S1. Preparation of blood cell suspension:

[0082] ① Place the collected human normal early - pregnancy decidual tissue in physiological saline containing 1% double - antibody, store it at 4 °C, and transport it back to the laboratory at low temperature. Transfer the decidual tissue to the clean workshop through the transfer window and operate on the decidual tissue in the biological safety cabinet. Prepare a sterilized stainless - steel lunch box in the biological safety cabinet, transfer the decidual tissue to the stainless - steel lunch box, add 500 ml of physiological saline containing 1% double - antibody to rinse the residual blood components on the outer surface of the tissue, and use a non - toothed forceps to remove the blood clots in the tissue without damaging the tissue structure as much as possible.

[0083] ② Use a non - toothed forceps to pick up the washed decidual tissue, drain the residual liquid on the surface, and transfer it to a new sterile stainless - steel lunch box. Use tissue scissors to cut the decidual tissue into pieces of 2 mm * 2 mm * 2 mm in size, add 100 ml of anticoagulant buffer to soak and rinse the tissue, and recover the blood components flowing out during this washing into a 225 ml centrifuge bottle with a 50 ml centrifuge tube to obtain the first blood component. Add 100 ml of anticoagulant buffer again to soak and rinse the tissue, pipette to suspend the tissue, and remove the tissue blocks and blood clots through a 200 - mesh filter, and recover the blood components flowing out this time into a 225 ml centrifuge bottle to obtain the second blood component;

[0084] ③Centrifuge the first blood component and the second blood component at 800 g for 10 min, with a ramp rate of 9 and a deceleration rate of 6. After centrifugation, remove the centrifugation supernatant with a pipette. The obtained cell pellet is blood cells. Resuspend the cell pellet with 60 ml of normal saline and combine them into one tube.

[0085] S2. Collect buffy coat cells: Dispense 60 ml of lymphocyte separation medium into 50-ml centrifuge tubes at 20 ml / tube. Slowly add 20 ml of cell suspension along the tube wall to each tube and perform the first centrifugation at 800 g for 20 min, with a ramp rate of 7 and a deceleration rate of 0. After centrifugation, aspirate the buffy coat cells with a Pasteur pipette into a 50-ml centrifuge tube, add 45 ml of phosphate buffer solution and mix well. Take a sample for counting and perform the second centrifugation at 800 g for 10 min, with a ramp rate of 9 and a deceleration rate of 9, to obtain a known number of buffy coat cells.

[0086] S3. Sort CD3 - cells: Resuspend the cell pellet by adding 80 μl of pre-cooled 4°C sorting buffer per 1×10 7 cells. Add 20 μl of CD3 magnetic beads per 1×10 7 cells, mix well, and incubate at 4°C for 20 min. After incubation, add 3 times the volume of sorting buffer, mix well, centrifuge at 300 g for 10 min, with a ramp rate of 9 and a deceleration rate of 9, discard the supernatant, and resuspend the cell pellet with sorting buffer to obtain a sample solution labeled with CD3 magnetic beads. Pass the sample solution labeled with CD3 magnetic beads through an LS column and wash the LS column 2 times with 6 ml of sorting buffer. Collect the outflowing cell suspension, add sorting buffer to make it up to 10 ml, take a sample for counting, centrifuge at 300 g for 10 min, with a ramp rate of 9 and a deceleration rate of 9, to obtain a known number of CD3 - cells.

[0087] S4. Sort CD3 - CD56 + cells: Resuspend the cell pellet by adding 80 μl of pre-cooled 4°C sorting buffer per 1×10 7 cells. Add... (The text seems incomplete here) 7Add 20 μl of CD56 magnetic beads to the cells, mix well, and incubate at 4°C for 20 min. After incubation, add three times the volume of sorting buffer, mix well, centrifuge at 300 g for 10 min (ramp up and down at a rate of 9), discard the supernatant, and resuspend the cell pellet in sorting buffer to obtain the CD56 magnetic bead-labeled sample solution. Pass the CD56 magnetic bead-labeled sample solution through an LS column, and wash the LS column twice with 6 ml of sorting buffer. Replace the labeled positive collection tube under the collection port, remove the LS column from the magnetic stand, ensure that the positive cell collection tube is aligned with the collection port, add 5 ml of pre-cooled sorting buffer to the inner wall of the LS column, collect the flowing cell suspension, quickly load it into the piston of a sterile syringe, push the piston to wash the LS column, and collect the flowing cell suspension, which is the cell suspension of decidual natural killer cells of CD3 - CD56 + .

[0088] S5. Amplification and culture: Mix the cell suspension of CD3 - CD56 + with 20 ml of NK cell complete medium, take a sample for counting and perform flow cytometry detection; centrifuge at 300 g for 10 min (ramp up and down at a rate of 9), discard the supernatant, resuspend the cell pellet in NK cell complete medium, and control the cell density after resuspension to be between 1×10 6 -2×10 6 cells / ml. Inoculate a T75 culture flask and place it in a carbon dioxide incubator with a saturated humidity of 37°C and a 5% CO2 concentration. Take a sample for counting every 2 days, and adjust the cell density to 1×10 6 cells / ml according to the counting results by adding an appropriate amount of NK complete medium. After culturing for 14 days, the required decidual natural killer cells are obtained.

[0089] Collect 1×10 6 cells in a flow cytometry tube, add 3 ml of PBS, mix well, centrifuge at 300 g for 5 min, discard the supernatant, resuspend the cell pellet in 100 μl of PBS, add antibodies CD3, CD56, CD16, and CD49a, incubate at room temperature for 15 min, then add 3 ml of BPS for washing, centrifuge at 300 g for 5 min, discard the supernatant, resuspend the cell pellet in 200 μl of PBS, and use a flow cytometer for detection. The detection results are as shown in Figure 1 and Figure 2 .

[0090] The detected cell phenotype is CD3 - CD56 + CD16 - CD49a + .

[0091] (III) Preparation of decidual natural killer cell composite hydrogel: Add 6×10 5Mix with decidual natural killer cells to obtain a decidual natural killer cell composite hydrogel.

[0092] Example Group 1

[0093] This example group detected the inhibitory effect of the hydrogel containing decidual natural killer cells on Staphylococcus aureus, and was specifically divided into an experimental group and a control group.

[0094] Under A-level environment, take 1 ml of Staphylococcus aureus bacterial liquid with a concentration of 50 CFU / mL, and inoculate it on a sterile tryptic soy agar (TSA) solid medium by the spreading method.

[0095] Experimental group: Take an appropriate amount of the prepared decidual natural killer cell composite hydrogel and evenly apply it on the tryptic soy agar (TSA) solid medium inoculated with Staphylococcus aureus;

[0096] Control group: Take the hydrogel prepared without decidual natural killer cells with the same volume as the experimental group and evenly apply it on the tryptic soy agar (TSA) solid medium inoculated with Staphylococcus aureus.

[0097] After culturing in a 37°C constant temperature incubator for 7 days, observe and count the number of colonies on the TSA medium. The results are as Figure 3 shown.

[0098] Example Group 2

[0099] This example group detected the inhibitory effect of the hydrogel containing decidual natural killer cells on Pseudomonas aeruginosa, and was specifically divided into an experimental group and a control group.

[0100] Under A-level environment, take 1 ml of Pseudomonas aeruginosa bacterial liquid with a concentration of 50 CFU / mL, and inoculate it on a sterile tryptic soy agar (TSA) solid medium by the spreading method.

[0101] Experimental group: Take an appropriate amount of the prepared decidual natural killer cell composite hydrogel and evenly apply it on the tryptic soy agar (TSA) solid medium inoculated with Pseudomonas aeruginosa;

[0102] Control group: Take the hydrogel prepared without decidual natural killer cells with the same volume as the experimental group and evenly apply it on the tryptic soy agar (TSA) solid medium inoculated with Pseudomonas aeruginosa.

[0103] After culturing in a 37°C constant temperature incubator for 7 days, observe and count the number of colonies on the TSA medium. The results are as Figure 4 shown.

[0104] Example Group 3

[0105] This example group detected the inhibitory effect of the hydrogel containing decidual natural killer cells on Escherichia coli, and was specifically divided into an experimental group and a control group.

[0106] Under A-level environment, 1 ml of Escherichia coli bacterial solution with a concentration of 50 CFU / mL was taken and inoculated on a sterile LB solid medium by the coating method.

[0107] Experimental group: An appropriate amount of the prepared decidual natural killer cell composite hydrogel was taken and evenly smeared on the LB solid medium inoculated with Escherichia coli.

[0108] Control group: A hydrogel of the same volume as the experimental group, prepared without decidual natural killer cells, was taken and evenly smeared on the LB solid medium inoculated with Escherichia coli.

[0109] After culturing in a 37°C constant temperature incubator for 7 days, the number of colonies on the LB solid medium was observed and counted. The results are as Figure 5 shown.

[0110] Example group 4

[0111] This example group detected the wound repair effect of the hydrogel containing decidual natural killer cells on bacterial-infected wounds.

[0112] SPF-grade male SD rats weighing 160 - 180 g were selected and fed with a high-fat diet (60% of energy from fat) for 4 weeks. At the 5th week, STZ was injected intraperitoneally at 35 - 40 mg / kg. 10% glucose solution was given 6 hours after injection to prevent acute hypoglycemia. Fasting blood glucose was measured at 72 h and 144 h after injection. Stable blood glucose ≥ 16.7 mmol / L for 2 times was regarded as diabetic rats.

[0113] After stabilizing for four weeks, the diabetic rats were anesthetized with isoflurane and the skin was excised to the fascia layer with a biopsy punch (diameter 8 mm). Two wounds were taken from the same rat, and the distance between the wounds was more than 4 cm. 1 ml of a bacterial solution of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli mixed at a ratio of 1:1:1 was dropped on the wound, and the concentration was 1×10 6 CFU / ml.

[0114] The two wounds of the same rat were divided into an experimental group and a control group. The experimental group was smeared with the hydrogel containing decidual natural killer cells, and the control group was smeared with the hydrogel without decidual natural killer cells.

[0115] The drug was administered every 3 days. On days 0, 3, 7, and 14, the longest transverse and longitudinal diameters of the wound were measured with a steel ruler (or an opaque ruler) and photographed (including the ruler) to calculate the wound healing rate. Wound healing percentage 1 = (transverse diameter of the wound on day 0 - transverse diameter of the wound on day n) / transverse diameter of the wound on day 0 × 100%; wound healing percentage 2 = (longitudinal diameter of the wound on day 0 - longitudinal diameter of the wound on day n) / transverse diameter of the wound on day 0 × 100%. The wound healing rate was calculated as the product of wound healing percentage 1 and wound healing percentage 2. The results are shown in Table 1. Figure 6 shown.

[0116] Depend on Figure 1 and Figure 2 It can be seen that the surface marker of the decidual natural killer cells obtained is CD3 - CD56 + CD16 - CD49a + , indicating that the cells obtained according to the preparation method of the present invention are decidual natural killer cells, which can secrete the antimicrobial peptide granulysin.

[0117] According to Example Groups 1-3, the results obtained are as follows Figures 3 to 5 As shown, it can be seen that the hydrogel containing decidual natural killer cells can effectively inhibit the pathogenic bacteria of IDFU, Staphylococcus aureus and Pseudomonas aeruginosa, as well as Escherichia coli. Figure 3 It can be seen that the inhibition rate of the hydrogel containing decidual natural killer cells on Staphylococcus aureus reached 90.19%; Figure 4 It can be seen that the inhibition rate of the hydrogel containing decidual natural killer cells on Pseudomonas aeruginosa reached 84.91%; Figure 5 It can be seen that the inhibition rate of the hydrogel containing decidual natural killer cells on Escherichia coli reached 91.84%.

[0118] Figure 6 This is a graph showing the changes in wound healing rate in diabetic mice. The experimental group was treated with hydrogel containing decidual natural killer cells. Compared with the control group treated with hydrogel without decidual natural killer cells, it can be seen that the wound healing rate of the experimental group was higher than that of the control group, and the repair speed was faster than that of the control group, which shows that the bacterial infection was effectively inhibited to achieve the purpose of anti-infection.

[0119] The technical principle of the present invention is described above in conjunction with specific embodiments. These descriptions are only for explaining the principle of the present invention and cannot be interpreted as limiting the scope of protection of the present invention in any way. Based on the explanations herein, those skilled in the art can associate other specific implementations of the present invention without paying creative labor, and these methods will fall within the scope of protection of the present invention.

Claims

1. A method for preparing decidual natural killer cells, characterized in that: It includes the following steps: S1. Preparation of blood cell suspension: Collect decidual tissues of normal early pregnancy in humans, extract blood cells therefrom, and resuspend them with physiological saline to obtain a blood cell suspension; S2. Collection of white membrane layer cells: Add the blood cell suspension into a centrifuge tube containing the same volume of lymphocyte separation solution. After the first centrifugation, take the white membrane layer cells, add phosphate buffer solution and mix well, take a sample for counting, and perform the second centrifugation to obtain a known number of white membrane layer cells; S3. Sort CD3 - Cells: Resuspend the buffy coat cells at 80 - 90 μl per 1 × 10 7 cells with pre-cooled sorting buffer, add 20 - 30 μl of CD3 magnetic beads per 1 × 10 7 cells, mix well, incubate at 3 - 5 °C for 20 min. After incubation, add three times the volume of sorting buffer, mix well and centrifuge, discard the supernatant, and resuspend with sorting buffer to obtain the sample solution labeled with CD3 magnetic beads; negatively sort to obtain the cell suspension of CD3 - , take samples for counting, centrifuge to obtain the CD3 - cells with known quantity; S4, sorting CD3 - CD56 + For cells: 80-90 μl / 1×10 7 cells, resuspend the CD3 - Add 20-30 μl of CD56 magnetic beads per 1×10 cells 7 cells, mix well, incubate at 3-5℃ for 20min, add 3 times sorting buffer after incubation, mix well, centrifuge, remove supernatant, and resuspend with sorting buffer to obtain sample solution labeled with CD56 magnetic beads; positive sorting obtains CD3 - CD56 + A cell suspension of decidual natural killer cells; S5. Amplification culture: Mix the cell suspension of CD3 - CD56 + with the complete medium for NK cells, take samples for counting and perform flow cytometry detection; centrifuge, discard the supernatant, resuspend with the complete medium for NK cells, and the cell density after resuspension is 1×10 6 -2×10 6 cells / ml; inoculate into a T75 culture flask, culture in a carbon dioxide incubator at 37°C with saturated humidity and 5% CO2 concentration, take samples for counting every two days, and adjust the cell density to 1×10 6 -2×10 6 cells / ml according to the counting results. After culturing for 14 days, the required decidual natural killer cells are obtained.

2. The preparation method of decidual natural killer cells according to claim 1, characterized in that: In step S1, the extraction of blood cells from the decidual tissues of normal early pregnancy in humans includes the following steps: ① Transfer the decidual tissue to a sterilized container, rinse it with physiological saline containing 1% double antibody to remove the residual blood components on the outer surface of the decidual tissue, and remove the blood clots in the decidual tissue with a non-toothed forceps without damaging the structure of the decidual tissue; ② After draining the residual liquid on the surface of the decidual tissue, cut the tissue into pieces, add anticoagulant buffer solution to soak and rinse, recover the liquid flowing out during washing to obtain the first blood component; add anticoagulant buffer solution again to soak and rinse the decidual tissue, pipette to suspend the tissue, filter it through a 200-mesh sieve to remove tissue blocks and blood clots, and recover the washed liquid to obtain the second blood component; ③ Centrifuge the first blood component and the second blood component at 750 - 850 g for 10 min, with a rise of 9 and a fall of 6. After centrifugation is completed, remove the supernatant, and the obtained cell precipitate is blood cells.

3. The preparation method of decidual natural killer cells according to claim 1, characterized in that: In step S2, the conditions for the first centrifugation are centrifugation at 800 g for 20 min, with the centrifuge rising at 7 and falling at 0; the conditions for the second centrifugation are centrifugation at 800 g for 10 min, with the centrifuge rising at 9 and falling at 9.

4. The preparation method of decidual natural killer cells according to claim 1, characterized in that: In steps S3 and S4, the conditions for centrifugation are centrifugation at 300 g for 10 min, with the centrifuge rising at 9 and falling at 9.

5. The method for preparing decidual natural killer cells according to claim 1, characterized in that: The decidual natural killer cells are stored in a cryopreservation protective solution at a temperature of -196 °C.

6. A decidual natural killer cell composite hydrogel, characterized in that: The decidual natural killer cell composite hydrogel includes decidual natural killer cells and a hydrogel, and 6×10 5 -1×10 6 decidual natural killer cells are added to each 1 ml of the hydrogel; The surface markers of the decidual natural killer cells are CD3 - CD56 + CD16 - CD49a + 。 7. A decidual natural killer cell composite hydrogel according to claim 6, characterized in that: The matrix material of the hydrogel is sodium alginate.

8. Use of a decidua natural killer cell composite hydrogel in the preparation of a drug for treating infectious diabetic foot ulcers, characterized in that: The decidual natural killer cell composite hydrogel includes decidual natural killer cells and a hydrogel, and 6×10 5 -1×10 6 decidual natural killer cells are added to each 1 ml of the hydrogel; The surface markers of the decidual natural killer cells are CD3 - CD56 + CD16 - CD49a + 。 9. Use of the decidual natural killer cell composite hydrogel according to claim 8 in the preparation of a medicament for treating infectious diabetic foot ulcers, characterized in that: The decidual natural killer cell composite hydrogel needs to be placed in a container and stored sealed at 4 °C.

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