A chemokine CCL28 immunomodulator for local injection in anti-rejection reaction of tissue cell transplantation and its application

Abstract

The present invention discloses a chemokine CCL28 immunomodulator for local injection in tissue cell transplantation anti-rejection reaction and its application, wherein the chemokine CCL28 immunomodulator comprises chemokine CCL28 and an adjuvant and / or carrier of chemokine CCL28. The adjuvant and / or carrier is a hydrogel, and the hydrogel comprises methacryloyl gelatin and a photoinitiator, and the concentration is 8-10% W / V of the mass of methacryloyl gelatin / the volume of PBS containing the photoinitiator. The chemokine CCL28 immunomodulator of the present invention can significantly improve the rejection reaction after transplantation by local injection of the transplant in tissue cell transplantation anti-rejection reaction.

Description

Technical Field

[0001] The present invention relates to the field of transplantation anti-rejection, and in particular to a chemokine CCL28 immunomodulator for local injection in transplantation anti-rejection reaction and its application. Background Art

[0002] Transplantation is an effective treatment method widely used in clinical practice, and postoperative immunosuppressive therapy is an essential measure to avoid immune rejection. Continuous treatment with immunosuppressants targeting T cell killing has always been a common strategy to inhibit transplant rejection, but the use of immunosuppressants may cause serious side effects, such as hepatotoxicity, hypertension, infection, and malignant tumors.

[0003] In recent years, Treg immunotherapy for graft protection has developed rapidly. It can form graft antigen-specific immune tolerance without destroying the immune balance, in the hope of minimizing drug toxicity and achieving long-term protection of the graft. However, the technology still has technical bottlenecks such as the small number of Treg cells in vivo and the difficulty of in vitro expansion, as well as how to gather a sufficient number of regulatory T cells locally in the graft to form graft protection and induce immune tolerance.

[0004] Therefore, there is currently a lack of a technical solution that can stably maintain a sufficient number of regulatory T cells locally aggregated in the graft to solve the problem of transplant anti-rejection reaction. Summary of the invention

[0005] The technical problem to be solved by the present invention is that a sufficient number of regulatory T cells cannot be locally gathered in the transplant, and the deficiencies and defects mentioned in the above background technology are overcome to provide a chemokine CCL28 immunomodulator for local injection in the anti-rejection reaction of tissue cell transplantation and its application.

[0006] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0007] A chemokine CCL28 immunomodulator for local injection in anti-rejection reaction of tissue cell transplantation, wherein the chemokine CCL28 immunomodulator comprises chemokine CCL28.

[0008] The local use of immunomodulators containing the chemokine CCL28 in the graft during transplant anti-rejection reaction can accurately and locally release CCL28 and recruit regulatory T cells locally in the graft.

[0009] The chemokine CCL28 immunomodulator comprises chemokine CCL28.

[0010] Preferably, the chemokine CCL28 immunomodulator comprises an auxiliary material and / or a carrier of chemokine CCL28.

[0011] Preferably, the auxiliary material and / or carrier is a hydrogel, and the hydrogel includes methacryloyl gelatin and a photoinitiator, and the concentration of the hydrogel is 8-10% (W / V) of the mass of the methacryloyl gelatin / the volume of PBS containing the photoinitiator.

[0012] The use of hydrogel allows CCL28 to be slowly released only locally, which can better increase the drug concentration in the local area of ​​the skin graft, enhance the ability to recruit local immunosuppressive cells, and improve the local anti-rejection ability of the skin graft. At the same time, it reduces the immunosuppression caused by its large-scale diffusion to the surrounding normal tissues, and alleviates the adverse effects on normal tissues.

[0013] Preferably, the tissue cell transplantation to prevent rejection is a xenogeneic skin transplantation, wherein the transplant is pig skin tissue.

[0014] The chemokine CCL28 immunomodulator of the present application can be used for a variety of xenotransplantation, but using pig organs as a source of human transplant organs is a way to solve the current severe shortage of human organs. The physiological tissues of pig organs such as the heart, kidneys, lungs, pancreatic islets, and corneas are similar to those of humans; there are relatively few diseases that are cross-transmitted between pigs and humans, which greatly reduces the risk of cross-species infection.

[0015] Under the same technical concept, the present invention also provides an application of a chemokine CCL28 immunomodulator for local injection in anti-rejection reaction of tissue cell transplantation. The chemokine CCL28 immunomodulator is locally injected at the transplant site of the tissue cell transplantation environment, and participates in regulating the local immune microenvironment of the transplant in the anti-rejection reaction of tissue cell transplantation.

[0016] Preferably, the regulation of the local immune microenvironment of the transplant is to transform the immune-promoting microenvironment dominated by effector T cells into an immunosuppressive microenvironment mediated by Treg (regulatory T cells).

[0017] Preferably, the method for preparing the chemokine CCL28 immunomodulator comprises:

[0018] (1) Prepare initiator standard solution;

[0019] (2) Preparation of auxiliary material solution: Take GelMA reagent, add initiator standard solution, heat and shake to obtain GelMA auxiliary material solution;

[0020] (3) Preparation of a mixture of chemokines and excipients: Take the chemokine CCL28, resuspend it in a preheated GelMA excipient solution, and solidify it under a light source to obtain a chemokine CCL28 immunomodulator.

[0021] Preferably, the light source curing time in step (3) is 12-18 seconds.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The chemokine CCL28 immunomodulator of the present invention can significantly improve the rejection reaction after transplantation by local injection into the transplanted tissue cell to prevent rejection reaction.

[0024] (2) The present invention uses a method of locally injecting chemokines into the graft to stably maintain a sufficient number of regulatory T cells locally aggregated in the graft; the chemokine CCL28 is used for the first time in skin transplantation, which can increase immunosuppressive cells Treg, reduce the local total CD4+T cell content, and reverse the local immune microenvironment of the graft.

[0025] (3) The present application combines chemokines with excipients. The excipient is hydrogel, and the use of hydrogel allows CCL28 to be slowly released only locally, thereby better increasing the drug concentration in the local area of ​​the skin transplant, enhancing the ability to recruit local immunosuppressive cells, and improving the local anti-rejection ability of the skin transplant. At the same time, it reduces the immunosuppression caused by its large-scale diffusion to the surrounding normal tissues, and mitigates the adverse effects on normal tissues.

[0026] (4) Compared with the method of using immunosuppressants, the local use of chemokines in the present invention has a better effect on the skin transplantation model, can ensure that a sufficient number of regulatory T cells are locally accumulated in the transplant, and has lower toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 is an immunohistochemical analysis of the pig skin xenograft tissue of the immunodeficient mouse of Example 1;

[0029] Figure 2 The photographs and immunohistochemical analysis of the local rejection reaction of the skin xenograft mice after human immune reconstitution in Example 1;

[0030] Figure 3 The photographs and immunohistochemical analysis of Example 1 show that the rejection reaction of skin xenografts was alleviated after subcutaneous local injection of CCL28;

[0031] Figure 4 is a graft survival curve diagram of Comparative Example 1;

[0032] Figure 5 This is the immunofluorescence staining display of Comparative Example 1;

[0033] Figure 6 This is the immunohistochemical staining display of Comparative Example 1;

[0034] Figure 7 is a curve diagram of the sustained release volume of the hydrogel of Example 2;

[0035] Figure 8 is a graft survival curve diagram of Example 1 and Example 2;

[0036] Fig. 9 It is the immunofluorescence staining display figure of Example 1 and Example 2;

[0037] Fig.10 It is a schematic diagram of the regulatory T cell recruitment multiples of Example 1 and Example 2. DETAILED DESCRIPTION

[0038] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0039] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0040] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0041] Embodiment 1:

[0042] The chemokine CCL28 immunomodulator used in this example includes chemokine CCL28.

[0043] The application of locally injected chemokine CCL28 immunomodulator in anti-rejection of tissue cell transplantation includes the following steps:

[0044] The first step is the transplantation of xenogeneic skin: Immunohistochemistry confirmed the successful xenotransplantation of pig skin in immunodeficient mice

[0045] The experimental steps are as follows:

[0046] We performed skin transplantation using NSG mice as recipients and skin tissue from SPF-grade newborn pigs as donors.

[0047] 1. After anesthetizing the newborn pig with 3% pentobarbital (3 mL / kg), shave the hair on the back, wash with warm water, disinfect with alcohol, and kill with blood drawn from the heart. Fix the pig on a small operating table in the clean bench, and disinfect the back skin again with alcohol and complex iodine.

[0048] 2. Select the appropriate width of the skin grafting machine, adjust the thickness to 200 μm, and remove several skin grafts from the back of newborn pigs and store them in pre-cooled saline;

[0049] 3. Soak the removed skin in complex iodine for 30 seconds, then wash twice with pre-cooled saline;

[0050] 4. Use sterile surgical instruments to trim the removed skin into 1x1cm size, remove the thin or damaged parts, and store in pre-cooled saline;

[0051] 5. Mice aged 6-8 weeks were anesthetized with 1% barbiturate (6 μL / g), the hair on the back was removed, and the mice were fixed on a small operating table in a clean bench and disinfected with iodine 3 times;

[0052] 6. Use sterile surgical instruments to remove skin about 1x1cm in size from the lower back of the mouse;

[0053] 7. Place the prepared xenogeneic skin graft on the skin defect on the lower back of the mouse, suture the four corners with cosmetic thread, and then glue the four sides with biological glue to ensure that the xenogeneic skin graft is aligned with the edge of the mouse skin;

[0054] 8. Use cotton and bandage to apply a pressure bandage to the graft on the mouse's back to fix it;

[0055] 9. After surgery, the mice were placed on a warm pad until they fully woke up. After xenogeneic skin transplantation, the mice were housed in single cages to prevent mutual damage to the skin grafts.

[0056] 10. Gentomycin (800 IU / mL, 5 μL / g) was injected intraperitoneally every day for 7 days after surgery to prevent infection; 4-5 weeks after surgery, the survival of the skin graft was evaluated to determine whether the model was successfully established. Stable xenogeneic skin grafts showed a smaller transplant area than when transplanted, and the appearance of xenogeneic skin grafts was black. When the skin color of the transplanted site turned lighter to pink and the edges contracted, it was considered that the graft was completely lost.

[0057] The experimental results are as follows Figure 1 As shown, the control group is the solvent PBS control group without CCL28, and the CCL28 group is the experimental group with the addition of chemokine CCL28 immunomodulator. Through immunohistochemical analysis, the survival of pig skin transplanted into NSG mice was good, and the pig skin was well connected with the surrounding tissues after transplantation, indicating that the immunodeficient mouse model of pig skin xenotransplantation was successfully established.

[0058] Step 2: Forming the basis for rejection: Immunohistochemistry confirmed that local rejection occurred in skin xenograft mice after human immune reconstitution

[0059] Normal human peripheral blood samples were obtained from the Third Xiangya Hospital of Central South University with approval from the Ethics Committee. Informed consent was obtained for all collected samples.

[0060] 1. Collect 50 mL of normal peripheral blood sample using an anticoagulant tube and perform subsequent operations in a clean bench;

[0061] 2. Preparation of stem cell protection solution: Take a clean and sterile 50mL centrifuge tube, weigh 2.5g BSA powder with an electronic balance, take a new bottle of 500mL 1×DPBS, use a 10mL pipette electric gun in the clean bench to draw 30mL of 1×DPBS and add it to the above 50mL centrifuge tube, shake vigorously and mix, then add 2mL 0.5M EDTA, invert and mix, use a sterile 50mL syringe and 0.22μM filter to filter the solution back into the above 1×DPBS bottle, invert and mix to make the stem cell protection solution (Buffer);

[0062] 3. Isolation of peripheral blood mononuclear cells (PBMC): Take 4 50mL centrifuge tubes, add 15mL of lymphocyte separation solution to each tube with a Pasteur pipette, then slowly add the cell resuspension to the lymphocyte separation solution, 35mL / tube, so that the total volume reaches about 50mL; after centrifugation at 300g without brake for 20min, carefully pipette the white mononuclear cell layer in the middle of the centrifuge tube into a new sterile 50mL centrifuge tube with a Pasteur pipette, then add buffer to 40mL, invert and mix, then pipette 10μl for cell counting, centrifuge at 300g for 10min and discard the supernatant;

[0063] 4. Using the immunodeficient mice that have undergone successful skin xenografting in the first step, 107 cells (100 ul) were taken from each mouse and injected into the tail vein to reconstruct the human immune system;

[0064] 5. One week later, peripheral blood was collected from NSG mice with human immune system reconstructed by orbital bleeding, and flow cytometry was performed to analyze human and mouse CD45+ cells, calculate the ratio of human and mouse CD45+ cells, and determine whether immune reconstruction was successful;

[0065] 6. Using mice with successful immune reconstruction, inject the solvent PBS into the skin graft site for 10 consecutive days;

[0066] 7. After 10 days, the pig skin transplantation site and the surrounding mouse skin tissue were taken for photographing, and immediately fixed in 4% paraformaldehyde for immunohistochemical staining.

[0067] The experimental results are as follows Figure 2As shown, the results showed that after local injection of PBS in immune-reconstructed skin xenograft mice, the local edges of the xenografts were rejected and detached, angiogenesis increased, and immunohistochemistry results showed vacuoles in the skin tissue structure, indicating that local rejection reactions occurred in skin xenograft mice after human immune reconstitution.

[0068] The third step is to use chemokine CCL28 immunomodulator to regulate rejection response

[0069] Normal human peripheral blood samples were obtained from the Third Xiangya Hospital of Central South University with approval from the Ethics Committee. Informed consent was obtained for all collected samples.

[0070] 1. Collect 50 mL of normal peripheral blood sample using an anticoagulant tube and perform subsequent operations in a clean bench.

[0071] 2. Preparation of stem cell protection solution: Take a clean and sterile 50mL centrifuge tube, weigh 2.5g BSA powder with an electronic balance, take a new bottle of 500mL 1×DPBS, use a 10mL pipette electric gun in the clean bench to draw 30mL of 1×DPBS and add it to the above 50mL centrifuge tube, shake vigorously and mix, then add 2mL 0.5M EDTA, invert and mix, use a sterile 50mL syringe and 0.22μM filter to filter the solution back into the above 1×DPBS bottle, invert and mix to make the stem cell protection solution (Buffer).

[0072] 3. Isolation of peripheral blood mononuclear cells (PBMC): Take 4 50mL centrifuge tubes, add 15mL of lymphocyte separation solution to each tube with a Pasteur pipette, then slowly pipette the cell resuspension into the lymphocyte separation solution, 35mL / tube, so that the total volume reaches about 50mL; after centrifugation at 300g without brake for 20min, use a Pasteur pipette to carefully pipette the white mononuclear cell layer in the middle of the centrifuge tube into a new sterile 50mL centrifuge tube, then add buffer to 40mL, invert to mix, then pipette 10μl for cell counting, centrifuge at 300g for 10min, and discard the supernatant.

[0073] 4. Using the immunodeficient mice that have successfully received skin xenografts in the first step, take 10 7 The cells (volume 100ul) were injected into the tail vein to reconstitute the human immune system.

[0074] 5. One week later, peripheral blood was collected from NSG mice with human immune system reconstructed by orbital bleeding, and flow cytometry was performed to analyze human and mouse CD45+ cells, calculate the ratio of human and mouse CD45+ cells, and determine whether the immune reconstruction was successful.

[0075] 6. Using mice with successful immune reconstitution, the chemokine CCL28 immunomodulator was injected locally around the skin graft for 10 consecutive days.

[0076] 7. After 10 days, the pig skin transplantation site and the surrounding mouse skin tissue were taken for photographing, and immediately fixed in 4% paraformaldehyde for immunohistochemical staining.

[0077] The experimental results are as follows Figure 3 As shown, the results showed that after local injection of chemokine CCL28 immunomodulator in immune-reconstructed skin xenograft mice, the xenografts were intact, with no obvious inflammatory response and no rejection. Immunohistochemistry results showed that the skin tissue structure was normal, indicating that subcutaneous local injection of chemokine CCL28 immunomodulator can alleviate the rejection reaction of skin xenografts.

[0078] Embodiment 2:

[0079] The remaining steps are the same as those in Example 1. When the chemokine CCL28 immunomodulator is locally injected, the auxiliary material hydrogel is combined to improve the degree of regulation of the chemokine CCL28 immunomodulator on the local anti-rejection reaction.

[0080] The hydrogel model we used was EFL-GM-60, with a concentration of 8% (W / V) (methacrylated gelatin GelMA mass W / PBS volume containing photoinitiator V), and the EFL-LS-1601-405 light source curing time was 15s. The amount of chemokine CCL28 used was 15ng / ul hydrogel, and the volume of hydrogel injected into each graft was 200ul.

[0081] The specific preparation steps are as follows:

[0082] 1. Prepare 0.25% (w / v) initiator standard solution. (1) Take 20 ml PBS and add it to the brown bottle containing initiator LAP (containing 0.05 g LAP); (2) Heat and dissolve in a 40-50°C water bath for 15 minutes, shaking several times during the process. The LAP standard solution can be stored at 4°C in the dark for 12 months.

[0083] 2. Prepare GelMA solution (GelMA concentration is 8-10% (w / v)) (1) Take the required mass of GelMA and put it into a centrifuge tube; (2) Take the initiator standard solution and add it to the above centrifuge tube, and shake it to make the GelMA fully infiltrated; (3) Heat and dissolve it in a 60-70℃ water bath in the dark for 20-30 minutes, shaking several times during the process; (4) Immediately sterilize the GelMA solution using a 0.22μm sterile syringe filter (to prevent low-temperature gelation).

[0084] 3. Preparation of chemokine and hydrogel mixture. (1) Take 3ug of chemokine CCL28 and resuspend it in 200ul of GelMA solution preheated at 37℃; (2) Use a 5ml syringe to aspirate the chemokine and hydrogel mixture, and irradiate with a 405nm light source for 15s to gel, thereby obtaining a hydrogel containing CCL28;

[0085] 4. Immediately inject the gel mixture around the graft and observe and measure the hydrogel volume.

[0086] The rejection reaction was regulated by using a hydrogel-containing chemokine CCL28 immunomodulator. The experimental results are as follows:

[0087] (1) Determine the sustained release rate of CCL28-containing hydrogel in vivo;

[0088] The experimental results are as follows Figure 7 Shown: The hydrogel containing CCL28 completely sustained release in vivo for 14 days.

[0089] (2) Record and count the survival status of skin tissue;

[0090] The experimental results are as follows Figure 8 As shown: Among them, the control group is the hydrogel control group without adding CCL28 in Example 1, and the CCL28+hydrogel group is the hydrogel experimental group containing the chemokine CCL28 immunomodulator in Example 2. After the skin transplanted mice were locally injected with the hydrogel containing CCL28, the grafts had a longer survival period.

[0091] (3) Subcutaneous local injection of CCL28 hydrogel around the graft was used for treatment, and skin tissue from the graft and surrounding areas was obtained for immunofluorescence staining of Treg cells;

[0092] The experimental results are as follows Fig. 9 As shown: The control group is the hydrogel control group without CCL28 in Example 1, and the CCL28 group + hydrogel group is the hydrogel experimental group containing the chemokine CCL28 immunomodulator in Example 2. Immunofluorescence staining shows that after the skin transplant mice were locally injected with the hydrogel containing CCL28, the local Treg cell content in the graft tissue increased. Compared with the group with single injection of chemokine CCL28, the results are as follows Fig.10 As shown, the recruitment effect of regulatory T cells was enhanced in the hydrogel group injected with the chemokine CCL28 immunomodulator.

[0093] Comparative Example 1:

[0094] The remaining steps were the same as those in Example 1, and the control group was kept without immunomodulator injection, and the other group was treated with subcutaneous local injection of CCL28 for control experiments.

[0095] (1) Record and count the survival status of skin tissue;

[0096] (2) Obtain skin tissue from the transplanted area and the area surrounding the transplanted area for immunofluorescence staining of Treg cells;

[0097] The experimental results are as follows Figure 4 , 5 As shown: Among them, the control group is the solvent PBS control group injected with no CCL28 in comparative example 1, and the CCL28 group is the subcutaneous local injection group of the CCL28 immunomodulator. After the immune reconstruction of the skin graft is successful, the skin graft of the local injection CCL28 group has a longer survival period. Immunofluorescence staining shows that after the immune-reconstructed skin xenograft mice are locally injected with CCL28, the local Treg cell content of the xenograft tissue is increased.

[0098] (3) Take skin tissue from the transplanted area and the surrounding area for CD4 + Immunohistochemical staining of T cells;

[0099] The experimental results are as follows Figure 6 As shown: the control group is the control group of comparative example 1 injected with solvent PBS without CCL28, and the CCL28 group is the subcutaneous local injection group of CCL28 immunomodulator. Immunohistochemical staining shows that after local injection of CCL28 in immune-reconstructed skin xenograft mice, the local CD4 + Increased T cell levels.

Claims

1. Use of a chemokine CCL28 immunomodulator in the preparation of a preparation for regulating the local immune microenvironment of a transplant for anti-rejection reaction of tissue cell transplantation, characterized in that: The chemokine CCL28 immunomodulator includes chemokine CCL28; the chemokine CCL28 immunomodulator includes an auxiliary material and / or a carrier of chemokine CCL28; the auxiliary material and / or the carrier is a hydrogel, the hydrogel includes methacryloyl gelatin and a photoinitiator, and the concentration of the hydrogel is 8-10% W / V of the mass of methacryloyl gelatin / the volume of PBS containing the photoinitiator; the tissue cell transplantation anti-rejection reaction is a xenogeneic skin transplantation, wherein the transplant is pig skin tissue; the regulation of the local immune microenvironment of the transplant is to transform the immune-promoting microenvironment dominated by effector T cells into an immunosuppressive microenvironment mediated by regulatory T cells.

2. The use according to claim 1, characterized in that The method for preparing the chemokine CCL28 immunomodulator comprises: (1) Prepare the initiator standard solution; (2) Preparation of auxiliary material solution: Take auxiliary material reagent, add initiator standard solution, heat and shake to obtain auxiliary material solution; (3) Preparation of the mixture of chemokine and excipients: Take the chemokine CCL28, resuspend it in the preheated excipient solution, and solidify it under a light source to obtain the chemokine CCL28 immunomodulator.

3. The use according to claim 2, characterized in that The light source curing time in step (3) is 12-18 seconds.

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