Manganese-containing stem cell coating having anticoagulant function, and preparation method therefor and use thereof

Abstract

The present application relates to the technical field of stem cell treatment, and provides a manganese-containing stem cell coating having an anticoagulant function, and a preparation method therefor and a use thereof. The preparation method comprises the following steps: simultaneously adding tannic acid, MnCl2·4H2O, and heparin sodium into a mesenchymal stem cell suspension, uniformly mixing, and incubating to obtain an incubated stem cell solution; and centrifuging, and collecting a precipitate to obtain the manganese-containing stem cell coating having an anticoagulant function. In the present application, natural tannic acid and manganese chloride tetrahydrate are mixed, so that deposition can occur on the surface film of mesenchymal stem cells; and the coating carries heparin sodium, so that a coagulation reaction caused by the stem cells can be effectively prevented. Results show that the manganese-containing stem cell coating having an anticoagulant function in the present application has a significant antithrombotic effect. In addition, the coating retains the original activity of the stem cells, has good biocompatibility, and significantly improves the effect of treating ulcerative colitis, thereby realizing safe and effective stem cell treatment.

Description

Manganese-containing stem cell coating with anticoagulant function and preparation method and application thereof Technical Field

[0001] The present application belongs to the field of stem cell therapy technology, and in particular relates to a manganese-containing stem cell coating with anti-coagulation function, and a preparation method and application thereof. Background Art

[0002] The systemic application of stem cells to modulate immune responses is becoming a new trend in cell therapy. Systemic infusion of mesenchymal stem cells (MSCs) has been shown to be a promising treatment for autoimmune diseases, vascular diseases, graft-versus-host disease (GVHD), and diabetes. Hundreds of clinical trials of MSC therapy are currently underway worldwide.

[0003] However, mesenchymal stem cells (MSCs) express tissue factor (TF), a powerful coagulation activator, and systemic infusion of stem cells can lead to diffuse coagulation. After intravenous infusion, the coagulation reaction of stem cells increases the obstruction of stem cells in the lungs, resulting in low treatment efficiency and even serious phenomena such as death. Currently, many strategies have been applied to improve the treatment of mesenchymal stem cells. Most of these strategies focus on promoting the cellular properties of mesenchymal stem cells. For example, certain genes are introduced into mesenchymal stem cells to overexpress key regulatory factors for cell therapy. However, the transfection rate of genetically engineered stem cells is low and there are risks associated with viral vectors.

[0004] Heparin is the drug of choice for the clinical treatment of disseminated intravascular coagulation (DIC) due to its strong anticoagulant effect. While surface heparinization has been shown to be a successful strategy in preventing thrombosis and improving the hemocompatibility of biomaterials in contact with blood, systemic use of heparin carries the risk of bleeding complications.

[0005] Plant-derived tannic acid is a naturally occurring, plant-derived antioxidant polyphenol green material. Natural polyphenols are plant secondary metabolites with numerous clinically demonstrated benefits, including strong antioxidant, free radical scavenging, anti-inflammatory, antiviral, antibacterial, immunomodulatory, neuroprotective, and cardioprotective properties. Tannic acid, due to its unique functional groups, can be used as an antioxidant, antimicrobial, and crosslinker in biomaterial products. It plays a significant role in improving the mechanical and physical properties of biomaterials, as its active phenolic groups can react with biomaterial functional groups, thereby enhancing performance. Due to its high biosafety, the mechanism by which tannic acid improves biomaterial performance as a natural crosslinker has been extensively studied, and its applications in tissue engineering, tissue adhesives, drug delivery, wound healing, and toxicity studies have been extensively investigated.

[0006] Manganese (Mn), one of the most common intermetallic compounds, is essential for a variety of physiological processes, including development, reproduction, immune regulation, and antioxidant defense. Its role in immune regulation is also receiving attention. Recent epidemiological studies have shown that deficiencies in trace minerals, including Mn, are potential risk factors for IBD. The gut microbiome is a highly dynamic system, influenced by environmental factors such as heavy metals and antibiotics. Manganese ions disrupt the gut microbiome in a sex-specific manner, altering the abundance of bacterial genes. In male mice treated with Mn, the abundance of Bacteroidetes bacteria increased, while the abundance of Lactobacilli decreased.

[0007] Summary of the Invention

[0008] In view of this, the purpose of the present application is to provide a manganese-containing stem cell coating with anti-coagulation function, and its preparation method and application. The manganese-containing stem cell coating with anti-coagulation function of the present application can effectively prevent the coagulation reaction caused by stem cells, and the manganese-containing stem cell coating with anti-coagulation function still retains the original activity of stem cells, and its activity in treating colitis is significantly improved compared with stem cells.

[0009] In order to achieve the above-mentioned invention objectives, this application provides the following technical solutions:

[0010] The present application provides a method for preparing a manganese-containing stem cell coating with anti-coagulation function, comprising the following steps:

[0011] Tannic acid, MnCl2·4H2O and sodium heparin are added to a mesenchymal stem cell suspension, mixed and incubated to obtain an incubated stem cell solution, centrifuged and the precipitate is collected to obtain a manganese-containing stem cell coating with anticoagulant function.

[0012] Preferably, the concentration of the mesenchymal stem cell suspension is 0.5 to 2×10 6 pieces / mL.

[0013] Preferably, the final concentration of the tannic acid is 10-50 μg / mL; the final concentration of the MnCl2·4H2O is 2.5-12.5 μg / mL; and the final concentration of the heparin sodium is 5-50 mg / mL.

[0014] Preferably, the incubation method is 37° C., 5% CO 2 for 3 to 7 minutes.

[0015] Preferably, the centrifugation is performed at 900-1100 rpm for 4-6 minutes, and the centrifugation is performed twice.

[0016] Preferably, the mesenchymal stem cells are mesenchymal stem cells derived from periodontal ligament.

[0017] The present application also provides a manganese-containing stem cell coating with anti-coagulation function prepared by the above preparation method.

[0018] The present application also provides an application of the above-mentioned preparation method or the manganese-containing stem cell coating with anticoagulant function, wherein the application includes at least one of the following:

[0019] (1) Use of the preparation method or the manganese-containing stem cell coating in the preparation of antithrombotic drugs;

[0020] (2) The preparation method or the manganese-containing stem cell coating is used for anti-thrombosis.

[0021] The present application also provides an application of the above-mentioned preparation method or the manganese-containing stem cell coating with anticoagulant function in the preparation of a drug for improving mesenchymal stem cell-induced coagulation, wherein the manganese-containing stem cell coating with anticoagulant function replaces mesenchymal stem cells for drug treatment.

[0022] The present application also provides the above-mentioned preparation method or the manganese-containing stem cell coating with anticoagulant function for improving the coagulation induced by mesenchymal stem cells, and the manganese-containing stem cell coating with anticoagulant function is used to replace mesenchymal stem cells for drug treatment.

[0023] The present application also provides an application of the above-mentioned preparation method or the manganese-containing stem cell coating with anticoagulant function, wherein the application includes at least one of the following:

[0024] (1) Use of the preparation method or the manganese-containing stem cell coating in the preparation of anti-colitis drugs;

[0025] (2) Application of the preparation method or the manganese-containing stem cell coating in anti-colitis.

[0026] Preferably, the colitis is ulcerative colitis.

[0027] The present application also provides a drug for improving mesenchymal stem cell-induced coagulation and anti-colitis, the drug comprising the above-mentioned manganese-containing stem cell coating with anti-coagulation function

[0028] Compared with the prior art, this application has the following beneficial effects:

[0029] The present application provides a manganese-containing stem cell coating with anticoagulant function, its preparation method and application. The present application mixes natural tannic acid and manganese chloride tetrahydrate, which can be deposited on the surface film of mesenchymal stem cells. The manganese-containing stem cell coating with anticoagulant function is equipped with sodium heparin, which can effectively prevent the coagulation reaction caused by stem cells. The results show that the manganese-containing stem cell coating with anticoagulant function of the present application has a significant anti-thrombotic effect. In addition, the manganese-containing stem cell coating with anticoagulant function still retains the original activity of stem cells, significantly improves the effect of the activity of stem cells in treating ulcerative colitis, and has good biocompatibility, thereby making stem cell therapy safe and effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG1 is a flow chart of the preparation of a manganese-containing stem cell coating with anticoagulant function;

[0031] FIG2 is a scanning electron micrograph of a manganese-containing stem cell coating having anticoagulant function;

[0032] FIG3 is a graph showing UV-visible absorption spectra of different stem cell coatings;

[0033] Figure 4 shows the zeta potential values ​​of different stem cell coatings;

[0034] Figure 5 shows the Mn-containing stem cell coating with anticoagulant function. 2+ Carrying capacity;

[0035] FIG6 shows the heparin loading capacity of the manganese-containing stem cell coating with anticoagulant function;

[0036] FIG7 is a light microscopic image of a manganese-containing stem cell coating having anticoagulant function;

[0037] Figure 8 shows live-dead staining of different stem cell coatings;

[0038] FIG9 shows the results of in vitro anti-thrombotic performance test of the manganese-containing stem cell coating with anti-coagulant function, where the yellow circle represents a blood clot;

[0039] FIG10 shows the results of in vivo anti-thrombotic performance test of the manganese-containing stem cell coating with anti-coagulant function, wherein the arrows represent local microthrombi;

[0040] FIG11 shows the effects of different stem cell coatings on the survival rate of colitis model mice;

[0041] FIG12 shows the H&E staining results of colon tissues with different stem cell coatings. DETAILED DESCRIPTION

[0042] The present application provides a method for preparing a manganese-containing stem cell coating with anti-coagulation function, comprising the following steps:

[0043] Tannic acid, MnCl2·4H2O and sodium heparin are added to a mesenchymal stem cell suspension, mixed and incubated to obtain an incubated stem cell solution, centrifuged and the precipitate is collected to obtain a manganese-containing stem cell coating with anticoagulant function.

[0044] In this application, tannic acid, MnCl2·4H2O and heparin sodium are added to the mesenchymal stem cell suspension and mixed. This application uses natural polyphenol tannic acid as an organic ligand, with Mn 2+ It is an inorganic cross-linking agent, which can instantly deposit a thin film on the surface of mesenchymal stem cells. The tannic acid and MnCl2·4H2O used are easy to obtain, inexpensive, and both are recognized as safe by the FDA. The metal-coordination bond formed is biodegradable, making stem cell therapy safe and effective. The tannic acid in this application has a universal surface binding affinity and can easily form a coating on the surface of stem cells. The metal polyphenol network formed with MnCl2·4H2O has a large porosity and adjustable pore size, shape and function, and can be used as a universal and efficient drug delivery platform. The manganese-containing stem cell coating with anticoagulant function successfully carries the anticoagulant drug sodium heparin, which can effectively prevent the coagulation reaction after intravenous infusion of stem cells, help stem cells escape from the lungs, and allow more stem cells to be distributed to other organs through the pulmonary capillary network. The concentration of the mesenchymal stem cell suspension is 0.5~2×10 6 / mL. The mesenchymal stem cell suspension can be prepared using 0.9% physiological saline or PBS. The final concentration of the tannic acid is preferably 10-50 μg / mL; the final concentration of the MnCl2·4H2O is preferably 2.5-12.5 μg / mL; and the final concentration of the heparin sodium is preferably 5-50 mg / mL. The tannic acid was purchased from Aladdin, item number T308008. The MnCl2·4H2O was purchased from Aladdin, item number M109464.

[0045] In the present application, the mixing method is not particularly limited and can be pipetting or vortex mixing. The incubation method is preferably 37°C, 5% CO2 for 3-7 minutes, further incubated for 4-6 minutes, and further incubated for 5 minutes. In the present application, the mesenchymal stem cells are preferably mesenchymal stem cells derived from the periodontal ligament.

[0046] In the present application, after incubation to obtain an incubated stem cell solution, centrifugation is performed to collect the precipitate to obtain a manganese-containing stem cell coating with anticoagulant function. The centrifugation is preferably performed at 900-1100 rpm for 4-6 minutes, preferably twice, to remove nonspecifically attached molecules.

[0047] The manganese-containing stem cell coating with anticoagulant function prepared by the present application still retains the original physiological activity of the stem cells, and the research of the present application found that the effect of the manganese-containing stem cell coating with anticoagulant function in treating colitis is significantly improved than that of stem cells that have not been functionally modified. In addition, the manganese-containing stem cell coating with anticoagulant function also has significant anti-thrombotic effect, and can be used to replace stem cells for drug treatment, thereby improving the adverse effects of coagulation reactions brought about by stem cells.

[0048] In view of this, the present application also provides a manganese-containing stem cell coating with anti-coagulation function prepared by the above preparation method.

[0049] The present application also provides an application of the above-mentioned preparation method or the manganese-containing stem cell coating with anticoagulant function in the preparation of antithrombotic drugs.

[0050] The present application also provides an application of the above-mentioned preparation method or the manganese-containing stem cell coating with anticoagulant function in the preparation of a drug for improving mesenchymal stem cell-induced coagulation, wherein the manganese-containing stem cell coating with anticoagulant function replaces mesenchymal stem cells for drug treatment.

[0051] The present application also provides an application of the above-mentioned preparation method or the manganese-containing stem cell coating with anti-coagulation function in the preparation of anti-colitis drugs.

[0052] In the present application, the colitis is preferably ulcerative colitis.

[0053] The present application also provides a drug for improving the coagulation-induced effect of mesenchymal stem cells, wherein the drug comprises the above-mentioned manganese-containing stem cell coating with anti-coagulation function.

[0054] In this application, the drug comprises a manganese-containing stem cell coating with anticoagulant function as the sole active ingredient, and the coating accounts for at least 95% of the drug by mass. The drug also includes pharmaceutically acceptable excipients, such as excipients. The drug can be administered in the form of a suspension or an injection. The drug can be administered intravenously.

[0055] The technical solutions provided in this application are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of this application.

[0056] Example 1

[0057] A method for preparing a manganese-containing stem cell coating with anticoagulant function comprises the following steps:

[0058] (1) digesting the periodontal ligament-derived mesenchymal stem cells with 0.25% trypsin to obtain digested periodontal ligament-derived mesenchymal stem cells;

[0059] (2) 1×10 6 The digested periodontal ligament-derived mesenchymal stem cells were suspended in 1 mL of 0.9% normal saline to obtain a cell suspension;

[0060] (3) Add tannic acid at a final concentration of 10 μg / mL, MnCl2·4H2O at a final concentration of 2.5 μg / mL, and heparin sodium at a final concentration of 10 mg / mL to the cell suspension, mix by pipetting for 15 seconds, and incubate in a 37°C 5% CO2 incubator for 5 minutes to obtain the incubated stem cell solution;

[0061] (4) After incubation, the stem cell solution was centrifuged at 1000 rpm for 5 min, and the precipitate a was collected and suspended in 1 mL of 0.9% saline. The mixture was gently pipetted for 15 s to remove nonspecific adhesion molecules to obtain a stem cell suspension solution. The stem cell suspension solution was centrifuged at 1000 rpm for 5 min, and the precipitate b was collected to obtain a functionalized stem cell coating (abbreviated as Hep-MPN@MSC or Hep-MPN).

[0062] Example 2

[0063] A method for preparing a manganese-containing stem cell coating with anticoagulant function comprises the following steps:

[0064] (1) digesting the periodontal ligament-derived mesenchymal stem cells with 0.25% trypsin to obtain digested periodontal ligament-derived mesenchymal stem cells;

[0065] (2) 1×10 6 The digested periodontal ligament-derived mesenchymal stem cells were suspended in 1 mL of 0.9% normal saline to obtain a cell suspension;

[0066] (3) Add tannic acid at a final concentration of 25 μg / mL, MnCl2·4H2O at a final concentration of 7.5 μg / mL, and heparin sodium at a final concentration of 25 mg / mL to the cell suspension, mix by pipetting for 15 seconds, and incubate in a 37°C 5% CO2 incubator for 5 minutes to obtain the incubated stem cell solution;

[0067] (4) After incubation, the stem cell solution was centrifuged at 1000 rpm for 5 min, and the precipitate a was collected and suspended in 1 mL of 0.9% saline. The mixture was gently pipetted for 15 s to remove nonspecific adhesion molecules to obtain a stem cell suspension solution. The stem cell suspension solution was centrifuged at 1000 rpm for 5 min, and the precipitate b was collected to obtain a functionalized stem cell coating (abbreviated as Hep-MPN@MSC or Hep-MPN).

[0068] Example 3

[0069] A method for preparing a manganese-containing stem cell coating with anticoagulant function comprises the following steps:

[0070] (1) digesting the periodontal ligament-derived mesenchymal stem cells with 0.25% trypsin to obtain digested periodontal ligament-derived mesenchymal stem cells;

[0071] (2) 2×10 6 The digested periodontal ligament-derived mesenchymal stem cells were suspended in 1 mL of 0.9% normal saline to obtain a cell suspension;

[0072] (3) Add tannic acid at a final concentration of 50 μg / mL, MnCl2·4H2O at a final concentration of 12.5 μg / mL, and heparin sodium at a final concentration of 50 mg / mL to the cell suspension, mix by pipetting for 15 seconds, and incubate in a 37°C 5% CO2 incubator for 6 minutes to obtain the incubated stem cell solution;

[0073] (4) After incubation, the stem cell solution was centrifuged at 1000 rpm for 5 min, and the precipitate a was collected and suspended in 1 mL of 0.9% saline. The mixture was gently pipetted for 15 s to remove nonspecific adhesion molecules to obtain a stem cell suspension solution. The stem cell suspension solution was centrifuged at 1000 rpm for 5 min, and the precipitate b was collected to obtain a functionalized stem cell coating (abbreviated as Hep-MPN@MSC or Hep-MPN).

[0074] Example 4

[0075] A method for preparing a manganese-containing stem cell coating with anticoagulant function comprises the following steps:

[0076] (1) digesting the periodontal ligament-derived mesenchymal stem cells with 0.25% trypsin to obtain digested periodontal ligament-derived mesenchymal stem cells;

[0077] (2) 1×10 6 The digested periodontal ligament-derived mesenchymal stem cells were suspended in 1 mL of 0.9% normal saline to obtain a cell suspension;

[0078] (3) Add tannic acid at a final concentration of 10 μg / mL, MnCl2·4H2O at a final concentration of 2.5 μg / mL, and heparin sodium at a final concentration of 5 mg / mL to the cell suspension, mix by pipetting for 15 seconds, and incubate in a 37°C 5% CO2 incubator for 5 minutes to obtain the incubated stem cell solution;

[0079] (4) After incubation, the stem cell solution was centrifuged at 1000 rpm for 5 min, and the precipitate a was collected and suspended in 1 mL of 0.9% saline. The mixture was gently pipetted for 15 s to remove nonspecific adhesion molecules to obtain a stem cell suspension solution. The stem cell suspension solution was centrifuged at 1000 rpm for 5 min, and the precipitate b was collected to obtain a functionalized stem cell coating (abbreviated as Hep-MPN@MSC or Hep-MPN).

[0080] Comparative Example 1

[0081] A method for preparing a tannic acid-modified stem cell coating comprises the following steps:

[0082] (1) digesting the periodontal ligament-derived mesenchymal stem cells with 0.25% trypsin to obtain digested periodontal ligament-derived mesenchymal stem cells;

[0083] (2) 1×10 6 The digested periodontal ligament-derived mesenchymal stem cells were suspended in 1 mL of 0.9% normal saline to obtain a cell suspension;

[0084] (3) Add tannic acid to a final concentration of 10 μg / mL to the cell suspension, mix by pipetting for 15 seconds, and incubate in a 37°C 5% CO2 incubator for 5 minutes to obtain an incubated stem cell solution;

[0085] (4) After incubation, the stem cell solution was centrifuged at 1000 rpm for 5 min, and the precipitate a was collected and suspended in 1 mL of 0.9% saline. The mixture was gently pipetted for 15 s to remove non-specific adhesion molecules to obtain a stem cell suspension solution. The stem cell suspension solution was centrifuged at 1000 rpm for 5 min, and the precipitate b was collected to obtain a tannic acid-modified stem cell coating (abbreviated as TA or TA@MSC).

[0086] Comparative Example 2

[0087] A MnCl2·4H2O modified stem cell coating, comprising the following steps:

[0088] (1) digesting the periodontal ligament-derived mesenchymal stem cells with 0.25% trypsin to obtain digested periodontal ligament-derived mesenchymal stem cells;

[0089] (2) 1×10 6 The digested periodontal ligament-derived mesenchymal stem cells were suspended in 1 mL of 0.9% normal saline to obtain a cell suspension;

[0090] (3) Add MnCl2·4H2O to a final concentration of 2.5 μg / mL to the cell suspension, mix by pipetting for 15 seconds, and incubate in a 37°C 5% CO2 incubator for 5 minutes to obtain the incubated stem cell solution;

[0091] (4) After the stem cell solution was incubated by centrifugation at 1000 rpm for 5 min, the precipitate a was collected and suspended in 1 mL of 0.9% saline. The mixture was gently pipetted for 15 s to remove non-specific adhesion molecules to obtain a stem cell suspension solution. The stem cell suspension solution was centrifuged at 1000 rpm for 5 min, and the precipitate b was collected to obtain the MnCl2·4H2O-modified stem cell coating (abbreviated as Mn 2+ ).

[0092] Comparative Example 3

[0093] A heparin sodium modified stem cell coating, comprising the following steps:

[0094] (1) digesting the periodontal ligament-derived mesenchymal stem cells with 0.25% trypsin to obtain digested periodontal ligament-derived mesenchymal stem cells;

[0095] (2) 1×10 6 The digested periodontal ligament-derived mesenchymal stem cells were suspended in 1 mL of 0.9% normal saline to obtain a cell suspension;

[0096] (3) Heparin sodium was added to the cell suspension at a final concentration of 10 mg / mL, mixed by pipetting for 15 seconds, and incubated in a 37°C 5% CO2 incubator for 5 minutes to obtain a stem cell solution;

[0097] (4) After incubation, the stem cell solution was centrifuged at 1000 rpm for 5 min, and the precipitate a was collected and suspended in 1 mL of 0.9% saline. The mixture was gently pipetted for 15 s to remove non-specific adhesion molecules to obtain a stem cell suspension solution. The stem cell suspension solution was centrifuged at 1000 rpm for 5 min, and the precipitate b was collected to obtain a sodium heparin-modified stem cell coating (heparin sodium@MSC).

[0098] Comparative Example 4

[0099] A method for preparing an MPN-modified stem cell coating comprises the following steps:

[0100] (1) digesting the periodontal ligament-derived mesenchymal stem cells with 0.25% trypsin to obtain digested periodontal ligament-derived mesenchymal stem cells;

[0101] (2) 1×10 6 The digested periodontal ligament-derived mesenchymal stem cells were suspended in 1 mL of 0.9% normal saline to obtain a cell suspension;

[0102] (3) Add tannic acid with a final concentration of 10 μg / mL and MnCl2·4H2O with a final concentration of 2.5 μg / mL to the cell suspension, mix by pipetting for 15 seconds, and incubate in a 37°C 5% CO2 incubator for 5 minutes to obtain the incubated stem cell solution;

[0103] (4) After incubation, the stem cell solution was centrifuged at 1000 rpm for 5 min, and the precipitate a was collected and suspended in 1 mL of 0.9% saline. The mixture was gently pipetted for 15 s to remove non-specific adhesion molecules to obtain a stem cell suspension solution. The stem cell suspension solution was centrifuged at 1000 rpm for 5 min, and the precipitate b was collected to obtain the MPN-modified stem cell coating (abbreviated as MPN or MPN@MSC).

[0104] Example 5

[0105] The manganese-containing stem cell coating with anti-coagulation function prepared in Example 1 was subjected to morphological observation and related characterization to explore whether the manganese-containing stem cell coating with anti-coagulation function was successfully prepared.

[0106] The Hep-MPN@MSCs prepared in Example 1 and unmodified periodontal ligament-derived mesenchymal stem cells (control group) were added to 2 mL of 2.5% glutaraldehyde electron microscopy fixative and fixed overnight at 4°C before SEM imaging. A flow chart for the preparation of the manganese-containing stem cell coating with anticoagulant function is shown in Figure 1.

[0107] As shown in Figure 2 , compared with the control group, the cell surface of Hep-MPN@MSC became rougher.

[0108] Tannic acid has ultraviolet absorption at 350 nm, so ultraviolet-visible absorption spectroscopy was performed to investigate whether the manganese-containing stem cell coating with anticoagulant function prepared in Example 1 was loaded with tannic acid.

[0109] The mesenchymal stem cells derived from the periodontal ligament without functional modification (control group), TA and Mn prepared in comparative examples 1 to 4 were respectively 2+ 2 mL of ddH2O was added to the heparin sodium@MSC, MPN, and the manganese-containing stem cell coating with anticoagulant function prepared in Example 1, and UV-visible absorption spectroscopy was performed. The results are shown in FIG3 .

[0110] As can be seen in FIG3 , the absorption of the manganese-containing stem cell coating with anticoagulant function at 350 nm is enhanced, indicating that the manganese-containing stem cell coating with anticoagulant function prepared in the present application is loaded with tannic acid.

[0111] The mesenchymal stem cells derived from the periodontal ligament without functional modification (control group), TA and Mn prepared in comparative examples 1 to 4 were respectively 2+2 mL of PBS was added to MSC, sodium heparin@MSC, MPN, and the manganese-containing stem cell coating with anticoagulant function prepared in Example 1 for zeta potential measurement. The results are shown in Figure 4. Three replicates were performed for each treatment group.

[0112] The results in FIG4 show that the surface of the manganese-containing stem cell coating with anticoagulant function prepared in the present application has a negative charge, and the Zeta potential is -53.52 mV.

[0113] This application further explored whether the manganese-containing stem cell coating with anticoagulant function prepared in Example 1 successfully carried manganese ions, and conducted a manganese ion detection on it.

[0114] ICP-MS analysis was performed by adding 2 mL of PBS to unmodified periodontal ligament-derived mesenchymal stem cells (control group) and the manganese-containing stem cell coating with anticoagulant function prepared in Example 1. Three replicates were performed for each treatment group.

[0115] As shown in the results of FIG5 , compared with the control group, the manganese ions on the manganese-containing stem cell coating with anticoagulant function increased significantly, indicating that the manganese-containing stem cell coating with anticoagulant function of the present application successfully carried manganese ions.

[0116] This application further investigated whether the manganese-containing stem cell coating with anticoagulant function prepared in Example 1 was successfully loaded with heparin. The toluidine blue colorimetric method was used to quantitatively determine the heparin loading amount of the manganese-containing stem cell coating with anticoagulant function. The experimental method is as follows:

[0117] A standard concentration gradient solution (1 mL) was prepared: 0, 1, 5, 10, 15, 20, 25, and 30 μg / mL heparin solution was used to construct a standard curve. The anticoagulant manganese-containing stem cell coating prepared in Example 1 and the sodium heparin-modified stem cell coating (control group) from Comparative Example 3 were added to 1 mL of toluidine blue solution (0.1 M hydrochloric acid, 2 mg / mL NaCl, 0.4 mg / mL toluidine blue O-zinc chloride complex salt) and incubated at room temperature for 4 h to allow the toluidine blue to complex with the heparin. The precipitate was collected by centrifugation at 3500 rpm for 10 min and rinsed twice with deionized water. The precipitate was dissolved in 2 mL of a mixed solution consisting of 80% (v / v) ethanol and 0.1 M NaOH in a volume ratio of 4:1. After the complex was completely dissolved, 200 μL of the supernatant was transferred to a 96-well plate and the absorbance was measured at 530 nm. The absorbance of the solution was measured at a wavelength of 631 nm using a spectrophotometric microplate reader, and the amount of heparin was calculated using a previously constructed calibration curve. Each treatment group had five replicates.

[0118] As can be seen from FIG6 , compared with the stem cell coating modified with heparin sodium alone, the manganese-containing stem cell coating with anticoagulant function prepared in the present application significantly increases the heparin loading capacity.

[0119] The present application further prepares a manganese-containing stem cell coating with anticoagulant function by using FITC-labeled heparin sodium. The specific preparation method is shown in Example 1. Fluorescence microscopy is then used to observe whether the manganese-containing stem cell coating with anticoagulant function is loaded with heparin sodium. The control group is mesenchymal stem cells derived from the periodontal ligament that have not undergone functional modification.

[0120] As shown in FIG7 , compared with the control group, the manganese-containing stem cell coating with anticoagulant function of the present application has significant green fluorescence, that is, the manganese-containing stem cell coating with anticoagulant function of the present application successfully carries sodium heparin.

[0121] In summary, the present application successfully prepared a manganese-containing stem cell coating with anti-coagulation function.

[0122] Example 6

[0123] The mesenchymal stem cells derived from the periodontal ligament without functional modification (control group), the MPN-modified stem cell coating prepared in comparative example 4, the manganese-containing stem cell coating with anticoagulant function prepared in example 1, and the manganese-containing stem cell coating with anticoagulant function prepared in example 4 were respectively prepared at a concentration of 1 to 5×10 5 After being seeded at a density of 100 μg / mL on a coverslip and cultured for 24 h, the cells grown on the coverslip were washed three times with PBS, and the live / dead working solution was added and incubated at room temperature in the dark for 5 min. The staining solution was removed, PBS was added, and the cells were observed under a fluorescence microscope.

[0124] The results in FIG8 show that both the manganese-containing stem cell coating with anticoagulant function prepared in Example 1 of the present application and the manganese-containing stem cell coating with anticoagulant function prepared in Example 4 have no obvious cytotoxicity.

[0125] Example 7

[0126] Verification of the antithrombotic performance of manganese-containing stem cell coating with anticoagulant function

[0127] 1. Verification of the antithrombotic performance of manganese-containing stem cell coating with anticoagulant function in vitro

[0128] According to the preparation methods of Comparative Example 3, Comparative Example 4, and Example 1, MPN-modified stem cell coatings and manganese-containing stem cell coatings with anticoagulant function were prepared on PS sheets, respectively. At the same time, mesenchymal stem cells derived from periodontal ligament that had not undergone functional modification were cultured on PS sheets as a control group, and a 10 mg / mL heparin sodium solution prepared with 0.9% saline was used as the heparin sodium-saline group (positive control group). Each treatment group was replicated three times, and the prepared cell suspensions were added to blood collection glass tubes. SD rat heart blood was collected into the blood collection tubes. The blood collection tubes were tilted 45 degrees every 15 seconds to observe the formation of blood clots and record the time.

[0129] As can be seen from Figure 9, the MPN-modified stem cell coating and the control group produced blood clots within 10 minutes, while the manganese-containing stem cell coating with anticoagulant function prepared by the present application did not produce blood clots within 20 minutes. The anticoagulant effect was equivalent to that of the sodium heparin anticoagulant tube. The manganese-containing stem cell coating with anticoagulant function prepared by the present application had a better anticoagulant effect within 20 minutes.

[0130] In summary, the manganese-containing stem cell coating with anti-coagulation function of the present application has anti-thrombotic properties.

[0131] 2. In vivo antithrombotic properties of Hep-MPN@MSC

[0132] An ulcerative colitis model was established in mice using 3% dextran sodium sulfate (DSS) as a free-feeding diet. On the third day, PBS was injected into the tail vein as a blank control group (PBS), periodontal ligament-derived mesenchymal stem cells were injected into the control group (MSC), and a manganese-containing stem cell coating group with anticoagulant function prepared in Example 1 was injected into the tail vein. Each treatment group had three replicates. Ten minutes after the tail vein injection, important organs of the mice were sampled for histological staining to observe the formation of microthrombi.

[0133] The results in Figure 10 show that compared with the blank control group, obvious microthrombosis can be seen in the microvessels and arterioles of various organs in the MSC group, and the manganese-containing stem cell coating with anticoagulant function prepared in this application can significantly reduce the formation of microthrombosis.

[0134] Example 8

[0135] To verify that the anticoagulant manganese-containing stem cell coating prepared in this application can still maintain the original activity of unfunctionalized periodontal ligament-derived mesenchymal stem cells, the efficacy of the anticoagulant manganese-containing stem cell coating in treating ulcerative colitis was studied. Interestingly, the study found that compared with unfunctionalized periodontal ligament-derived mesenchymal stem cells, the anticoagulant manganese-containing stem cell coating prepared in this application was significantly more effective in treating ulcerative colitis.

[0136] A mouse ulcerative colitis model was established using 3% DSS free feeding. The healthy group was not fed with 3% DSS free feeding, and PBS without 3% DSS was injected into the tail vein on the third day; after free feeding with 3% DSS, PBS (abbreviated as DSS+PBS) was injected into the tail vein on the third day, and periodontal ligament-derived mesenchymal stem cells were used as a control group (abbreviated as DSS+MSC), tannic acid-modified stem cell coating prepared in comparative example 1 (abbreviated as DSS+TA@MSC), MPN-modified stem cell coating prepared in comparative example 4 (abbreviated as DSS+MPN@MSC), and manganese-containing stem cell coating group with anticoagulant function prepared in Example 1 (abbreviated as DSS+Hep-MPN@MSC) were injected into the tail vein respectively. Each treatment group had 6 replicates, and body weight and fecal occult blood were measured every day. On the 10th day, samples were taken for histological staining of the colon.

[0137] The results in Figure 11 show that compared with DSS+MSC, DSS+TA@MSC, and DSS+MPN@MSC, the survival rate of mice in the DSS+Hep-MPN@MSC treatment group increased after being treated with the manganese-containing stem cell coating with anticoagulant function prepared in the present application.

[0138] Histological analysis in Figure 12 shows that compared with the DSS+MSC, DSS+TA@MSC, and DSS+MPN@MSC groups, the DSS+Hep-MPN@MSC group showed significantly reduced inflammatory cell infiltration in the lamina propria, reduced mucosal ulceration, and reduced mucosal collapse and granulation tissue formation. The manganese-containing stem cell coating with anticoagulant function of this application effectively improved the effectiveness of mesenchymal stem cells in preventing ulcerative colitis and tissue damage.

[0139] The above is only a preferred embodiment of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A method for preparing a manganese-containing stem cell coating with anticoagulant function, characterized in that: The following steps are involved: Tannic acid, MnCl2·4H2O and sodium heparin are added to a mesenchymal stem cell suspension, mixed and incubated to obtain an incubated stem cell solution, centrifuged and the precipitate is collected to obtain a manganese-containing stem cell coating with anticoagulant function.

2. The preparation method according to claim 1, characterized in that The concentration of the mesenchymal stem cell suspension is 0.5 to 2×10 6 pieces / mL.

3. The preparation method according to claim 1, characterized in that The final concentration of the tannic acid is 10 to 50 μg / mL; the final concentration of the MnCl2·4H2O is 2.5 to 12.5 μg / mL; and the final concentration of the heparin sodium is 5 to 50 mg / mL.

4. The preparation method according to claim 1, characterized in that The incubation method is 37° C., 5% CO 2 , incubation for 3 to 7 minutes.

5. The preparation method according to claim 1, characterized in that The centrifugation method is 900-1100 rpm for 4-6 minutes; the centrifugation frequency is 2 times.

6. The preparation method according to claim 1, characterized in that The mesenchymal stem cells are mesenchymal stem cells derived from periodontal ligament.

7. A manganese-containing stem cell coating with anti-coagulation function prepared according to the preparation method according to any one of claims 1 to 6.

8. Use of the preparation method according to any one of claims 1 to 6 or the manganese-containing stem cell coating with anticoagulant function according to claim 7, characterized in that: The application includes at least one of the following: (1) Use of the preparation method or the manganese-containing stem cell coating in the preparation of antithrombotic drugs; (2) The preparation method or the manganese-containing stem cell coating is used for anti-thrombosis.

9. Use of the preparation method according to any one of claims 1 to 6 or the manganese-containing stem cell coating with anticoagulant function according to claim 7 in the preparation of a drug for improving the coagulogenic effect of mesenchymal stem cells, characterized in that: The manganese-containing stem cell coating with anti-coagulation function is used to replace mesenchymal stem cells for drug treatment.

10. The preparation method according to any one of claims 1 to 6 or the manganese-containing stem cell coating with anticoagulant function according to claim 7 is used to improve the coagulation of mesenchymal stem cells, characterized in that: The manganese-containing stem cell coating with anti-coagulation function is used to replace mesenchymal stem cells for drug treatment.

11. Use of the preparation method according to any one of claims 1 to 6 or the manganese-containing stem cell coating with anticoagulant function according to claim 7, characterized in that: The application includes at least one of the following: (1) application of the preparation method or the manganese-containing stem cell coating in the preparation of anti-colitis drugs; (2) application of the preparation method or the manganese-containing stem cell coating in anti-colitis.

12. The use according to claim 11, characterized in that The colitis is ulcerative colitis.

13. A drug for improving the coagulation and anti-colitis effects of mesenchymal stem cells, characterized in that: The medicine comprises the manganese-containing stem cell coating with anti-coagulation function according to claim 7.

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