Mesenchymal stem cell drug prescription and preparation process thereof

Abstract

The invention provides a mesenchymal stem cell drug prescription and a preparation process thereof, and belongs to the technical field of biological medicines. The mesenchymal stem cell drug prescription combination provided by the invention comprises a mesenchymal stem cell solution and a self-assembly solution, the mesenchymal stem cell solution is composed of mesenchymal stem cells, human serum albumin, heparin, dextran 40 and a compound electrolyte solution, and the self-assembly solution is composed of protamine, dipotassium glycyrrhizinate, rosmarinic acid and normal saline. The mesenchymal stem cell drug prescription combination provided by the invention can improve the cell viability and stability of the mesenchymal stem cells, highly express CD73, CD90, CD105, HGF and sTNFR1, has a better immune regulation function, and provides a new thought for clinical application of the mesenchymal stem cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a mesenchymal stem cell medicine prescription and a preparation process thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are adult stem cells of matrix origin, mainly existing in connective tissues and organ stroma such as bone marrow, dental pulp, fat, and umbilical cord. Mesenchymal stem cells have many unique advantages, such as the ability to self-renew and effectively regulate immune function, and the source of these stem cells is very rich and easy to culture. According to current medical research, mesenchymal stem cell preparations have been widely used, covering neurological diseases, damage to various organ tissues, and autoimmune diseases. They have not only greatly improved the success rate of various organ transplants, but also effectively reduced the rejection reaction of patients after organ transplantation, and have an application value that cannot be ignored in clinical practice. Due to the influence of many factors and the complexity of different patient treatment plans, the technology for preparing stem cell preparations needs to comprehensively consider many factors in the specific operation process. The prepared stem cells cannot be cultured and applied immediately, so it is necessary to strengthen the research on the stability of stem cell preservation in order to improve the activity and survival rate of cells.

[0003] In the prescription of mesenchymal stem cell injection, low molecular weight heparin calcium (or heparin sodium) is usually added to prevent cell aggregation, and human serum albumin is added to provide nutrition, maintain osmotic pressure, promote cell adhesion and proliferation, etc. However, the use of heparin calcium may also be accompanied by some side effects, mainly including active bleeding or increased risk of bleeding related to abnormal hemostasis, organic damage that may cause bleeding (such as active peptic ulcers), hemorrhagic cerebrovascular accidents, acute infective endocarditis, and patients with severe renal impairment (creatinine clearance less than 30ml / min). In addition, the addition of human serum albumin may also be accompanied by some side effects, including allergic reactions, increased oxidative activity, etc., manifested as skin itching, erythema, and even difficulty breathing and fever.

[0004] Adding protamine, rosmarinic acid, etc. to the stem cell prescription can reduce the side effects of the above ingredients. It has the advantages of high safety, good antiseptic performance, and high thermal stability, and is a natural preservative. In addition, protamine also has high nutritional and functional properties. For example, protamine can inhibit the formation of vascular tumors, inhibit the occurrence of vascular inflammation, limit the proliferation of tumor cells, and induce their apoptosis, and has anti-tumor activity; adding protamine to the mesenchymal stem cell prescription can be used to neutralize the anticoagulant effect of heparin, thereby reducing the risk of bleeding in patients. Rosmarinic acid is a natural antioxidant with strong anti-allergic and antioxidant activity, which helps prevent cell damage caused by free radicals. In addition, rosmarinic acid also has strong anti-inflammatory, antibacterial, antiviral, and anti-tumor activities. Chinese medicine self-assemblies not only have the same drug delivery ability as synthetic nanocarriers, but also have the characteristics of degradability, compatibility, and safety that synthetic nanomaterials do not have. Studies have found that dipotassium glycyrrhizinate is amphiphilic and can aggregate to form self-assembled micelles. It is widely used as a multifunctional drug carrier to improve drug absorption and enhance drug stability.

[0005] Therefore, a new mesenchymal stem cell drug prescription combination is provided, which utilizes dipotassium glycyrrhizinate, protamine, and rosmarinic acid to improve the cell activity and stability of mesenchymal stem cells, providing a new idea for the clinical application of mesenchymal stem cells. Summary of the invention

[0006] In view of the above-mentioned deficiencies, the present invention provides a mesenchymal stem cell drug prescription and its preparation process. The mesenchymal stem cell drug prescription combination provided by the present invention includes a mesenchymal stem cell solution and a self-assembly solution; the mesenchymal stem cell solution is composed of mesenchymal stem cells, human serum albumin, heparin, dextran 40, and a compound electrolyte solution, and the self-assembly solution is composed of protamine, dipotassium glycyrrhizinate, rosmarinic acid and physiological saline. The mesenchymal stem cell drug prescription combination provided by the present invention can improve the cell activity and stability of mesenchymal stem cells, highly express CD73, CD90, CD105, HGF and sTNFR1, have good immune regulation function, and provide new ideas for the clinical application of mesenchymal stem cells.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] In one aspect, the present invention provides a mesenchymal stem cell drug prescription combination, wherein the mesenchymal stem cell drug prescription combination comprises a mesenchymal stem cell solution and a self-assembly solution;

[0009] The mesenchymal stem cell solution is composed of mesenchymal stem cells, human serum albumin, heparin, dextran 40, and a compound electrolyte solution;

[0010] The self-assembly solution consists of protamine, dipotassium glycyrrhizinate, rosmarinic acid and physiological saline.

[0011] Specifically, the self-assembly solution contains 0.55-1 mg / mL protamine, 2.75-4.7 mg / mL dipotassium glycyrrhizinate, and 1.65-3.4 mg / mL rosmarinic acid.

[0012] Preferably, the self-assembly solution contains 0.8-0.85 mg / mL protamine, 4.2 mg / mL dipotassium glycyrrhizinate, and 2.8 mg / mL rosmarinic acid.

[0013] More preferably, the self-assembly solution contains 0.8 mg / mL protamine, 4.2 mg / mL dipotassium glycyrrhizinate, and 2.8 mg / mL rosmarinic acid.

[0014] Specifically, the concentration ratio of protamine, dipotassium glycyrrhizinate and rosmarinic acid in the self-assembly solution is 1:5-5.5:3-4.

[0015] Preferably, the concentration ratio of protamine, dipotassium glycyrrhizinate and rosmarinic acid in the self-assembly solution is 1:5.25:3.5.

[0016] Preferably, the volume ratio of the mesenchymal stem cell solution to the self-assembly solution is 1:1.

[0017] Specifically, the concentration of mesenchymal stem cells in the mesenchymal stem cell solution is 1×10 5 -1×10 7 cells / mL, the concentration of human serum albumin is 10-40 mg / mL, the concentration of heparin is 50-200 IU / mL, and the concentration of dextran 40 is 120-240 mg / mL.

[0018] Preferably, the concentration of mesenchymal stem cells in the mesenchymal stem cell solution is 2×10 5 cells / mL, the concentration of human albumin was 20 mg / mL, the concentration of heparin was 100 IU / mL, and the concentration of dextran 40 was 240 mg / mL.

[0019] Specifically, the mesenchymal stem cells are selected from one or a combination of two or more of umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, placental mesenchymal stem cells, dental pulp mesenchymal stem cells, amniotic membrane mesenchymal stem cells, and skin mesenchymal stem cells.

[0020] In a second aspect, the present invention provides a method for preparing the above-mentioned mesenchymal stem cell drug prescription combination, the preparation method comprising the following steps:

[0021] S1, dissolving protamine, dipotassium glycyrrhizinate and rosmarinic acid in physiological saline, stirring to obtain a self-assembly solution;

[0022] S2, resuspending the mesenchymal stem cells in a compound electrolyte solution containing human serum albumin, heparin and dextran 40 to obtain a mesenchymal stem cell solution;

[0023] S3. Add the mesenchymal stem cell solution into the self-assembly solution and stir to obtain a mesenchymal stem cell drug prescription combination.

[0024] Specifically, the stirring speed in step S1 is 600-1000 rpm, and the stirring time is 45-60 min.

[0025] Preferably, the stirring speed in step S1 is 800 rpm and the stirring time is 50 min.

[0026] Specifically, the stirring in step S3 is at 4° C., 400-600 rpm, and stirring for 15-20 min.

[0027] Preferably, the stirring in step S3 is at 4° C., 400 rpm, and for 20 min.

[0028] In a third aspect, the present invention provides use of the above-mentioned mesenchymal stem cell drug prescription combination in the preparation of a drug containing mesenchymal stem cells.

[0029] Preferably, the content of mesenchymal stem cells in the drug is ≥ 0.5×10 5 / mL.

[0030] Specifically, the medicine containing mesenchymal stem cells is used to prevent or treat tumors or autoimmune diseases.

[0031] Preferably, the tumor includes, but is not limited to, a digestive system tumor, a respiratory system tumor, a reproductive system tumor, a musculoskeletal system tumor, a nervous system tumor, an endocrine system tumor, a circulatory system tumor, a urinary system tumor or a reproductive system tumor.

[0032] Preferably, the autoimmune diseases include but are not limited to systemic lupus erythematosus, Sjogren's syndrome, Wegener's granulomatosis, sarcoidosis, Reiter's syndrome, Behcet's syndrome; respiratory diseases include but are not limited to asthma, chronic obstructive pulmonary disease, and adult respiratory distress syndrome.

[0033] Specifically, the dosage form of the mesenchymal stem cell-containing drug includes, but is not limited to, tablets, capsules, injections, solutions, syrups, emulsions, suspensions, granules, ointments, suppositories, aerosols or pills.

[0034] Preferably, the dosage form of the mesenchymal stem cell-containing drug is an injection.

[0035] In a third aspect, the present invention provides a medicine containing mesenchymal stem cells, wherein the medicine is prepared by the above-mentioned mesenchymal stem cell medicine prescription combination.

[0036] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0037] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, one or more of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, and buffers.

[0038] The beneficial effects of the present invention are:

[0039] The mesenchymal stem cell drug prescription combination provided by the present invention can improve the cell activity and stability of mesenchymal stem cells, highly express CD73, CD90, CD105, HGF and sTNFR1, and has good immune regulation function, providing a new idea for the clinical application of mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Cell number detection results

[0041] Figure 2 The proliferation percentage of PBMCs stimulated by CD3 / CD28 antibodies; *** in the figure represents P<0.001.

[0042] Figure 3 The inhibition rate of hUC-MSC on the proliferation of PBMC stimulated by CD3 / CD28 antibodies; * in the figure represents P<0.05.

[0043] Figure 4 is the concentration of TNF-α secreted by PBMC; *** in the figure represents P<0.001.

[0044] Figure 5 is the concentration of IFN-γ secreted by PBMC; *** in the figure represents P<0.001. DETAILED DESCRIPTION

[0045] The present invention is described below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention so that the technical solution of the present invention is easier to understand and grasp. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0046] The experimental materials used in the present invention are shown in Table 1:

[0047] Table 1 Experimental materials

[0048]

[0049] Basic Experimental Example 1 Cultivation and Passaging of Mesenchymal Stem Cells

[0050] Mesenchymal stem cells were cultured to the fifth generation and cryopreserved in the following culture system and environment: DMEM / F12 culture medium containing 10% v / v FBS, 37° C., 5% CO 2 .

[0051] Example 1 Preparation of Mesenchymal Stem Cell Drug Prescription Combination 1

[0052] 1. Dissolve protamine, dipotassium glycyrrhizinate and rosmarinic acid in 10 mL of normal saline, and stir at 800 rpm for 50 min to obtain a self-assembly solution; the self-assembly solution contains 0.8 mg / mL protamine, 4.2 mg / mL dipotassium glycyrrhizinate and 2.8 mg / mL rosmarinic acid.

[0053] 2. The fifth generation mesenchymal stem cells obtained in Basic Experimental Example 1 were revived and cultured to 80% cell confluence, then digested with trypsin, centrifuged, washed with physiological saline three times, and resuspended with a compound electrolyte solution containing human albumin, heparin and dextran 40 to obtain 10 mL of mesenchymal stem cell solution; the mesenchymal stem cell solution contained 2×10 6 cell mesenchymal stem cells, 0.2g human serum albumin, 1000IU heparin, 2.4g dextran 40.

[0054] 3. Add the mesenchymal stem cell solution to the self-assembly solution at a ratio of mesenchymal stem cell solution: self-assembly solution = 1:1 (v / v), stir at 400 rpm at 4°C for 20 min, and obtain 20 mL of mesenchymal stem cell drug prescription combination 1 (P1).

[0055] Example 2 Preparation of Mesenchymal Stem Cell Drug Prescription Combination 2

[0056] 1. Dissolve protamine, dipotassium glycyrrhizinate and rosmarinic acid in 10 mL of normal saline, and stir at 800 rpm for 50 min to obtain a self-assembly solution; the self-assembly solution contains 0.55 mg / mL protamine, 2.75 mg / mL dipotassium glycyrrhizinate and 1.65 mg / mL rosmarinic acid.

[0057] 2. The fifth generation mesenchymal stem cells obtained in Basic Experimental Example 1 were revived and cultured to 80% cell confluence, then digested with trypsin, centrifuged, washed with physiological saline three times, and resuspended with a compound electrolyte solution containing human albumin, heparin and dextran 40 to obtain 10 mL of mesenchymal stem cell solution; the mesenchymal stem cell solution contained 2×106 cell mesenchymal stem cells, 0.1g human serum albumin, 500IU heparin, 1.2g dextran 40.

[0058] 3. Add the mesenchymal stem cell solution to the self-assembly solution at a ratio of mesenchymal stem cell solution: self-assembly solution = 1:1 (v / v), stir at 4°C and 400 rpm for 20 min, and obtain 20 mL of mesenchymal stem cell drug prescription combination 2 (P2).

[0059] Example 3 Preparation of Mesenchymal Stem Cell Drug Prescription Combination 3

[0060] 1. Dissolve protamine, dipotassium glycyrrhizinate and rosmarinic acid in 10 ml of physiological saline, and stir at 800 rpm for 50 min to obtain a self-assembly solution; the self-assembly solution contains 0.85 mg / mL protamine, 4.675 mg / mL dipotassium glycyrrhizinate and 3.4 mg / mL rosmarinic acid.

[0061] 2. The fifth generation mesenchymal stem cells obtained in Basic Experimental Example 1 were revived and cultured to 80% cell confluence, then digested with trypsin, centrifuged, washed with physiological saline three times, and resuspended with a compound electrolyte solution containing human albumin, heparin and dextran 40 to obtain 10 mL of mesenchymal stem cell solution; the mesenchymal stem cell solution contained 2×10 6 cell mesenchymal stem cells, 0.4g human serum albumin, 2000IU heparin, 2.4g dextran 40.

[0062] 3. Add the mesenchymal stem cell solution to the self-assembly solution at a ratio of mesenchymal stem cell solution: self-assembly solution = 1:1 (v / v), stir at 400 rpm at 4°C for 20 min, and obtain 20 mL of mesenchymal stem cell drug prescription combination 3 (P3).

[0063] Comparative Example 1 Preparation of Mesenchymal Stem Cell Drug Prescription Combination 4

[0064] 1. After the 5th generation mesenchymal stem cells obtained in Basic Experimental Example 1 were revived and cultured to 80% cell confluence, they were digested with trypsin, centrifuged, washed with physiological saline for 3 times, and resuspended with physiological saline to obtain 10 mL of mesenchymal stem cell solution; the mesenchymal stem cell solution contained 2×10 6 cellmesenchymal stem cells.

[0065] 2. Add the mesenchymal stem cell solution to the physiological saline at a ratio of mesenchymal stem cell solution: physiological saline = 1:1 (v / v), stir at 400 rpm at 4°C for 20 min, and obtain 20 mL of mesenchymal stem cell drug prescription combination 4 (P4).

[0066] Comparative Example 2 Preparation of Mesenchymal Stem Cell Drug Prescription Combination 5

[0067] 1. The fifth generation mesenchymal stem cells obtained in Basic Experimental Example 1 were revived and cultured to 80% cell confluence, then digested with trypsin, centrifuged, and washed with physiological saline three times, and resuspended with a compound electrolyte solution containing human albumin, heparin, and dextran 40 to obtain 10 mL of a mesenchymal stem cell solution; the mesenchymal stem cell solution contained 2×10 6 cell mesenchymal stem cells, 0.2g human serum albumin, 1000IU heparin, 2.4g dextran 40.

[0068] 2. Add the mesenchymal stem cell solution to the physiological saline at a ratio of mesenchymal stem cell solution: physiological saline = 1:1 (v / v), stir at 400 rpm at 4°C for 20 min, and obtain 20 mL of mesenchymal stem cell drug prescription combination 5 (P5).

[0069] Comparative Example 3 Preparation of Mesenchymal Stem Cell Drug Prescription Combination 6

[0070] 1. Dissolve protamine, dipotassium glycyrrhizinate and rosmarinic acid in 10 mL of normal saline, and stir at 800 rpm for 50 min to obtain a self-assembly solution; the self-assembly solution contains 0.8 mg / mL protamine, 4.2 mg / mL dipotassium glycyrrhizinate and 2.8 mg / mL rosmarinic acid.

[0071] 2. The fifth generation mesenchymal stem cells obtained in Basic Experimental Example 1 were revived and cultured to 80% cell confluence, then digested with trypsin, centrifuged, washed with saline for 3 times, and resuspended with saline to obtain 10 mL of mesenchymal stem cell solution; the mesenchymal stem cell solution contained 2×10 6 cellmesenchymal stem cells.

[0072] 3. Add the mesenchymal stem cell solution to the self-assembly solution at a ratio of mesenchymal stem cell solution: self-assembly solution = 1:1 (v / v), stir at 4°C and 400 rpm for 20 min, and obtain 20 mL of mesenchymal stem cell drug prescription combination 6 (P6).

[0073] Comparative Example 4 Preparation of Mesenchymal Stem Cell Drug Prescription Combination 7

[0074] 1. Dissolve protamine and dipotassium glycyrrhizinate in physiological saline, and stir at 800 rpm for 50 min to obtain a self-assembly solution; the self-assembly solution contains 0.8 mg / mL protamine and 7 mg / mL dipotassium glycyrrhizinate.

[0075] 2. The fifth generation mesenchymal stem cells obtained in Basic Experimental Example 1 were revived and cultured to 80% cell confluence, then digested with trypsin, centrifuged, washed with physiological saline three times, and resuspended with a compound electrolyte solution containing human albumin, heparin and dextran 40 to obtain 10 mL of mesenchymal stem cell solution; the mesenchymal stem cell solution contained 2×10 6 cell mesenchymal stem cells, 0.2g human serum albumin, 1000IU heparin, 2.4g dextran 40.

[0076] 3. Add the mesenchymal stem cell solution to the self-assembly solution at a ratio of mesenchymal stem cell solution: self-assembly solution = 1:1 (v / v), stir at 4°C and 400 rpm for 20 min, and obtain 20 mL of mesenchymal stem cell drug prescription combination 7 (P7).

[0077] Comparative Example 5 Preparation of Mesenchymal Stem Cell Drug Prescription Combination 8

[0078] 1. Dissolve dipotassium glycyrrhizinate and rosmarinic acid in physiological saline, and stir at 800 rpm for 50 min to obtain a self-assembly solution; the self-assembly solution contains 5 mg / mL dipotassium glycyrrhizinate and 2.8 mg / mL rosmarinic acid.

[0079] 2. The fifth generation mesenchymal stem cells obtained in Basic Experimental Example 1 were revived and cultured to 80% cell confluence, then digested with trypsin, centrifuged, washed with physiological saline three times, and resuspended with a compound electrolyte solution containing human albumin, heparin and dextran 40 to obtain 10 mL of mesenchymal stem cell solution; the mesenchymal stem cell solution contained 2×10 6 cell mesenchymal stem cells, 0.2g human serum albumin, 1000IU heparin, 2.4g dextran 40.

[0080] 3. Add the mesenchymal stem cell solution to the self-assembly solution at a ratio of mesenchymal stem cell solution: self-assembly solution = 1:1 (v / v), stir at 400 rpm at 4°C for 20 min, and obtain 20 mL of mesenchymal stem cell drug prescription combination 8 (P8).

[0081] Experimental Example 1 Cell Number

[0082] The mesenchymal stem cell drug prescription combination (P1-P8) prepared in Examples 1-3 and Comparative Examples 1-5 was packaged and stored in a 4°C refrigerator. The cell viability was measured on the day of packaging D0 (i.e., the day of preparation), D1, D2, D3, and D4. The cell number determination method is as follows: 100 μL of 0.4% trypan blue dye was mixed with 100 μL of the mesenchymal stem cell drug composition (P1-P8) prepared in Examples 1-3 and Comparative Examples 1-5, and 20 μL was dripped into the well of the cell counting plate. The cell number, cell viability, and cell agglomeration rate were detected by Countstar cell counter.

[0083] The results of cell number detection in each mesenchymal stem cell drug prescription combination (all 20mL) are as follows Figure 1 As shown in Table 2, there was no significant difference in the number of cells in the P1-P3 groups at D0 and D1, and it was still maintained above 85% at D2. From D3 onwards, the number of cells in the P1-P3 groups was lower than 80%; while the number of cells in the P5-P8 groups decreased to about 80% at D2 and decreased to less than 25% at D4.

[0084] The results of mesenchymal stem cell preparations stored at different times showed that the number of cells in the P1-P3 groups could be maintained at more than 85% within 48 hours of storage, and then gradually decreased. The number of cells in the P5-P8 groups decreased to 80% after 48 hours of storage, and the number of cells in the P4 group decreased to 50% after 48 hours of storage.

[0085] Table 2 Statistics of cell numbers of mesenchymal stem cell drug prescription combinations at different times (*10 6 / 20mL)

[0086]

[0087] Experimental Example 2 Cell Viability

[0088] The mesenchymal stem cell drug prescription combinations (P1-P8) prepared in Examples 1-3 and Comparative Examples 1-5 were stored in a 4°C refrigerator, and the cell viability was measured on the day of packaging D0 (i.e., the day of preparation), D1, D2, D3, and D4.

[0089] The cell viability determination method is as follows: 100 μL of 0.4% trypan blue dye is mixed with 100 μL of the mesenchymal stem cell pharmaceutical composition (P1-P8) prepared in Examples 1-3 and Comparative Examples 1-5, respectively, and 20 μL is dropped into the well of a cell counting plate, and the cell viability is detected by a Countstar cell counter.

[0090] The results are shown in Table 3. There was no significant change in the cell activity of all P1-P8 groups at D1 and D2. The cell activity began to decrease from D2, and the cell activity of P1-P3 group decreased the least. The cell activity was more than 90% after 24 hours of storage at 4℃, and more than 80% after 48 hours of storage. There were still more than 40% live cells on D4. The cell activity of P7-P8 was slightly worse than that of P1-P3 group. The cell activity was more than 85% after 24 hours of storage, and more than 80% after 48 hours. The cell activity of P5-P6 group was the next worst. The most serious decrease in activity was in P4 group, and the cell activity could not be detected on D4.

[0091] Table 3 Statistics of cell viability of mesenchymal stem cell drug prescription combinations (%)

[0092]

[0093] Note: “—” in the table means the cell viability is less than 5%.

[0094] Experimental Example 3 Cell Aggregation Rate

[0095] The mesenchymal stem cell drug prescription combination (P1-P8) prepared in Examples 1-3 and Comparative Examples 1-5 was taken, and the cell viability was measured on the day of packaging D0 (i.e., the day of preparation), D1, D2, D3, and D4. The cell viability determination method is as follows: 100 μL of 0.4% trypan blue dye was mixed with 100 μL of the mesenchymal stem cell drug composition (P1-P8) prepared in Examples 1-3 and Comparative Examples 1-5, respectively, and 20 μL was dropped into the well of the cell counting plate, and the cell agglomeration rate was detected by Countstar cell counter.

[0096] The results are shown in Figure 4. During the detection process from D0 to D4, the cell agglomeration rate of the P1-P3 group did not change significantly, and at D4, the agglomeration rate was still less than 10%; P7-P8 was second; P5-P6 was third, and the agglomeration rate began to increase from D2, reaching 25% at D4; and the agglomeration rate of P4 increased most significantly, reaching as high as 70% at D4.

[0097] Table 4 Statistics of cell agglomeration rate of mesenchymal stem cell drug prescription combinations (%)

[0098]

[0099]

[0100] Note: “—” in the table means the clumping rate is higher than 25%.

[0101] Experimental Example 4 Flow cytometry phenotyping

[0102] The mesenchymal stem cell drug prescription combinations (P1-P8) prepared in Examples 1-3 and Comparative Examples 1-5 were taken, and the following operations were performed after being packaged and stored for 24 hours: the cells were collected and evenly divided into 10 centrifuge tubes, and the supernatant was discarded after centrifugation at 300 g for 5 minutes, and the following antibodies were added respectively: PE-IgG1, PE-CD73, PE CD90, PE-CD105, PE-CD45, PE-HLA-DR, PE-CD11b, FITC-IgG1, FITC-CD19 and FITC-CD34 (antibodies from BD Company, USA), and the cell surface antigens were detected by BD Calibur flow cytometer.

[0103] The measurement results are shown in Table 5: The mesenchymal stem cell drug prescription combinations (P1-P8) all highly expressed CD73, CD90, and CD105, and did not express CD19, CD34, CD45, CD11b, and HLA-DR.

[0104] Table 5 Flow cytometry phenotype of mesenchymal stem cell drug prescription combination (%)

[0105]

[0106] Experimental Example 5 Biological Efficacy Detection

[0107] The mesenchymal stem cell drug prescription combinations (P1-P8) prepared in Examples 1-3 and Comparative Examples 1-5 were packaged and stored in a refrigerator at 4°C. The biological functions of the mesenchymal stem cells were determined after packaging and storage for 24 hours. The determination method was as follows: 1*10 cells were added to each group. 4 / cm 2 Inoculate T75 culture flasks, culture for 48 hours, harvest the supernatant, digest and count the cells, and detect the HGF and sTNFR1 content in the samples according to the instructions of the HGF and sTNFR1 kits. The results of HGF and sTNFR1 detection are shown in Table 6. The detection results show that the HGF and sTNFR1 secreted by cells in the P1 group are the highest, followed by P2 and P3, P7-P8, P5-P6, and P4 is the lowest.

[0108] Table 6 HGF, sTNFR1 content

[0109]

[0110] Experimental Example 6 Detection of the immune regulation function of mesenchymal stem cells

[0111] The mesenchymal stem cell drug prescription combinations (P1-P8) prepared in Examples 1-3 and Comparative Examples 1-5 were packaged and stored in a 4°C refrigerator for 24 hours to determine the immune regulation function of the mesenchymal stem cells. The determination method is as follows:

[0112] 1. Isolation and CFSE labeling of peripheral blood mononuclear cells

[0113] Peripheral blood was collected from healthy adults, and peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density gradient centrifugation. The cells were re-selected in RPMI1640 medium containing 10% FBS. After cell counting, the concentration was adjusted to 1*10 6 / mL, add CFSE staining (final concentration of 0.2μmol / L), incubate at 37℃ in the dark for 1h, then wash with RPMI1640 complete medium to remove excess dye and set aside.

[0114] 2. Effect of mesenchymal stem cells on PBMC proliferation stimulated by CD3 / CD28 antibodies

[0115] The experiment was divided into the following groups: PBMC negative control group (PBMC-: indicating that PBMC was not stimulated with CD3 / CD28 antibodies), PBMC positive control group (PBMC+: indicating that PBMC was stimulated with CD3 / CD28 antibodies), and PBMC stimulated with CD3 / CD28 antibodies + mesenchymal stem cell drug prescription combination (P1-P8) co-culture group, a total of ten groups.

[0116] The mesenchymal stem cell pharmaceutical compositions (P1-P8) prepared in Examples 1-3 and Comparative Examples 1-5 were placed in a 4°C refrigerator for 24 h, and the cell number and viability were calculated using a Countstar cell counter. The mesenchymal stem cells in each group were counted at 2*10 5 Live cells / well were inoculated in a 6-well plate, and 2 wells were left to add an equal volume of a pharmaceutical excipient composition without mesenchymal stem cells. After culture overnight and until the cells were completely attached to the wall, the supernatant was discarded, and 1 mL of CFSE-labeled PBMC suspension was added to each well of each group, so that the ratio of mesenchymal stem cells and PBMC in the co-culture group was 1:5. Subsequently, anti-CD3 / CD28 antibodies (final concentration of 1 μg / ml) were added to the proliferation group and the co-culture group to stimulate the PBMC, and no CD3 / CD28 antibodies were added to the PBMC single culture group. Continue to culture for 72 hours, collect PBMC cells and cell culture fluid (inflammatory factors to be tested) from each group, and use flow cytometry FITC channel detection to analyze the proliferation rate of PBMC in each group.

[0117] MSCs inhibition rate on PBMC proliferation (%) = (PBMC division index - PBMC & MSCs division index) / PBMC division index * 100%

[0118] The results are as follows Figure 2-3As shown in the results, anti-CD3 / CD28 antibody stimulation can significantly promote the proliferation of peripheral blood PBMC cells (PBMC positive cells, among which the activated cells are mainly lymphocytes); compared with the PBMC cell positive control group, when mesenchymal stem cells and PBMC cells were co-cultured at a ratio of 1:5, the proliferation of PBMC cells could be significantly inhibited (P<0.001, Figure 2 ); Among them, the proliferation inhibition effect of P1, P2, and P3 co-culture groups was the most obvious, exceeding 50%, followed by P7 and P8, and the proliferation inhibition effect of P5 and P6 was weaker, while P4 had the weakest ability to inhibit the proliferation of PBMCs, less than 40%; compared with P1-P3, the ability of P4 group to inhibit lymphocyte proliferation was significantly decreased (P<0.05, Figure 3 ).

[0119] 3. Levels of inflammatory factors in peripheral blood mononuclear cells honey

[0120] The cell culture fluids of each group in Experimental Example 6, "2. Effect of mesenchymal stem cells on CD3 / CD28 antibody-stimulated PBMC cell proliferation", were used to detect the concentrations of TNF-α and IFN-γ in the supernatant. The results are as follows Figure 4-5 As shown in the data, CD3 / CD28 antibodies can significantly activate Th1 cells in PBMC and secrete high levels of TNF-α and IFN-γ (PBMC positive control group), while compared with the positive control group, the concentrations of TNF-α and IFN-γ in the supernatant of the co-culture groups (P1-P8) were significantly reduced (P<0.001), indicating that the mesenchymal stem cell drug prescription combination can significantly inhibit the activation of Th1 cells, among which the inhibitory effect of the P1, P2, and P3 co-culture groups was the most obvious, followed by P5-P8, and the inhibitory effect of P4 was the worst.

[0121] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A mesenchymal stem cell drug prescription combination, characterized in that: The mesenchymal stem cell drug prescription combination comprises a mesenchymal stem cell solution and a self-assembly solution; The mesenchymal stem cell solution is composed of mesenchymal stem cells, human serum albumin, heparin, dextran 40, and a compound electrolyte solution; The self-assembly solution consists of protamine, dipotassium glycyrrhizinate, rosmarinic acid and physiological saline.

2. The mesenchymal stem cell drug prescription combination according to claim 1, characterized in that: The self-assembly solution contains 0.55-1 mg / mL protamine, 2.75-4.7 mg / mL dipotassium glycyrrhizinate and 1.65-3.4 mg / mL rosmarinic acid.

3. The mesenchymal stem cell drug prescription combination according to claim 2, characterized in that: The concentration ratio of protamine, dipotassium glycyrrhizinate and rosmarinic acid in the self-assembly solution is 1:5-5.5:3-4.

4. The mesenchymal stem cell drug prescription combination according to claim 3, characterized in that: The concentration ratio of protamine, dipotassium glycyrrhizinate and rosmarinic acid in the self-assembly solution is 1:5.25:3.

5.

5. The mesenchymal stem cell drug prescription combination according to claim 1, characterized in that: The volume ratio of the mesenchymal stem cell solution to the self-assembly solution is 1:

1.

6. The mesenchymal stem cell drug prescription combination according to claim 1, characterized in that: The concentration of mesenchymal stem cells in the mesenchymal stem cell solution is 1×10 5 -1×10 7 cells / mL, the concentration of human serum albumin is 10-40 mg / mL, the concentration of heparin is 50-200 IU / mL, and the concentration of dextran 40 is 120-240 mg / mL.

7. The mesenchymal stem cell drug prescription combination according to claim 1, characterized in that: The mesenchymal stem cells are selected from one or a combination of two or more of umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, placental mesenchymal stem cells, dental pulp mesenchymal stem cells, amniotic membrane mesenchymal stem cells, and skin mesenchymal stem cells.

8. The method for preparing the mesenchymal stem cell drug prescription combination according to any one of claims 1 to 7, characterized in that: The preparation method comprises the following steps: S1, dissolving protamine, dipotassium glycyrrhizinate and rosmarinic acid in physiological saline, stirring to obtain a self-assembly solution; S2, resuspending the mesenchymal stem cells in a compound electrolyte solution containing human serum albumin, heparin and dextran 40 to obtain a mesenchymal stem cell solution; S3. Add the mesenchymal stem cell solution into the self-assembly solution and stir to obtain a mesenchymal stem cell drug prescription combination.

9. The preparation method according to claim 8, characterized in that: The stirring in step S1 is 600-1000 rpm, and the stirring time is 45-60 min.

10. The preparation method according to claim 8, characterized in that: The stirring in step S3 is at 4° C., 400-600 rpm, and stirring for 15-20 min.

11. Use of the mesenchymal stem cell drug prescription combination according to any one of claims 1 to 8 in the preparation of a drug containing mesenchymal stem cells.

12. The use according to claim 11, characterized in that: The content of mesenchymal stem cells in the drug is ≥ 0.5×10 5 / mL.

13. The use according to claim 11, characterized in that: The medicine containing mesenchymal stem cells is used for preventing or treating tumors or autoimmune diseases.

14. The use according to claim 13, characterized in that The dosage forms of the mesenchymal stem cell-containing drug include: tablets, capsules, injections, solutions, syrups, emulsions, suspensions, granules, ointments, suppositories, aerosols or pills.

15. A drug containing mesenchymal stem cells, characterized in that: The medicine is prepared by the mesenchymal stem cell medicine prescription combination according to any one of claims 1 to 7.

16. The drug according to claim 15, characterized in that The medicine also includes pharmaceutically acceptable excipients.

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