A mesenchymal stem cell drug prescription and its preparation process

Abstract

The present invention provides a mesenchymal stem cell drug formulation and its preparation process, belonging to the field of biomedical technology. The mesenchymal stem cell drug formulation 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 formulation 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 a good immune regulation function, providing a new idea for the clinical application of mesenchymal stem cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a mesenchymal stem cell drug formulation and its preparation process. Background Art

[0002] Mesenchymal stem cells (MSCs) are adult stem cells derived from stroma, mainly present in connective tissues and organ interstitium such as bone marrow, dental pulp, adipose tissue, umbilical cord, etc. Mesenchymal stem cells have many unique advantages, such as the ability to self-renew, effectively regulate immune functions, etc., and the sources of such stem cells are very rich and easy to culture. From the existing medical research, mesenchymal stem cell preparations have been widely used, covering neurological diseases, damage to various organs and tissues, and autoimmune diseases, etc. They not only greatly improve the success rate of various organ transplants, but also effectively reduce the rejection reaction of patients after organ transplantation, and have inappreciable application value clinically. Due to the influence of various factors and the complexity of treatment plans for different patients, the technology for preparing stem cell preparations needs to comprehensively consider various factors in the specific operation process. The prepared stem cells cannot be immediately cultured and applied, so it is necessary to strengthen the research on the preservation stability of stem cells to improve the cell activity and survival rate.

[0003] In the formulation of mesenchymal stem cell injection, low molecular weight calcium heparin (or sodium heparin) 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 calcium heparin may also be accompanied by some side effects, mainly including active bleeding or an increased risk of bleeding related to abnormal hemostasis, organic damage that may cause bleeding (such as active peptic ulcer), hemorrhagic cerebrovascular accident, acute infective endocarditis, and patients with severe renal impairment (creatinine clearance rate less than 30 ml / min). In addition, the addition of human blood albumin may also be accompanied by some side effects, including allergic reactions, increased oxidative activity, etc., manifested as skin itching, erythema, and even dyspnea and increased body temperature.

[0004] Adding protamine, rosmarinic acid, etc. to the stem cell prescription reduces the side effects of the above components. It has the advantages of high safety, good anti-corrosion performance, high thermal stability, etc., 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, having anti-tumor activity; adding protamine to the mesenchymal stem cell prescription can be used to neutralize the anticoagulant effect of heparin, thereby reducing the bleeding risk of patients. Rosmarinic acid is a natural antioxidant with strong anti-allergic and antioxidant activities, which helps prevent cell damage caused by free radicals. And rosmarinic acid also has strong anti-inflammatory, antibacterial, antiviral, and anti-tumor activities. The self-assembled body of traditional Chinese medicine not only has the same drug delivery ability as artificially synthesized nanocarriers, but also has characteristics such as degradability, compatibility, and safety that artificial synthetic nanomaterials do not have. It is found that dipotassium glycyrrhizinate has amphiphilicity and can aggregate to form self-assembled micelles, which are widely used as multifunctional drug carriers to improve drug absorption and enhance drug stability.

[0005] Therefore, a new combination of mesenchymal stem cell drug prescriptions is provided, using 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 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-assembled solution; the mesenchymal stem cell solution is composed of mesenchymal stem cells, human albumin, heparin, dextran 40, and compound electrolyte solution, and the self-assembled 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, and has good immune regulation function, providing a new idea for the clinical application of mesenchymal stem cells.

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

[0008] On the one hand, the present invention provides a mesenchymal stem cell drug prescription combination, and the mesenchymal stem cell drug prescription combination includes a mesenchymal stem cell solution and a self-assembled solution;

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

[0010] The self-assembled solution described above is composed of protamine, dipotassium glycyrrhizinate, rosmarinic acid and normal saline.

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

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

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

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

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

[0016] Preferably, the volume ratio of the mesenchymal stem cell solution to the self-assembled 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 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 is 20 mg / mL, the concentration of heparin is 100 IU / mL, and the concentration of dextran 40 is 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 mesenchymal stem cells, and skin mesenchymal stem cells.

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

[0021] S1. Dissolve protamine, dipotassium glycyrrhizinate, and rosmarinic acid in physiological saline and stir to obtain a self-assembled solution;

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

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

[0024] Specifically, the stirring in step S1 is at 600 - 1000 rpm for 45 - 60 minutes.

[0025] Preferably, the stirring in step S1 is at 800 rpm for 50 minutes.

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

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

[0028] In a third aspect, the present invention provides the application 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 ≥ 0.5×10 5 / mL.

[0030] Specifically, the drug containing mesenchymal stem cells is used for preventing or treating tumors or autoimmune diseases.

[0031] Preferably, the tumors include but are not limited to: digestive system tumors, respiratory system tumors, reproductive system tumors, locomotor system tumors, nervous system tumors, endocrine system tumors, circulatory system tumors, urinary system tumors, or reproductive system tumors.

[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; the respiratory system diseases include but are not limited to: asthma, chronic obstructive pulmonary disease, adult respiratory distress syndrome.

[0033] Specifically, the dosage forms of the drug containing mesenchymal stem cells include but are not limited to: tablets, capsules, injections, solutions, syrups, emulsions, suspensions, granules, ointments, suppositories, aerosols, or pills.

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

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

[0036] Specifically, the drug further 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, solubilizers, solubilizing agents, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, and buffers.

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

[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 functions, providing new ideas for the clinical application of mesenchymal stem cells. Description of the Drawings

[0040] Figure 1 Is the detection result of cell number

[0041] Figure 2 Is the proliferation percentage of PBMC stimulated by CD3 / CD28 antibody; *** in the figure represents P < 0.001.

[0042] Figure 3 Is the inhibition rate of hUC-MSC on the proliferation of PBMC stimulated by CD3 / CD28 antibody; * 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 Embodiments

[0045] The present invention will be described below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention, so that the technical solutions of the present invention can be more easily understood and grasped. The experimental methods described in the following embodiments are all 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: Culture and passage of mesenchymal stem cells

[0050] The mesenchymal stem cells were cultured to the 5th generation and cryopreserved. The culture system and environment were: DMEM / F12 culture medium containing 10% v / v FBS, 37 °C, 5% CO2.

[0051] Example 1: Preparation of mesenchymal stem cell drug formulation combination 1

[0052] 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-assembled solution; the self-assembled solution contains 0.8 mg / mL protamine, 4.2 mg / mL dipotassium glycyrrhizinate and 2.8 mg / mL rosmarinic acid.

[0053] 2. After resuscitating and culturing the 5th generation of mesenchymal stem cells obtained in Basic Experimental Example 1 to 80% cell confluence, digest with trypsin, centrifuge, wash 3 times with physiological saline, and resuspend 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 contains 2×10 6 cells of mesenchymal stem cells, 0.2 g of human albumin, 1000 IU of heparin, 2.4 g of dextran 40.

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

[0055] Example 2: Preparation of mesenchymal stem cell drug formulation combination 2

[0056] 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-assembled solution; the self-assembled solution contains 0.55 mg / mL protamine, 2.75 mg / mL dipotassium glycyrrhizinate and 1.65 mg / mL rosmarinic acid.

[0057] 2. After resuscitating and culturing the 5th generation of mesenchymal stem cells obtained in Basic Experimental Example 1 to 80% cell confluence, digest with trypsin, centrifuge, wash 3 times with physiological saline, and resuspend 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 contains 2×106 Mesenchymal stem cells, 0.1 g of human albumin, 500 IU of heparin, 1.2 g of dextran 40.

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

[0059] Preparation of mesenchymal stem cell drug formulation combination 3 in Example 3

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

[0061] 2. After resuscitating and culturing the 5th-generation mesenchymal stem cells obtained in Basic Experimental Example 1 until 80% cell confluence, digest with trypsin, centrifuge, wash 3 times with physiological saline, and resuspend 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 contains 2×10 6 Mesenchymal stem cells, 0.4 g of human albumin, 2000 IU of heparin, 2.4 g of dextran 40.

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

[0063] Preparation of mesenchymal stem cell drug formulation combination 4 in Comparative Example 1

[0064] 1. After resuscitating and culturing the 5th-generation mesenchymal stem cells obtained in Basic Experimental Example 1 until 80% cell confluence, digest with trypsin, centrifuge, wash 3 times with physiological saline, and resuspend with physiological saline to obtain 10 mL of mesenchymal stem cell solution; the mesenchymal stem cell solution contains 2×10 6 Mesenchymal stem cells.

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

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

[0067] 1. After resuscitating and culturing the 5th generation of mesenchymal stem cells obtained in Basic Experimental Example 1 until 80% cell confluence, digest them with trypsin, centrifuge, wash them 3 times with physiological saline, and resuspend them 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 contains 2×10 6 cells of mesenchymal stem cells, 0.2 g of human albumin, 1000 IU of heparin, and 2.4 g of dextran 40.

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

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

[0070] 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-assembled solution; the self-assembled solution contains 0.8 mg / mL of protamine, 4.2 mg / mL of dipotassium glycyrrhizinate, and 2.8 mg / mL of rosmarinic acid.

[0071] 2. After resuscitating and culturing the 5th generation of mesenchymal stem cells obtained in Basic Experimental Example 1 until 80% cell confluence, digest them with trypsin, centrifuge, wash them 3 times with physiological saline, and resuspend them with physiological saline to obtain 10 mL of mesenchymal stem cell solution; the mesenchymal stem cell solution contains 2×10 6 cells of mesenchymal stem cells.

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

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

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

[0075] 2. After resuscitating and culturing the 5th-generation mesenchymal stem cells obtained in Basic Experimental Example 1 until 80% cell confluence, they were digested with trypsin, centrifuged, and washed 3 times with physiological saline, and then resuspended with a compound electrolyte solution containing human albumin, heparin, and dextran 40 to obtain a 10 mL mesenchymal stem cell solution; the mesenchymal stem cell solution contained 2×10 6 cells of mesenchymal stem cells, 0.2 g of human albumin, 1000 IU of heparin, and 2.4 g of dextran 40.

[0076] 3. According to the ratio of mesenchymal stem cell solution: self-assembled solution = 1:1 (v / v), the mesenchymal stem cell solution was added to the self-assembled solution, and stirred at 4°C and 400 rpm for 20 min to obtain 20 mL of mesenchymal stem cell drug formulation 7 (P7).

[0077] Preparation of mesenchymal stem cell drug formulation 8 in Comparative Example 5

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

[0079] 2. After resuscitating and culturing the 5th-generation mesenchymal stem cells obtained in Basic Experimental Example 1 until 80% cell confluence, they were digested with trypsin, centrifuged, and washed 3 times with physiological saline, and then resuspended with a compound electrolyte solution containing human albumin, heparin, and dextran 40 to obtain a 10 mL mesenchymal stem cell solution; the mesenchymal stem cell solution contained 2×10 6 cells of mesenchymal stem cells, 0.2 g of human albumin, 1000 IU of heparin, and 2.4 g of dextran 40.

[0080] 3. According to the ratio of mesenchymal stem cell solution: self-assembled solution = 1:1 (v / v), the mesenchymal stem cell solution was added to the self-assembled solution, and stirred at 4°C and 400 rpm for 20 min to obtain 20 mL of mesenchymal stem cell drug formulation 8 (P8).

[0081] Cell number in Experimental Example 1

[0082] The mesenchymal stem cell drug prescription combinations (P1 - P8) prepared in Examples 1 - 3 and Comparative Examples 1 - 5 were aliquoted and stored in a 4°C refrigerator. The cell viability was measured on the day of aliquoting D0 (i.e., the day of preparation completion), D1, D2, D3, and D4. The method for measuring the cell number was as follows: 100 μL of 0.4% trypan blue dye was mixed with 100 μL of the mesenchymal stem cell drug compositions (P1 - P8) prepared in Examples 1 - 3 and Comparative Examples 1 - 5. Then, 20 μL was taken and dropped into the wells of a cell counting chamber, and the cell number, cell viability, and cell aggregation rate were detected using a Countstar cell counter.

[0083] The detection results of the cell numbers in each mesenchymal stem cell drug prescription combination (all 20 mL) are as Figure 1 shown in Table 1 and Table 2. There were no significant differences in the cell numbers of the P1 - P3 groups at D0 and D1, and they could still be maintained above 85% at D2. Starting from D3, the cell numbers of the P1 - P3 groups were all below 80%. For the P5 - P8 groups, the cell numbers began to decrease to about 80% at D2 and decreased to below 25% at D4.

[0084] From the results of storing the mesenchymal stem cell preparations for different times, it can be seen that: for the P1 - P3 groups, the cell numbers could be maintained above 85% within 48 hours of storage and then gradually decreased. For the P5 - P8 groups, the cell numbers all decreased to 80% within 48 hours of storage, and for the P4 group, it decreased to 50% within 48 hours of storage.

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

[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 aliquoting D0 (i.e., the day of preparation completion), D1, D2, D3, and D4.

[0089] The method for measuring the cell viability was as follows: 100 μL of 0.4% trypan blue dye was mixed with 100 μL of the mesenchymal stem cell drug compositions (P1 - P8) prepared in Examples 1 - 3 and Comparative Examples 1 - 5 respectively. Then, 20 μL was taken and dropped into the wells of a cell counting chamber, and the cell viability was detected using a Countstar cell counter.

[0090] The results are shown in Table 3. There were no significant changes in the cell viability of all P1-P8 groups at D1 and D2. The cell viability began to decrease starting from D2. Among them, the cell viability of the P1-P3 groups decreased the least. After being stored at 4°C for 24 h, the cell viability was above 90%, and after 48 h, it was above 80%. By D4, there were still more than 40% viable cells. The cell viability of the P7-P8 groups was slightly worse than that of the P1-P3 groups. After being stored for 24 h, the cell viability was above 85%, and by 48 h, it was above 80%. The cell viability of the P5-P6 groups was the second. The most serious decrease in viability was in the P4 group, and the cell viability could not be detected by D4.

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

[0092]

[0093] Note: The "-" in the table represents that the cell viability is less than 5%.

[0094] Experimental Example 3 Cell aggregation rate

[0095] Take the mesenchymal stem cell drug prescription combinations (P1-P8) prepared in Examples 1-3 and Comparative Examples 1-5, and measure the cell viability on the day of sub-packaging D0 (i.e., the day of preparation completion), D1, D2, D3, and D4 respectively. The method for measuring cell viability is as follows: Mix 100 μL of 0.4% trypan blue dye with 100 μL of the mesenchymal stem cell drug compositions (P1-P8) prepared in Examples 1-3 and Comparative Examples 1-5 respectively. Take 20 μL and drop it into the wells of a cell counting chamber, and use a Countstar cell counter to detect the cell aggregation rate.

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

[0097] Table 4 Statistical results of the cell aggregation rate of the mesenchymal stem cell drug prescription combinations (%)

[0098]

[0099]

[0100] Note: The "-" in the table represents that the aggregation rate is higher than 25%.

[0101] Experimental Example 4 Flow cytometry phenotype detection

[0102] Take the mesenchymal stem cell drug prescription combinations (P1 - P8) prepared in Examples 1 - 3 and Comparative Examples 1 - 5. After aliquoting and storing for 24 h, the following operations were carried out: Collect the cells, evenly distribute them into 10 centrifuge tubes, centrifuge at 300 g for 5 minutes, then aspirate and discard the supernatant. Add the following antibodies 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 Biosciences, USA), and detect cell surface antigens by a BDCalibur 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 cytometric phenotypes of mesenchymal stem cell drug prescription combinations (%)

[0105]

[0106] Experimental Example 5 Detection of biological potency

[0107] Take the mesenchymal stem cell drug prescription combinations (P1 - P8) prepared in Examples 1 - 3 and Comparative Examples 1 - 5, aliquot them, store them in a 4 °C refrigerator, and measure the biological functions of mesenchymal stem cells after aliquoting and storing for 24 h. The measurement method is as follows: Cells in each group were inoculated into a T75 culture flask at a density of 1×10 4 / cm 2 . After culturing for 48 h, harvest the supernatant, digest and count the cells, and operate according to the instructions of the HGF and sTNFR1 kits to detect the contents of HGF and sTNFR1 in the samples. The detection results of HGF and sTNFR1 are shown in Table 6. The detection results showed that the levels of HGF and sTNFR1 secreted by the cells in Group P1 were the highest, followed by P2 and P3, then P7 - P8, lower in P5 - P6, and the lowest in P4.

[0108] Table 6 Contents of HGF and sTNFR1

[0109]

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

[0111] Take the mesenchymal stem cell drug prescription combinations (P1 - P8) prepared in Examples 1 - 3 and Comparative Examples 1 - 5, aliquot them, store them in a 4 °C refrigerator for 24 h, and measure the immune regulation function of mesenchymal stem cells. The measurement method is as follows:

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

[0113] Take healthy adult peripheral blood, and isolate peripheral blood mononuclear cells (PBMC) by Ficoll density gradient centrifugation. Resuspend them in RPMI1640 medium containing 10% FBS. After cell counting, adjust the concentration to 1×10 6 / mL, add CFSE staining (final concentration 0.2 μmol / L), incubate at 37 °C in the dark for 1 h, then wash with complete RPMI1640 medium to remove excess dye, and set aside for use.

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

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

[0116] Take the mesenchymal stem cell drug compositions (P1 - P8) prepared in Examples 1 - 3 and Comparative Examples 1 - 5, store them in a 4 °C refrigerator for 24 h, then use a Countstar cell counter to calculate the cell number and viability, and place the mesenchymal stem cells in each group at 2×10 5 viable cells / well, inoculate them into 6-well plates, and leave 2 wells to add an equal volume of drug excipient composition without mesenchymal stem cells. Culture overnight until the cells are completely adherent, aspirate the supernatant, add 1 mL of CFSE-labeled PBMC suspension to each well in each group, so that the ratio of mesenchymal stem cells to PBMC in the co-culture group is 1:5. Subsequently, add anti-CD3 / CD28 antibody (final concentration 1 μg / ml) to stimulate PBMC in the proliferation group and co-culture group, and the PBMC single culture group does not add CD3 / CD28 antibody. Continue to culture for 72 h, collect PBMC cells and cell culture supernatants (for detecting inflammatory factors) in each group, and use the FITC channel of a flow cytometer to detect and analyze the proliferation rate of PBMC in each group.

[0117] Inhibitory rate of MSCs on PBMC proliferation (%) = (PBMC division index - PBMC&MSCs division index) / PBMC division index × 100%

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

[0119] 3. Inflammatory factor levels in peripheral blood mononuclear cell honey

[0120] The cell culture media of each group in "2. Effect of mesenchymal stem cells on the proliferation of PBMC cells stimulated by anti-CD3 / CD28 antibody" in Experimental Example 6 were used to detect the concentrations of TNF-α and IFN-γ in the supernatant by ELISA method. The results are as Figures 4 - 5 shown. Anti-CD3 / CD28 antibody 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) are significantly reduced (P<0.001), indicating that the mesenchymal stem cell drug prescription combination can significantly inhibit the activation of Th1 cells. Among them, the inhibitory effect in the co-culture groups of P1, P2, and P3 is the most obvious, followed by P5-P8, and the inhibitory effect of P4 is the worst.

[0121] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and this embodiment is not used to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of this invention patent should be subject to the appended claims.

Claims

1. A mesenchymal stem cell drug combination, characterized in that, The described mesenchymal stem cell drug combination includes a mesenchymal stem cell solution and a self-assembling solution; The mesenchymal stem cell solution is composed of mesenchymal stem cells, human albumin, heparin, dextran 40, and a compound electrolyte solution; The self-assembling solution is composed of protamine, dipotassium glycyrrhizinate, rosmarinic acid, and physiological saline; the self-assembling 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.

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

3. The mesenchymal stem cell drug combination according to claim 2, wherein, In the self-assembling solution, the concentration ratio of protamine, dipotassium glycyrrhizinate, and rosmarinic acid is 1:5.25:3.

5.

4. The mesenchymal stem cell drug combination according to claim 1, wherein The volume ratio of the mesenchymal stem cell solution to the self-assembling solution is 1:

1.

5. The mesenchymal stem cell drug 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.

6. The mesenchymal stem cell drug combination according to claim 1, wherein, The described 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 mesenchymal stem cells, and skin mesenchymal stem cells.

7. A method for preparing the mesenchymal stem cell drug combination according to any one of claims 1-6, characterized in that, The described preparation method includes the following steps: S1. Dissolve protamine, dipotassium glycyrrhizinate, and rosmarinic acid in physiological saline and stir to obtain a self-assembling solution; S2. Resuspend mesenchymal stem cells in a compound electrolyte solution containing human albumin, heparin, and dextran 40 to obtain a mesenchymal stem cell solution; S3. Add the mesenchymal stem cell solution to the self-assembling solution and stir to obtain a mesenchymal stem cell drug combination.

8. The preparation method according to claim 7, characterized in that, The stirring in step S1 is at 600 - 1000 rpm for 45 - 60 min.

9. The preparation method according to claim 7, wherein, The stirring in step S3 is at 4°C, 400 - 600 rpm for 15 - 20 min.

10. A drug containing mesenchymal stem cells, characterized in that, The described drug is prepared from the mesenchymal stem cell drug combination according to any one of claims 1 - 6.

11. The drug according to claim 10, characterized in that, The described drug further includes pharmaceutically acceptable excipients.

12. The drug according to claim 10, characterized in that, The dosage forms of the described drug include: tablets, capsules, injections, solutions, syrups, emulsions, suspensions, granules, ointments, suppositories, aerosols, or pills.

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