CN112190596A - Mesenchymal stem cell preparation for treating arthritis and preparation method thereof - Google Patents
Mesenchymal stem cell preparation for treating arthritis and preparation method thereof Download PDFInfo
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- CN112190596A CN112190596A CN202010959571.3A CN202010959571A CN112190596A CN 112190596 A CN112190596 A CN 112190596A CN 202010959571 A CN202010959571 A CN 202010959571A CN 112190596 A CN112190596 A CN 112190596A
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- metformin
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Abstract
The application provides a mesenchymal stem cell preparation for treating arthritis, which comprises mesenchymal stem cells, metformin-pretreated mesenchymal stem cells, platelet-rich plasma and a dispersion medium. The mesenchymal stem cells pretreated by the metformin can improve the osteogenic differentiation capacity of the mesenchymal stem cells, and are beneficial to repairing damage. The mixed use of the mesenchymal stem cells and the mesenchymal stem cells pretreated by the metformin can not only ensure the secretion of the anti-inflammatory factors of the mesenchymal stem cells, but also ensure the tissue repair capability of the mesenchymal stem cells. The mesenchymal stem cell preparation provided by the invention is suitable for relieving inflammation in osteoarthritis and repairing damaged tissues, and can realize the relief of symptoms and functional recovery of patients.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a mesenchymal stem cell preparation for treating arthritis and a preparation method thereof.
Background
Arthritis generally refers to inflammatory diseases caused by inflammation, infection, degeneration, trauma or other factors, and can be divided into tens of kinds. Arthritis patients in China are more than 1 hundred million, and the number of the arthritis patients is increasing continuously. The clinical manifestations are red, swelling, heat, pain, dysfunction and joint deformity of joints, and severe patients cause joint disability and affect the life quality of patients. According to statistics, half of people over 50 years old in China suffer from osteoarthritis, and 90% of women and 80% of men suffer from osteoarthritis in people over 65 years old. The prevalence rate of China is 0.34-0.36%, and the life of serious people is shortened by about 10-15 years.
Joint replacement is a method of replacing a painful joint that has lost joint function with an artificially produced joint, and is generally applied to severe osteonecrosis, persons who cannot be restored by comminuted fracture dislocation, painful and dyskinetic osteoarthropathy, rheumatoid arthritis that is stiff or difficult to move, bone tumors, and the like. Joint replacement has complications such as prosthesis loosening, wear or fracture, deep infection, foreign body reaction, and calcification of soft tissues that impede mobility.
The mesenchymal stem cells are cells with self-renewal and multidirectional differentiation capabilities, and are easily obtained and widely available. Can be differentiated into various tissues such as bone, cartilage, fat and the like, has immunoregulation function and has great potential for regenerative medicine and autoimmune diseases. The mesenchymal stem cells are a new treatment mode for treating osteoarthritis, and a large number of preclinical researches prove that the mesenchymal stem cells have good effects on treating rheumatoid arthritis. The Chinese patent with application number of 201010152461.2 discloses a cell injection for treating bone injury and a preparation method thereof, wherein the injection is a compound consisting of mesenchymal stem cells from autologous bone marrow, temperature-sensitive gel and effective nutritional factors extracted by autologous blood separation. However, the composite biocompatible polymer or temperature-sensitive gel described in the application document forms a semisolid or gel after being implanted into a body, which is not beneficial to the dispersion of mesenchymal stem cells in a joint cavity, and prevents the application of the process of improving the joint cavity microenvironment through the immune regulation function of the stem cells in cells; the mesenchymal stem cells or the chondrocytes only contain single cell types, and the mesenchymal stem cells are only applied to have paracrine and immune regulation functions, but the tissue repair is realized by in-vivo induced differentiation, so the treatment time is long; the induced mesenchymal stem cells are used for treatment, only have the function of tissue repair, do not have the function of anti-inflammatory factor secretion, and are not beneficial to the repair of arthritis.
Disclosure of Invention
Aiming at the problems that the arthritis treatment preparation or method in the prior art has single cell type, long treatment time, can not fully exert the biology of the mesenchymal stem cells, is not beneficial to the recovery of arthritis and the like, the invention provides the mesenchymal stem cell preparation for treating the arthritis and the preparation method thereof.
In order to realize the purpose of the invention, the invention is realized by adopting the following technical scheme:
a mesenchymal stem cell preparation for treating arthritis comprises mesenchymal stem cells, metformin-pretreated mesenchymal stem cells, platelet-rich plasma and a dispersion medium.
Metformin promotes osteoblast activity, increases mineral precipitation, and simultaneously inhibits osteoclast activity and prevents bone mass reduction. Metformin induces the growth and differentiation of osteoblast-like cell lines (UMR 106, MC3T 3) with the ability to increase mineralization of the extracellular matrix. Furthermore, metformin also has the effects of promoting the differentiation of bone marrow stromal cells to osteoblasts and inhibiting the differentiation to adipocytes. The metformin is used for pretreating the mesenchymal stem cells, so that the osteogenesis capacity of the mesenchymal stem cells can be improved, and the tissue repair is facilitated.
The platelet-rich plasma can maintain the viability of the mesenchymal stem cells in the joint cavity and prolong the anti-inflammatory action time of the mesenchymal stem cells. In addition, platelet-rich plasma contains a large amount of growth factors such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), insulin-like growth factor 1 (IGF-1) and other rich growth factors, and can promote the regeneration of articular cartilage.
Preferably, the mesenchymal stem cells are bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells and dental pulp mesenchymal stem cells, and the final cell concentration of the mesenchymal stem cell preparation is 106～108one/mL.
Preferably, the ratio of the mesenchymal stem cells to the metformin-pretreated mesenchymal stem cells is 1: 1-4: 1.
Preferably, the concentration of platelets in the platelet-rich plasma is 2-3 × 106one/uL.
Preferably, the volume ratio of the platelet-rich plasma to the dispersion medium is 1: 1-2: 1.
Preferably, the dispersion medium comprises human serum albumin, low molecular heparin, compound amino acid, vitamin C and compound electrolyte. The mass-volume ratio of human serum albumin is 1-5%, the mass-volume ratio of low molecular heparin is 0.5%, the mass-volume ratio of compound amino acid is 1-10%, the mass-volume ratio of vitamin C is 0.3-0.7%, and the balance is compound electrolyte.
The human serum albumin and the compound amino acid adopted by the invention are all components of clinical injection, can provide nutrition for cells and are beneficial to metabolism of the cells. The addition of vitamin C can maintain the activity of various peroxidases and is also beneficial to the maintenance of cell metabolism and activity. The addition of the low-molecular heparin ensures that cells maintain a good cell dispersion state in the preservation process, reduces the phenomena of intercellular adhesion agglomeration and cell wall adhesion, reduces the risk of intravascular cell agglomeration embolism possibly occurring during clinical cell infusion, and simultaneously reduces cell loss caused by cell aggregation and filtration by an infusion filter, and the addition of trace heparin does not cause adverse reactions such as clinical bleeding. The compound electrolyte solution can maintain the osmotic pressure of cells and is beneficial to the survival of the cells.
The invention provides a preparation method of a mesenchymal stem cell preparation for treating arthritis, which comprises the following steps:
(1) and (3) isolated culture of mesenchymal stem cells: after mesenchymal stem cells were isolated, the cells were inoculated into an α -MEM medium containing 10% fetal bovine serum and placed in CO2Culturing in an incubator, and digesting and passing by adopting 0.2 percent of pancreatin when the fusion degree of the mesenchymal stem cells is 80 to 85 percent;
(2) metformin pretreatment of mesenchymal stem cells: inoculating the mesenchymal stem cells passaged to P4 generation into alpha-MEM medium containing metformin and 10% fetal calf serum, and culturing in CO2Culturing in an incubator;
(3) preparation of platelet-rich plasma: taking venous blood added with anticoagulant, concentrating platelets by adopting a two-step centrifugation method, carrying out low-speed centrifugation at 100 Xg for 10 minutes in the first step to separate blood plasma and platelets from red blood cells and white blood cells, carrying out high-speed centrifugation at 400 Xg for 10 minutes in the second step to further concentrate platelet-containing blood plasma and separate platelet-containing components, and obtaining the platelet with the concentration of 2-3X 106Plasma per uL;
(4) the preparation of the mesenchymal stem cell preparation comprises the steps of weighing human serum albumin, low molecular heparin, compound amino acid and vitamin C according to the mass-volume ratio, preparing a dispersion medium, pre-cooling at 4 ℃ for standby, digesting and counting the mesenchymal stem cells pretreated by the mesenchymal stem cells and the metformin, mixing the platelet-rich plasma and the dispersion medium according to the volume ratio of 1: 1-2: 1, suspending the standby cells, and adjusting the final cell concentration to 106～108one/mL.
Preferably, the concentration of the metformin in the step (2) is 50-100 mu M/L. Preferably, the concentration of the metformin in the step (2) is 50-80 μ M.
Preferably, the culture time in the step (2) is 24-48 hours.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a mesenchymal stem cell preparation for treating arthritis and a preparation method thereof. The mesenchymal stem cells pretreated by the metformin can improve the osteogenic differentiation capacity of the mesenchymal stem cells, and are beneficial to repairing damage. The mixed use of the mesenchymal stem cells and the mesenchymal stem cells pretreated by the metformin can not only ensure the secretion of the anti-inflammatory factors of the mesenchymal stem cells, but also ensure the tissue repair capability of the mesenchymal stem cells. The platelet-rich plasma can maintain the viability of the mesenchymal stem cells in the joint cavity and prolong the anti-inflammatory action time of the mesenchymal stem cells. It is rich in platelet and plasma, and growth factor, and can promote the regeneration of articular cartilage. In addition, the prepared cell preparation is in a suspension form, and can ensure that cells, various growth factors and active substances are dispersed in the whole joint cavity after injection in the joint cavity, improve the microenvironment of the joint cavity and facilitate the recovery of arthritis.
Drawings
Fig. 1A is a flow detection result of a positive surface marker of umbilical cord mesenchymal stem cells CD90 in an embodiment of the present invention;
fig. 1B is a flow detection result of an umbilical cord mesenchymal stem cell CD105 positive surface marker in an embodiment of the present invention;
fig. 1C is a flow detection result of a positive surface marker of umbilical cord mesenchymal stem cells CD73 in an embodiment of the present invention;
fig. 1D is a flow detection result of an umbilical cord mesenchymal stem cell CD34 negative surface marker in an embodiment of the present invention;
fig. 1E is a flow detection result of an umbilical cord mesenchymal stem cell CD45 negative surface marker in an embodiment of the present invention;
fig. 1F is a flow detection result of an umbilical cord mesenchymal stem cell CD14 negative surface marker in an embodiment of the present invention;
fig. 1G is a flow detection result of an umbilical cord mesenchymal stem cell CD19 negative surface marker in an embodiment of the present invention;
FIG. 1H shows the result of flow detection of HLA-DR negative surface marker of umbilical cord mesenchymal stem cells according to an embodiment of the present invention;
FIG. 2A shows alizarin red staining results of osteogenic differentiation of umbilical cord mesenchymal stem cells of a control group in the example of the present invention;
fig. 2B is a result of alizarin red staining for osteogenic differentiation of umbilical cord mesenchymal stem cells in the metformin group in the example of the present invention.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Description of the reagents:
alpha-MEM medium was purchased from Sigma;
metformin was purchased from Sigma;
human serum albumin: purchased from jertebain biologicals ltd, switzerland, production lot number P10009968;
low molecular weight heparin calcium injection: purchased from Shenzhen Sailur biological pharmacy Co., Ltd, production lot 1809103;
compound amino acid: purchased from tokyo corporation, east asia pharmaceutical ltd, jiang xie, pharmaceutical standard H19993208;
vitamin C: procurement from sienlijun pharmaceutical llc company, production lot A8L 708;
compound electrolyte: purchased from Shanghai Baite medical supplies, Inc., drug Standard H2000475.
Example 1
The embodiment provides a preparation of mesenchymal stem cells for treating arthritis, which comprises the mesenchymal stem cells, the metformin-pretreated mesenchymal stem cells, platelet-rich plasma and a dispersion medium.
In some embodiments, the mesenchymal stem cell is a bone marrow mesenchymal stem cell, an umbilical cord mesenchymal stem cell, an adipose mesenchymal stem cell, a dental pulp mesenchymal stem cell, the final cell concentration of the mesenchymal stem cell preparation is 106～108Per mL, e.g. 106one/mL, 107one/mL, 108one/mL.
In some embodiments, the ratio of mesenchymal stem cells to metformin-pretreated mesenchymal stem cells is 1:1 to 4:1, such as 1:1, 2:1, 3:1, 4: 1.
In some casesIn the examples, the concentration of platelets in platelet-rich plasma is 2 to 3X 106a/uL of, for example, 2X 1062.5 × 10 units/uL6mu/uL, 3X 106one/uL.
In some embodiments, the volume ratio of platelet rich plasma to dispersion medium is from 1:1 to 2:1, e.g., 1:1, 1.5:1, 2: 1.
In some embodiments, the dispersion medium comprises human serum albumin, low molecular heparin, complexed amino acids, vitamin C, and complexed electrolytes.
In some embodiments, the human serum albumin has a mass to volume ratio of 1 to 5%, such as 1%, 2%, 3%, 4%, 5%, the low molecular heparin has a mass to volume ratio of 0.5%, the compound amino acid has a mass to volume ratio of 1 to 10%, such as 1%, 3%, 6%, 8%, 10%, the vitamin C has a mass to volume ratio of 0.3 to 0.7%, such as 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, and the balance is the compound electrolyte.
Example 2
The embodiment provides a preparation method of a mesenchymal stem cell preparation for treating arthritis, which comprises the following steps:
isolated culture of mesenchymal stem cells
A conventional method is adopted to extract 50mL of marrow liquid from the puncture point of the anterior iliac spine by using a bone puncture needle, and the marrow liquid is injected into a sterile centrifuge tube of 50 mL. Diluted with an equal volume of 20uL/mL heparin in PBS and an equal volume of lymphocyte separator solution of specific gravity 1.073g/mL was added along the walls of the tubes. Centrifugation at 2000rmp for 30min, and aspiration of the mononuclear cell layer. Centrifuging and washing with sterile physiological saline for 2 times, centrifuging at 1000rmp for 5min, discarding supernatant, and collecting precipitate. The mesenchymal stem cells obtained above were suspended in an α -MEM medium containing 10% fetal bovine serum, and the number of cells was counted at 5 × 106one/mL of the cells were inoculated in a culture flask containing 10% fetal bovine serum in alpha-MEM, and the flask was placed at 37 ℃ with a volume fraction of 5% CO2And (3) in a saturated humidity incubator, replacing the culture medium after 3 days of culture, discarding adherent cells, and replacing the culture medium 1 time every 3 days later. After 7-9 days, when 80% of the cells had grown and fused, they were passaged by digestion with 0.20% trypsin.
Di-metformin and metformin pretreatment mesenchymal stem cells
Taking the subcultured P4-substituted mesenchymal stem cells as 105Inoculating each/mL of the cells into a culture dish, culturing in alpha-MEM containing 10% FBS, adding metformin to the culture solution to a final concentration of 50-100. mu.M, standing at 37 deg.C and 5% CO by volume2Culturing in a saturated humidity incubator for 24-48 hours.
Identification of umbilical cord mesenchymal stem cells
1. Identification of surface markers of mesenchymal stem cells
The P4-generation mesenchymal stem cells obtained by the subculture are digested with 0.25% trypsin, and stably proliferating cells are collected. Will 106The collected human umbilical cord mesenchymal stem cells are suspended in 100mL PBS buffer solution containing 1% bovine serum albumin, referring to FIGS. 1A-H, FIGS. 1A-H are the loss detection results of umbilical cord mesenchymal stem cells CD90, CD73, CD34, CD45, CD14, CD19 and HLA-DR surface markers, and the results show that the positive surface marker of the umbilical cord mesenchymal stem cells obtained in the embodiment is greater than 95% and the negative surface marker is less than 2%, so the cultured umbilical cord mesenchymal stem cells meet the stem cell standard.
2. Identification of osteogenic capacity of mesenchymal stem cells
Digesting the P4 umbilical cord mesenchymal stem cells obtained by subculture with 0.20% trypsin to obtain the product with a concentration of 1 × 106The individual/mL cell suspension is inoculated in a 12-well plate and divided into a control group and a metformin group pretreatment group, liquid is changed every three days, the density change of the cells is observed, osteogenic inducing liquid (10% FBS, 50ug/mL ascorbic acid, 100nM dexamethasone and alpha-MEM of 10mM beta sodium glycerophosphate) is added after the cell density is more than 90%, the liquid is completely changed every two days, the formation of bone nodules is identified by alizarin red staining after 21 days, the cells are observed and photographed under a microscope, and referring to fig. 2A-2B, fig. 2A is the result of alizarin red staining of osteogenic differentiation of umbilical cord stem cells in the control group, fig. 2B is the result of alizarin red staining of osteogenic differentiation of umbilical cord stem cells in the metformin group, and it can be seen that the alizarin red staining of umbilical cord cells in the metformin pretreatment group is deepened, which illustrates that the umbilical cord obtained in the embodiment is obtainedThe osteogenic capacity of the mesenchymal stem cells is improved.
Preparation of four, mesenchymal stem cell preparation
In the invention, the mesenchymal stem cell preparation comprises mesenchymal stem cells, mesenchymal stem cells pretreated by metformin, platelet concentration in platelet-rich plasma and a dispersion medium.
Weighing human serum albumin stock solution, low molecular heparin, compound amino acid and vitamin C, dissolving in compound electrolyte, preparing into dispersion medium, and pre-cooling at 4 deg.C for use. And (4) digesting and cleaning the mesenchymal stem cells and the mesenchymal stem cells pretreated by the metformin, and counting for later use. Mixing the platelet-rich plasma with a dispersion medium according to the volume ratio of 1: 1-2: 1, and suspending the cells to be used to ensure that the final cell concentration of the mesenchymal stem cells is 106～108one/mL.
If 200mL of the mesenchymal stem cell preparation for treating arthritis is prepared, the preparation comprises 25mL of human serum albumin stock solution, 0.5mL of low molecular heparin, 10mL of compound amino acid, 0.5g of vitamin C, 64 mL of compound electrolyte and 100mL of platelet-rich plasma and mesenchymal stem cells. Preparing other components in preparation except for mesenchymal stem cells in advance, precooling at 4 ℃ for standby, finally resuspending the mesenchymal stem cells in the solution to prepare single cell suspension, wherein the number of the mesenchymal stem cells in each milliliter of preparation is 1 multiplied by 107The mesenchymal stem cells are 5 x 106Mesenchymal stem cells pretreated with metformin 5X 106And (4) respectively. The mesenchymal stem cells are still kept in a single cell suspension state within 48 hours at the environmental temperature of 2-15 ℃, and the cell activity (trypan blue staining activity) is kept above 90%.
The above description is only an embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions within the technical scope of the present disclosure should be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
Claims (10)
1. A mesenchymal stem cell preparation for treating arthritis is characterized by comprising mesenchymal stem cells, metformin-pretreated mesenchymal stem cells, platelet-rich plasma and a dispersion medium.
2. The mesenchymal stem cell preparation for treating arthritis according to claim 1, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, and the final cell concentration of the mesenchymal stem cell preparation is 106～108one/mL.
3. A mesenchymal stem cell preparation for use in the treatment of arthritis according to claim 1, wherein the ratio of mesenchymal stem cells to metformin-pretreated mesenchymal stem cells is from 1:1 to 4: 1.
4. Mesenchymal stem cell preparation for use in the treatment of arthritis according to claim 1, wherein the concentration of platelets in platelet rich plasma is 2-3 x 106one/uL.
5. Mesenchymal stem cell preparation for use in the treatment of arthritis according to claim 1, wherein the volume ratio of platelet rich plasma to dispersing medium is from 1:1 to 2: 1.
6. The mesenchymal stem cell preparation for treating arthritis according to claim 1, wherein the dispersion medium comprises human serum albumin, low molecular heparin, complexed amino acid, vitamin C and complexed electrolyte.
7. The mesenchymal stem cell preparation for treating arthritis according to claim 6, wherein the mass-to-volume ratio of human serum albumin is 1-5%, the mass-to-volume ratio of low molecular heparin is 0.5%, the mass-to-volume ratio of compound amino acid is 1-10%, the mass-to-volume ratio of vitamin C is 0.3-0.7%, and the balance is compound electrolyte.
8. A method of preparing a mesenchymal stem cell preparation for use in the treatment of arthritis according to claim 1, comprising the steps of:
(1) and (3) isolated culture of mesenchymal stem cells: after mesenchymal stem cells were isolated, the cells were inoculated into an α -MEM medium containing 10% fetal bovine serum and placed in CO2Culturing in an incubator, and digesting and passing by adopting 0.2 percent of pancreatin when the fusion degree of the mesenchymal stem cells is 80 to 85 percent;
(2) metformin pretreatment of mesenchymal stem cells: inoculating the mesenchymal stem cells passaged to P4 generation into alpha-MEM medium containing metformin and 10% fetal calf serum, and culturing in CO2Culturing in an incubator;
(3) preparation of platelet-rich plasma: taking venous blood added with anticoagulant, concentrating platelets by adopting a two-step centrifugation method to obtain the platelets with the concentration of 2-3 multiplied by 106Plasma per uL;
(4) weighing human serum albumin, low molecular heparin, compound amino acid and vitamin C, preparing into a dispersion medium, pre-cooling at 4 ℃ for later use, digesting and counting the mesenchymal stem cells and the mesenchymal stem cells pretreated by metformin, mixing platelet-rich plasma and the dispersion medium according to the volume ratio of 1: 1-2: 1, suspending the cells for later use, and adjusting the final cell concentration to 106～108one/mL.
9. The method for preparing a mesenchymal stem cell preparation for treating arthritis according to claim 8, wherein the concentration of metformin in step (2) is 50 to 100 μ M.
10. Use of a mesenchymal stem cell preparation according to any one of claims 1 to 7 in the preparation of a medicament for the treatment of arthritis.
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| CN114990065A (en) * | 2022-05-16 | 2022-09-02 | 康妍葆(北京)干细胞科技有限公司 | Directional inducer of neural stem cells, induction method and application |
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