A stem cell preparation for treating arthritis and its preparation method

Abstract

The present invention belongs to the field of biomedicine technology and discloses a stem cell preparation for treating arthritis and a preparation method thereof. The stem cell preparation for treating arthritis of the present invention comprises umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide, and a cell protection solution. The umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide can effectively inhibit inflammatory responses. The stem cell preparation provided by the present invention can promote the expression of Collagen II and Aggrecan, proteins related to cartilage repair in mice, and is suitable for alleviating inflammation and repairing cartilage tissue in arthritis diseases, thereby achieving symptom relief for patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a stem cell preparation for treating arthritis and a preparation method thereof. Background Art

[0002] Arthritis refers to an inflammatory disease that primarily occurs in the joints and surrounding tissues, caused by inflammation, infection, degeneration, and trauma. Clinical manifestations include redness, swelling, heat, pain, dysfunction, and deformity. In severe cases, it can lead to joint disability and compromise the patient's quality of life. The mechanism of action of arthritis involves the immune system mistakenly attacking the joints, leading to inflammation and thickening of the joint capsule and promoting bone and cartilage damage in these areas. Complex cytokines and chemokines also regulate the inflammatory environment, particularly interleukin-6, interleukin-1, and tumor necrosis factor TNF-α, exacerbating the inflammatory response and promoting synovial cell proliferation and inflammatory cell infiltration.

[0003] Common arthritis currently includes rheumatoid arthritis and osteoarthritis. Rheumatoid arthritis is a systemic autoimmune disease characterized by chronic inflammation and joint damage, characterized by multi-joint involvement, which can progress to joint destruction and deformity, and often involves peripheral joints. Osteoarthritis is a chronic, irreversible joint disease characterized by joint degeneration. It is one of the degenerative diseases that affects the largest number of people worldwide. It occurs with age and affects the fingers, knees and hips. Both types of arthritis are types of rheumatic diseases, and their causes involve genetic inheritance and environmental factors. Appropriate early treatment can improve joint symptoms and function, reduce mortality and reduce complications. Currently, there is a lack of effective non-surgical treatment methods in clinical practice. The only way to improve the quality of life of patients is through joint replacement, but it also brings problems such as poor postoperative efficacy and postoperative complications. There is a need for a non-surgical treatment method that can effectively improve the symptoms and prognosis of arthritis.

[0004] In recent years, developments in the field of stem cells have brought new options for the treatment of arthritis. Stem cell technology slows down the progression of the disease and improves patients' quality of life by regenerating cartilage tissue and repairing joint structure. There are many sources of clinically relevant stem cells, such as bone marrow, placenta, and fat. Mesenchymal stem cells are a type of cell with certain self-renewal and multidirectional differentiation capabilities, and have great potential for regenerative medicine and autoimmune diseases. The effect of using mesenchymal stem cells to treat arthritis is not obvious. Therefore, there is an urgent need for a new stem cell preparation on the market for the treatment of arthritis. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a stem cell preparation for treating arthritis, which can be used to alleviate inflammation and repair cartilage tissue in arthritis.

[0006] A second object of the present invention is to provide a method for preparing a stem cell preparation for treating arthritis.

[0007] One of the purposes of the present invention is achieved by the following technical solution:

[0008] A stem cell preparation for treating arthritis, comprising umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide, and a cell protection solution.

[0009] Furthermore, the final cell concentration of umbilical cord mesenchymal stem cells in the stem cell preparation is 1×10 7 -1×10 8 pieces / mL.

[0010] Furthermore, the preparation steps of the umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide are: inoculating the umbilical cord mesenchymal stem cells into a culture medium containing protocatechuic acid and bilobalide, culturing for 24-48 hours, and collecting the cells; the protocatechuic acid concentration in the culture medium is 10-20 μg / mL, and the bilobalide concentration is 12-16 μg / mL.

[0011] Furthermore, the seeding density of the umbilical cord mesenchymal stem cells is 1×10 4 -3×10 4 pieces / mL.

[0012] Furthermore, the umbilical cord mesenchymal stem cells are P3-P5 generation umbilical cord mesenchymal stem cells.

[0013] Furthermore, the culture medium also contains 80-100 μg / mL penicillin and 90-100 μg / mL streptomycin.

[0014] Furthermore, the cell preservation solution is prepared from the following components in percentage by mass: 1-5% human serum albumin, 3-10% compound amino acid injection, 0.5-1.5% glutamine, 0.5-1.5% glucose, and the remainder is compound electrolyte injection.

[0015] The second object of the present invention is achieved by adopting the following technical solution:

[0016] The method for preparing any of the above stem cell preparations for treating arthritis comprises the following steps:

[0017] (1) Mix the compound amino acid injection, glutamine, and glucose evenly according to the proportion;

[0018] (2) adding human serum albumin and compound electrolyte injection to the solution obtained in step (1), mixing them evenly to obtain a cell protection solution;

[0019] (3) The umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide are added to the cell protection solution obtained in step (2) to obtain a stem cell preparation.

[0020] Compared to existing technologies, the present invention offers the following advantages: A stem cell preparation for treating arthritis is prepared, comprising umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide, and a cell protection solution. The umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide can effectively inhibit inflammatory responses. The stem cell preparation provided by the present invention can promote the expression of Collagen II and Aggrecan, proteins associated with cartilage repair in mice, and is suitable for alleviating inflammation and repairing damaged tissue in arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the morphology of P3 umbilical cord mesenchymal stem cells;

[0022] Figure 2 This is the morphology of P4 umbilical cord mesenchymal stem cells;

[0023] Figure 3 This is the morphology of P5 umbilical cord mesenchymal stem cells;

[0024] Figure 4 The expression level of Collagen II, a cartilage repair-related protein in mice, under different stem cell preparation treatment conditions;

[0025] Figure 5 The expression levels of Aggrecan, a cartilage repair-related protein in mice under different stem cell preparation treatment conditions. DETAILED DESCRIPTION

[0026] The following is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it is not intended that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art of the present invention, without departing from the inventive concept, several simple deductions or replacements can also be made, all of which should be considered to belong to the scope of protection of the present invention. The specific conditions not indicated in the examples are carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0027] Example 1:

[0028] A stem cell preparation for treating arthritis, comprising umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide, and a cell protection solution. The final cell concentration of the umbilical cord mesenchymal stem cells in the stem cell preparation is 1×10 7 / mL; the cell preservation solution is composed of the following components in percentage by mass: 1% human serum albumin, 3% compound amino acid injection, 0.5% glutamine, 0.5% glucose, and the balance is compound electrolyte injection.

[0029] A method for preparing a stem cell preparation for treating arthritis, comprising the following steps:

[0030] The umbilical cord was rinsed with sterile saline in a clean bench and cut into pieces less than 1 mm with surgical scissors. 3 The tissue blocks were washed three times with normal saline, and the tissue blocks were added to DMEM culture medium containing 5% FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL) for culture. They were placed in a constant temperature incubator at 37°C and 5% carbon dioxide saturated humidity. The culture medium was completely replaced after 6 days. The umbilical cord mesenchymal stem cells appeared around the tissue blocks. The culture medium was replaced every 3 days. The cells were cultured until the cell confluence reached 80%. The cells were digested with 0.25% EDTA trypsin solution, centrifuged to remove the digestion solution, and then resuspended in DMEM culture medium for subculture. The subculture ratio was 1:2 to obtain P3 umbilical cord mesenchymal stem cells. The morphology of umbilical cord mesenchymal stem cells was observed under a microscope, as shown in the following figure. Figure 1 shown.

[0031] (1) Preparation of umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide: The prepared P3 umbilical cord mesenchymal stem cells were cultured at a concentration of 1×10 4 The cells were inoculated into DMEM medium containing 10 μg / mL protocatechuic acid, 12 μg / mL bilobalide, 80 μg / mL penicillin and 90 μg / mL streptomycin, and cultured for 24 h, and the cells were collected.

[0032] (2) Preparation of cell protection solution: Mix the compound amino acid injection, glutamine, and glucose in proportion;

[0033] Human serum albumin and compound electrolyte injection were added to the above solution, mixed evenly, and a cell protection solution was obtained.

[0034] (3) Preparation and storage of stem cell preparations: The umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide in step (1) are added to the cell protection solution obtained in step (2) to obtain a stem cell preparation for treating arthritis. The stem cell preparation is placed in a cell freezing bag and stored in liquid nitrogen.

[0035] Example:

[0036] A stem cell preparation for treating arthritis, comprising umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide, and a cell protection solution. The final cell concentration of the umbilical cord mesenchymal stem cells in the stem cell preparation is 5×10 7 / mL; the cell preservation solution is composed of the following components in mass percentage: 3% human serum albumin, 7% compound amino acid injection, 1.0% glutamine, 1.0% glucose, and the balance is compound electrolyte injection.

[0037] A method for preparing a stem cell preparation for treating arthritis, comprising the following steps:

[0038] (1) The culture method of umbilical cord mesenchymal stem cells is the same as that in Example 1. P4 umbilical cord mesenchymal stem cells are collected. The morphology of umbilical cord mesenchymal stem cells is observed under a microscope. Figure 2 shown.

[0039] (2) Preparation of umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide: The prepared P4 umbilical cord mesenchymal stem cells were cultured at 2×10 4 The cells were inoculated into DMEM medium containing 15 μg / mL protocatechuic acid, 14 μg / mL bilobalide, 90 μg / mL penicillin and 95 μg / mL streptomycin, and cultured for 36 h, and the cells were collected.

[0040] (3) Preparation of cell protection solution: Mix the compound amino acid injection, glutamine, and glucose in proportion;

[0041] Human serum albumin and compound electrolyte injection were added to the above solution, mixed evenly, and a cell protection solution was obtained.

[0042] (4) Preparation and storage of stem cell preparations: The umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide in step (2) are added to the cell protection solution obtained in step (3) to obtain a stem cell preparation for treating arthritis. The stem cell preparation is placed in a cell freezing bag and stored in liquid nitrogen.

[0043] Example 3:

[0044] A stem cell preparation for treating arthritis, comprising umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide, and a cell protection solution. The final cell concentration of the umbilical cord mesenchymal stem cells in the stem cell preparation is 1×10 8 / mL; the cell preservation solution is composed of the following components in percentage by mass: 5% human serum albumin, 10% compound amino acid injection, 1.5% glutamine, 1.5% glucose, and the balance is compound electrolyte injection.

[0045] A method for preparing a stem cell preparation for treating arthritis, comprising the following steps:

[0046] (1) The culture method of umbilical cord mesenchymal stem cells is the same as that in Example 1. P5 umbilical cord mesenchymal stem cells are collected. The morphology of umbilical cord mesenchymal stem cells is observed under a microscope. Figure 3 shown.

[0047] (2) Preparation of umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide: The prepared P5 umbilical cord mesenchymal stem cells were cultured at 3×10 4 The cells were inoculated into DMEM medium containing 20 μg / mL protocatechuic acid, 16 μg / mL bilobalide, 100 μg / mL penicillin and 100 μg / mL streptomycin, and cultured for 48 h, and the cells were collected.

[0048] (3) Preparation of cell protection solution: Mix the compound amino acid injection, glutamine, and glucose in proportion;

[0049] Human serum albumin and compound electrolyte injection were added to the above solution, mixed evenly, and a cell protection solution was obtained.

[0050] (4) Preparation and storage of stem cell preparations: The umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide in step (2) are added to the cell protection solution obtained in step (3) to obtain a stem cell preparation for treating arthritis. The stem cell preparation is placed in a cell freezing bag and stored in liquid nitrogen.

[0051] Comparative Example 1:

[0052] Comparative Example 1 provides a stem cell preparation for treating arthritis, which differs from Example 1 in that protocatechuic acid is omitted during the umbilical cord mesenchymal stem cell culture process, and all other aspects are the same as Example 1.

[0053] Comparative Example 2:

[0054] Comparative Example 2 provides a stem cell preparation for treating arthritis, which differs from Example 1 in that bilobalide is omitted during the umbilical cord mesenchymal stem cell culture process, and all other aspects are the same as Example 1.

[0055] Comparative Example 3:

[0056] Comparative Example 3 provides a stem cell preparation for treating arthritis, which differs from Example 1 in that protocatechuic acid is omitted during the umbilical cord mesenchymal stem cell culture process, and the dosage of bilobalide is adjusted to 22 μg / mL. Other aspects are the same as Example 1.

[0057] Comparative Example 4:

[0058] Comparative Example 4 provides a stem cell preparation for treating arthritis, which differs from Example 1 in that bilobalide is omitted during the umbilical cord mesenchymal stem cell culture process, and the dosage of protocatechuic acid is adjusted to 22 μg / mL. Other aspects are the same as Example 1.

[0059] Comparative Example 5:

[0060] Comparative Example 5 provides a stem cell preparation for treating arthritis, and the P4 umbilical cord mesenchymal stem cells prepared in Example 1 are directly taken without any treatment.

[0061] Test Example 1:

[0062] ELISA to detect cytokine levels:

[0063] The stem cell preparations prepared in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Comparative Example 5 were revived in a constant temperature water bath at 37°C. Human chondrocytes were cultured in DMEM culture medium containing 10% FBS, and 200 μL of the revived stem cell preparations were added to each of them, and cultured in a constant temperature incubator at 37°C and 5% carbon dioxide saturated humidity for 48 hours. According to the instructions of the ELISA kit, the levels of TNF-α, IL-6, TGF-β1, and IGF-1 were detected respectively. The standard concentration was used as the horizontal axis and the corresponding OD value was used as the vertical axis. A linear regression curve of the standard was drawn, and the concentration value of each sample was calculated according to the curve equation. The test results are shown in Table 1.

[0064] Table 1 Cytokine level determination results:

[0065] ;

[0066] As can be seen from Table 1, compared with Comparative Examples 1, 2, 3, 4 and 5, the expression levels of TNF-α and IL-6 in Example 1, Example 2 and Example 3 were significantly reduced, while the expression levels of TGF-β1 and IGF-1 were significantly increased, which indicates that the combined pretreatment of umbilical cord mesenchymal stem cells with protocatechuic acid and biloba lactone can effectively inhibit the inflammatory response.

[0067] Test Example 2:

[0068] Effects of stem cell preparations on the expression of cartilage repair proteins in mice:

[0069] (1) Forty eight-week-old mice were selected for the experiment. The mice were anesthetized and 1 mg of monoclonal antibody mixture was injected into the unilateral knee joint cavity to establish an osteoarthritis model. The mice with osteoarthritis were randomly divided into four groups, with 10 mice in each group.

[0070] (2) 200 μL of the stem cell preparations prepared in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Comparative Example 5 of the present invention were injected into the tail veins of four groups of mice suffering from osteoarthritis.

[0071] (3) At week 8, the mice were sacrificed and the knee joints were harvested for histological examination. The collected knee joint tissues were fixed with formaldehyde for 30 hours, dehydrated with alcohol, and soaked in naphtha for 2 hours. They were then immersed in wax for 4 hours and cut into sections.

[0072] (4) The sections were incubated overnight in an incubation solution containing anti-collagen II and anti-agrecan antibodies diluted 1:1000. The sections were then detected with a fluorescently labeled secondary antibody diluted 1:500 and incubated at room temperature for 1 hour. The expression levels of collagen II and aggrecan were quantitatively analyzed using ImageJ software.

[0073] The results are as follows Figure 4 and Figure 5 The figures show the expression levels of cartilage repair-related proteins in mice treated with different stem cell preparations. Compared with Comparative Examples 1, 2, 3, 4, and 5, the expression levels of cartilage repair-related proteins Collagen II and Aggrecan were significantly increased in Examples 1, 2, and 3, indicating that combined pretreatment of umbilical cord mesenchymal stem cells with protocatechuic acid and bilobalide significantly promotes cartilage repair. This indicates that the stem cell preparations prepared in these examples can treat arthritis.

[0074] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. The basic principles and main features of the present invention have been described above using specific implementation schemes. Modifications or substitutions may be made based on the present invention, but such modifications or substitutions do not detract from the essence of the corresponding technical solutions from the scope of protection claimed by the present invention.

Claims

1. A stem cell preparation for treating arthritis, characterized in that: The invention comprises umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide, and a cell protection solution. The preparation steps of the umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide are as follows: inoculating the umbilical cord mesenchymal stem cells into a culture medium containing protocatechuic acid and bilobalide, culturing for 24-48 hours, and collecting the cells; the protocatechuic acid concentration in the culture medium is 10-20 μg / mL, and the bilobalide concentration is 12-16 μg / mL.

2. The stem cell preparation for treating arthritis according to claim 1, characterized in that: The final cell concentration of umbilical cord mesenchymal stem cells in the stem cell preparation is 1×10 7 -1×10 8 pieces / mL.

3. The stem cell preparation for treating arthritis according to claim 1, characterized in that: The seeding density of the umbilical cord mesenchymal stem cells is 1×10 4 -3×10 4 pieces / mL.

4. The stem cell preparation for treating arthritis according to claim 1, characterized in that: The umbilical cord mesenchymal stem cells are P3-P5 generation umbilical cord mesenchymal stem cells.

5. The stem cell preparation for treating arthritis according to claim 1, characterized in that: The culture medium also contains 80-100 μg / mL penicillin and 90-100 μg / mL streptomycin.

6. The stem cell preparation for treating arthritis according to claim 1, characterized in that: The cell preservation solution is prepared from the following components according to mass percentage: 1-5% human serum albumin, 3-10% compound amino acid injection, 0.5-1.5% glutamine, 0.5-1.5% glucose, and the balance is compound electrolyte injection.

7. The method for preparing a stem cell preparation for treating arthritis according to claim 6, wherein: The following steps are involved: (1) Mix the compound amino acid injection, glutamine, and glucose evenly according to the proportion; (2) adding human serum albumin and compound electrolyte injection to the solution obtained in step (1), mixing them evenly to obtain a cell protection solution; (3) The umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide are added to the cell protection solution obtained in step (2) to obtain a stem cell preparation.

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