Stem cell preparation for treating arthritis and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicines, and discloses a stem cell preparation for treating arthritis and a preparation method thereof. The stem cell preparation for treating arthritis comprises umbilical cord mesenchymal stem cells pretreated by protocatechuic acid and bilobalide, and a cell protection solution. Wherein the umbilical cord mesenchymal stem cells pretreated by the protocatechuic acid and the bilobalide can effectively inhibit inflammatory response. The stem cell preparation provided by the invention can promote expression of mouse cartilage repair related proteins Collagen II and Aggrecan, and is suitable for relieving inflammation in arthritis diseases and repairing cartilage tissues, so that symptoms of patients are relieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical 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 occurs mainly in human joints and surrounding tissues, caused by inflammation, infection, degeneration, and trauma. Clinical manifestations include redness, swelling, heat, pain, dysfunction, and joint deformity. In severe cases, it can lead to joint disability and affect the patient's quality of life. The mechanism of arthritis includes the human immune system mistakenly attacking the joints, causing inflammation and thickening of the joint capsule, and promoting bone and cartilage damage in these areas. Some complex cytokines and chemokines also regulate the inflammatory environment, especially interleukin-6, interleukin-1, and tumor necrosis factor TNF-α, which aggravate the inflammatory response and promote the proliferation of synovial cells and the infiltration of inflammatory cells.

[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. At present, 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. At present, there is a need for a non-surgical treatment method that can effectively improve the symptoms and prognosis of arthritis.

[0004] In recent years, the development of the stem cell field has brought new options for the treatment of arthritis. Stem cell technology slows down the progression of the disease and improves the quality of life of patients 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 type of 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 relieve 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: A stem cell preparation for treating arthritis, comprising umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide, and a cell protection solution.

[0008] 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.

[0009] 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.

[0010] Furthermore, the seeding density of the umbilical cord mesenchymal stem cells is 1×10 4 -3×10 4 Pieces / mL.

[0011] Furthermore, the umbilical cord mesenchymal stem cells are P3-P5 generation umbilical cord mesenchymal stem cells.

[0012] Furthermore, the culture medium also contains 80-100 μg / mL penicillin and 90-100 μg / mL streptomycin.

[0013] Furthermore, the cell preservation solution is made of the following components in terms of 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 remainder is compound electrolyte injection.

[0014] The second object of the present invention is achieved by adopting the following technical solution: The method for preparing any of the above stem cell preparations for treating arthritis comprises the following steps: (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 evenly, and obtaining a cell protection solution; (3) adding the umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide to the cell protection solution obtained in step (2) to obtain a stem cell preparation.

[0015] Compared with the prior art, the beneficial effect of the present invention is that: the present invention prepares 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 umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide can effectively inhibit inflammatory response. The stem cell preparation provided by the present invention can promote the expression of mouse cartilage repair-related proteins Collagen II and Aggrecan, and is suitable for relieving inflammation and repairing damaged tissues in arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the morphology of P3 generation umbilical cord mesenchymal stem cells; Figure 2 This is the morphology of P4 generation umbilical cord mesenchymal stem cells; Figure 3 This is the morphology of P5 umbilical cord mesenchymal stem cells; Figure 4 The expression level of Collagen II, a protein related to cartilage repair in mice, under different stem cell preparation treatment conditions; Figure 5 The expression level of Aggrecan, a cartilage repair-related protein in mice under different stem cell preparation treatment conditions. DETAILED DESCRIPTION

[0017] The following is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or replacements can also be made, all of which should be deemed to belong to the protection scope of the present invention. The specific conditions not indicated in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0018] Embodiment 1: 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 terms of mass percentage: 1% human serum albumin, 3% compound amino acid injection, 0.5% glutamine, 0.5% glucose, and the remainder is compound electrolyte injection.

[0019] A method for preparing a stem cell preparation for treating arthritis, comprising the following steps: Rinse the umbilical cord with sterile saline in a clean bench and cut it into pieces less than 1 mm with surgical scissors. 3The tissue blocks were washed three times with saline, and the tissue blocks were added to DMEM culture medium containing 5% FBS, penicillin (100 U / mL) and streptomycin (100μg / 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. Umbilical cord mesenchymal stem cells were observed to appear around the tissue blocks. The culture medium was replaced every 3 days thereafter. The cells were cultured until the cell growth confluence reached 80%. The cells were digested with 0.25% EDTA trypsin solution, the digestion solution was removed by centrifugation, and DMEM culture medium was added for resuspending. The subculture was performed at a subculture ratio of 1:2 to obtain P3 umbilical cord mesenchymal stem cells. The morphology of umbilical cord mesenchymal stem cells was observed under a microscope, such as Figure 1 shown.

[0020] (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 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.

[0021] (2) Preparation of cell protection solution: Mix the compound amino acid injection solution, glutamine and glucose in proportion; Human serum albumin and compound electrolyte injection are added to the above-obtained solution, mixed evenly, and a cell protection solution is obtained.

[0022] (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.

[0023] Example: 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 terms of mass percentage: 3% human serum albumin, 7% compound amino acid injection, 1.0% glutamine, 1.0% glucose, and the remainder is compound electrolyte injection.

[0024] A method for preparing a stem cell preparation for treating arthritis, comprising the following steps: (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.

[0025] (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.

[0026] (3) Preparation of cell protection solution: Mix the compound amino acid injection solution, glutamine and glucose in proportion; Human serum albumin and compound electrolyte injection are added to the above-obtained solution, mixed evenly, and a cell protection solution is obtained.

[0027] (4) Preparation and storage of stem cell preparations: The umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and biloba lactone 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.

[0028] Embodiment 3: 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 terms of mass percentage: 5% human serum albumin, 10% compound amino acid injection, 1.5% glutamine, 1.5% glucose, and the remainder is compound electrolyte injection.

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

[0030] (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.

[0031] (3) Preparation of cell protection solution: Mix the compound amino acid injection solution, glutamine and glucose in proportion; Human serum albumin and compound electrolyte injection are added to the above-obtained solution, mixed evenly, and a cell protection solution is obtained.

[0032] (4) Preparation and storage of stem cell preparations: The umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and biloba lactone 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.

[0033] Comparative Example 1: Comparative Example 1 provides a stem cell preparation for treating arthritis, which differs from Example 1 in that protocatechuic acid is omitted during the culturing of umbilical cord mesenchymal stem cells, and the rest is the same as Example 1.

[0034] Comparative Example 2: Comparative Example 2 provides a stem cell preparation for treating arthritis, which differs from Example 1 in that bilobalide is omitted during the culturing of umbilical cord mesenchymal stem cells, and the rest is the same as Example 1.

[0035] Comparative Example 3: 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, and the rest is the same as Example 1.

[0036] Comparative Example 4: Comparative Example 4 provides a stem cell preparation for treating arthritis, which differs from Example 1 in that bilobalide is omitted during the culturing of umbilical cord mesenchymal stem cells, and the dosage of protocatechuic acid is adjusted to 22 μg / mL, and the rest is the same as Example 1.

[0037] Comparative Example 5: Comparative Example 5 provides a stem cell preparation for treating arthritis, and the P4 generation umbilical cord mesenchymal stem cells prepared in Example 1 are directly taken without any treatment.

[0038] Test Example 1: ELISA to detect cytokine levels: 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 resuscitated in a 37°C constant temperature water bath. Human chondrocytes were cultured in DMEM medium containing 10% FBS, and 200 μL of the resuscitated stem cell preparations were added respectively, 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, with the concentration of the standard as the horizontal axis and the corresponding OD value as the vertical axis, and a linear regression curve of the standard was drawn, and the concentration values ​​of each sample were calculated according to the curve equation. The test results are shown in Table 1.

[0039] Table 1 Cytokine level determination results: ; As can be seen from Table 1, compared with Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Comparative Example 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.

[0040] Test Example 2: Effects of stem cell preparations on the expression of cartilage repair proteins in mice: (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.

[0041] (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 vein of four groups of mice suffering from osteoarthritis.

[0042] (3) At the 8th week, the mice were killed and the knee joints were collected 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 slices.

[0043] (4) The sections were incubated overnight in an incubation solution containing 1:1000 dilutions of anti-Collagen II and anti-Aggrecan antibodies. Then, the sections were detected with 1:500 dilutions of fluorescently labeled secondary antibodies and incubated at room temperature for 1 hour. The expression levels of Collagen II and Aggrecan were quantitatively analyzed using ImageJ software.

[0044] The results are as follows Figure 4 and Figure 5 As shown, the expression levels of cartilage repair-related proteins in mice under different stem cell preparation treatment conditions. Compared with Comparative Examples 1, 2, 3, 4, and 5, the expression levels of cartilage repair-related proteins Collagen II and Aggrecan in Example 1, Example 2, and Example 3 were significantly increased, indicating that the combined pretreatment of protocatechuic acid and bilobalide with umbilical cord mesenchymal stem cells to promote cartilage repair is more significant. This shows that the stem cell preparation prepared in this example can treat arthritis.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. On the basis of the present invention, some modifications or replacements may be made thereto, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection claimed by the present invention.

Claims

1. A stem cell preparation for treating arthritis, characterized in that: It includes umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide, and cell protection solution.

2. A 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 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 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.

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

5. The stem cell preparation for treating arthritis according to claim 3, characterized in that: The umbilical cord mesenchymal stem cells are P3-P5 generation umbilical cord mesenchymal stem cells.

6. The stem cell preparation for treating arthritis according to claim 3, characterized in that: The culture medium also contains 80-100 μg / mL penicillin and 90-100 μg / mL streptomycin.

7. 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 remainder is compound electrolyte injection.

8. The method for preparing a stem cell preparation for treating arthritis according to any one of claims 1 to 7, characterized in that: 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 evenly, and obtaining a cell protection solution; (3) adding the umbilical cord mesenchymal stem cells pretreated with protocatechuic acid and bilobalide to the cell protection solution obtained in step (2) to obtain a stem cell preparation.

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