Mesenchymal stem cell suitable for osteoarthritis diseases and cultured by optimized drug combination

Abstract

The invention discloses a mesenchymal stem cell suitable for osteoarthritis diseases and cultured by adopting an optimized medicine combination, and belongs to the technical field of stem cells. The technical problems to be solved are that in the existing mesenchymal stem cell culture process, stemness loss, differentiation potential weakening, multiplication capacity reduction and the like are easily caused by increase of culture generations. According to the key point of the technical scheme, the pharmaceutical composition is provided and comprises betamethasone dipropionate, betamethasone sodium phosphate, interleukin-10 (IL-10) and a hepatocyte growth factor (HGF), by adding the composition into a mesenchymal stem cell culture medium, the technical problems of stemness loss, differentiation potential weakening, multiplication capacity reduction and the like caused by increase of culture generations can be effectively solved.

Description

Technical Field

[0001] This invention belongs to the field of stem cell technology, specifically relating to a mesenchymal stem cell cultured using an optimized drug combination, suitable for use in osteoarthritis. Background Technology

[0002] Stem cell therapy, as an emerging regenerative medicine technology, repairs damaged tissues and organs through the self-renewal and differentiation of stem cells and the secretion of cytokines. For example, in the treatment of arthritis, stem cells can differentiate into chondrocytes to directly participate in cartilage synthesis and secrete a variety of cytokines to promote the proliferation and differentiation of chondrocytes and increase the synthesis of cartilage matrix.

[0003] In stem cell therapy applications, a large number of stem cells are often required, necessitating extensive in vitro expansion. Studies have shown that with prolonged in vitro culture time, stem cells undergo functional deterioration and gradually lose their stemness, thus limiting their application prospects. This process is also known as stem cell senescence. Stem cell senescence is recognized as a complex and comprehensive process involving multiple signaling pathways and molecules, with intricate mechanisms. Therefore, delaying stem cell senescence and maintaining their stemness and proliferative properties is crucial.

[0004] Glucocorticoids, as powerful anti-inflammatory drugs, are commonly used to treat autoimmune diseases and are considered important differentiation inducers in vitro, in addition to their application in stem cells. However, recent studies have shown that under low concentration conditions, they can maintain stem cell stemness, proliferation, and pluripotency, allowing cells to maintain a more youthful state. Summary of the Invention

[0005] The purpose of this invention is to provide: A mesenchymal stem cell cultured with an optimized drug combination suitable for osteoarthritis, and related technologies, to solve the technical problems that easily occur in the existing mesenchymal stem cell culture process, such as loss of stemness, weakened differentiation potential, and reduced proliferation capacity due to the increase of culture passages, or a combination thereof.

[0006] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0007] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0008] On one hand, the present invention provides a pharmaceutical composition comprising betamethasone dipropionate, betamethasone sodium phosphate, interleukin-10 (IL-10), and hepatocyte growth factor (HGF).

[0009] Specifically, the pharmaceutical composition comprises 4-6 mg / mL betamethasone dipropionate, 1-3 mg / mL betamethasone sodium phosphate, 8-12 ng / mL IL-10, and 15-25 ng / mL HGF.

[0010] More specifically, the pharmaceutical composition comprises 4-5 mg / mL betamethasone dipropionate.

[0011] More specifically, the pharmaceutical composition comprises 5-6 mg / mL betamethasone dipropionate.

[0012] More specifically, the pharmaceutical composition comprises 1-2 mg / mL betamethasone sodium phosphate.

[0013] More specifically, the pharmaceutical composition comprises 2-3 mg / mL betamethasone sodium phosphate.

[0014] More specifically, the pharmaceutical composition comprises 8-10 ng / mL IL-10.

[0015] More specifically, the pharmaceutical composition comprises 10-12 ng / mL IL-10.

[0016] More specifically, the pharmaceutical composition comprises 15-20 ng / mL HGF.

[0017] More specifically, the pharmaceutical composition comprises 20-25 ng / mL HGF.

[0018] Specifically, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0019] More specifically, the pharmaceutically acceptable carrier includes, but is not limited to, any one or more of the following: binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

[0020] Preferably, the pharmaceutically acceptable carrier is physiological saline.

[0021] Specifically, the dosage form of the pharmaceutical composition includes: liquid dosage form, gas dosage form, solid dosage form, or semi-solid dosage form.

[0022] More specifically, the liquid dosage form includes: solvent-based, aromatic aqueous solutions, tinctures, elixirs, colloidal solutions, pastes, suspensions, or emulsifiers.

[0023] More specifically, the gaseous dosage form includes: aerosol or spray.

[0024] More specifically, the solid dosage form includes: powder, pill, tablet or film.

[0025] More specifically, the semi-solid dosage form includes: ointment, suppository or paste.

[0026] In another aspect, the present invention provides the use of any of the above-mentioned pharmaceutical compositions in the culture of mesenchymal stem cells.

[0027] The application includes at least one of the following: (1) It is applied to improve the stem cell retention potential; (2) It plays an important role in improving the chondrogenic differentiation potential of stem cells; (3) It is applied to reduce the potential of stem cells to differentiate into adipocytes.

[0028] In another aspect, the present invention provides a method for culturing mesenchymal stem cells, comprising adding any of the above-mentioned drug compositions to a mesenchymal stem cell culture system.

[0029] Specifically, the ratio of the amount of the pharmaceutical composition added to the mass of the culture system is 1:45-55.

[0030] More specifically, the ratio of the amount of the pharmaceutical composition added to the mass of the culture system is 1:45-50.

[0031] More specifically, the ratio of the amount of the pharmaceutical composition added to the mass of the culture system is 1:50-55.

[0032] In another aspect, the present invention provides mesenchymal stem cells cultured by any of the above methods.

[0033] In another aspect, the present invention provides the application of any of the above-mentioned mesenchymal stem cells in the preparation of drugs for treating osteoarthritis and rheumatoid arthritis.

[0034] In another aspect, the present invention provides a treatment method comprising administering a therapeutically effective amount of any of the aforementioned mesenchymal stem cells to a subject.

[0035] The term "subject" includes living organisms (e.g., mammals) that can elicit an immune response. Examples of subjects include humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0036] The terms "treat" or "ameliorate" refer to the medical management of a subject's disease, condition, or undesirable condition. Benefits of treatment or prevention include improved clinical outcomes; reduction or alleviation of symptoms associated with the disease, condition, or undesirable condition; decreased symptom occurrence; improved quality of life; longer disease-free status; reduction in the severity of the disease, condition, or undesirable condition; stabilization of the disease state; delay in disease progression; remission; survival; prolonged survival; or any combination thereof.

[0037] The term "therapeutic effective dose" refers to a pharmaceutically considered effective dosage, that is, an amount of active drug sufficient to significantly improve the condition without causing serious side effects. Dosage depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality, and individual response of the patient or animal, the route of administration, frequency of administration, and therapeutic purpose; therefore, the dosage of this invention can vary widely.

[0038] The present invention has at least the following beneficial effects: 1. The pharmaceutical composition provided by the present invention can solve the problem of stem cell loss due to the increase of culture passage number.

[0039] 2. The pharmaceutical composition provided by this invention can solve the problem of weakened differentiation potential of stem cells due to increased culture passages.

[0040] 3. The pharmaceutical composition provided by the present invention can solve the problem of reduced proliferation capacity of stem cells due to increased culture passages.

[0041] 4. The pharmaceutical composition provided by this invention can solve the problem of stem cells differentiating into adipocytes due to the increase in the number of culture generations. Attached Figure Description

[0042] Figure 1 The cell proliferation rate induced by different amounts of the drug composition.

[0043] Figure 2 To reduce the cell proliferation rate after passage culture of different amounts of drug compositions.

[0044] Figure 3 The drug composition was used to induce cell proliferation rates at different times.

[0045] Figure 4To eliminate the rapid proliferation rate of passaged cells induced by the drug composition. Detailed Implementation

[0046] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0048] Example 1: Experiment with different amounts of the drug composition Betamethasone dipropionate, betamethasone sodium phosphate, IL-10, and HGF were mixed in a mass ratio of 500,000:200,000:1:2 and named as compound betamethasone, to be used later.

[0049] Cell culture: Resuscitate one P5 passage (5E6) cell line, culture it in 50% Huakan-50% source culture medium, seed it in T175 cells, and incubate it at 37°C in a 5% CO2 incubator. After 2 days of culture, digest and collect the cells (P6). Cell grouping and induction: P6 generation cells were seeded at a rate of 0.8E4 / CM2. The next day, a compound betamethasone combination was added, with four groups added at different amounts: 0.1 mL, 0.5 mL, and 1 mL / 5 mL. Cell morphology was observed and counted after 4 and 8 days of culture. The compound was then removed and transferred to a new T75 flask for further culture. The cells were then incubated at 37°C in a 5% CO2 incubator for 4 and 8 days. Cell morphology was observed and counted after digestion. The counting results are shown in Table 1. RNA was extracted for RT-qPCR, and the experimental results are shown in Table 5.

[0050] Table 1

[0051] As shown in Table 1, compound betamethasone reduced MSC proliferation by about 2 times during the induction phase. After removing the effects of compound betamethasone on subculture, the high concentration of compound betamethasone group had a greater proliferation rate. However, in the overall 0-8 days, the greater the amount of compound betamethasone added, the slower the cell proliferation.

[0052] Table 2. Primer Sequences

[0053] Configure the reaction system according to the table below. Table 3. Reaction System

[0054] Mix thoroughly before the reaction and proceed with the reverse transcription reaction according to the following reaction procedure. Table 4. Reaction conditions

[0055] Table 5

[0056] Note: Compared with the control group, **: P < 0.01; ***: P < 0.001.

[0057] As shown in Table 5, compared with the control group, the 0.1 mL group showed increased expression of chondrogenic differentiation-related genes SOX9, Collagen-II, and BMP2, with Collagen-II and BMP2 showing significant differences; osteogenic differentiation-related genes RUNX2 and BGLAP also showed significant increases; adipogenic differentiation gene PPAR-γ expression decreased; immune regulation gene TGF-β1 expression increased; and stem gene TWIST expression remained unchanged.

[0058] Compared with the control group, the 0.5 mL group showed increased expression of chondrogenic differentiation-related genes Collagen-II and BMP2, and slightly increased expression of stemness maintenance genes TWIST and ID1, but none of these differences were statistically significant.

[0059] In summary, the addition of compound betamethasone at a concentration of 0.1 ml / 5 mL significantly enhanced the expression of chondrocyte / bone differentiation genes. Subsequent experiments were conducted using 0.1 ml / 5 mL at different induction times.

[0060] Example 2: Experiment with different induction times of the drug composition Cell culture: Resuscitate one P5 passage (5E6) cell line, culture it in 50% Huakan-50% source culture medium, seed it in T175 cells, and incubate it at 37°C in a 5% CO2 incubator. After 2 days of culture, digest and collect the cells (P6). Cell grouping and induction: P6 generation cells were seeded at a rate of 0.8E4 / CM2. The next day, a compound betamethasone combination was added. Four groups were established with different induction times: 24h, 48h, and 96h. After 4 and 8 days of culture, cell morphology was observed, cells were digested and counted, and flow cytometry was performed. Half of the cells were collected, frozen, and RNA extracted. The other half of the cells were then removed from the drug combination and transferred to a new T75 flask for culture. Cell morphology was observed and cells were digested and counted after 4 and 8 days of culture at 37℃ with 5% CO2. The experimental results are shown in Table 6, and the flow cytometry results are shown in Tables 7 and 8. Table 7 shows the initial flow cytometry results for P6 cells. RNA was extracted for RT-qPCR, and the results are shown in Tables 9 and 10.

[0061] Table 6

[0062] As shown in Table 6, there was no difference in cell status among the groups. The cell density was lower in the 96h group after 8 days. Based on the current results, compound betamethasone induction for 24h and 48h had almost no effect on cell proliferation.

[0063] Table 7

[0064] Table 8

[0065] The flow cytometry results for the 96h induction group were unreliable due to low cell count and residual betamethasone agonists, while the results for the other groups were normal. Flow cytometry results showed that the triple positivity rate of betamethasone agonists decreased in the first 4 days of induction, but after another 4 days of culture, there was little difference between the groups.

[0066] Table 9

[0067] Note: Compared with the control group, *: p<0.05; **: P<0.01; ***: P<0.001.

[0068] At different time points after induction with compound betamethasone, the expression of the chondrogenic gene BMP2 was significantly increased in the 24h and 96h groups, and the expression of the adipogenic gene PPAR-γ was significantly decreased in the 24h group. The expression of the other genes was decreased, indicating that the presence of compound betamethasone has a certain impact on stem cells.

[0069] Table 10

[0070] Note: Compared with the control group, *: p<0.05; **: P<0.01; ***: P<0.001.

[0071] Compared with the control group, the expression of chondrogenic differentiation genes SOX9, Collagen-II, and BMP2 in the 24h induction group showed no significant changes; the osteogenic differentiation gene BGLAP showed a significant increase; the adipogenic differentiation gene PPAR-γ showed a significant decrease; and the expression of the immune regulatory gene TGF-β1 and the stem maintenance genes TWIST and ID1 were all reduced, but without significant changes.

[0072] Compared with the control group, the expression of chondrogenic differentiation-related genes SOX9, Collagen-II, and BMP2 was increased in the 48h induction group, with significant differences in SOX9 and BMP2; osteogenic differentiation gene BGLAP was significantly increased; adipogenic differentiation gene PPAR-γ was significantly decreased; immune regulation gene TGF-β1 expression was increased; and stemness maintenance genes TWIST and ID1 were both significantly increased.

[0073] Compared with the control group, the expression of chondrogenic differentiation genes SOX9, Collagen-II, and BMP2 in the 96h induction group was increased, but not significantly; the expression of osteogenic differentiation gene Runx2 was significantly increased; the expression of adipogenic differentiation gene PPAR-γ was decreased, but not significantly; the expression of immune regulation gene TGF-β1 was increased, but not significantly; the expression of stemness maintenance gene TWIST was increased, and the expression of ID1 was decreased.

[0074] To demonstrate the rationality of the component ratio in this compound betamethasone group and its advantages over current technologies, the following groups were set up for comparison, with hydrocortisone serving as the positive control group. Different components of the compound betamethasone group were removed. All groups were induced for 48 hours at a dosage of 0.1 mL / 5 mL. Gene expression results are shown in the table below: Table 11

[0075] Note: Compared with the control group, *: p<0.05; **: p<0.01.

[0076] As shown in the table above, compared with the control group, the compound betamethasone group (betamethasone + IL-10 + HGF) showed significantly increased expression of chondrogenic differentiation-related genes SOX9, Collagen-II, and BMP2; significantly increased expression of osteogenic differentiation gene BGLAP; significantly decreased expression of adipogenic differentiation gene PPAR-γ; significantly increased expression of immune regulation gene TGF-β1; and increased expression of stemness maintenance genes TWIST and ID1, with TWIST showing a significant increase.

[0077] Compared with the positive control group and the compound betamethasone group with different components removed, the added cytokines IL-10 and HGF enhanced the effect of betamethasone in different aspects. Moreover, the two components added together had a synergistic effect with betamethasone, achieving the best effect.

[0078] In summary, pretreatment with compound betamethasone at a concentration of 0.1 mL / 5 mL for 48 hours significantly enhances the chondrogenic differentiation potential of stem cells and reduces their adipogenic differentiation; it also enhances stem cell stemness and anti-inflammatory immune effects without affecting stem cell viability and morphology. Therefore, pretreatment with compound betamethasone can enhance stem cell stemness, proliferative characteristics, and pluripotency. It also provides a simple and effective strategy for large-scale in vitro preparation of sufficient quantities of highly functional stem cells, greatly expanding the potential application value of stem cells in regenerative medicine and cell therapy.

[0079] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises betamethasone dipropionate, betamethasone sodium phosphate, IL-10, and HGF.

2. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition comprises 4-6 mg / mL betamethasone dipropionate, 1-3 mg / mL betamethasone sodium phosphate, 8-12 ng / mL IL-10, and 15-25 ng / mL HGF.

3. The pharmaceutical composition according to any one of claims 1-2, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutically acceptable carriers include any one or more of the following: binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutically acceptable carrier is physiological saline.

6. The use of the pharmaceutical composition according to any one of claims 1-5 in the culture of mesenchymal stem cells.

7. A method for culturing mesenchymal stem cells, characterized in that, This includes adding the pharmaceutical composition according to any one of claims 1-5 to a mesenchymal stem cell culture system.

8. The cultivation method according to claim 7, characterized in that, The ratio of the amount of the pharmaceutical composition added to the mass of the culture system is 1:45-55.

9. Mesenchymal stem cells cultured by the culture method according to any one of claims 7-8.

10. The use of the mesenchymal stem cells according to claim 9 in the preparation of a medicament for treating osteoarthritis and rheumatoid arthritis.

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