CN117106709A - Method for culturing bone marrow mesenchymal stem cells and application of bone marrow mesenchymal stem cells in treatment of temporomandibular arthritis - Google Patents
Method for culturing bone marrow mesenchymal stem cells and application of bone marrow mesenchymal stem cells in treatment of temporomandibular arthritis Download PDFInfo
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- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
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- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/37—Parathyroid hormone [PTH]
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- Organic Chemistry (AREA)
- Developmental Biology & Embryology (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
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- General Chemical & Material Sciences (AREA)
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- Orthopedic Medicine & Surgery (AREA)
- Pain & Pain Management (AREA)
- Virology (AREA)
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Abstract
The application belongs to the technical field of biological medicines, and particularly relates to application of bone marrow mesenchymal stem cells (BMSCs) induced and cultured by PTH in preparation of medicines for treating temporomandibular arthritis. The application has the beneficial effects that the PTH can obviously improve the proliferation capability of BMSC, improve the tolerance of the BMSC to active oxygen and hypoxia, and the bone marrow mesenchymal stem cells induced and cultured by the PTH have the potential of being developed into medicines for treating temporomandibular arthritis, and finally enhance the treatment effect of BMSC on TMJOA.
Description
Technical Field
The application relates to the technical field of biological medicines, in particular to a method for culturing bone marrow mesenchymal stem cells and application thereof in preparing medicines for treating temporomandibular arthritis.
Background
Temporomandibular joint osteoarthritis (Temporomandibuar joint osteoarthrits, TMJOA) is a joint degenerative disease characterized mainly by articular cartilage damage, accompanied by synovial and subchondral bone changes, with high incidence (reportedly about 18.27% -42.9% of domestic incidence), which can cause pain and dyskinesia in the maxillofacial region, thus causing a great psychological and physiological stress in patients. The occurrence and development of TMJOA are complex processes involving multiple functions such as tissue injury, repair and inflammation, so that the treatment of TMJOA is difficult. At present, clinical treatment of TMJOA mainly aims at controlling disease progress, relieving pain of patients, treating medicines, physically treating, controlling inflammation and the like, and medicines and methods for effectively controlling TMJOA progress are not available. In recent years, the rise of stem cell, especially mesenchymal stem cell (Mesenchymal Stem Cell, MSC), technology has brought new promise for the treatment of many diseases. Compared with the traditional treatment method, the method has a plurality of unique advantages: firstly, stem cells can reach the damaged part through the homing effect so as to differentiate into corresponding tissue cells, thereby repairing the damage; secondly, the stem cells can secrete a large amount of cytokines, and the cytokines can promote proliferation and differentiation of transplanted stem cells, can regulate local inflammatory reaction, reduce inflammatory pressure and better promote injury repair; thirdly, many stem cells can be obtained by self, thereby greatly reducing rejection reaction. In view of the good effect of stem cells in the treatment of other diseases, some scholars have also begun to explore their use in the treatment of Osseointegrated (OA) and have shown better therapeutic effects. However, since the temporomandibular joint has a relatively small space, the blood and oxygen supply is often insufficient due to the relatively large joint, and once the inflammatory injury occurs, the supply of blood oxygen is more difficult, so that the transplanted MSC is difficult to survive in the damaged temporomandibular joint cavity, and the proliferation and differentiation abilities of the transplanted MSC are inhibited. Therefore, how to increase the proliferation and differentiation capacity of MSC and the survival time of MSC in the temporomandibular joint cavity becomes the key point of MSC for treating TMJOA.
Parathyroid hormone (parathyroid hormone, PTH) is a basic single-chain polypeptide hormone secreted by parathyroid cells. Its main function is to regulate the metabolism of calcium and phosphorus in vertebrate body, promote the rise of blood calcium level and the decrease of blood phosphorus level, so it has important functions in bone development, bone formation, etc. Whereas parathyroid hormone concentrations are significant in cells, we have explored to investigate the effect of PTH-induced MSCs on TMJOA.
Disclosure of Invention
The application aims to provide a method for culturing bone marrow mesenchymal stem cells and application thereof in preparing medicines for treating temporomandibular arthritis. Experiments show that PTH can remarkably improve proliferation capacity of bone marrow mesenchymal stem cells (BMSCs), improve tolerance of the BMSCs to active oxygen and hypoxia, finally enhance treatment effect of the BMSCs on TMJOA, and has better safety.
The first object of the present application is to provide a method for culturing bone marrow mesenchymal stem cells, which adopts PTH induction culture.
Preferably, the PTH concentration is 20-100ng/ml. Preferably, the PTH concentration is 50ng/ml.
The second object of the application is the use of the bone marrow mesenchymal stem cells cultured by the method in preparing a medicine for treating temporomandibular joint osteoarthritis.
Preferably, the medicament comprises a therapeutically effective amount of bone marrow mesenchymal stem cells.
Preferably, the medicament further comprises auxiliary materials or carriers acceptable to human bodies.
A third object of the present application is to provide a pharmaceutical composition comprising a therapeutic amount of bone marrow mesenchymal stem cells cultured by the above method and other pharmaceutically acceptable excipients.
The dosage forms of the medicine comprise oral preparations and injection preparations, and can be prepared into tablets, capsules or injection preparations by a conventional method; in particular, injection formulations are included.
The application has the beneficial effects that the PTH can obviously improve the proliferation capability of BMSC, improve the tolerance of the BMSC to active oxygen and hypoxia, and the bone marrow mesenchymal stem cells induced and cultured by the PTH have the potential of being developed into medicines for treating temporomandibular joint osteoarthritis, and finally enhance the treatment effect of BMSC on TMJOA.
Drawings
FIG. 1A is a graph showing the effect of varying PTH concentrations on BMSC activity in example 2 of the present application;
FIG. 1B is a Edu experimental plot of the application for promoting BMSC proliferation by PTH (50 ng/ml) of example 2;
FIG. 1C is a graph showing the quantitative analysis of the BMSC proliferation Edu assay promoted by PTH (50 ng/ml) according to example 2 of the present application;
FIG. 2A is a graph of the cell viability of BMSCs under hypoxic conditions enhanced by example 3PTH (50 ng/ml) of the present application;
FIG. 2B is a graph of the proliferation potency of BMSC under hypoxic conditions enhanced by PTH (50 ng/ml) according to example 3 of the present application;
FIG. 2C is a graph of a Edu quantitative analysis of the proliferation capacity of example 3PTH (50 ng/ml) of the present application under conditions of elevated BMSC hypoxia;
FIG. 2D is a PI stain of example 3 of the present application showing PTH (50 ng/ml) alleviation of reactive oxygen species (H 2 O 2 ) A map of resulting BMSC apoptosis;
FIG. 2E is a quantitative chart of PI staining for example 3 of the present application;
fig. 3 is a graph showing the therapeutic effect of the PTH of example 4 of the present application on increasing BMSC to TMJOA.
Detailed Description
The present application is further described in detail below with reference to examples, but is not limited thereto.
Example 1: research method
Establishment of TMJOA animal model
After weighing the rats, they were anesthetized with 10% chloral hydrate (0.3 ml/100 g). After disinfecting the skin in the area with iodophor, 1mg sodium iodoacetate (MIA) was injected into the upper cavity of the temporomandibular joint of a side rat using a 50 μl microinjector, and an animal model of temporomandibular joint osteoarthritis was established. Control rats were anesthetized and operated in the same manner. The difference is that the equal amount of physiological saline for injection is injected into the upper cavity of the temporomandibular joint, and after three weeks, the model modeling of the rat TMJOA model is completed.
2. Experimental grouping and processing
Rats were divided into 5 groups, namely, normal control group, model group (MIA-induced), BMSC treatment group (simple BMSC treatment group), PTH treatment group (simple PTH treatment group) and PTH-combined BMSC treatment group (PTH-combined BMSC treatment group). In this experiment, MIA induction was performed with 1mg MIA. After lesion determination, BMSC (1X 10) 6 ) Or PTH (50 ng/ml) treated BMSC infusion therapy. After 5 days use H&E, detecting damage condition, and displaying tissue apoptosis condition through TUNEL staining, so as to comprehensively evaluate the condition of rat temporomandibular arthritis and the treatment effect of PTH combined with BMSC.
BMSC culture
Bone marrow from rats of 12 weeks old was collected and the attached soft tissue was removed. Digestion with 3mg/mL type I collagenase and 4mg/mL dispase II was performed for 60 minutes at 37 ℃. Cells were cultured in alpha-MEM (Gibco). After 1 week of culture, the growth was observed.
Edu proliferation assay
Edu is a thymidine analogue which can be incorporated into DNA being synthesized instead of thymine, and which is luminescent and chromogenic through its own fluorescent group, thereby indirectly reflecting the proliferation capacity of the cell. The stronger the fluorescent signal, the more vigorous the proliferation, and conversely, the inhibition of proliferation. Cells were incubated for 8 hours with EdU prior to treatment, washed, stained with DAPI, and then observed under a fluorescent microscope or quantitatively detected using a flow cytometer.
5. Flow cytometer for detecting BMSC surface mark
The cells were digested with pancreatin, centrifuged at 1000rpm for 5min, resuspended in PBS to a concentration of about 1X 10 6 And each ml. Then 100. Mu.L was transferred to another EP tube, and an appropriate amount of antibody (typically 2. Mu.L) was added thereto and incubated at room temperature for 30min in the absence of light. Finally, 500 mu L of Stain Buffer is added into each tube, and after the liquid is uniformly blown by a liquid-transfering device, the detection is carried out by an up-flow cytometer.
Example 2: PTH-promoted BMSC proliferation assay
First, we examined the drug toxicity of PTH to BMSC. BMSCs were treated with different concentrations of PTH and their cell viability was examined after 24 hours. The results show that PTH only showed significant toxicity to BMSCs at a concentration of 100ng/ml. However, at smaller doses, the effect of promoting cell viability was exhibited, and was most pronounced at 50ng/ml (FIG. 1A). To further determine the pro-proliferative effect of PTH on BMSCs, we examined the proliferation of BMSCs by the Edu experiment. The results showed that PTH increased the Edu positive rate of BMSC, indicating that PTH increased the proliferative capacity of BMSC (Fig 1B, C).
Example 3: PTH enhances hypoxia tolerance and active oxygen resistance experiments of BMSC
Limitations on BMSC treatment of TMJOA are mainly derived from the hypoxic, high reactive oxygen environment created when temporomandibular arthritis occurs. To this end, we further validated the tolerance of BMSCs to hypoxia and to reactive oxygen species. First, BMSCs were treated with different oxygen concentrations (21%, 5%, 1%) and cell viability was examined at different time points. The results showed 1% O 2 Significantly inhibited the proliferation and viability of BMSCs, which decreased by 85% after 48 hours (Fig 2A). Thus, at 1% O 2 As conditions for subsequent experiments. Next, we tested PTH vs hypoxia (1% O) using the Edu experiment 2 ) BMSC proliferation potency effects under conditions. The results showed 1% O 2 Proliferation of BMSCs was significantly inhibited, but PTH increased Edu positive rate (FIG. 2B-C) of BMSCs, indicating that PTH increased hypoxia tolerance of BMSCs.
Next, we again add H 2 O 2 The active oxygen environment was simulated. Apoptosis was detected by flow-through, and H was found as a result 2 O 2 (100. Mu.M) significantly induced apoptosis, but PTH reduced the rate of apoptosis, indicating that PTH increased resistance of BMSC to active oxygen (FIG 2C-D).
Example 4: PTH promotes BMSC to TMJOA's treatment effect experiment
In the previous experiments, PTH has been demonstrated to increase the proliferation capacity of BMSC, enhance its resistance to hypoxia and active oxygen, and further we studied whether PTH-induced BMSC could increase the therapeutic effect on TMJOA. First, we injected MIA into the temporomandibular joint of the rat, constructing the TMJOA model. On this basis, BMSCs and PTH-induced BMSCs were treated separately. After 2 weeks the temporomandibular joint of the rat was harvested and H & E stained. The results show that: MIA can induce TMJOA to occur, and is characterized by irregular arrangement and inconsistent thickness of cartilage layers of condyles, osteophyte formation and thinning of cartilage layers. The BMSC treatment group showed no significant improvement over the model group, but the PTH-induced cultured BMSC treatment group significantly reduced the condylar tissue damage (FIG 3).
All documents referred to herein are incorporated by reference in their entirety as if each were individually incorporated by reference.
Claims (7)
1. A method for culturing mesenchymal stem cells, which is characterized by adopting PTH induction culture.
2. The method of claim 1, wherein the PTH concentration is 20-100ng/ml.
3. The method of claim 1, wherein the PTH concentration is 50ng/ml.
4. Use of bone marrow mesenchymal stem cells cultured according to the method of any one of claims 1 to 3 in the preparation of a medicament for the treatment of temporomandibular arthritis.
5. The use according to claim 4, wherein the medicament comprises a therapeutically effective amount of bone marrow mesenchymal stem cells.
6. The use according to claim 5, wherein the medicament further comprises a human acceptable adjuvant or carrier.
7. A pharmaceutical composition comprising a therapeutic amount of mesenchymal stem cells cultured according to the method of any one of claims 1 to 3, together with a pharmaceutically acceptable adjuvant.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2023109973744 | 2023-08-09 | ||
| CN202310997374 | 2023-08-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117106709A true CN117106709A (en) | 2023-11-24 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN202311067940.8A Pending CN117106709A (en) | 2023-08-09 | 2023-08-23 | Method for culturing bone marrow mesenchymal stem cells and application of bone marrow mesenchymal stem cells in treatment of temporomandibular arthritis |
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| CN (1) | CN117106709A (en) |
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- 2023-08-23 CN CN202311067940.8A patent/CN117106709A/en active Pending
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