CN116376815B - Culture medium for promoting osteoblast differentiation of mesenchymal stem cells - Google Patents
Culture medium for promoting osteoblast differentiation of mesenchymal stem cells Download PDFInfo
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- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 47
- 239000001963 growth medium Substances 0.000 title claims abstract description 36
- 230000001737 promoting effect Effects 0.000 title claims abstract description 11
- 230000004072 osteoblast differentiation Effects 0.000 title claims abstract description 6
- 229920001184 polypeptide Polymers 0.000 claims abstract description 37
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 37
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 37
- 230000009818 osteogenic differentiation Effects 0.000 claims abstract description 14
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 5
- 210000001185 bone marrow Anatomy 0.000 claims description 27
- 210000004027 cell Anatomy 0.000 claims description 22
- 108010024682 Core Binding Factor Alpha 1 Subunit Proteins 0.000 claims description 13
- 102000015775 Core Binding Factor Alpha 1 Subunit Human genes 0.000 claims description 12
- 230000000694 effects Effects 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 230000002308 calcification Effects 0.000 claims description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 3
- 239000000463 material Substances 0.000 claims 1
- 125000001651 cyanato group Chemical group [*]OC#N 0.000 description 11
- 239000000243 solution Substances 0.000 description 8
- 239000002609 medium Substances 0.000 description 7
- 230000011164 ossification Effects 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 4
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 208000001132 Osteoporosis Diseases 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 210000000963 osteoblast Anatomy 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000006386 Bone Resorption Diseases 0.000 description 1
- 206010065687 Bone loss Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000269913 Pseudopleuronectes americanus Species 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000024279 bone resorption Effects 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 230000000431 effect on proliferation Effects 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000107 myocyte Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 230000002188 osteogenic effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
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- 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
- C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
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- 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
- C12N2501/998—Proteins not provided for elsewhere
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1353—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides a culture medium for promoting osteoblast differentiation of mesenchymal stem cells, and belongs to the technical field of mesenchymal stem cells. The culture medium provided by the invention has the effective components of Pleurocidin polypeptide, and is supplemented with a DMEM culture medium, wherein the use concentration of the Pleurocidin polypeptide in the culture medium is 4-32 mug/ml. The osteogenic differentiation capacity of the mesenchymal stem cells can be effectively improved after the mesenchymal stem cells are pretreated by using the culture medium.
Description
Technical Field
The invention belongs to the technical field of mesenchymal stem cells, and particularly relates to a culture medium for promoting osteoblast differentiation of mesenchymal stem cells.
Background
Mesenchymal stem cells are a stem cell with self-renewal and multiple differentiation potential originating from mesoderm. It has strong proliferation ability and multiple differentiation lineages, and can be differentiated into osteoblasts, chondrocytes, myocytes, stromal cells and nerve cells under different induction conditions. Bone marrow mesenchymal stem cells play an important role in the maintenance of bone homeostasis, which is achieved by bone resorption by osteoclasts and bone formation by osteoblasts. When bone formation is insufficient, serious bone mass loss occurs to cause osteoporosis. Existing studies indicate that bone loss can be alleviated by injecting bone marrow mesenchymal stem cells into an animal model of osteoporosis. However, there is still a disadvantage that the bone formation efficiency is low, so that the improvement of the bone formation ability of the bone marrow mesenchymal stem cells is a problem to be solved at present.
Pleurocidin is a natural polypeptide isolated from the epidermis of Pleuronectes americanus. The amino acid sequence is as follows: GWGSFFKKAAHVGKHVGKAALTHYL. It has broad-spectrum antibacterial activity and can resist various bacteria and fungi. However, there is no report on whether Pleurocidin plays a role in bone marrow mesenchymal stem cell osteogenesis.
Disclosure of Invention
The invention aims to provide a culture medium for promoting the osteogenic differentiation of mesenchymal stem cells, so that the osteogenic differentiation and application of the mesenchymal stem cells are better realized.
In order to achieve the above object, in a first aspect, the present application provides a culture medium for promoting osteoblast differentiation of mesenchymal stem cells, which is characterized in that the culture medium comprises a Pleurocidin polypeptide as an active ingredient, and a DMEM culture medium is supplemented.
Preferably, the content of Pleurocidin polypeptide in the culture medium is 4-32 mug/ml.
Preferably, the culture medium promotes alpase activity in bone marrow mesenchymal stem cells.
Preferably, the culture medium promotes cell calcification in bone marrow mesenchymal stem cells.
Preferably, the medium promotes the expression of RUNX2 and OCN in bone marrow mesenchymal stem cells.
Preferably, the mesenchymal stem cells are bone marrow mesenchymal stem cells.
In a second aspect, the invention provides the use of a Pleurocidin polypeptide in the preparation of a medium for promoting osteogenic differentiation of mesenchymal stem cells.
Preferably, the content of Pleurocidin polypeptide in the culture medium is 4-32 mug/ml.
Preferably, the culture medium promotes alpase activity in bone marrow mesenchymal stem cells.
Preferably, the culture medium promotes cell calcification in bone marrow mesenchymal stem cells.
Preferably, the medium promotes the expression of RUNX2 and OCN in bone marrow mesenchymal stem cells.
The beneficial effects of the invention are as follows:
The invention provides a novel culture medium capable of effectively promoting bone marrow mesenchymal stem cell osteogenic differentiation, which consists of Pleurocidin polypeptide and DMEM culture medium, wherein mRNA expression of RUNX2 and OCN in the bone marrow mesenchymal stem cell can be effectively promoted in the osteogenic differentiation induction process when the concentration of the Pleurocidin polypeptide is between 4 mu g/ml and 32 mu g/ml; meanwhile, the activity of ALP enzyme in the mesenchymal stem cells and the calcification of the cells can be effectively promoted.
Drawings
FIG. 1 shows the results of staining and quantification of ALP enzyme in bone marrow mesenchymal stem cells upon osteogenic differentiation after pretreatment with a Pleurocidin polypeptide;
FIG. 2 shows the result of staining and quantification of alizarin red in bone marrow mesenchymal stem cells after pretreatment with a Pleurocidin polypeptide.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
Example 1
(1) The Pleurocidin polypeptides were formulated as polypeptide solutions of 2. Mu.g/ml, 4. Mu.g/ml, 8. Mu.g/ml, 16. Mu.g/ml, 32. Mu.g/ml, 64. Mu.g/ml, 128. Mu.g/ml using serum-free DMEM medium;
(2) The mesenchymal stem cells of the P3 generation are inoculated into a 96-well plate, and each hole is 1000 cells and 100 mu l;
(3) After overnight incubation, the medium was removed and the cells were treated with the prepared polypeptide solutions, respectively, with 3 replicates for each concentration, while a control group without polypeptide was set;
(4) After 48 hours of incubation, absorbance at 450nm was measured at each concentration with reference to CCK-8 assay protocol, and the results obtained are shown in Table 1.
TABLE 1 proliferation of cells following Plaurocidin polypeptide treatment
From table 1 we can conclude that: the polypeptide has no obvious effect on proliferation of bone marrow mesenchymal stem cells when the concentration of the polypeptide is 2-32 mug/ml, and the long-time treatment can generate a certain inhibition effect on cells when the concentration of the polypeptide is 64 and 128 mug/ml.
Example 2
(1) Primers for osteogenic differentiation related genes RUNX2 and OCN were designed and synthesized, and specific primer sequences were as follows:
the upstream primer sequence of RUNX2 is: 5'-cttcaaggtggtagccctcg-3';
the sequence of the downstream primer of RUNX2 is: 5'-taacagcagaggcattccgg-3';
The upstream primer sequences of OCNs are: 5'-tgaagagacccaggcgcta-3';
The upstream primer sequences of OCNs are: 5'-cacagtccggattgagctca-3';
(2) Inoculating the bone marrow mesenchymal stem cells of the P3 generation into a 6-hole plate, and when the cell density reaches more than 80, respectively treating the cells for 48 hours by using polypeptide solutions of 2 mug/ml, 4 mug/ml, 8 mug/ml, 16 mug/ml and 32 mug/ml;
(3) The medium was removed, and an osteogenic differentiation medium was added thereto, and the mRNA expression levels of RUNX2 and OCN were measured by extracting RNA at 7d, and the results obtained are shown in tables 2 and 3.
TABLE 2 relative expression of mRNA of RUNX2 after treatment with Pleurocidin polypeptide
| Concentration of Pleurocidin polypeptide | Relative expression level of RUNX2 |
| Control group | 1.000±0.036 |
| 2μg/ml | 1.075±0.051 |
| 4μg/ml | 1.286±0.085** |
| 8μg/ml | 1.580±0.048*** |
| 16μg/ml | 2.022±0.093*** |
| 32μg/ml | 2.113±0.061*** |
TABLE 3 relative mRNA expression of OCN after treatment with Pleurocidin polypeptide
| Concentration of Pleurocidin polypeptide | Relative expression level of OCN |
| Control group | 1.000±0.053 |
| 2μg/ml | 1.098±0.079 |
| 4μg/ml | 1.420±0.129** |
| 8μg/ml | 2.086±0.124*** |
| 16μg/ml | 2.715±0.179*** |
| 32μg/ml | 3.055±0.159*** |
As can be seen from tables 2 and 3, the polypeptide solution of 2. Mu.g/ml had no significant effect on the relative expression levels of RUNX2 and OCN, whereas the relative expression levels of RUNX2 and OCN were significantly promoted when the polypeptide concentration was 4. Mu.g/ml, 8. Mu.g/ml, 16. Mu.g/ml, and 32. Mu.g/ml.
Example 3
(1) Inoculating the P3 generation bone marrow mesenchymal stem cells into a 6-hole plate, and when the cell density reaches more than 80%, respectively using 32 mug/ml polypeptide solution to pretreat the cells for 48 hours, wherein the control group is not treated;
(2) Removing the culture medium, adding an osteogenic differentiation culture medium, culturing for 7 days, and then staining cells by referring to an ALP staining kit, and photographing under a microscope; the ALP enzyme activity was also measured by referring to the ALP enzyme quantitative kit, and the results obtained are shown in FIG. 1.
As can be seen from FIG. 1, the ALP enzyme activity in the cells was significantly enhanced after pretreatment of bone marrow mesenchymal stem cells with 32. Mu.g/ml of the polypeptide solution.
Example 4
(1) Inoculating the P3 generation bone marrow mesenchymal stem cells into a 6-hole plate, and when the cell density reaches more than 80%, respectively using 32 mug/ml polypeptide solution to pretreat the cells for 48 hours, wherein the control group is not treated;
(2) Removing the culture medium, adding an osteogenic differentiation culture medium, and after 14 days of osteogenic culture, adding paraformaldehyde to fix the cells;
(3) Removing paraformaldehyde after 30min, cleaning cells by using PBS, and adding alizarin red staining solution to stain the cells for 30min at 37 ℃;
(4) Removing the staining solution after staining, cleaning cells by using PBS, and taking pictures under a microscope; absorbance was measured at 562nm after dissolving alizarin red using 10% CPC.
As can be seen from fig. 2, when the bone marrow mesenchymal stem cells were induced by cell osteogenic differentiation after pretreatment with the polypeptide solution, the number of calcified nodules was significantly greater than that of the control group, indicating that Pleurocidin was effective in promoting calcification of cells.
From the results of the above examples, it is demonstrated that pretreatment of mesenchymal stem cells with a Pleurocidin polypeptide can effectively enhance the osteogenic differentiation ability of mesenchymal stem cells.
It should be understood that while the application has been described in connection with the above specific forms, it is not intended to be limited to the specific form set forth herein. It will be obvious to those skilled in the art that various equivalent changes can be made to the technical features contained in the application as described without departing from the spirit of the application, and these changes shall fall within the scope of the application.
Claims (2)
1. A culture medium for promoting osteoblast differentiation of mesenchymal stem cells, which is characterized in that the effective component in the culture medium is Pleurocidin polypeptide, and a DMEM culture medium is used as an auxiliary material;
the amino acid sequence of the Pleurocidin polypeptide is as follows: GWGSFFKKAAHVGKHVGKAALTHYL;
the content of the Pleurocidin polypeptide in the culture medium is 4-32 mug/ml;
the culture medium promotes ALP enzyme activity in bone marrow mesenchymal stem cells;
The culture medium promotes cell calcification in bone marrow mesenchymal stem cells;
The culture medium promotes the expression of RUNX2 and OCN in bone marrow mesenchymal stem cells.
Application of pleurocidin polypeptide in preparing a culture medium for promoting osteogenic differentiation of mesenchymal stem cells is characterized in that,
The amino acid sequence of the Pleurocidin polypeptide is as follows: GWGSFFKKAAHVGKHVGKAALTHYL;
the content of the Pleurocidin polypeptide in the culture medium is 4-32 mug/ml;
the culture medium promotes ALP enzyme activity in bone marrow mesenchymal stem cells;
The culture medium promotes cell calcification in bone marrow mesenchymal stem cells;
The culture medium promotes the expression of RUNX2 and OCN in bone marrow mesenchymal stem cells.
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