CN111088225A - Method for promoting directional differentiation of mesenchymal stem cells - Google Patents
Method for promoting directional differentiation of mesenchymal stem cells Download PDFInfo
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- 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
Abstract
The invention relates to the field of stem cells, and provides a method for promoting directional differentiation of mesenchymal stem cells, which is used for solving the problem of low differentiation efficiency of the stem cells to bone cells and comprises the steps of culturing the mesenchymal stem cells by adopting a DMEM culture medium, performing adherent culture in a 6-well plate after the cells are recovered, digesting and passaging the cells by using 0.25% pancreatin every 2 days, inoculating the mesenchymal stem cells obtained by culture to the differentiation culture medium for induced differentiation culture, wherein the differentiation culture medium comprises 3-5 mu g/ml of insulin, 0.05-0.08 mu g/ml of transferrin, 1-3 ng/ml of growth hormone, 80-100 mu g/ml of glutathione, 0.5-2 mM of L-glutamine, 20-50 mu M of β -mercaptoethanol, 0.5-0.7 mM of sodium pyruvate, 0.05-0.1 mM of unnecessary amino acid, 8-12 mu M of nano ferric oxide, the balance of DMEM culture medium, and the balance of serum which is not added, and can be used for promoting the differentiation of the nano bone cells to bone cells obviously.
Description
Technical Field
The invention relates to the field of stem cells, in particular to a method for promoting directional differentiation of mesenchymal stem cells.
Background
Stem cells are a cell type with self-renewal capacity and multi-differentiation potential, and are the first cells in a series of processes from proliferation, migration, differentiation and maturation of human neonatal cells. Research on stem cells has become one of the most biologically challenging and attractive areas today. Stem cells are classified into embryonic stem cells and adult stem cells according to their source. Mesenchymal Stem Cells (MSCs) are one of the adult stem cells. Mesenchymal stem cells, a class of adult stem cells with self-renewal capacity, can differentiate into a variety of cell tissue types. It can differentiate not only into a variety of mesenchymal-derived cell lineages, such as adipocytes, osteoblasts, chondrocytes, myoblasts, etc., but also into other cell lineages, such as astrocytes, myoblasts, cardiomyocytes, and nerve cells, etc. Since MSCs isolated and cultured in vitro have no obvious change in cell phenotype and no loss of function, they are considered to be of great significance for cell therapy and tissue repair engineering.
How to promote the directional differentiation of the mesenchymal stem cells is a problem to be solved urgently.
Disclosure of Invention
The invention provides a method for promoting directional differentiation of mesenchymal stem cells, aiming at solving the problem of low differentiation efficiency of stem cells to osteocytes.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a method for promoting directional differentiation of mesenchymal stem cells comprises the steps of adopting a DMEM culture medium to culture the mesenchymal stem cells, recovering the cells, performing adherent culture in a 6-well plate, digesting and passaging the cells with 0.25% pancreatin every 2 days, inoculating the mesenchymal stem cells obtained by culture to the differentiation culture medium, and performing induced differentiation culture, wherein the differentiation culture medium comprises 3-5 mu g/ml of insulin, 0.05-0.08 mu g/ml of transferrin, 1-3 ng/ml of growth hormone, 80-100 mu g/ml of glutathione, 0.5-2 mM of L-glutamine, 20-50 mu M of β -mercaptoethanol, 0.5-0.7 mM of sodium pyruvate, 0.05-0.1 mM of unnecessary amino acid, 8-12 mu M of nano ferric oxide and the balance of the DMEM culture medium.
The nano ferric oxide is introduced into the culture medium of the stem cells, so that the differentiation of the stem cells to the bone cells can be promoted to a certain extent.
Biological serum is not added into the culture medium, and the nano oxide is introduced, so that the differentiation of the mesenchymal stem cells to bone cells can be remarkably promoted.
Preferably, the culture medium comprises 4-5 mu g/ml of insulin, 0.06-0.08 mu g/ml of transferrin, 2-3 ng/ml of growth hormone, 90-100 mu g/ml of glutathione, 1.5-2 mM of L-glutamine, 25-50 mu M of β -mercaptoethanol, 0.6-0.7 mM of sodium pyruvate, 0.08-0.1 mM of unnecessary amino acid, 10-12 mu M of nano ferric oxide and the balance of a DMEM culture medium.
Preferably, the composition comprises 4 mu g/ml of insulin, 0.06 mu g/ml of transferrin, 2ng/ml of growth hormone, 90 mu g/ml of glutathione, 1.5mM of L-glutamine, 25 mu M of β -mercaptoethanol, 0.6mM of sodium pyruvate, 0.08mM of non-essential amino acid, 10 mu M of nano ferric oxide and the balance of DMEM culture medium.
Preferably, the nano ferric oxide is modified nano ferric oxide. The modified ferric oxide can further promote the differentiation of stem cells to osteocytes.
Preferably, the preparation method of the modified ferric oxide comprises the following steps: taking 20-25 parts by mass of ferric nitrate, 1-3 parts by mass of silver nitrate, 15-20 parts by mass of sodium hydroxide and 3-5 parts by mass of carbon nano tubes; dissolving ferric nitrate in 40 times of absolute ethyl alcohol, dissolving silver nitrate in 50 times of deionized water, and mixing the aqueous solution of the silver nitrate with the ethanol solution of the ferric nitrate to obtain a mixed solution; dissolving sodium hydroxide in 5 times of deionized water to obtain a sodium hydroxide solution, mixing the sodium hydroxide solution with the mixed solution, stirring, performing ultrasonic treatment for 20min, heating to 150 ℃, performing heat preservation reaction for 12h, taking a product, washing the product with deionized water and absolute ethyl alcohol for multiple times, drying, and sintering at 600-800 ℃ for 2h to obtain powder; ball-milling the powder and the carbon nano tube for 2 hours, adding deionized water in the ball-milling process to obtain slurry, drying the slurry to obtain a dried product, and calcining the product at 400-600 ℃ to obtain the modified ferric oxide. The carbon nano tube is added to modify the nano ferric oxide, so that the differentiation of the stem cells to the bone cells can be further promoted.
Preferably, the carbon nano tube coating comprises 21-25 parts by mass of ferric nitrate, 2-3 parts by mass of silver nitrate, 17-20 parts by mass of sodium hydroxide and 4-5 parts by mass of carbon nano tubes.
Preferably, the carbon nano tube comprises 21 parts by mass of ferric nitrate, 2 parts by mass of silver nitrate, 17 parts by mass of sodium hydroxide and 4 parts by mass of carbon nano tubes.
Preferably, the carbon nanotube is a modified carbon nanotube, and the modification method of the carbon nanotube is as follows; taking 1-2 parts by mass of a carbon nano tube, 200-400 parts by mass of dimethylformamide and 5-10 parts by mass of zinc acetate; ablating the carbon nano tube for 3 hours at 500-600 ℃, carrying out reflux acid washing on the carbon nano tube by using nitric acid after ablation, and then washing the carbon nano tube by using deionized water until filtrate is neutral; dispersing the ablated carbon nano tube into dimethylformamide, carrying out ultrasonic treatment for 1 hour, then stirring, slowly adding zinc acetate in a stirring state, carrying out ultrasonic treatment for 2 hours again, transferring the obtained solution into a hydrothermal reaction kettle, adding the solution into the hydrothermal reaction kettle for 10-15 hours, and heating the solution at the temperature of 60-90 ℃ to obtain a product; and alternately washing the obtained product with ethanol and deionized water for 5-8 times to obtain the modified carbon nanotube. The nano ferric oxide is modified by the modified carbon nano tube, so that the nano ferric oxide can better promote the differentiation of stem cells to osteocytes.
Preferably, the carbon nano tube comprises 1.5-2 parts by mass of dimethylformamide and 8-10 parts by mass of zinc acetate.
Preferably, the carbon nano tube comprises 1.5 parts by mass, 300 parts by mass of dimethylformamide and 8 parts by mass of zinc acetate.
Compared with the prior art, the invention has the beneficial effects that: biological serum is not added into the culture medium, and the carbon nano tube is introduced, so that the differentiation of the mesenchymal stem cells to bone cells can be remarkably promoted; can greatly improve the osteogenic differentiation efficiency of stem cells.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
A method for promoting directional differentiation of mesenchymal stem cells comprises the steps of culturing mesenchymal stem cells by adopting a DMEM culture medium, recovering the cells, carrying out adherent culture in a 6-pore plate, digesting and passaging the cells by using 0.25% pancreatin every 2d, inoculating the mesenchymal stem cells obtained by culture to a differentiation culture medium, carrying out induced differentiation culture, wherein the differentiation culture medium comprises 4 mu g/ml of insulin, 0.06 mu g/ml of transferrin, 2ng/ml of growth hormone, 90 mu g/ml of glutathione, 1.5mM of L-glutamine, 25 mu M of β -mercaptoethanol, 0.6mM of sodium pyruvate, 0.08mM of non-essential amino acid, 10 mu M of nano ferric oxide and the balance of DMEM culture medium, wherein the nano ferric oxide is modified nano ferric oxide, the preparation method comprises the steps of taking 21 parts by mass of ferric nitrate, 2 parts by mass of sodium hydroxide, 17 parts by mass of carbon nanotubes, 4 parts by dissolving the ferric nitrate in 40 times of absolute ethanol, dissolving the modified nano ferric oxide in modified nano ferric nitrate, drying the modified nano ferric nitrate, heating the modified carbon nanotube in a solution of the modified carbon nanotube after being stirred for 2 hours, carrying out a ball-milling reaction with the modified carbon nanotube, heating the modified carbon nanotube after being stirred with the deionized water, stirring for a temperature of 1.5 hours, adding the modified carbon nanotube in a modified carbon nanotube solution of the deionized water, heating and stirring, carrying out a ball-milling reaction for a time, obtaining a reaction, drying process, adding the modified carbon nanotube-carbon nanotube-carbon nanotube.
The nano ferric oxide is introduced into the culture medium of the stem cells, so that the differentiation of the stem cells to the bone cells can be promoted to a certain extent. Biological serum is not added into the culture medium, and the nano oxide is introduced, so that the differentiation of the mesenchymal stem cells to bone cells can be remarkably promoted. The modified ferric oxide can further promote the differentiation of stem cells to osteocytes. The carbon nano tube is added to modify the nano ferric oxide, so that the differentiation of the stem cells to the bone cells can be further promoted. The nano ferric oxide is modified by the modified carbon nano tube, so that the nano ferric oxide can better promote the differentiation of stem cells to osteocytes.
Example 2
A method for promoting directional differentiation of mesenchymal stem cells comprises the steps of culturing mesenchymal stem cells by adopting a DMEM culture medium, recovering the cells, carrying out adherent culture in a 6-pore plate, digesting and passaging the cells by using 0.25% pancreatin every 2 days, inoculating the mesenchymal stem cells obtained by culture to a differentiation culture medium, carrying out induced differentiation culture, wherein the differentiation culture medium comprises 3 mu g/ml of insulin, 0.05 mu g/ml of transferrin, 1ng/ml of growth hormone, 80 mu g/ml of glutathione, 0.5mM of L-glutamine, β -mercaptoethanol 20 mu M of sodium pyruvate, 0.5mM of non-essential amino acid, 0.05mM of nano ferric oxide, 8 mu M of nano ferric oxide and the balance of DMEM culture medium, wherein the nano ferric oxide is modified nano ferric oxide, the preparation method comprises the steps of taking 20 parts by mass of ferric nitrate, 1 part by mass of sodium hydroxide, 15 parts by mass of carbon nanotubes, 3 parts by mass of carbon nanotubes, dissolving the ferric nitrate in 40 times of absolute ethanol, dissolving the modified nano ferric oxide in modified nano ferric oxide, stirring the silver nitrate, heating the modified nano ferric oxide in a modified nano ferric nitrate solution, carrying out a ball-milling reaction for 2 hours, heating the modified carbon nanotube after the modified carbon nanotube is dissolved in a reaction, adding the deionized water with the deionized water, stirring, the deionized water with the deionized water, carrying out a reaction for 2 hours, carrying out a ball-milling reaction, carrying out a reaction for 2-time, carrying out a reaction, carrying out a thermal-time reaction, carrying out a thermal-milling reaction, heating, carrying out a thermal-time reaction, carrying out a thermal-grinding reaction, adding the thermal-grinding reaction, obtaining a thermal-grinding reaction, obtaining a thermal-grinding reaction, obtaining.
Example 3
A method for promoting directional differentiation of mesenchymal stem cells comprises the steps of culturing mesenchymal stem cells by adopting a DMEM culture medium, recovering the cells, carrying out adherent culture in a 6-pore plate, digesting and passaging by 0.25% of pancreatin every 2 days, inoculating the mesenchymal stem cells obtained by culture to a differentiation culture medium, carrying out induced differentiation culture, wherein the differentiation culture medium comprises 5 mu g/ml of insulin, 0.08 mu g/ml of transferrin, 3ng/ml of growth hormone, 100 mu g/ml of glutathione, 2mM of L-glutamine, 50 mu M of β -mercaptoethanol, 0.7mM of sodium pyruvate, 0.1mM of non-essential amino acid, 12 mu M of nano ferric oxide and the balance of DMEM culture medium, wherein the nano ferric oxide is modified nano ferric oxide, the preparation method comprises the steps of taking 25 parts by mass of ferric nitrate, 3 parts by mass of sodium hydroxide, 5 parts by mass of the carbon nanotubes, dissolving the ferric nitrate in 40 times of absolute ethanol, dissolving the modified nano ferric oxide in 50 times of the modified nano ferric nitrate, carrying out a ball-milling reaction with the modified nano ferric nitrate, heating the modified carbon nanotube in a modified carbon nanotube solution at a temperature of 2 ℃ of the deionized water, carrying out a ball-milling reaction, carrying out a ball-milling reaction at a temperature of 10 ℃ of the modified carbon nanotube-2-carbon nanotube-carbon nanotube carbon-carbon nanotube carbon-carbon nanotube carbon-carbon nanotube carbon-carbon nanotube carbon.
Example 4
Example 4 is different from example 1 in that the nano ferric oxide is not modified, and the nano ferric oxide is commercially available nano ferric oxide.
Example 5
Example 5 differs from example 1 in that the carbon nanotubes are not modified.
Example 6
The embodiment 6 is different from the embodiment 1 in that the modification method of the nano ferric oxide comprises the following steps: and dispersing the nano ferric oxide and the carbon nano tube into deionized water together, performing ultrasonic treatment for 1h, drying and roasting to obtain the modified nano ferric oxide.
Comparative example 1
Comparative example 1 differs from example 1 in that it comprises 4. mu.g/ml of insulin, 0.06. mu.g/ml of transferrin, 2ng/ml of growth hormone, 90. mu.g/ml of glutathione, 1.5mM of L-glutamine, 25. mu.M of β -mercaptoethanol, 0.6mM of sodium pyruvate, 0.08mM of an optional amino acid, and the balance being DMEM medium.
Comparative example 2
Comparative example 2 the difference from example 1 was that the mesenchymal stem cells were cultured in DMEM medium, after recovery, they were cultured in 6-well plates adherent to each other, digested with 0.25% trypsin every 2 days and passaged, and the mesenchymal stem cells obtained by the culture were inoculated into a differentiation medium comprising 2. mu.g/ml of insulin, 0.03. mu.g/ml of transferrin, 0.5ng/ml of growth hormone, 60. mu.g/ml of glutathione, 0.2mM of L-glutamine, 15. mu.M of β -mercaptoethanol, 0.3mM of sodium pyruvate, 0.02mM of non-essential amino acids, 5. mu.M of nano-iron trioxide, and the balance being DMEM medium.
Comparative example 3
Comparative example 3 the difference from example 1 was that the mesenchymal stem cells were cultured in DMEM medium, after recovery, they were cultured in 6-well plates adherent to each other, digested with 0.25% trypsin every 2 days and passaged, and the mesenchymal stem cells obtained by the culture were inoculated into a differentiation medium comprising 10. mu.g/ml of insulin, 0.1. mu.g/ml of transferrin, 5ng/ml of growth hormone, 120. mu.g/ml of glutathione, 3mM of L-glutamine, 60. mu.M of β -mercaptoethanol, 0.8mM of sodium pyruvate, 0.2mM of an optional amino acid, 15. mu.M of nano iron sesquioxide, and the balance DMEM medium, to induce differentiation culture.
Examples of the experiments
Calcium deposition of the extracellular matrix is dyed by 0.5% alizarin red liquid for 1 hour, washed by PBS and incubated by 500ul1% hydrogen chloride, and a light absorption value is detected at 420nm by a multifunctional microplate reader to analyze the result of calcium deposition amount of the osteogenic differentiation extracellular matrix, so that the effect of the culture medium on achievement differentiation is analyzed. As shown in table 1.
TABLE 1 osteogenic differentiation Rate
In examples 1 to 6, on the basis of comparative example 1, the proliferation rate is increased by 11.5 to 98.5%, which indicates that the introduction of nano ferric oxide into the culture medium can significantly improve the differentiation efficiency of stem cells and provide conditions for large-scale osteogenic differentiation of stem cells.
The stem cell differentiation effect in examples 1 to 3 is relatively remarkable, which indicates that the nano ferric oxide treated by the modified carbon nanotube can effectively promote the stem cell differentiation, and the stem cell differentiation effect of example 1 is obviously superior to that of examples 2 and 3, which indicates that the stem cell differentiation rate can be further improved by adding a certain amount of nano ferric oxide.
The differentiation rate of the nano ferric oxide in the embodiment 4 is not modified, but the improvement effect is weaker than that of the embodiment 1-3, which shows that the modified ferric oxide can further improve the differentiation rate of the stem cells.
In the embodiment 5, the effect of compounding unmodified carbon nanotubes and nano ferric oxide is weaker than that of the embodiment 1; the modification method of nano-ferric oxide in example 6 is different from that of example 1, and the effect is weaker than that of example 1, which shows that only nano-ferric oxide treated by carbon nanotubes modified by a certain method can promote the differentiation of stem cells into osteocytes.
The comparative example 1 is not added with the nano ferric oxide, the addition amount of the nano ferric oxide in the comparative examples 2 and 3 is greatly different from that in the examples 1-3, and the effect of the comparative examples 2 and 3 on promoting the proliferation of the stem cells is not obviously changed compared with the comparative example 1, which shows that only a certain addition amount of the nano ferric oxide can promote the differentiation of the stem cells.
The above detailed description is specific to possible embodiments of the present invention, and the above embodiments are not intended to limit the scope of the present invention, and all equivalent implementations or modifications that do not depart from the scope of the present invention should be included in the present claims.
Claims (8)
1. A method for promoting directional differentiation of mesenchymal stem cells, comprising:
culturing mesenchymal stem cells by adopting a DMEM culture medium, recovering the cells, carrying out adherent culture in a 6-well plate, digesting by using 0.25% pancreatin every 2 days, and carrying out passage;
inoculating the mesenchymal stem cells obtained by culture to a differentiation culture medium for induced differentiation culture;
the differentiation culture medium comprises 3-5 mu g/ml of insulin, 0.05-0.08 mu g/ml of transferrin, 1-3 ng/ml of growth hormone, 80-100 mu g/ml of glutathione, 0.5-2 mM of L-glutamine, 20-50 mu M of β -mercaptoethanol, 0.5-0.7 mM of sodium pyruvate, 0.05-0.1 mM of unnecessary amino acid, 8-12 mu M of nano ferric oxide and the balance of a DMEM culture medium;
the nano ferric oxide is modified nano ferric oxide;
the preparation method of the modified nano ferric oxide comprises the following steps:
taking 20-25 parts by mass of ferric nitrate, 1-3 parts by mass of silver nitrate, 15-20 parts by mass of sodium hydroxide and 3-5 parts by mass of carbon nano tubes;
dissolving ferric nitrate in 40 times of absolute ethyl alcohol, dissolving silver nitrate in 50 times of deionized water, and mixing the aqueous solution of the silver nitrate with the ethanol solution of the ferric nitrate to obtain a mixed solution; dissolving sodium hydroxide in 5 times of deionized water to obtain a sodium hydroxide solution, mixing the sodium hydroxide solution with the mixed solution, stirring, performing ultrasonic treatment for 20min, heating to 150 ℃, performing heat preservation reaction for 12h, taking a product, washing the product with deionized water and absolute ethyl alcohol for multiple times, drying, and sintering at 600-800 ℃ for 2h to obtain powder;
ball-milling the powder and the carbon nano tube for 2 hours, adding deionized water in the ball-milling process to obtain slurry, drying the slurry to obtain a dried product, and calcining the product at 400-600 ℃ to obtain the modified nano ferric oxide.
2. The method for promoting directional differentiation of mesenchymal stem cells according to claim 1, wherein the method comprises 4-5 μ g/ml of insulin, 0.06-0.08 μ g/ml of transferrin, 2-3 ng/ml of growth hormone, 90-100 μ g/ml of glutathione, 1.5-2 mM of L-glutamine, 25-50 μ M of β -mercaptoethanol, 0.6-0.7 mM of sodium pyruvate, 0.08-0.1 mM of unnecessary amino acids, 10-12 μ M of nano ferric oxide, and the balance of DMEM medium.
3. The method of claim 2, wherein the method comprises 4 μ g/ml of insulin, 0.06 μ g/ml of transferrin, 2ng/ml of growth hormone, 90 μ g/ml of glutathione, 1.5mM of L-glutamine, 25 μ M of β -mercaptoethanol, 0.6mM of sodium pyruvate, 0.08mM of non-essential amino acids, 10 μ M of nano ferric oxide, and the balance DMEM medium.
4. The method for promoting directional differentiation of mesenchymal stem cells according to claim 1, wherein the amount of ferric nitrate is 21 to 25 parts by mass, the amount of silver nitrate is 2 to 3 parts by mass, the amount of sodium hydroxide is 17 to 20 parts by mass, and the amount of carbon nanotubes is 4 to 5 parts by mass.
5. The method for promoting directional differentiation of mesenchymal stem cells according to claim 4, wherein the iron nitrate is 21 parts by mass, the silver nitrate is 2 parts by mass, the sodium hydroxide is 17 parts by mass, and the carbon nanotubes are 4 parts by mass.
6. The method for promoting directional differentiation of mesenchymal stem cells according to claim 1, wherein the carbon nanotubes are modified carbon nanotubes, and the modification method of the carbon nanotubes comprises:
taking 1-2 parts by mass of a carbon nano tube, 200-400 parts by mass of dimethylformamide and 5-10 parts by mass of zinc acetate;
ablating the carbon nano tube for 3 hours at 500-600 ℃, carrying out reflux acid washing on the carbon nano tube by using nitric acid after ablation, and then washing the carbon nano tube by using deionized water until filtrate is neutral;
dispersing the ablated carbon nano tube into dimethylformamide, carrying out ultrasonic treatment for 1 hour, then stirring, slowly adding zinc acetate in a stirring state, carrying out ultrasonic treatment for 2 hours again, transferring the obtained solution into a hydrothermal reaction kettle, adding the solution into the hydrothermal reaction kettle for 10-15 hours, and heating the solution at the temperature of 60-90 ℃ to obtain a product;
and alternately washing the obtained product with ethanol and deionized water for 5-8 times to obtain the modified carbon nanotube.
7. A method for promoting directional differentiation of mesenchymal stem cells according to claim 6, wherein the mass ratio of carbon nanotubes is 1.5-2, dimethylformamide is 300-400, and zinc acetate is 8-10.
8. The method for promoting directional differentiation of mesenchymal stem cells according to claim 7, wherein the mass parts of carbon nanotubes comprise 1.5 parts by mass, 300 parts by mass of dimethylformamide and 8 parts by mass of zinc acetate.
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