CN110157663B - Method for culturing myocardial cells by stem cell in vitro induction - Google Patents

Method for culturing myocardial cells by stem cell in vitro induction Download PDF

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CN110157663B
CN110157663B CN201910314980.5A CN201910314980A CN110157663B CN 110157663 B CN110157663 B CN 110157663B CN 201910314980 A CN201910314980 A CN 201910314980A CN 110157663 B CN110157663 B CN 110157663B
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陈忠平
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Lancy Purcell Biotechnology Guangzhou Co ltd
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Abstract

The invention relates to the field of stem cells, and provides a method for culturing cardiomyocytes by stem cell in vitro induction, which is used for solving the problem of low differentiation rate of stem cells when differentiating the cardiomyocytes. The invention provides a method for culturing myocardial cells by stem cell in vitro induction, which comprises the steps of preparing single cell suspension from autologous stem cells and preparing embryoid bodies; transferring the prepared embryoid bodies to a gelatin-coated pore plate, adding an induced differentiation culture medium to each embryoid body, and performing intervening differentiation culture; the induced differentiation culture medium comprises 0.7-1.2 mg/L of salvianolic acid B, 0.5-1.5 mg/mL of ginsenoside and 0.8-1.2 mg/L of icariin. Sufficient cardiomyocytes can be obtained which can effectively improve the success rate of cell transplantation therapy.

Description

Method for culturing myocardial cells by stem cell in vitro induction
Technical Field
The invention relates to the field of stem cells, in particular to a method for culturing myocardial cells by stem cell in vitro induction.
Background
Heart failure caused by myocardial cell dysfunction is a major disease worldwide, and the leading cause of death in heart failure patients is loss of cardiac pumping function or arrhythmia. The annual mortality rate of patients with severe heart failure is over 50%. Although later patients may undergo heart transplantation, the donor organ is then in a severe short-supply condition at all times. Due to the objective status quo of high morbidity and mortality of heart failure, complications such as death of transplanted heart, immune rejection and the like, and late failure of transplanted heart, development of new therapeutic methods to improve the function of cardiomyocytes and prevent the occurrence of heart failure is urgently needed. The current treatment method for heart failure mainly focuses on early revascularization and inhibition of further cardiomyocyte loss, and the cardiomyocytes are terminally differentiated cells with extremely limited proliferation capacity and extremely difficult recovery after loss.
The myocardial cell transplantation therapy focuses on the reconstruction of myocardial function and the restoration of the blood pumping capacity of regenerated myocardial tissue, and studies have also found that myocardial cells injected into the heart can form new myocardial tissue. A key element in cell transplantation therapy is the determination of the appropriate donor cell type. Therefore, how to induce the stem cells to directionally differentiate into the cardiomyocytes and how to improve the differentiation rate of the stem cells to the cardiomyocytes is an urgent technical problem to be solved for medical treatment.
Disclosure of Invention
The invention solves the technical problem of low differentiation rate when stem cells are differentiated into myocardial cells, and provides a method for culturing the myocardial cells by stem cell in vitro induction.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a method for culturing myocardial cells by stem cell in vitro induction comprises S11, preparing single cell suspension from autologous stem cells; s12, after counting the cells of the single cell suspension, diluting the single cell suspension into a cell concentration of 10 by using a differentiation culture medium-5~10-3cell/mL stem cell suspension, inoculating on the inner surface of the cover of the bacteria culture dish, turning the cover of the culture dish to enable the stem cells on the cover to form hanging drops, adding water or aqueous solution into the culture dish to keep the humidity of the hanging drops in the incubation process, and placing the culture dish in 5% CO2Culturing for 1-3 days in an incubator at 37 ℃; s13, collecting the embryoid bodies formed in the hanging drops on the culture dish cover, transferring the embryoid bodies into a bacterial culture dish containing a differentiation culture medium, and putting the bacterial culture dish into an incubator to continue to suspend so as to promote the further proliferation of the embryoid bodies; s14, transferring the prepared embryoid bodies to a gelatin-coated pore plate, adding an induced differentiation culture medium to each embryoid body, and performing intervening differentiation culture; the induced differentiation culture medium comprises 0.7-1.2 mg/L of salvianolic acid B, 0.5-1.5 mg/mL of ginsenoside and 0.8-1.2 mg/L of icariin.
The autologous stem cells are induced and differentiated into the myocardial cells, and the obtained myocardial cells are used as cell donors of a myocardial cell transplantation therapy, so that the rejection reaction of original cells to the transplanted myocardial cells can be reduced, and the success rate of myocardial cell transplantation is improved; the salvianolic acid B, ginsenoside and icariin are used for inducing the stem cells to be converted into the myocardial cells, so that the differentiation rate of the stem cells converted into the myocardial cells can be improved.
The autologous stem cells are induced and differentiated in a culture medium by the mixture of salvianolic acid B, ginsenoside and icariin, so that sufficient cardiac muscle cells which can effectively improve the success rate of cell transplantation therapy can be obtained.
Preferably, the differentiation induction culture medium further comprises GMEM culture medium, fetal bovine serum, non-essential amino acids, 50mmol/L sodium pyruvate water solution and 0.1mol/L dimercaptoethanol water solution, wherein the GMEM culture medium: fetal bovine serum: non-essential amino acids: 50mmol/L aqueous sodium pyruvate solution: 0.1mol/L aqueous dimercaptoethanol =73.5:24:1:1.3: 0.2. The differentiation rate of stem cells into cardiomyocytes can be improved by optimizing the content of each main substance in the culture medium.
Preferably, 50-60% of the ginsenoside is prepared into microcapsules, and the microcapsules containing the ginsenoside are added into a culture medium, wherein the preparation method of the microcapsules containing the ginsenoside comprises the following steps: s31, preparing a mixed solution by glutathione, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and deionized water according to a ratio of 1:1: 100-120, adding a phosphate buffer solution with pH =6 into the mixed solution, and stirring for 24 hours under an anaerobic condition to obtain a wall material, wherein the volume ratio of the mixed solution to the phosphate buffer solution is 1: 6-8; s32, dispersing ginsenoside into hydroxyl silicone oil, wherein the ratio of the ginsenoside to the hydroxyl silicone oil is 1: 100-120, so as to obtain a core material; s33, mixing the wall material and the core material according to the volume ratio of 1:5, carrying out ultrasonic treatment on an oil-water two-phase interface for 5-10 min by using an ultrasonic probe in an ice-water bath, and carrying out multiple adsorption and washing on a product to obtain the microcapsule. After the ginsenoside is microencapsulated, the content of the ginsenoside in a culture medium can be kept relatively stable, and particularly, the ginsenoside can be kept to play a relatively stable role in promoting stem cell differentiation in the process of stem cell proliferation.
Preferably, the preparation method of the ginsenoside comprises the following steps: s41, adding cellulase and pectinase into deionized water, mixing with sliced ginseng, and reacting at 45 ℃ for 1h to obtain an enzymolysis extracting solution; s42, adding 95% ethanol into the enzymolysis extracting solution for the first time, refluxing at 55 ℃ for 3 hours, filtering to obtain a primary extracting solution and residues, adding 95% ethanol into the residues for the second time for extraction, refluxing at 55 ℃ for 2 hours, and mixing the primary extracting solution and the residues to obtain an alcohol extracting solution; s43, concentrating the alcohol extract at 50 ℃ under reduced pressure to obtain an extract, recovering ethanol in the concentration process, and drying the obtained extract in vacuum to obtain the ginsenoside. The ginsenoside extracted by enzymolysis can be used together with icariin and salvianolic acid B to further improve the differentiation rate of stem cells into myocardial cells.
Preferably, the mass ratio of the cellulase to the pectinase to the deionized water to the ginseng is 5 x 10-4:10-3:1:4。。
Preferably, the mass ratio of the ethanol to the enzymolysis extracting solution is 1: 0.4: 1.6. .
Preferably, the wall material further comprises modified nano ferroferric oxide particles, and the preparation method of the wall material added with the modified nano ferroferric oxide comprises the following steps: preparing a mixed solution by glutathione, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and deionized water according to a ratio of 1:1: 100-120, and adding a phosphate buffer solution with pH =6 into the mixed solution; modified nano ferroferric oxide particles are dispersed in the phosphate buffer solution, the mass ratio of the modified nano ferroferric oxide to the phosphate buffer solution is 1:600, and the modified nano ferroferric oxide is dispersed in the phosphate buffer solution in an ultrasonic dispersion mode; stirring for 24 hours under an anaerobic condition, repeatedly adsorbing and washing to obtain the wall material, wherein the volume ratio of the mixed solution to the phosphate buffer solution is 1: 6-8. The modified nano ferroferric oxide is added into the wall material, so that the probability of combination of the microcapsules and stem cells can be enhanced, the microcapsules dispersed in the culture medium can continuously provide nutritional factors for the stem cells at the periphery, and the differentiation rate is further improved.
Preferably, the modification method of the modified nano ferroferric oxide comprises the following steps: dispersing nano ferroferric oxide in deionized water, adding nano layered double hydroxide, performing ultrasonic dispersion, and drying to obtain nano layered hydroxide modified nano ferroferric oxide, wherein the nano ferroferric oxide comprises the following components in parts by weight: nano layered hydroxide: deionized water =1: 0.2-0.5: 20. the nano ferroferric oxide modified by the nano layered hydroxide can be compounded with biological macromolecules, so that certain targeting property can be generated; the targeted microcapsule can be combined with stem cells, so as to continuously provide an environment for inducing differentiation for surrounding stem cells.
Preferably, the method for preparing the nano-layered hydroxide comprises the following steps: dissolving aluminum nitrate and aluminum nitrate in deionized water, adding the solution into 1.25% sodium hydroxide solution which is vigorously stirred, carrying out hydrothermal reaction, drying a reaction product, and crushing to obtain nano layered hydroxide; the aluminum nitrate: magnesium nitrate: deionized water =1:0.75: 30. After the nano ferroferric oxide modified by the nano layered hydroxide prepared in a certain proportion is mixed into the wall material, the combination effect of the microcapsule and the biomacromolecule can be effectively improved.
Preferably, the conditions of the hydrothermal reaction are: reacting for 48 hours at 80-120 ℃. When the reaction time is sufficient at a certain temperature, the obtained nano layered hydroxide used for modifying the nano tetraoxide can be more uniformly dispersed into the wall material.
Compared with the prior art, the invention has the beneficial effects that: enough cardiac muscle cells which can effectively improve the success rate of cell transplantation therapy can be obtained; after the ginsenoside is microencapsulated, the content of the ginsenoside in a culture medium can be kept relatively stable, and particularly, the ginsenoside can be kept to play a relatively stable role in promoting stem cell differentiation in the process of stem cell proliferation.
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 culturing myocardial cells by stem cell in vitro induction comprises S11, preparing single cell suspension from autologous stem cells; s12, after counting the cells of the single cell suspension, diluting the single cell suspension into a cell concentration of 10 by using a differentiation culture medium-5~10-3cell/mL stem cell suspension, inoculating on the inner surface of the cover of the bacteria culture dish, turning the cover of the culture dish to enable the stem cells on the cover to form hanging drops, adding water or aqueous solution into the culture dish to keep the humidity of the hanging drops in the incubation process, and placing the culture dish in 5% CO2Culturing in 37 deg.C incubator for 2 days; s13, collecting the embryoid bodies formed in the hanging drops on the culture dish cover, transferring the embryoid bodies into a bacterial culture dish containing a differentiation culture medium, and putting the bacterial culture dish into an incubator to continue to suspend so as to promote the further proliferation of the embryoid bodies; s14, transferring the prepared embryoid bodies to a gelatin-coated pore plate, adding an induced differentiation culture medium to each embryoid body, and performing intervening differentiation culture; the induced differentiation culture medium comprises 0.8mg/L of salvianolic acid B, 1mg/mL of ginsenoside and 0.9mg/L of icariin. The culture medium for inducing differentiation also comprises a GMEM culture medium, fetal calf serum, non-essential amino acids, 50mmol/L sodium pyruvate water solution and 0.1mol/L dimercaptoethanol water solution, wherein the MEM culture medium: fetal bovine serum:non-essential amino acids: 50mmol/L aqueous sodium pyruvate solution: 0.1mol/L aqueous dimercaptoethanol =73.5:24:1:1.3: 0.2. Preparing 55% of the ginsenoside into microcapsules, and adding the microcapsules containing the ginsenoside into a culture medium, wherein the preparation method of the microcapsules containing the ginsenoside comprises the following steps: s31, preparing a mixed solution by glutathione, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and deionized water according to a ratio of 1:1:110, adding a phosphate buffer solution with pH =6 into the mixed solution, and stirring for 24 hours under an anaerobic condition to obtain a wall material, wherein the volume ratio of the mixed solution to the phosphate buffer solution is 1: 7; s32, dispersing ginsenoside into hydroxyl silicone oil, wherein the ratio of the ginsenoside to the hydroxyl silicone oil is 1:110, so as to obtain a core material; s33, mixing the wall material and the core material according to the volume ratio of 1:5, carrying out ultrasonic treatment on an oil-water two-phase interface for 8min by using an ultrasonic probe in an ice-water bath, and carrying out multiple adsorption and washing on a product to obtain the microcapsule. The preparation method of the ginsenoside comprises the following steps: s41, adding cellulase and pectinase into deionized water, mixing with sliced ginseng, and reacting at 45 ℃ for 1h to obtain an enzymolysis extracting solution; s42, adding 95% ethanol into the enzymolysis extracting solution for the first time, refluxing at 55 ℃ for 3 hours, filtering to obtain a primary extracting solution and residues, adding 95% ethanol into the residues for the second time for extraction, refluxing at 55 ℃ for 2 hours, and mixing the primary extracting solution and the residues to obtain an alcohol extracting solution; s43, concentrating the alcohol extract at 50 ℃ under reduced pressure to obtain an extract, recovering ethanol in the concentration process, and drying the obtained extract in vacuum to obtain the ginsenoside. The mass ratio of the cellulase to the pectinase to the deionized water to the ginseng is 5 multiplied by 10-4:10-3:1:4. The mass ratio of the ethanol to the enzymolysis extracting solution is 1: 0.4: 1.6. the wall material also comprises modified nano ferroferric oxide particles, and the preparation method of the wall material added with the modified nano ferroferric oxide comprises the following steps: preparing a mixed solution by glutathione, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and deionized water according to a ratio of 1:1:108, and adding a phosphate buffer solution with pH =6 into the mixed solution; modified nano ferroferric oxide particles are dispersed in the phosphate buffer solution, the mass ratio of the modified nano ferroferric oxide to the phosphate buffer solution is 1:600, and the modified nano ferroferric oxide is dispersed in phosphoric acidThe mode in the salt buffer solution is ultrasonic dispersion; stirring for 24 hours under an anaerobic condition, repeatedly adsorbing and washing to obtain the wall material, wherein the volume ratio of the mixed solution to the phosphate buffer solution is 1: 6.5. The modification method of the modified nano ferroferric oxide comprises the following steps: dispersing nano ferroferric oxide in deionized water, adding nano layered double hydroxide, performing ultrasonic dispersion, and drying to obtain nano layered hydroxide modified nano ferroferric oxide, wherein the nano ferroferric oxide comprises the following components in parts by weight: nano layered hydroxide: deionized water =1: 0.4: 20. the preparation method of the nano layered hydroxide comprises the following steps: dissolving aluminum nitrate and aluminum nitrate in deionized water, adding the solution into 1.25% sodium hydroxide solution which is vigorously stirred, carrying out hydrothermal reaction, drying a reaction product, and crushing to obtain nano layered hydroxide; the aluminum nitrate: magnesium nitrate: deionized water =1:0.75: 30. The conditions of the hydrothermal reaction are as follows: the reaction was carried out at 100 ℃ for 48 h.
The autologous stem cells are induced and differentiated into the myocardial cells, and the obtained myocardial cells are used as cell donors of a myocardial cell transplantation therapy, so that the rejection reaction of original cells to the transplanted myocardial cells can be reduced, and the success rate of myocardial cell transplantation is improved; the salvianolic acid B, ginsenoside and icariin are used for inducing the stem cells to be converted into the myocardial cells, so that the differentiation rate of the stem cells converted into the myocardial cells can be improved.
The autologous stem cells are induced and differentiated in a culture medium by the mixture of salvianolic acid B, ginsenoside and icariin, so that sufficient cardiac muscle cells which can effectively improve the success rate of cell transplantation therapy can be obtained. The differentiation rate of stem cells into cardiomyocytes can be improved by optimizing the content of each main substance in the culture medium. After the ginsenoside is microencapsulated, the content of the ginsenoside in a culture medium can be kept relatively stable, and particularly, the ginsenoside can be kept to play a relatively stable role in promoting stem cell differentiation in the process of stem cell proliferation. The ginsenoside extracted by enzymolysis can be used together with icariin and salvianolic acid B to further improve the differentiation rate of stem cells into myocardial cells. . . The modified nano ferroferric oxide is added into the wall material, so that the probability of combination of the microcapsules and stem cells can be enhanced, the microcapsules dispersed in the culture medium can continuously provide nutritional factors for the stem cells at the periphery, and the differentiation rate is further improved. The nano ferroferric oxide modified by the nano layered hydroxide can be compounded with biological macromolecules, so that certain targeting property can be generated; the targeted microcapsule can be combined with stem cells, so as to continuously provide an environment for inducing differentiation for surrounding stem cells. After the nano ferroferric oxide modified by the nano layered hydroxide prepared in a certain proportion is mixed into the wall material, the combination effect of the microcapsule and the biomacromolecule can be effectively improved. When the reaction time is sufficient at a certain temperature, the obtained nano layered hydroxide used for modifying the nano tetraoxide can be more uniformly dispersed into the wall material.
Example 2
A method for culturing myocardial cells by stem cell in vitro induction comprises S11, preparing single cell suspension from autologous stem cells; s12, after counting the cells of the single cell suspension, diluting the single cell suspension into a cell concentration of 10 by using a differentiation culture medium-5~10-3cell/mL stem cell suspension, inoculating on the inner surface of the cover of the bacteria culture dish, turning the cover of the culture dish to enable the stem cells on the cover to form hanging drops, adding water or aqueous solution into the culture dish to keep the humidity of the hanging drops in the incubation process, and placing the culture dish in 5% CO2Culturing in 37 deg.C incubator for 1 day; s13, collecting the embryoid bodies formed in the hanging drops on the culture dish cover, transferring the embryoid bodies into a bacterial culture dish containing a differentiation culture medium, and putting the bacterial culture dish into an incubator to continue to suspend so as to promote the further proliferation of the embryoid bodies; s14, transferring the prepared embryoid bodies to a gelatin-coated pore plate, adding an induced differentiation culture medium to each embryoid body, and performing intervening differentiation culture; the induced differentiation culture medium comprises 0.7mg/L of salvianolic acid B, 0.5mg/mL of ginsenoside and 0.8mg/L of icariin. The culture medium for inducing differentiation also comprises a GMEM culture medium, fetal calf serum, non-essential amino acids, 50mmol/L sodium pyruvate water solution and 0.1mol/L dimercaptoethanol water solution, wherein the MEM culture medium: fetal bovine serum: non-essential amino acids: 50mmol/L aqueous sodium pyruvate solution: 0.1mol/L aqueous dimercaptoethanol =73.5:24:1:1.3: 0.2. Making 50% of the ginsenoside into microcapsule, and mixing with the above microcapsuleThe microcapsule is added into a culture medium, and the preparation method of the microcapsule containing ginsenoside comprises the following steps: s31, preparing a mixed solution by glutathione, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and deionized water according to a ratio of 1:1:100, adding a phosphate buffer solution with pH =6 into the mixed solution, and stirring for 24 hours under an anaerobic condition to obtain a wall material, wherein the volume ratio of the mixed solution to the phosphate buffer solution is 1: 6; s32, dispersing ginsenoside into hydroxyl silicone oil, wherein the ratio of the ginsenoside to the hydroxyl silicone oil is 1:100, so as to obtain a core material; s33, mixing the wall material and the core material according to the volume ratio of 1:5, carrying out ultrasonic treatment on an oil-water two-phase interface for 5min by using an ultrasonic probe in an ice-water bath, and carrying out multiple adsorption and washing on a product to obtain the microcapsule. The preparation method of the ginsenoside comprises the following steps: s41, adding cellulase and pectinase into deionized water, mixing with sliced ginseng, and reacting at 45 ℃ for 1h to obtain an enzymolysis extracting solution; s42, adding 95% ethanol into the enzymolysis extracting solution for the first time, refluxing at 55 ℃ for 3 hours, filtering to obtain a primary extracting solution and residues, adding 95% ethanol into the residues for the second time for extraction, refluxing at 55 ℃ for 2 hours, and mixing the primary extracting solution and the residues to obtain an alcohol extracting solution; s43, concentrating the alcohol extract at 50 ℃ under reduced pressure to obtain an extract, recovering ethanol in the concentration process, and drying the obtained extract in vacuum to obtain the ginsenoside. The mass ratio of the cellulase to the pectinase to the deionized water to the ginseng is 5 multiplied by 10-4:10-3:1:4. The mass ratio of the ethanol to the enzymolysis extracting solution is 1: 0.4: 1.6. the wall material also comprises modified nano ferroferric oxide particles, and the preparation method of the wall material added with the modified nano ferroferric oxide comprises the following steps: preparing a mixed solution by glutathione, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and deionized water according to a ratio of 1:1:100, and adding a phosphate buffer solution with pH =6 into the mixed solution; modified nano ferroferric oxide particles are dispersed in the phosphate buffer solution, the mass ratio of the modified nano ferroferric oxide to the phosphate buffer solution is 1:600, and the modified nano ferroferric oxide is dispersed in the phosphate buffer solution in an ultrasonic dispersion mode; stirring for 24 hours under an anaerobic condition, repeatedly adsorbing and washing to obtain the wall material, wherein the volume ratio of the mixed solution to the phosphate buffer solution is 1:6. What is needed isThe modification method of the modified nano ferroferric oxide comprises the following steps: dispersing nano ferroferric oxide in deionized water, adding nano layered double hydroxide, performing ultrasonic dispersion, and drying to obtain nano layered hydroxide modified nano ferroferric oxide, wherein the nano ferroferric oxide comprises the following components in parts by weight: nano layered hydroxide: deionized water =1: 0.2: 20. the preparation method of the nano layered hydroxide comprises the following steps: dissolving aluminum nitrate and aluminum nitrate in deionized water, adding the solution into 1.25% sodium hydroxide solution which is vigorously stirred, carrying out hydrothermal reaction, drying a reaction product, and crushing to obtain nano layered hydroxide; the aluminum nitrate: magnesium nitrate: deionized water =1:0.75: 30. The conditions of the hydrothermal reaction are as follows: the reaction was carried out at 80 ℃ for 48 h.
Example 3
A method for culturing myocardial cells by stem cell in vitro induction comprises S11, preparing single cell suspension from autologous stem cells; s12, after counting the cells of the single cell suspension, diluting the single cell suspension into a cell concentration of 10 by using a differentiation culture medium-5~10-3cell/mL stem cell suspension, inoculating on the inner surface of the cover of the bacteria culture dish, turning the cover of the culture dish to enable the stem cells on the cover to form hanging drops, adding water or aqueous solution into the culture dish to keep the humidity of the hanging drops in the incubation process, and placing the culture dish in 5% CO2Culturing in 37 deg.C incubator for 3 days; s13, collecting the embryoid bodies formed in the hanging drops on the culture dish cover, transferring the embryoid bodies into a bacterial culture dish containing a differentiation culture medium, and putting the bacterial culture dish into an incubator to continue to suspend so as to promote the further proliferation of the embryoid bodies; s14, transferring the prepared embryoid bodies to a gelatin-coated pore plate, adding an induced differentiation culture medium to each embryoid body, and performing intervening differentiation culture; the induced differentiation culture medium comprises 1.2mg/L salvianolic acid B, 1.5mg/mL ginsenoside and 1.2mg/L icariin. The culture medium for inducing differentiation also comprises a GMEM culture medium, fetal calf serum, non-essential amino acids, 50mmol/L sodium pyruvate water solution and 0.1mol/L dimercaptoethanol water solution, wherein the MEM culture medium: fetal bovine serum: non-essential amino acids: 50mmol/L aqueous sodium pyruvate solution: 0.1mol/L aqueous dimercaptoethanol =73.5:24:1:1.3: 0.2. Making 60% of the ginsenoside into microcapsule, and mixing with microcapsule containing ginsenosideThe microcapsule is added into a culture medium, and the preparation method of the microcapsule containing ginsenoside comprises the following steps: s31, preparing a mixed solution by glutathione, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and deionized water according to a ratio of 1:1:120, adding a phosphate buffer solution with pH =6 into the mixed solution, and stirring for 24 hours under an anaerobic condition to obtain a wall material, wherein the volume ratio of the mixed solution to the phosphate buffer solution is 1: 8; s32, dispersing ginsenoside into hydroxyl silicone oil, wherein the ratio of the ginsenoside to the hydroxyl silicone oil is 1:120, so as to obtain a core material; s33, mixing the wall material and the core material according to the volume ratio of 1:5, carrying out ultrasonic treatment on an oil-water two-phase interface for 10min by using an ultrasonic probe in an ice-water bath, and carrying out multiple adsorption and washing on a product to obtain the microcapsule. The preparation method of the ginsenoside comprises the following steps: s41, adding cellulase and pectinase into deionized water, mixing with sliced ginseng, and reacting at 45 ℃ for 1h to obtain an enzymolysis extracting solution; s42, adding 95% ethanol into the enzymolysis extracting solution for the first time, refluxing at 55 ℃ for 3 hours, filtering to obtain a primary extracting solution and residues, adding 95% ethanol into the residues for the second time for extraction, refluxing at 55 ℃ for 2 hours, and mixing the primary extracting solution and the residues to obtain an alcohol extracting solution; s43, concentrating the alcohol extract at 50 ℃ under reduced pressure to obtain an extract, recovering ethanol in the concentration process, and drying the obtained extract in vacuum to obtain the ginsenoside. The mass ratio of the cellulase to the pectinase to the deionized water to the ginseng is 5 multiplied by 10-4:10-3:1:4. The mass ratio of the ethanol to the enzymolysis extracting solution is 1: 0.4: 1.6. the wall material also comprises modified nano ferroferric oxide particles, and the preparation method of the wall material added with the modified nano ferroferric oxide comprises the following steps: preparing a mixed solution by glutathione, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and deionized water according to a ratio of 1:1:120, and adding a phosphate buffer solution with pH =6 into the mixed solution; modified nano ferroferric oxide particles are dispersed in the phosphate buffer solution, the mass ratio of the modified nano ferroferric oxide to the phosphate buffer solution is 1:600, and the modified nano ferroferric oxide is dispersed in the phosphate buffer solution in an ultrasonic dispersion mode; stirring for 24 hours under an anaerobic condition, repeatedly adsorbing and washing to obtain the wall material, wherein the volume ratio of the mixed solution to the phosphate buffer solution is 1: 8. The changeThe modification method of the nanometer ferroferric oxide comprises the following steps: dispersing nano ferroferric oxide in deionized water, adding nano layered double hydroxide, performing ultrasonic dispersion, and drying to obtain nano layered hydroxide modified nano ferroferric oxide, wherein the nano ferroferric oxide comprises the following components in parts by weight: nano layered hydroxide: deionized water =1: 0.5: 20. the preparation method of the nano layered hydroxide comprises the following steps: dissolving aluminum nitrate and aluminum nitrate in deionized water, adding the solution into 1.25% sodium hydroxide solution which is vigorously stirred, carrying out hydrothermal reaction, drying a reaction product, and crushing to obtain nano layered hydroxide; the aluminum nitrate: magnesium nitrate: deionized water =1:0.75: 30. The conditions of the hydrothermal reaction are as follows: the reaction was carried out at 120 ℃ for 48 h.
Example 4
Example 4 is different from example 1 in that the ginsenoside is entirely added to the medium.
Example 5
Example 5 the difference from example 1 is that all ginsenosides in the ginsenoside microcapsules are commercially available ginsenosides.
Example 6
Example 6 is different from example 1 in that the nano-modified ferroferric oxide is not modified by nano-layered tetraoxide.
Comparative example 1
Comparative example 1 the same as example 1 except that the culture medium contained no icariin.
Examples of the experiments
The embodiment is preferably performed by analyzing the differentiation rate of stem cells into cardiomyocytes and the survival rate after transplantation according to the methods of the different examples and comparative examples.
The differentiation rate is the proportion of cardiomyocytes to the total number of cells after a period of differentiation;
the survival rate after transplantation is: after cardiomyocytes were prepared according to the methods of examples 1 to 6 and comparative examples using a mouse as a study subject, the cardiomyocytes were transplanted into the heart of the mouse, and after 3 weeks, the mouse was dissected and the number of transplanted cells in the heart of the mouse was counted.
TABLE 1 Stem cell differentiation culture and transplantation Effect
Figure DEST_PATH_IMAGE001
As can be seen from Table 1, the differentiation rates of the stem cells in examples 1 to 3 were all higher than that in the comparative example, indicating that the combination of icariin, ginsenoside and salvianolic acid B as the components of the culture medium can effectively promote the differentiation of the stem cells to the cardiomyocytes.
In example 4, part of the ginsenosides is not microencapsulated, and the differentiation rate is obviously lower than that in examples 1 to 3, which shows that the microencapsulated ginsenosides can durably and effectively promote the differentiation of stem cells to cardiac muscle cells; the ginsenoside in the embodiment 5 is commercially available ginsenoside, and the ginsenosides in the embodiments 1 to 3 are extracted by enzymolysis, which shows that the ginsenoside obtained by the enzymolysis has a certain effect of improving the differentiation of stem cells to cardiac muscle; the nano ferroferric oxide in example 6 is not modified by the nano layered hydroxide, and the differentiation rate is lower than that in examples 1 to 3, which shows that the wall material of the microcapsule of the microencapsulated ginsenoside F may influence the capability of the microcapsule of promoting stem cell differentiation if the wall material is not treated by the modified nano ferroferric oxide.
In examples 1 to 3, ginsenoside, salvianolic acid B and epimedium are used as main induction factors for inducing stem cells to differentiate into myocardial cells, partial ginsenoside is subjected to microencapsulation treatment, and the wall material of the microcapsule is further treated, so that the differentiation rate is improved.
The survival rate of the transplanted cells in examples 1-3 is significantly higher than that of the comparative example and higher than that of examples 4-6, which shows that the survival rate of the transplanted cells can be effectively improved by using the culture medium in examples 1-3.
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 (5)

1. A method for culturing myocardial cells by stem cell in vitro induction is characterized in that S11, autologous stem cells are taken to prepare single cell suspension; s12, after counting the cells of the single cell suspension, diluting the single cell suspension into a cell concentration of 10 by using a differentiation culture medium-5~10-3cell/mL stem cell suspension, inoculating on the inner surface of the cover of the bacteria culture dish, turning the cover of the culture dish to enable the stem cells on the cover to form hanging drops, adding water or aqueous solution into the culture dish to keep the humidity of the hanging drops in the incubation process, and placing the culture dish in 5% CO2Culturing for 1-3 days in an incubator at 37 ℃; s13, collecting the embryoid bodies formed in the hanging drops on the culture dish cover, transferring the embryoid bodies into a bacterial culture dish containing a differentiation culture medium, and putting the bacterial culture dish into an incubator to continue to suspend so as to promote the further proliferation of the embryoid bodies; s14, transferring the prepared embryoid bodies to a gelatin-coated pore plate, adding an induced differentiation culture medium to each embryoid body, and performing intervening differentiation culture; the induced differentiation culture medium comprises 0.7-1.2 mg/L of salvianolic acid B, 0.5-1.5 mg/mL of ginsenoside and 0.8-1.2 mg/L of icariin;
50% -60% of the ginsenoside is prepared into microcapsules, the microcapsules containing the ginsenoside are added into a culture medium, and the preparation method of the microcapsules containing the ginsenoside comprises the following steps: s31, preparing a mixed solution from glutathione, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and deionized water according to a ratio of 1:1: 100-120, and adding a phosphate buffer solution with pH =6 into the mixed solution; modified nano ferroferric oxide particles are dispersed in the phosphate buffer solution, the mass ratio of the modified nano ferroferric oxide to the phosphate buffer solution is 1:600, and the modified nano ferroferric oxide is dispersed in the phosphate buffer solution in an ultrasonic dispersion mode; stirring for 24 hours under an anaerobic condition, repeatedly adsorbing and washing to obtain a wall material, wherein the volume ratio of the mixed solution to the phosphate buffer solution is 1: 6-8; s32, dispersing ginsenoside into hydroxyl silicone oil, wherein the ratio of the ginsenoside to the hydroxyl silicone oil is 1: 100-120, so as to obtain a core material; s33, mixing the wall material and the core material according to the volume ratio of 1:5, carrying out ultrasonic treatment on an oil-water two-phase interface for 5-10 min by using an ultrasonic probe in an ice-water bath, and carrying out multiple adsorption and washing on a product to obtain a microcapsule;
the preparation method of the ginsenoside comprises the following steps: s41, adding cellulase and pectinase into deionized water, mixing with sliced ginseng, and reacting at 45 ℃ for 1h to obtain an enzymolysis extracting solution; s42, adding 95% ethanol into the enzymolysis extracting solution for the first time, refluxing at 55 ℃ for 3 hours, filtering to obtain a primary extracting solution and residues, adding 95% ethanol into the residues for the second time for extraction, refluxing at 55 ℃ for 2 hours, and mixing with the primary extracting solution to obtain an ethanol extracting solution, wherein the adding amount of the ethanol for the first time is as follows: the second ethanol addition =1: 4; s43, concentrating the alcohol extract at 50 ℃ under reduced pressure to obtain an extract, recovering ethanol in the concentration process, and drying the obtained extract in vacuum to obtain ginsenoside;
the modification method of the modified nano ferroferric oxide comprises the following steps: dispersing nano ferroferric oxide in deionized water, adding nano layered double hydroxide, performing ultrasonic dispersion, and drying to obtain nano layered hydroxide modified nano ferroferric oxide, wherein the nano ferroferric oxide comprises the following components in parts by weight: nano layered hydroxide: deionized water =1: 0.2-0.5: 20;
the preparation method of the nano layered hydroxide comprises the following steps: dissolving aluminum nitrate and aluminum nitrate in deionized water, adding the solution into 1.25% sodium hydroxide solution which is vigorously stirred, carrying out hydrothermal reaction, drying a reaction product, and crushing to obtain nano layered hydroxide; the aluminum nitrate: magnesium nitrate: deionized water =1:0.75: 30.
2. The method for inducing and culturing cardiomyocytes according to claim 1, wherein the differentiation-inducing culture medium further comprises GMEM culture medium, fetal bovine serum, non-essential amino acids, 50mmol/L sodium pyruvate solution and 0.1mol/L dimercaptoethanol solution, and the GMEM culture medium: fetal bovine serum: non-essential amino acids: 50mmol/L aqueous sodium pyruvate solution: 0.1mol/L aqueous dimercaptoethanol =73.5:24:1:1.3: 0.2.
3. The method for culturing cardiomyocytes induced by stem cells in vitro as claimed in claim 1, wherein the mass ratio of the cellulase, pectinase, deionized water and ginseng isIs 5 x 10-4 :10-3 :1:4。
4. The method for culturing cardiomyocytes induced by stem cells in vitro according to claim 1, wherein the mass ratio of the ethanol to the enzymolysis extracting solution is 1: 0.4.
5. The method for inducing and culturing myocardial cells in vitro according to claim 1, wherein the hydrothermal reaction conditions are as follows: reacting for 48 hours at 80-120 ℃.
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