CN110157663A - A kind of stem cell external evoked method for cultivating cardiac muscle cell - Google Patents
A kind of stem cell external evoked method for cultivating cardiac muscle cell Download PDFInfo
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- CN110157663A CN110157663A CN201910314980.5A CN201910314980A CN110157663A CN 110157663 A CN110157663 A CN 110157663A CN 201910314980 A CN201910314980 A CN 201910314980A CN 110157663 A CN110157663 A CN 110157663A
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Abstract
The present invention relates to stem cell fields, provide a kind of stem cell external evoked method for cultivating cardiac muscle cell, differentiation rate is low when for solving the problems, such as stem cell myocardiac differentiation.The stem cell external evoked method for cultivating cardiac muscle cell of one kind provided by the invention, is derived from somatic stem cell and single cell suspension is made, prepare embryoid body；The embryoid body prepared is transferred to the coated orifice plate of gelatin, one, every hole embryoid body adds inductive differentiation medium, carries out intervening differentiation culture；It include 0.7 ~ 1.2mg/L of tanshin polyphenolic acid B, ginsengenin 20 .5 ~ 1.5mg/mL, 0.8 ~ 1.2mg/L of icariin in the inductive differentiation medium.Cardiac muscle cell that is available enough and can effectively improve cell transplantation therapy success rate.
Description
Technical field
The present invention relates to stem cell fields, and in particular to a kind of stem cell external evoked method for cultivating cardiac muscle cell.
Background technique
The heart failure as caused by cardiac myocyte dysiunction is a kind of worldwide main disease, patients with heart failure
The main reason for dead is cardiac pumping function forfeiture or heart murmur.The serious patients with heart failure one term death rate is more than 50%.
Although patients with terminal can carry out heart transplant, then since donor organ is constantly in the serious state that supply falls short of demand.By
In complication and the heart transplant such as the high incidence and the death rate of heart failure, the death of heart transplant, immunological rejection
The objective statuses such as later period failure, it would be highly desirable to develop new treatment method to improve the function of cardiac muscle cell, prevent the hair of heart failure
It is raw.Early stage revascularization is focused primarily upon to the treatment method of heart failure at present and inhibits further cardiomyocyte loss,
And cardiac muscle cell is a kind of cell of terminal differentiation, proliferative capacity is extremely limited, extremely difficult recovery after loss.
Cardiomyocyte transplantation therapy focuses on reconstruction myocardial function, restores the pump blood ability of regeneration cardiac muscular tissue, also grinds
The cardiac muscle cell for studying carefully discovery injection heart is capable of forming new cardiac muscular tissue.But cellular transplantation therapy key factor is to determine properly
Donor cell types.Therefore, stem cell directional how is induced to be divided into cardiac muscle cell, and it is thin how to improve stem cell Cardiomyocytes
The differentiation rate of born of the same parents be technical problem urgently to be resolved for medical treatment.
Summary of the invention
Present invention solves the technical problem that differentiation rate low problem when being stem cell myocardiac differentiation, provides a kind of dry
The method of cells in vitro induction culturing cardiac muscle cell.
In order to solve the above technical problem, the present invention provides technical solution are as follows:
A kind of stem cell external evoked method for cultivating cardiac muscle cell, S11. are derived from somatic stem cell and single cell suspension are made；S12.
It is 10 by cell concentration is diluted to differential medium after single cell suspension cell count-5~10-3Cells/mL's is dry thin
Born of the same parents' suspension, is inoculated on the cover inner surface of Micro-Organism Culture Dish, overturns culture dish lid, forms the stem cell on lid outstanding
It drips, water or aqueous solution is added in culture dish to keep humidity of hanging drop during incubation, culture dish is placed in 5%CO2, 37 DEG C of trainings
It supports and is cultivated 1 ~ 3 day in case；S13. the embryoid body formed in hanging drop on culture dish lid is collected, is transferred into and fills differentiation culture
In the Micro-Organism Culture Dish of base, it is put into and continues to suspend in incubator, embryoid body is promoted further to be proliferated；S14. the embryoid that will be prepared
Body is transferred to the coated orifice plate of gelatin, and one, every hole embryoid body adds inductive differentiation medium, carries out intervening differentiation culture；It is described
In inductive differentiation medium include 0.7 ~ 1.2mg/L of tanshin polyphenolic acid B, ginsengenin 20 .5 ~ 1.5mg/mL, icariin 0.8 ~
1.2mg/L。
Autologous stem cells are induced to differentiate into cardiac muscle cell, using obtained cardiac muscle cell as cardiomyocyte transplantation therapy
Cell donor can reduce original cell to the rejection of the cardiac muscle cell of transplanting, improve the success rate of cardiomyocyte transplantation；
It is changed into cardiac muscle cell using tanshin polyphenolic acid B, ginsenoside and icariin induction stem cell, it is the heart that stem cell transformation, which can be improved,
The differentiation rate of myocyte.
Mixture of the autologous stem cells through tanshin polyphenolic acid B, ginsenoside and icariin induces differentiation in the medium, can be with
Obtain cardiac muscle cell that is enough and can effectively improve cell transplantation therapy success rate.
Preferably, further include in the inductive differentiation medium GMEM culture medium, fetal calf serum, nonessential amino acid,
The Sodium Pyruvate aqueous solution of 50mmol/L and the 3-mercaptoethanol aqueous solution of 0.1mol/L, the GMEM culture medium: fetal calf serum:
Nonessential amino acid: the Sodium Pyruvate aqueous solution of 50mmol/L: 3-mercaptoethanol aqueous solution=73.5:24:1 of 0.1mol/L:
1.3:0.2.By the content of main matter each in Optimal Medium, the differentiation that stem cell is divided into cardiac muscle cell can be improved
Rate.
Preferably, microcapsules are made in the 50% ~ 60% of the ginsenoside, and the microcapsules containing ginsenoside are added to training
Support in base, the microcapsules containing ginsenoside the preparation method comprises the following steps: S31. by glutathione, 1-(3- dimethylamino third
Base) -3- ethyl-carbodiimide hydrochloride, deionized water press 1:1:100 ~ 120, be made mixed solution, be added in mixed solution
The phosphate buffer solution of pH=6 stirs for 24 hours under oxygen free condition, obtains wall material, the same phosphate buffer solution of mixed solution
Volume ratio be 1:6 ~ 8；S32. it disperses ginsenoside in hydroxy silicon oil, the ginsenoside is with the ratio of hydroxy silicon oil
1:100 ~ 120 obtain core material；S33. the ratio of 1:5 mixes wall material and core material by volume, is visited under ice-water bath using ultrasound
Head carries out 5 ~ 10min of ultrasound to oil-water two-phase interfaces, is repeatedly adsorbed, is washed to product, obtains microcapsules.By ginsenoside
After microencapsulation, the content of ginsenoside in the medium can keep a more stable content, especially in stem cell
In breeding, ginsenoside can be kept to play the role of more stable promotion stem cell differentiation always.
Preferably, the ginsenoside the preparation method comprises the following steps: cellulase, pectase are added to deionized water by S41.
In, with the ginseng mixing thinly sliced, 1h is reacted at 45 DEG C, obtains enzyme-extracting solution；S42. for the first time into enzyme-extracting solution
95% ethyl alcohol, 55 DEG C of reflux 3h is added, filtering obtains first extract and residue, and the ethyl alcohol of second of addition 95% of residue is mentioned
It takes, 55 DEG C of reflux 2h, merges with first extract, obtain alcohol extract；S43. alcohol extract is concentrated under reduced pressure at 50 DEG C at medicinal extract shape, dense
Ethyl alcohol is recycled in compression process, obtained medicinal extract vacuum drying both obtains ginsenoside.The ginsenoside obtained through enzymolysis and extraction is the same as excessive
Sheep leaves of pulse plants glycosides, tanshin polyphenolic acid B, which are used in conjunction with, can be further improved the differentiation rate that stem cell is divided into cardiac muscle cell.
Preferably, the cellulase, pectase, deionized water, ginseng mass ratio be 5 × 10-4:10-3:1:4。。
Preferably, the ethyl alcohol is 1:0.4:1.6 with the mass ratio of enzyme-extracting solution.
Preferably, further include modified Nano ferriferrous oxide particles in the wall material, joined the oxidation of modified Nano four three
The wall material of iron the preparation method comprises the following steps: by glutathione, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, go from
Sub- water presses 1:1:100 ~ 120, and mixed solution is made, and the phosphate buffer solution of pH=6 is added in mixed solution；The phosphoric acid
Modified Nano ferriferrous oxide particles are dispersed in salt buffer solution, the modified Nano ferroso-ferric oxide is molten with phosphate-buffered
The mass ratio of liquid is 1:600, and the modified Nano ferroso-ferric oxide is distributed to the mode in phosphate buffer solution as ultrasound point
It dissipates；It is stirred under oxygen free condition for 24 hours, adsorbs, washs repeatedly, obtain wall material, body of the mixed solution with phosphate buffer solution
Product is than being 1:6 ~ 8.Modified Nano ferroso-ferric oxide is added in wall material can be enhanced the probability that microcapsules are combined with stem cell, point
The microcapsules being dispersed in culture medium can persistently be that the stem cell on periphery provides trophic factors, and then improves differentiation rate.
Preferably, the method for modifying of the modified Nano ferroso-ferric oxide are as follows: gone what nano ferriferrous oxide was dispersed in
In ionized water, nano layered double hydroxides are added, drying after ultrasonic disperse obtains the hydroxide-modified nanometer of nano lamellar
Ferroso-ferric oxide, the nanometer four aoxidize three-body: nano lamellar hydroxide: deionized water=1:0.2 ~ 0.5:20.Nano lamellar
Nano ferriferrous oxide can be compound with large biological molecule after hydroxide-modified, and then can produce certain targeting；Tool
Having the microcapsules of targeting can combine with stem cell, and then continue to provide the environment of induction differentiation to peripheral stem.
Preferably, the manufacturing method of the nano lamellar hydroxide are as follows: aluminum nitrate and aluminum nitrate are dissolved in deionized water
Afterwards, it is added in 1.25% sodium hydroxide solution being vigorously stirred, carries out hydro-thermal reaction, reaction product crushed after being dried obtains
Nano lamellar hydroxide；The aluminum nitrate: magnesium nitrate: deionized water=1:0.75:30.The nanometer made of certain proportion
After the modified nano ferriferrous oxide of layered hydroxide is mixed into wall material, microcapsules can be effectively improved with large biological molecule
In conjunction with effect.
Preferably, the condition of the hydro-thermal reaction are as follows: react 48h at 80 ~ 120 DEG C.Reaction time at a certain temperature
When sufficient, obtained nano lamellar hydroxide is distributed to wall material for what modified Nano four aoxidized that three-body can be more uniform
In.
Compared with prior art, the device have the advantages that are as follows: it is available enough and can effectively improve cell
The cardiac muscle cell of transplantation therapy success rate；After ginsenoside microencapsulation, the content of ginsenoside in the medium can be protected
A more stable content is held, especially during stem cells hyperplasia, ginsenoside can be kept to play comparison always steady
The fixed effect for promoting stem cell differentiation.
Specific embodiment
Following implementation column is to further explanation of the invention, is not limitation of the present invention.
Embodiment 1
A kind of stem cell external evoked method for cultivating cardiac muscle cell, S11. are derived from somatic stem cell and single cell suspension are made；S12.
It is 10 by cell concentration is diluted to differential medium after single cell suspension cell count-5~10-3Cells/mL's is dry thin
Born of the same parents' suspension, is inoculated on the cover inner surface of Micro-Organism Culture Dish, overturns culture dish lid, forms the stem cell on lid outstanding
It drips, water or aqueous solution is added in culture dish to keep humidity of hanging drop during incubation, culture dish is placed in 5%CO2, 37 DEG C of trainings
It supports and is cultivated 2 days in case；S13. the embryoid body formed in hanging drop on culture dish lid is collected, is transferred into and fills differential medium
Micro-Organism Culture Dish in, be put into and continue to suspend in incubator, embryoid body is promoted further to be proliferated；S14. the embryoid body that will be prepared
It is transferred to the coated orifice plate of gelatin, one, every hole embryoid body adds inductive differentiation medium, carries out intervening differentiation culture；It is described to lure
Lead includes tanshin polyphenolic acid B 0.8mg/L, ginsenoside 1mg/mL, icariin 0.9mg/L in differential medium.The induction differentiation
Further include in culture medium GMEM culture medium, fetal calf serum, nonessential amino acid, 50mmol/L Sodium Pyruvate aqueous solution and
The 3-mercaptoethanol aqueous solution of 0.1mol/L, the MEM culture medium: fetal calf serum: nonessential amino acid: the acetone of 50mmol/L
Acid sodium aqueous solution: 3-mercaptoethanol aqueous solution=73.5:24:1:1.3:0.2 of 0.1mol/L.The 55% of the ginsenoside is made
Microcapsules containing ginsenoside are added in culture medium by microcapsules, the preparation side of the microcapsules containing ginsenoside
Method are as follows: glutathione, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, deionized water are pressed 1:1 by S31.:
110, mixed solution is made, the phosphate buffer solution of pH=6 is added in mixed solution, stirs for 24 hours, obtains under oxygen free condition
Wall material, the mixed solution are 1:7 with the volume ratio of phosphate buffer solution；S32. hydroxy silicon oil is dispersed by ginsenoside
In, the ginsenoside is 1:110 with the ratio of hydroxy silicon oil, obtains core material；S33. by volume the ratio of 1:5 by wall material and
Core material mixing carries out ultrasound 8min to oil-water two-phase interfaces using ultrasonic probe under ice-water bath, product is repeatedly adsorbed,
Washing, obtains microcapsules.The ginsenoside the preparation method comprises the following steps: cellulase, pectase are added to deionized water by S41.
In, with the ginseng mixing thinly sliced, 1h is reacted at 45 DEG C, obtains enzyme-extracting solution；S42. for the first time into enzyme-extracting solution
95% ethyl alcohol, 55 DEG C of reflux 3h is added, filtering obtains first extract and residue, and the ethyl alcohol of second of addition 95% of residue is mentioned
It takes, 55 DEG C of reflux 2h, merges with first extract, obtain alcohol extract；S43. alcohol extract is concentrated under reduced pressure at 50 DEG C at medicinal extract shape, dense
Ethyl alcohol is recycled in compression process, obtained medicinal extract vacuum drying both obtains ginsenoside.The cellulase, pectase, deionized water,
The mass ratio of ginseng is 5 × 10-4:10-3:1:4.The ethyl alcohol is 1:0.4:1.6 with the mass ratio of enzyme-extracting solution.The wall
Further include modified Nano ferriferrous oxide particles in material, joined the wall material of modified Nano ferroso-ferric oxide the preparation method comprises the following steps:
Glutathione, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, deionized water are pressed into 1:1:108, mixing is made
The phosphate buffer solution of pH=6 is added in solution in mixed solution；Modified Nano is dispersed in the phosphate buffer solution
Ferriferrous oxide particles, the modified Nano ferroso-ferric oxide are 1:600, the modification with the mass ratio of phosphate buffer solution
It is ultrasonic disperse that nano ferriferrous oxide, which is distributed to the mode in phosphate buffer solution,；It stirs under oxygen free condition for 24 hours, inhales repeatedly
Attached, washing, obtains wall material, and the mixed solution is 1:6.5 with the volume ratio of phosphate buffer solution.Four oxygen of modified Nano
Change the method for modifying of three-iron are as follows: in the deionized water for being dispersed in nano ferriferrous oxide, nano layered double hydroxides are added,
It is dry after ultrasonic disperse, the hydroxide-modified nano ferriferrous oxide of nano lamellar is obtained, the nanometer four aoxidizes three-body: receiving
Rice layered hydroxide: deionized water=1:0.4:20.The manufacturing method of the nano lamellar hydroxide are as follows: by aluminum nitrate and
It after aluminum nitrate is dissolved in deionized water, is added in 1.25% sodium hydroxide solution being vigorously stirred, carries out hydro-thermal reaction, reaction
Product crushed after being dried obtains nano lamellar hydroxide；The aluminum nitrate: magnesium nitrate: deionized water=1:0.75:30.It is described
The condition of hydro-thermal reaction are as follows: react 48h at 100 DEG C.
Autologous stem cells are induced to differentiate into cardiac muscle cell, using obtained cardiac muscle cell as cardiomyocyte transplantation therapy
Cell donor can reduce original cell to the rejection of the cardiac muscle cell of transplanting, improve the success rate of cardiomyocyte transplantation；
It is changed into cardiac muscle cell using tanshin polyphenolic acid B, ginsenoside and icariin induction stem cell, it is the heart that stem cell transformation, which can be improved,
The differentiation rate of myocyte.
Mixture of the autologous stem cells through tanshin polyphenolic acid B, ginsenoside and icariin induces differentiation in the medium, can be with
Obtain cardiac muscle cell that is enough and can effectively improve cell transplantation therapy success rate.Pass through main matter each in Optimal Medium
Content, the differentiation rate that stem cell is divided into cardiac muscle cell can be improved.After ginsenoside microencapsulation, ginsenoside is being trained
The content supported in base can keep a more stable content, especially during stem cells hyperplasia, can keep ginseng
Saponin(e plays the role of always more stable promotion stem cell differentiation.The same Herba Epimedii of the ginsenoside obtained through enzymolysis and extraction
Glycosides, tanshin polyphenolic acid B, which are used in conjunction with, can be further improved the differentiation rate that stem cell is divided into cardiac muscle cell.It adds and changes in wall material
The probability that microcapsules are combined with stem cell can be enhanced in property nano ferriferrous oxide, and the microcapsules being dispersed in culture medium can be held
Continuous is that the stem cell on periphery provides trophic factors, and then improves differentiation rate.Four oxygen of nanometer after nano lamellar is hydroxide-modified
Changing three-iron can be compound with large biological molecule, and then can produce certain targeting；Microcapsules with targeting can be same
Stem cell combines, and then continues to provide the environment of induction differentiation to peripheral stem.The nano lamellar made of certain proportion
After hydroxide-modified nano ferriferrous oxide is mixed into wall material, combination of the microcapsules with large biological molecule can be effectively improved
Effect.At a certain temperature when reaction time abundance, obtained nano lamellar hydroxide aoxidizes three-body for modified Nano four
Can be more uniform be distributed in wall material.
Embodiment 2
A kind of stem cell external evoked method for cultivating cardiac muscle cell, S11. are derived from somatic stem cell and single cell suspension are made；S12.
It is 10 by cell concentration is diluted to differential medium after single cell suspension cell count-5~10-3Cells/mL's is dry thin
Born of the same parents' suspension, is inoculated on the cover inner surface of Micro-Organism Culture Dish, overturns culture dish lid, forms the stem cell on lid outstanding
It drips, water or aqueous solution is added in culture dish to keep humidity of hanging drop during incubation, culture dish is placed in 5%CO2, 37 DEG C of trainings
It supports and is cultivated 1 day in case；S13. the embryoid body formed in hanging drop on culture dish lid is collected, is transferred into and fills differential medium
Micro-Organism Culture Dish in, be put into and continue to suspend in incubator, embryoid body is promoted further to be proliferated；S14. the embryoid body that will be prepared
It is transferred to the coated orifice plate of gelatin, one, every hole embryoid body adds inductive differentiation medium, carries out intervening differentiation culture；It is described to lure
Lead includes tanshin polyphenolic acid B 0.7mg/L, ginsengenin 20 .5mg/mL, icariin 0.8mg/L in differential medium.The induction point
Change culture medium in further include GMEM culture medium, fetal calf serum, nonessential amino acid, 50mmol/L Sodium Pyruvate aqueous solution and
The 3-mercaptoethanol aqueous solution of 0.1mol/L, the MEM culture medium: fetal calf serum: nonessential amino acid: the acetone of 50mmol/L
Acid sodium aqueous solution: 3-mercaptoethanol aqueous solution=73.5:24:1:1.3:0.2 of 0.1mol/L.The 50% of the ginsenoside is made
Microcapsules containing ginsenoside are added in culture medium by microcapsules, the preparation side of the microcapsules containing ginsenoside
Method are as follows: glutathione, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, deionized water are pressed 1:1 by S31.:
100, mixed solution is made, the phosphate buffer solution of pH=6 is added in mixed solution, stirs for 24 hours, obtains under oxygen free condition
Wall material, the mixed solution are 1:6 with the volume ratio of phosphate buffer solution；S32. hydroxy silicon oil is dispersed by ginsenoside
In, the ginsenoside is 1:100 with the ratio of hydroxy silicon oil, obtains core material；S33. by volume the ratio of 1:5 by wall material and
Core material mixing carries out ultrasound 5min to oil-water two-phase interfaces using ultrasonic probe under ice-water bath, product is repeatedly adsorbed,
Washing, obtains microcapsules.The ginsenoside the preparation method comprises the following steps: cellulase, pectase are added to deionized water by S41.
In, with the ginseng mixing thinly sliced, 1h is reacted at 45 DEG C, obtains enzyme-extracting solution；S42. for the first time into enzyme-extracting solution
95% ethyl alcohol, 55 DEG C of reflux 3h is added, filtering obtains first extract and residue, and the ethyl alcohol of second of addition 95% of residue is mentioned
It takes, 55 DEG C of reflux 2h, merges with first extract, obtain alcohol extract；S43. alcohol extract is concentrated under reduced pressure at 50 DEG C at medicinal extract shape, dense
Ethyl alcohol is recycled in compression process, obtained medicinal extract vacuum drying both obtains ginsenoside.The cellulase, pectase, deionized water,
The mass ratio of ginseng is 5 × 10-4:10-3:1:4.The ethyl alcohol is 1:0.4:1.6 with the mass ratio of enzyme-extracting solution.The wall
Further include modified Nano ferriferrous oxide particles in material, joined the wall material of modified Nano ferroso-ferric oxide the preparation method comprises the following steps:
Glutathione, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, deionized water are pressed into 1:1:100, mixing is made
The phosphate buffer solution of pH=6 is added in solution in mixed solution；Modified Nano is dispersed in the phosphate buffer solution
Ferriferrous oxide particles, the modified Nano ferroso-ferric oxide are 1:600, the modification with the mass ratio of phosphate buffer solution
It is ultrasonic disperse that nano ferriferrous oxide, which is distributed to the mode in phosphate buffer solution,；It stirs under oxygen free condition for 24 hours, inhales repeatedly
Attached, washing, obtains wall material, and the mixed solution is 1:6 with the volume ratio of phosphate buffer solution.The modified Nano four aoxidizes
The method of modifying of three-iron are as follows: in the deionized water for being dispersed in nano ferriferrous oxide, nano layered double hydroxides are added, surpass
It is dry after sound dispersion, the hydroxide-modified nano ferriferrous oxide of nano lamellar is obtained, the nanometer four aoxidizes three-body: nanometer
Layered hydroxide: deionized water=1:0.2:20.The manufacturing method of the nano lamellar hydroxide are as follows: by aluminum nitrate and nitre
It after sour aluminium is dissolved in deionized water, is added in 1.25% sodium hydroxide solution being vigorously stirred, carries out hydro-thermal reaction, reaction produces
Object crushed after being dried obtains nano lamellar hydroxide；The aluminum nitrate: magnesium nitrate: deionized water=1:0.75:30.The water
The condition of thermal response are as follows: react 48h at 80 DEG C.
Embodiment 3
A kind of stem cell external evoked method for cultivating cardiac muscle cell, S11. are derived from somatic stem cell and single cell suspension are made；S12.
It is 10 by cell concentration is diluted to differential medium after single cell suspension cell count-5~10-3Cells/mL's is dry thin
Born of the same parents' suspension, is inoculated on the cover inner surface of Micro-Organism Culture Dish, overturns culture dish lid, forms the stem cell on lid outstanding
It drips, water or aqueous solution is added in culture dish to keep humidity of hanging drop during incubation, culture dish is placed in 5%CO2, 37 DEG C of trainings
It supports and is cultivated 3 days in case；S13. the embryoid body formed in hanging drop on culture dish lid is collected, is transferred into and fills differential medium
Micro-Organism Culture Dish in, be put into and continue to suspend in incubator, embryoid body is promoted further to be proliferated；S14. the embryoid body that will be prepared
It is transferred to the coated orifice plate of gelatin, one, every hole embryoid body adds inductive differentiation medium, carries out intervening differentiation culture；It is described to lure
Lead includes tanshin polyphenolic acid B 1.2mg/L, ginsenoside 1.5mg/mL, icariin 1.2mg/L in differential medium.The induction point
Change culture medium in further include GMEM culture medium, fetal calf serum, nonessential amino acid, 50mmol/L Sodium Pyruvate aqueous solution and
The 3-mercaptoethanol aqueous solution of 0.1mol/L, the MEM culture medium: fetal calf serum: nonessential amino acid: the acetone of 50mmol/L
Acid sodium aqueous solution: 3-mercaptoethanol aqueous solution=73.5:24:1:1.3:0.2 of 0.1mol/L.The 60% of the ginsenoside is made
Microcapsules containing ginsenoside are added in culture medium by microcapsules, the preparation side of the microcapsules containing ginsenoside
Method are as follows: glutathione, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, deionized water are pressed 1:1 by S31.:
120, mixed solution is made, the phosphate buffer solution of pH=6 is added in mixed solution, stirs for 24 hours, obtains under oxygen free condition
Wall material, the mixed solution are 1:8 with the volume ratio of phosphate buffer solution；S32. hydroxy silicon oil is dispersed by ginsenoside
In, the ginsenoside is 1:120 with the ratio of hydroxy silicon oil, obtains core material；S33. by volume the ratio of 1:5 by wall material and
Core material mixing carries out ultrasound 10min to oil-water two-phase interfaces using ultrasonic probe under ice-water bath, is repeatedly inhaled to product
Attached, washing, obtains microcapsules.The ginsenoside the preparation method comprises the following steps: cellulase, pectase are added to deionization by S41.
In water, with the ginseng mixing thinly sliced, 1h is reacted at 45 DEG C, obtains enzyme-extracting solution；S42. into enzyme-extracting solution first
The ethyl alcohol of secondary addition 95%, 55 DEG C of reflux 3h, filtering obtain first extract and residue, and the ethyl alcohol of second of addition 95% of residue carries out
It extracts, 55 DEG C of reflux 2h, merges with first extract, obtain alcohol extract；S43. alcohol extract is concentrated under reduced pressure at 50 DEG C into medicinal extract shape,
Ethyl alcohol is recycled in concentration process, obtained medicinal extract vacuum drying both obtains ginsenoside.The cellulase, pectase, deionization
Water, ginseng mass ratio be 5 × 10-4:10-3:1:4.The ethyl alcohol is 1:0.4:1.6 with the mass ratio of enzyme-extracting solution.It is described
Further include modified Nano ferriferrous oxide particles in wall material, joined the preparation method of the wall material of modified Nano ferroso-ferric oxide
Are as follows: glutathione, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, deionized water are pressed into 1:1:120, are made
The phosphate buffer solution of pH=6 is added in mixed solution in mixed solution；Modification is dispersed in the phosphate buffer solution
Nano ferriferrous oxide granule, the modified Nano ferroso-ferric oxide is 1:600 with the mass ratio of phosphate buffer solution, described
It is ultrasonic disperse that modified Nano ferroso-ferric oxide, which is distributed to the mode in phosphate buffer solution,；It is stirred under oxygen free condition for 24 hours, instead
Attached, washing is relapsed, wall material is obtained, the mixed solution is 1:8 with the volume ratio of phosphate buffer solution.The modified Nano four
The method of modifying of Fe 3 O are as follows: in the deionized water for being dispersed in nano ferriferrous oxide, the double hydroxides of nano lamellar are added
Object, it is dry after ultrasonic disperse, obtain the hydroxide-modified nano ferriferrous oxide of nano lamellar, the oxidation of nanometer four three
Body: nano lamellar hydroxide: deionized water=1:0.5:20.The manufacturing method of the nano lamellar hydroxide are as follows: by nitric acid
It after aluminium and aluminum nitrate are dissolved in deionized water, is added in 1.25% sodium hydroxide solution being vigorously stirred, carries out hydro-thermal reaction,
Reaction product crushed after being dried obtains nano lamellar hydroxide；The aluminum nitrate: magnesium nitrate: deionized water=1:0.75:30.
The condition of the hydro-thermal reaction are as follows: react 48h at 120 DEG C.
Embodiment 4
Embodiment 4 with embodiment 1 the difference is that, the ginsenoside is all added directly into culture medium.
Embodiment 5
Embodiment 5 with embodiment 1 the difference is that, all commercially available ginsengs of ginsenoside in the ginsenoside microcapsules
Saponin(e.
Embodiment 6
Embodiment 6 with embodiment 1 the difference is that, the nano modification ferroso-ferric oxide without nano lamellar four oxidation three
It is modifies.
Comparative example 1
Comparative example 1 with embodiment 1 the difference is that, in the culture medium do not contain icariin.
Experimental example
After stem cell by analyzing different embodiment and comparative example corresponding methods is divided into the differentiation rate and transplanting of cardiac muscle cell
Survival rate, preferred embodiment.
Differentiation rate refers to that cardiac muscle cell accounts for the ratio of the total number of cells after differentiation a period of time；
Survival rate after transplanting are as follows: using mouse as research object, cardiac muscle cell is made according to the method in embodiment 1 ~ 6 and comparative example
Afterwards, it is transplanted at the heart of mouse, after 3 weeks, dissects mouse, the transplanted cells in mouse heart are counted.
1 stem cell of table differentiation culture and transplantation effect
As it can be seen from table 1 the differentiation rate of stem cell is above the differentiation rate in comparative example in embodiment 1 ~ 3, show excessive sheep
The same ginsenoside of leaves of pulse plants glycosides, tanshin polyphenolic acid B compounding can effectively promote stem cell cells into cardiomyocytes as the constituent of culture medium
Differentiation.
A part in embodiment 4 in ginsenoside without microencapsulation processing, differentiation rate significantly lower than embodiment 1 ~
3, show that the ginsenoside of microencapsulation can persistently effectively promote stem cell myocardiac differentiation；People in embodiment 5
Ginseng saponin(e is commercially available ginsenoside, and the ginsenoside in embodiment 1 ~ 3 is the ginsenoside of enzymolysis and extraction, is shown using enzymatic hydrolysis
The mode of extraction obtains ginsenoside and has certain effect to the differentiation of stem cell Cardiomyocytes is improved；Four oxygen of nanometer in embodiment 6
Change three-iron is hydroxide-modified without nano lamellar, and differentiation rate shows the ginsenoside of microencapsulation also below embodiment 1 ~ 3
If if microcapsules wall material it is non-modified nano ferriferrous oxide processing, may will affect its promote stem cell differentiation energy
Power.
Ginsenoside, tanshin polyphenolic acid B and Herba Epimedii are used in embodiment 1 ~ 3 as induction stem cell myocardiac differentiation
Main inducible factor, and microencapsulation processing has been carried out to part ginsenoside, and further carry out to the wall material of microcapsules
Processing, improves differentiation rate.
The survival rate of cell is significantly higher than comparative example after transplanting in embodiment 1 ~ 3, and is higher than embodiment 4 ~ 6, shows
Using culture medium in embodiment 1 ~ 3, the survival rate of cell after transplanting can be effectively improved.
Above-listed detailed description is illustrating for possible embodiments of the present invention, and above embodiments are not to limit this
The scope of the patents of invention, all equivalence enforcements or change without departing from carried out by the present invention, is intended to be limited solely by the scope of the patents of this case.
Claims (10)
1. a kind of stem cell external evoked method for cultivating cardiac muscle cell, which is characterized in that S11. is derived from somatic stem cell and list is made
Cell suspension；It S12. is 10 by cell concentration is diluted to differential medium after single cell suspension cell count-5~10- 3The stem cell suspension of cells/mL, is inoculated on the cover inner surface of Micro-Organism Culture Dish, overturns culture dish lid, makes on lid
Stem cell form hanging drop, water or aqueous solution are added in culture dish to keep humidity of hanging drop during incubation, culture dish is set
In 5%CO2, cultivate 1 ~ 3 day in 37 DEG C of incubators；S13. the embryoid body formed in hanging drop on culture dish lid is collected, is shifted
Into the Micro-Organism Culture Dish for filling differential medium, it is put into and continues to suspend in incubator, embryoid body is promoted further to be proliferated；S14.
The embryoid body prepared is transferred to the coated orifice plate of gelatin, one, every hole embryoid body adds inductive differentiation medium, done
Pre- differentiation culture；In the inductive differentiation medium include 0.7 ~ 1.2mg/L of tanshin polyphenolic acid B, ginsengenin 20 .5 ~ 1.5mg/mL,
0.8 ~ 1.2mg/L of icariin.
2. the stem cell external evoked method for cultivating cardiac muscle cell of one kind according to claim 1, which is characterized in that described
It further include that GMEM culture medium, fetal calf serum, nonessential amino acid, the Sodium Pyruvate of 50mmol/L are water-soluble in inductive differentiation medium
The 3-mercaptoethanol aqueous solution of liquid and 0.1mol/L, the GMEM culture medium: fetal calf serum: nonessential amino acid: 50mmol/L
Sodium Pyruvate aqueous solution: 3-mercaptoethanol aqueous solution=73.5:24:1:1.3:0.2 of 0.1mol/L.
3. the stem cell external evoked method for cultivating cardiac muscle cell of one kind according to claim 1, which is characterized in that described
Microcapsules are made in the 50% ~ 60% of ginsenoside, and the microcapsules containing ginsenoside are added in culture medium, described to contain ginseng
The microcapsules of saponin(e the preparation method comprises the following steps: S31. by glutathione, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloric acid
Salt, deionized water press 1:1:100 ~ 120, and mixed solution is made, and the phosphate buffer solution of pH=6, nothing are added in mixed solution
It is stirred under the conditions of oxygen for 24 hours, obtains wall material, the mixed solution is 1:6 ~ 8 with the volume ratio of phosphate buffer solution；S32. by people
Ginseng saponin(e is scattered in hydroxy silicon oil, and the ginsenoside is 1:100 ~ 120 with the ratio of hydroxy silicon oil, obtains core material；S33.
The ratio of 1:5 mixes wall material and core material by volume, is surpassed using ultrasonic probe to oil-water two-phase interfaces under ice-water bath
5 ~ 10min of sound repeatedly adsorbs product, is washed, obtains microcapsules.
4. the stem cell external evoked method for cultivating cardiac muscle cell of one kind according to claim 3, which is characterized in that described
Ginsenoside the preparation method comprises the following steps: cellulase, pectase are add to deionized water by S41., it is mixed with the ginseng thinly sliced
It closes, reacts 1h at 45 DEG C, obtain enzyme-extracting solution；S42. 95% ethyl alcohol, 55 DEG C of reflux are added for the first time into enzyme-extracting solution
3h, filtering obtain first extract and residue, and the ethyl alcohol of second of addition 95% of residue extracts, 55 DEG C of reflux 2h, same to first extract
Merge, obtains alcohol extract, the first time amount of alcohol added: second of amount of alcohol added=1:4；S43. by alcohol extract at 50 DEG C
Medicinal extract shape is concentrated under reduced pressure into, ethyl alcohol is recycled in concentration process, obtained medicinal extract vacuum drying both obtains ginsenoside.
5. the stem cell external evoked method for cultivating cardiac muscle cell of one kind according to claim 4, which is characterized in that described
Cellulase, pectase, deionized water, ginseng mass ratio be 5 × 10-4:10-3:1:4。
6. the stem cell external evoked method for cultivating cardiac muscle cell of one kind according to claim 4, which is characterized in that described
Ethyl alcohol is 1:0.4 with the mass ratio of enzyme-extracting solution.
7. the stem cell external evoked method for cultivating cardiac muscle cell of one kind according to claim 4, which is characterized in that described
Further include modified Nano ferriferrous oxide particles in wall material, joined the preparation method of the wall material of modified Nano ferroso-ferric oxide
Are as follows: glutathione, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, deionized water are pressed into 1:1:100 ~ 120,
Mixed solution is made, the phosphate buffer solution of pH=6 is added in mixed solution；It is dispersed in the phosphate buffer solution
Modified Nano ferriferrous oxide particles, the modified Nano ferroso-ferric oxide are 1:600 with the mass ratio of phosphate buffer solution,
It is ultrasonic disperse that the modified Nano ferroso-ferric oxide, which is distributed to the mode in phosphate buffer solution,；It is stirred under oxygen free condition
For 24 hours, it adsorbs, wash repeatedly, obtain wall material, the mixed solution is 1:6 ~ 8 with the volume ratio of phosphate buffer solution.
8. the stem cell external evoked method for cultivating cardiac muscle cell of one kind according to claim 7, which is characterized in that described
The method of modifying of modified Nano ferroso-ferric oxide are as follows: in the deionized water for being dispersed in nano ferriferrous oxide, nanometer layer is added
Shape double-hydroxide, it is dry after ultrasonic disperse, obtain the hydroxide-modified nano ferriferrous oxide of nano lamellar, the nanometer
Four oxidation three-bodies: nano lamellar hydroxide: deionized water=1:0.2 ~ 0.5:20.
9. the stem cell external evoked method for cultivating cardiac muscle cell of one kind according to claim 8, which is characterized in that described
The manufacturing method of nano lamellar hydroxide are as follows: after aluminum nitrate and aluminum nitrate are dissolved in deionized water, are added to and are vigorously stirred
In 1.25% sodium hydroxide solution, hydro-thermal reaction is carried out, reaction product crushed after being dried obtains nano lamellar hydroxide；Institute
State aluminum nitrate: magnesium nitrate: deionized water=1:0.75:30.
10. the stem cell external evoked method for cultivating cardiac muscle cell of one kind according to claim 9, which is characterized in that institute
State the condition of hydro-thermal reaction are as follows: react 48h at 80 ~ 120 DEG C.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113662985A (en) * | 2021-09-07 | 2021-11-19 | 李红梅 | Traditional Chinese medicine compound, traditional Chinese medicine effective component composition, compound and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425763A (en) * | 2002-12-30 | 2003-06-25 | 浙江大学 | Use of icariin in inducing external oriented differentiation of embryo stem cells |
| CN102994446A (en) * | 2012-12-04 | 2013-03-27 | 罗国安 | Inductive agent for inducing embryonic stem cells to directionally differentiate into myocardial cells and culture medium |
| CN103142669A (en) * | 2013-03-07 | 2013-06-12 | 安徽丰润生物技术有限公司 | Enzyme method for extracting ginsenoside and obtained ginsenoside extractive |
| CN103735531A (en) * | 2013-12-23 | 2014-04-23 | 吉林大学 | Preparation method for microcapsules with magnetic nanoparticles-containing capsule walls |
| CN108855117A (en) * | 2018-07-13 | 2018-11-23 | 盐城工学院 | Core-shell photocatalyst and its preparation method and application |
-
2019
- 2019-04-18 CN CN201910314980.5A patent/CN110157663B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425763A (en) * | 2002-12-30 | 2003-06-25 | 浙江大学 | Use of icariin in inducing external oriented differentiation of embryo stem cells |
| CN102994446A (en) * | 2012-12-04 | 2013-03-27 | 罗国安 | Inductive agent for inducing embryonic stem cells to directionally differentiate into myocardial cells and culture medium |
| CN103142669A (en) * | 2013-03-07 | 2013-06-12 | 安徽丰润生物技术有限公司 | Enzyme method for extracting ginsenoside and obtained ginsenoside extractive |
| CN103735531A (en) * | 2013-12-23 | 2014-04-23 | 吉林大学 | Preparation method for microcapsules with magnetic nanoparticles-containing capsule walls |
| CN108855117A (en) * | 2018-07-13 | 2018-11-23 | 盐城工学院 | Core-shell photocatalyst and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| 孙双双: "四氧化三铁基复合材料的制备及其在水处理中的应用研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
| 孙灵娜等: "层状双氢氧化物纳米材料的研究进展", 《中国西部科技》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113662985A (en) * | 2021-09-07 | 2021-11-19 | 李红梅 | Traditional Chinese medicine compound, traditional Chinese medicine effective component composition, compound and application thereof |
| CN113662985B (en) * | 2021-09-07 | 2022-06-24 | 北京中医药大学 | Traditional Chinese medicine composition with effect of promoting directional differentiation of stem cells into myocardial cells, traditional Chinese medicine effective component composition and application thereof |
| WO2023035479A1 (en) * | 2021-09-07 | 2023-03-16 | 北京中医药大学 | Traditional chinese medicine composite, traditional chinese medicine effective component composition, compound and applications thereof |
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Denomination of invention: A Method of Stem Cells Inducing and Culturing Cardiomyocytes in Vitro Effective date of registration: 20230629 Granted publication date: 20210727 Pledgee: Hunan Pingjiang Rural Commercial Bank Co.,Ltd. Pledgor: Chen Zhongping|Lancy Purcell Biotechnology (Guangzhou) Co.,Ltd. Registration number: Y2023980046730 |