CN110368401A - The activation method and stem cell medicine of mescenchymal stem cell and its application - Google Patents
The activation method and stem cell medicine of mescenchymal stem cell and its application Download PDFInfo
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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- C12N2501/999—Small molecules not provided for elsewhere
Abstract
The invention belongs to biomedicine technical fields, and in particular to the activation method and stem cell medicine of mescenchymal stem cell and its application.The activation method of mescenchymal stem cell provided by the present invention, comprising: mescenchymal stem cell is provided, the mescenchymal stem cell is subjected to the first culture using the first culture medium, the second culture is carried out using the second culture medium later；Wherein, first culture medium includes Radix Astragali water extract, and second culture medium includes cucoline.It is detected by experiment; relative to the mescenchymal stem cell before activation; osteoprotegerin (OPG) expression of mescenchymal stem cell after carrying out activation culture by the activation method increased significantly, and the expression of RANKL and prostaglandin E2 (PGE2) is substantially reduced.Thus, thus it is speculated that the mescenchymal stem cell that activation method obtains through the invention or the stem cell medicine comprising the mescenchymal stem cell can be applied in preparation prevention or treatment bone disease drug.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to the activation method and stem cell medicine of mescenchymal stem cell
And its application.
Background technique
Currently, the significant increase of disease incidence of the bone diseases such as Osteoarthritis, rheumatoid arthritis and osteoporosis,
The means for clinically being used to treat this kind of disease have to be controlled using progress drugs such as anodyne, chondroitin sulfate or combination Chinese materia medica preparations
It treats, or using joint replacement etc..In recent years, stem-cell therapy is as a kind for the treatment of means also gradually applied to bone disease
In clinical research, however, its effect is not very satisfactory.
Summary of the invention
In view of this, the main purpose of the present invention is to provide a kind of activation method of mescenchymal stem cell, it is of the invention
Another object is to provide a kind of mescenchymal stem cell preparation, it is intended to provide new way for preparation treatment bone disease drug.
In order to achieve the above-mentioned object of the invention, an aspect of of the present present invention provides a kind of activation method of mescenchymal stem cell,
Include: offer mescenchymal stem cell, the mescenchymal stem cell is subjected to the first culture using the first culture medium, later using the
Two culture mediums carry out the second culture；
Wherein, first culture medium includes Radix Astragali water extract, and second culture medium includes cucoline.
Another aspect of the present invention provides a kind of mescenchymal stem cell preparation, comprising: between above-mentioned activation method obtains
At least one of mesenchymal stem cells and pharmaceutically acceptable auxiliary material, carrier and additive.
Another aspect of the present invention, provides mescenchymal stem cell that aforementioned activation method obtains or above-mentioned mesenchyma is dry thin
Application of born of the same parents' preparation in preparation prevention or treatment bone disease drug.
In conclusion the present invention is successively using the first culture medium comprising Radix Astragali water extract and the comprising cucoline second training
It supports base and activation culture is carried out to mescenchymal stem cell.It is detected by experiment, relative to the mescenchymal stem cell before activation, by the work
Change method, which carries out osteoprotegerin (OPG) expression of mescenchymal stem cell after activation culture, increased significantly, RANKL (core because
Sub- κ B receptor activation factor ligand) and the expression of prostaglandin E2 (PGE2) be substantially reduced.The expression of OPG increases,
The expression of RANKL and PGE2 reduces, and can inhibit the formation and activation of osteoclast, promotes mescenchymal stem cell to bone injury
The repairing effect at position.Thus, the mescenchymal stem cell or include the mescenchymal stem cell that activation method obtains through the invention
Stem cell medicine can be applied to preparation prevention or treatment bone disease drug in, be particularly suitable for preparation inhibit bone resorption medicine
Object.
Detailed description of the invention
Fig. 1 is in test case 1 of the present invention using RANKL expression in each group mescenchymal stem cell of ELISA method detection
Histogram；
Fig. 2 is in test case 1 of the present invention using PGE2 expression in each group mescenchymal stem cell of ELISA method detection
Histogram；
Fig. 3 be test case 1 of the present invention in using RT-PCR method detect each group mescenchymal stem cell in RANKL, PGEES3,
The histogram of OPG expression.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
In order to provide new way to preparation treatment bone disease drug, on the one hand, the embodiment of the present invention provides an inter-species and fills
The activation method of matter stem cell, comprising: mescenchymal stem cell is provided, the mescenchymal stem cell is carried out using the first culture medium
First culture carries out the second culture using the second culture medium later；
Wherein, first culture medium includes Radix Astragali water extract, and second culture medium includes cucoline.
Specifically, mescenchymal stem cell refers under the growth conditions, non-freezing state or in rigid anabiotic state
Under mescenchymal stem cell.
Preferably, the mescenchymal stem cell is the third generation to the 5th generation mescenchymal stem cell.
Preferably, the mescenchymal stem cell is between mesenchymal stem cell, umbilical cord mesenchymal stem cells and fat
At least one of mesenchymal stem cells.This kind of mescenchymal stem cell materials are convenient, abundance, in vitro energy massive amplification, and exempt from
Epidemic focus is low, and distribution type is not had to when use, does not involve ethics and the question of morality, devoid of risk and side effect.
The embodiment of the present invention is not specifically limited the source of the mescenchymal stem cell, can be commercial goods, can also
The product obtained using conventional technical means in the art.
Specifically, the mescenchymal stem cell is carried out the first culture, the tool of first culture using the first culture medium
Body process refers to this field routine operation.In an inventive embodiments, third generation mescenchymal stem cell is taken, with certain density (example
Such as 4 × 105/ mL) it is inoculated in 24 orifice plates for being placed with coverslip, the first culture medium is added, is placed in cell incubator and carries out
Culture.
Preferably, the incubation time of the first culture is 22-26 hours, condition of culture is 37 DEG C, 5%CO2。
In embodiments of the present invention, the first culture medium includes Radix Astragali water extract.The Radix Astragali water extract can be commercial goods,
Conventional technical means in the art can also be used to be prepared.In one embodiment, Radix Astragali water extract is purchase in Shan Xijia
The Radix Astragali water body object of standing grain biotechnology company.
Preferably, the working concentration of the Radix Astragali water extract is more than 20 μ L/mL in first culture medium.When
When the working concentration of Radix Astragali water extract is more than 20 μ L/mL, the expression of RANKL has decreasing trend.
Further, in first culture medium, the working concentration of the Radix Astragali water extract is 20-200 μ L/mL.?
In this concentration range of 20-200 μ L/mL, the activation effect of mescenchymal stem cell increases with concentration and is enhanced, still, when its is dense
When degree is greater than 200 μ L/mL, activation effect increases unobvious.
In a preferred embodiment, the first culture medium includes: Radix Astragali water extract, fetal calf serum and basal medium；It is described
The working concentration of Radix Astragali water extract is 20-200 μ L/mL, and the mass volume ratio concentration of the fetal calf serum is 10%-20%.Its
In, the basal medium is DMEM culture medium and/or F12 culture medium.
Specifically, carrying out the second culture using the second culture medium later, the detailed process of second culture refers to ability
Domain routine operation.In an inventive embodiments, the mescenchymal stem cell culture after carrying out the first culture is taken, the second culture is added
Base is placed in and continues to cultivate in cell incubator.
Preferably, the incubation time of the second culture is 22-26 hours, condition of culture is 37 DEG C, 5%CO2。
In embodiments of the present invention, the second culture medium includes cucoline.The cucoline can be commercial goods, can also be used
Conventional technical means in the art is prepared.
Preferably, the working concentration of the cucoline is at 100 μM or more in second culture medium.In above-mentioned Huang
Under the working concentration range of stilbene water extract, at 100 μM or more, the expression of RANKL significantly drops the working concentration of cucoline
Low, the expression of OPG significantly improves.
Further, in second culture medium, the working concentration of the cucoline is 100-500 μM.In 100-500
In μM this concentration range, the activation effect of mescenchymal stem cell increases with concentration and is enhanced, still, when its concentration is greater than 500 μ
When M, activation effect increase degree is unobvious.
In a preferred embodiment, the second culture medium includes: cucoline, fetal calf serum and basal medium；The sinomenium acutum
100-500 μM of the working concentration of alkali, the mass volume ratio concentration of the fetal calf serum are 10%-20%.Wherein, the basis training
Supporting base is DMEM culture medium and/or F12 culture medium.
To sum up, in the activation method of the mescenchymal stem cell provided by the embodiment of the present invention, by successively using comprising
First culture medium of Radix Astragali water extract and the second culture medium comprising cucoline carry out activation culture to mescenchymal stem cell, are having
Have under the Radix Astragali water extract of above-mentioned specific working concentration range and the comprehensive function of cucoline, between being obtained by the activation method
The OPG expression of mesenchymal stem cells increased significantly, and the expression of RANKL and PGE2 are substantially reduced.On the one hand, RANKL/
The expression of OPG can be used as the key index for determining the bone resorption degree of Osteoclasts mediate, and RANKL is raw as osteoclast
At initial signal, mainly promote osteoclast to generate by interaction with RANK.As the soluble recepter of RANKL,
Osteoprotegerin with RANL competitive binding RANKL and can hinder the differentiation and activation of osteoclast, to inhibit bone resorption.It is another
The expression of aspect, PGE2 reduces, and inducibility of the mescenchymal stem cell in T cell proliferation can be reduced, to inhibit T thin
The proliferation of born of the same parents, enhances the function of Treg cell, and hinders the secretion of a variety of inflammatory factors such as IL-1 and IL-7, further blocks
The activation of RANKL and the differentiation for inhibiting precursor osteoclast, to reduce the formation of mature osteoclast and mitigate bone injury.This
The expression of OPG, RANKL and PGE2 of the activation method adjustable berth mesenchymal stem cells that inventive embodiments provide, pass through suppression
The formation and activation of osteoclast processed promote mescenchymal stem cell to the repairing effect of bone injury site.Thus, it is living by the present invention
The mescenchymal stem cell that change method obtains or the stem cell medicine comprising the mescenchymal stem cell can be applied to preparation prevention or control
It treats in bone disease drug, is particularly suitable for preparation and inhibits bone resorption drug.
On the other hand, the embodiment of the invention also provides a kind of stem cell medicines, comprising: between above-mentioned activation method obtains
At least one of mesenchymal stem cells and pharmaceutically acceptable auxiliary material, carrier and additive.
Preferably, the cell concentration of the mescenchymal stem cell is 5 × 105-2×106A/mL.
Preferably, the stem cell medicine is injection.
In a preferred embodiment, the stem cell medicine includes: mescenchymal stem cell 5 × 105-2×106A/mL, matter
Measure the human serum albumin stoste that volume by volume concentration is 1%, formula mannitol injection liquid 2mL, Compound vitamine injection 3mL, 50% Portugal
Grape sugar injection 5mL, physiological saline 85mL.Human serum albumin, composite vitamin, mannitol, glucose are clinical injection liquid
Ingredient can be cells with nutrient, the metabolism conducive to cell, the good stem cell in vitro microenvironment of maintenance.
Another aspect, the mescenchymal stem cell obtained the embodiment of the invention also provides aforementioned activation method or above-mentioned dry thin
Application of born of the same parents' preparation in preparation prevention or treatment bone disease drug.Preferably, the prevention or treatment bone disease drug
Preferably inhibit bone resorption drug.
To make, the above-mentioned implementation detail of the present invention and operation can be clearly readily appreciated by one skilled in the art and the present invention is real
Apply a mescenchymal stem cell activation method and stem cell medicine and its progress performance of application it is significant embody, below by way of reality
Example is applied implementation of the invention is illustrated.
Embodiment 1
The activation method for present embodiments providing a kind of mescenchymal stem cell, specifically comprises the following steps:
S11, mesenchymal stem cell is prepared
Using conventional method in anterior superior spine point of puncture, extract 50mL bone marrow fluid with medullo-puncture needle, inject 50mL it is sterile from
In heart pipe；It is diluted with the PBS liquid of equivalent heparin containing 20u/mL, and the leaching that isometric specific gravity is 1.073g/ml is added along tube wall
Bar cell separating liquid；2000rmp is centrifuged 30min, draws mononuclearcell layer；Sterile saline centrifuge washing 2 times, every time
1000rpm is centrifuged 5min, discards supernatant liquid, takes precipitating, obtains marrow MSC；
It by marrow MSC obtained above, is suspended with serum free medium, cell is counted, by 5 × 106A/mL is inoculated in
In culture bottle, 37 DEG C, 5%CO are set2, saturated humidity incubator in cultivate.After culture 3 days, serum-free medium is replaced, is discarded
Non- attached cell, later every 3 days 1 time replacement culture solution；After 7~9 days, when marrow MSC fusion is up to 50%~60%, first is carried out
Secondary passage.Serum-free medium is removed, is respectively 0.25% and 0.1% 37 DEG C of trypsase-EDTA digestion using concentration
1min, marrow MSC, which are shunk, is detached from bottle wall, and the culture supernatant previously removed is added, and neutralizes trypsase-EDTA, and use and move
Liquid pipe is gently blown and beaten, and medulla mesenchyma cell suspension is moved into 50mL sterile centrifugation tube.1000rpm is centrifuged 8min, discards
Clear liquid；Serum-free complete medium resuspension is rejoined, is counted, the culture dish for passing 1-1.5 inoculation 10cm specification according to 1；To bone
Marrow MSC cultivate to cell fusion degree be 70% when continue using the above method carry out secondary culture, collect third generation marrow between fill
Matter stem cell, for use.
S12, the first culture is obtained
Third generation mesenchymal stem cell is taken, with density 4 × 105/ mL is inoculated in 24 orifice plates for being placed with coverslip,
The first culture medium is added, is placed in 37 DEG C, 5%CO2It is cultivated in cell incubator for 24 hours, obtains the first culture.
Wherein, the first culture medium includes: Radix Astragali water extract, fetal calf serum and basal medium；The Radix Astragali water extract
Working concentration is 100 μ L/mL, and the mass volume ratio concentration of the fetal calf serum is 15%.Wherein, the basal medium is L-
DMEM culture medium.
S13, the second culture is obtained
The first culture is taken, the second culture medium is added, is placed in 37 DEG C, 5%CO2It is cultivated for 24 hours, is obtained in cell incubator
To the second culture.Through detecting, the mescenchymal stem cell concentration in the second culture reaches 1 × 106-1×107A/mL.
Wherein, the second culture medium includes: cucoline, fetal calf serum and basal medium；The working concentration of the cucoline
250 μM, the mass volume ratio concentration of the fetal calf serum is 15%.Wherein, the basal medium is L-DMEM culture medium.
Comparative example 1
This comparative example the difference from embodiment 1 is that: in first culture medium, the work of the Radix Astragali water extract is dense
Degree is 0 μ L/mL；Remaining place is substantially the same manner as Example 1, no longer repeats one by one herein.
Comparative example 2
This comparative example the difference from embodiment 1 is that: in second culture medium, the working concentration of the cucoline is
0μM；Remaining place is substantially the same manner as Example 1, no longer repeats one by one herein.
Embodiment 2
The present embodiment the difference from embodiment 1 is that: in first culture medium, the work of the Radix Astragali water extract is dense
Degree is 200 μ L/mL；In second culture medium, the working concentration of the cucoline is 100 μM；Remaining place and embodiment 1
It is essentially identical, it no longer repeats one by one herein.
Embodiment 3
The present embodiment the difference from embodiment 1 is that: in first culture medium, the work of the Radix Astragali water extract is dense
Degree is 50 μ L/mL；In second culture medium, the working concentration of the cucoline is 500 μM；Remaining place and embodiment 1
It is essentially identical, it no longer repeats one by one herein.
Embodiment 4
The present embodiment the difference from embodiment 1 is that: activation object be umbilical cord mesenchymal stem cells, remaining place with implement
Example 1 is essentially identical, no longer repeats one by one herein.
Wherein, the step of preparing umbilical cord mesenchymal stem cells is as follows:
Operated in superclean bench, take fresh mature healthy fetus umbilical cord, with containing 2 times of 100U/mL penicillin and
The normal saline flushing of streptomysin removes bloodstain, and Wal Tong Shi glue is removed, and is cut into 1 cubic millimeter or less blocky, even spread,
It is slowly added to 10mL serum free medium, 37 DEG C is set, is cultivated in the carbon dioxide that volume fraction is 5%, saturated humidity incubator.
After primitive cell culture 7 days, serum-free medium is replaced, culture reaches 70% fusion to cell, removes free serum culture
Liquid, addition concentration are respectively 0.25% and 0.1% trypsin-EDTA sodium (EDTA) digestion 1min, umbilical cord MSC
It shrinks and is detached from bottle wall, the culture supernatant previously removed is added at this time, neutralize trypsin-EDTA solutions, and use pipette
It gently blows and beats, umbilical cord MSC suspension is moved into 50mL centrifuge tube.1000rpm is centrifuged 8min, removes supernatant；Rejoin nothing
MSC is resuspended serum complete medium, and counts.1.5 inoculation 10cm culture dishes are passed according to 1.When MSC culture fusion 70% or so
Referring to above method secondary culture.
The MSC for selecting third generation culture, in logarithmic growth phase, and cell fusion degree when being no more than 80% according to passage
It is required that digestion, neutralization, washing, collection cell, using cells frozen storing liquid (90% fetal calf serum and 10% dimethyl sulfoxide (DMSO) weight
Outstanding MSC, mixes well, every pipe 1mL, which is sub-packed in, freezes the seal of tube.Cryopreservation tube is placed in the freezing storing box equipped with isopropanol, in -80
DEG C refrigerator overnight, next day move into liquid nitrogen container and save.
The step of MSC recovery are as follows: take out MSC cryopreservation tube, immerse in 37 DEG C of warm water dissolve immediately；Take appropriate free serum culture
Base suspension cell is simultaneously centrifuged, and discards supernatant liquid；Cell is resuspended in serum free medium, and is inoculated in culture dish, in 37 DEG C,
Carbon dioxide that volume fraction is 5% is cultivated in saturated humidity incubator.
Embodiment 5
The present embodiment the difference from embodiment 1 is that: activation object be fat mesenchymal stem cell, remaining place with implement
Example 1 is essentially identical, no longer repeats one by one herein.
Wherein, the step of preparing fat mesenchymal stem cell is as follows:
Abdomen white adipose tissue is conventionally separated, visible capilary and musculature, sterile PBS washing are rejected
Three times, then adipose tissue is shredded to 1 cubic millimeter of paste below with sterile scissors, sterile digestive juice is added and (contains
The serum-free DME culture medium of 0.1% Collagenase I type and 0.05% trypsase), 45-50min is at the uniform velocity stirred in 37 DEG C of water-baths,
It is clean to tissue block digestion；Filtrate is collected by filtration in 200 mesh screens, then using the low sugar for containing 10% fetal calf serum (FBS)
DMEM culture medium equivalent neutralizes, and 1500rpm is centrifuged 10min, discards supernatant liquid；With 1 × 106The cell density of a/mL, with no blood
Fat MSC is resuspended in clear culture medium, and is inoculated in culture bottle, 37 DEG C, volume fraction be 5% carbon dioxide, saturated humidity culture
It is cultivated in case, when fatty MSC, which is grown to, to be merged close to 80%, digests MSC cell using 0.25% trypsin solution, with 1:
3 inoculative proportion, which is inoculated into new culture bottle, carries out secondary culture.Fatty MSC is fused to be centrifuged no more than 80%, 1000rpm
8min discards supernatant liquid, rejoins Serum-free complete medium and MSC is resuspended, count, and passes 1-1.5 inoculation 10cm culture according to 1
Ware.Fatty MSC is cultivated when merging 70% or so such as above method secondary culture.
Embodiment 6
The mesenchymal stem cell that Example 1 activates prepares stem cell medicine.The stem cell medicine fills between including:
Matter stem cell 5 × 105-2×106A/mL, the human serum albumin stoste that mass volume ratio concentration is 1%, formula mannitol injection liquid
2mL, Compound vitamine injection 3mL, 50% glucose injection 5mL, physiological saline 85mL.
When preparation, in addition to mescenchymal stem cell, remaining ingredient need to be prepared in advance, and 4 DEG C of pre- cold standbies.
The stem cell medicine prepared is injection, which is stored in 2-10 DEG C of condition, in 2 hours
Cell remains single cell suspension state, and cell viability (Trypan Blue meter vigor) is maintained at 95% or more.
Test case 1
As shown in table 1, the third generation mesenchymal stem cell prepared using the step S11 of embodiment 1 is as negative right
According to group, the mescenchymal stem cell handled using the activation method of embodiment 1 is as experimental group, using the method for comparative example 1
The mescenchymal stem cell that processing obtains is as positive controls 1, the mescenchymal stem cell handled using the method for comparative example 2
As positive controls 2.
Then, Radix Astragali water extract, cucoline and combinations thereof are studied respectively using ELISA method in mescenchymal stem cell
The influence of RANKL and inflammatory factor PGE2 expression.And it is respectively used to using RT-PCR method detection each group mescenchymal stem cell
Express the mRNA level in-site of RANKL, OPG, PGEES3 (pGE2 synthase).
Table 1
| Investigation factor | Negative control group | Experimental group | Positive controls 1 | Positive controls 2 |
| Radix Astragali water extract | - | + | - | + |
| Cucoline | - | + | + | - |
Note: "-" expression is not investigated, and "+" indicates to investigate.
As depicted in figs. 1 and 2, compared with negative control group, experimental group (combination of Radix Astragali water extract and cucoline) can be shown
Write the expression for reducing inflammatory factor PGE2 and RANKL.
As shown in figure 3, compared with negative control group, experimental group is expressed the horizontal of RANKL and PGEES3 and is significantly reduced, expression
The mRNA level in-site of OPG significantly increases, consistent with the testing result of ELISA.
Test case 2
Patients with Knee Osteoarthritis 20 are chosen, wherein male 7, female 13；Age 41-78 years old, average (51.3 ± 4.2)；
The course of disease is -18 years Mays, and average (2.9 ± 0.7 years) have different degrees of gonalgia, limitation of activity medical history.By all trouble
Person is randomly divided into 2 groups, including treatment group and control group.
Treatment group's setting 10, average age (49.6 ± 5 years old), average course of disease (3.1 ± 0.8 years).Control group setting 10
Example, average age (52 ± 5.5 years old), average course of disease (2.5 ± 0.6 years).No statistical difference between treatment group and control group
Meaning (P > 0.05).
Cell infusion is carried out by side kneecap approach, is guided without radiology.It is injected using 19G needle, every side leg note
Penetrate 1.5mL.Injection in 14 days is primary, continuous injection 3 times.Treatment group's injection uses 6 stem cell medicine of embodiment, control group injection etc.
Measure the mescenchymal stem cell of 1 step S11 of embodiment.Number is specified to be commented using the standards of grading lequesne of international osteoarthritis
Point, including knee joint rest pain, motion pain, tenderness, swelling, morning stiffness, walking ability.
As shown in table 2, compared with the control group, the lequesne score for the treatment of group significantly reduces, and illustrates real comprising the present invention
The stem cell medicine for applying the mescenchymal stem cell of the activation method processing of example offer can be obviously improved Human Osteoarthritis
Lequesne score.
Table 2
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of activation method of mescenchymal stem cell characterized by comprising provide mescenchymal stem cell, will be described between fill
Matter stem cell carries out the first culture using the first culture medium, carries out the second culture using the second culture medium later；
Wherein, first culture medium includes Radix Astragali water extract, and second culture medium includes cucoline.
2. activation method according to claim 1, which is characterized in that in first culture medium, the Radix Astragali water is mentioned
The working concentration of object is more than 20 μ L/mL；And/or
In second culture medium, the working concentration of the cucoline is at 100 μM or more.
3. activation method according to claim 1, which is characterized in that the time of first culture is 22-26 hours；
And/or
The time of second culture is 22-26 hours.
4. activation method according to any one of claims 1 to 3, which is characterized in that first culture medium includes: Radix Astragali
Water extract, fetal calf serum and basal medium；
The working concentration of the Radix Astragali water extract is 20-200 μ L/mL, and the mass volume ratio concentration of the fetal calf serum is 10%-
20%.
5. activation method according to any one of claims 1 to 3, which is characterized in that second culture medium includes: sinomenium acutum
Alkali, fetal calf serum and basal medium；
The working concentration of the cucoline is 100-500 μM, and the mass volume ratio concentration of the fetal calf serum is 10%-20%.
6. activation method according to any one of claims 1 to 3, which is characterized in that the mescenchymal stem cell is marrow
At least one of mescenchymal stem cell, umbilical cord mesenchymal stem cells and fat mesenchymal stem cell；And/or
The mescenchymal stem cell is the third generation to the 5th generation mescenchymal stem cell.
7. a kind of stem cell medicine characterized by comprising between activation method as claimed in any one of claims 1 to 6 obtains
At least one of mesenchymal stem cells and pharmaceutically acceptable auxiliary material, carrier and additive.
8. stem cell medicine according to claim 7, which is characterized in that the cell concentration of the mescenchymal stem cell is 5
×105-2×106A/mL.
9. mescenchymal stem cell preparation according to claim 7, which is characterized in that the stem cell medicine is injection.
10. any one of mescenchymal stem cell or claim 7 to 9 that activation method as claimed in any one of claims 1 to 6 obtains
Application of the stem cell medicine in preparation prevention or treatment bone disease drug.
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| KR20190084710A (en) * | 2018-01-09 | 2019-07-17 | (주)오스힐 | Three-dimensional cell aggregates of stem cell comprising nano particle encapsulating drugs for treatment of soft tissue diseases and preparation method of the same |
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