CN110257324A - A kind of culture medium and abductive approach for fat mesenchymal stem cell adipogenic induction - Google Patents
A kind of culture medium and abductive approach for fat mesenchymal stem cell adipogenic induction Download PDFInfo
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- 230000006698 induction Effects 0.000 title claims abstract description 27
- 230000002293 adipogenic effect Effects 0.000 title claims abstract description 25
- 238000013459 approach Methods 0.000 title abstract description 7
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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
- C12N5/0653—Adipocytes; Adipose tissue
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- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1384—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from adipose-derived stem cells [ADSC], from adipose stromal stem cells
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Abstract
The invention discloses a kind of culture mediums and abductive approach for fat mesenchymal stem cell adipogenic induction, the culture medium includes the first culture medium and the second culture medium, abductive approach includes first cultivating fat mesenchymal stem cell in the first culture medium, then cultivated in the second culture medium.The present invention can effectively improve the efficiency that fat mesenchymal stem cell breaks up at rouge, shorten the adipogenic induction time, improve it in the feasibility of clinical application.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of cultures for fat mesenchymal stem cell adipogenic induction
Base and abductive approach.
Background technique
It for a long time, is all that the common of department of plastic surgery is asked to the treatment of soft tissue defects caused by the reasons such as wound and tumour
Topic.Currently, the measure mainly taken mainly includes artificial material filling, autologous fat transplantation etc., still, artificial material filling is deposited
In certain foreign body reaction risk, and the tissue survival rate of autologous fat transplantation is low, and fat absorption necrosis rate is high, is also easy to cause
Complication limits its use clinically.
Fat mesenchymal stem cell is present in adipose tissue, and the stem cell with multinomial differentiation potential has and
Source enriches, convenient advantage of drawing materials, therefore is considered as the ideal seed cell of regeneration and reparation.Based on fat mesenchymal
Stem cell constructs tissue engineering fat at rouge differentiation capability, can effectively improve the effect of fat transfer, is the defect of soft tissue
Reconstruction provides new approach, has a good application prospect in organizational project and regenerative medicine field.But it is fatty at present
Mescenchymal stem cell is lower at the efficiency that rouge breaks up, and the adipogenic induction time is long, it is difficult to meet clinically to adipose tissue
Wilderness demand limits its application clinically.
Summary of the invention
Technical problems based on background technology, the invention proposes one kind to lure for fat mesenchymal stem cell at rouge
The culture medium and abductive approach led improve the efficiency that fat mesenchymal stem cell breaks up at rouge, shorten the adipogenic induction time, mention
It is high its clinical application feasibility.
A kind of culture medium for fat mesenchymal stem cell adipogenic induction, including the first culture medium and the second culture medium；
First culture medium includes the component of the DMEM/F12 culture medium containing 10% fetal calf serum and following final concentration:
1-1.2 μm of ol/L of dexamethasone, insulin 5-8 μm of ol/L, Indomethacin 5-6nmol/L, 4-6 μm of ol/L of astaxanthin, hexichol second
1-2 μm of ol/L of alkene glycosides；
Second culture medium includes the component of the DMEM/F12 culture medium containing 10% fetal calf serum and following final concentration:
1-1.2 μm of ol/L of dexamethasone, insulin 4-6 μm of ol/L, Indomethacin 5-6nmol/L, 2-2.5 μm of ol/L of astaxanthin, hexichol
0.6-1.2 μm of ol/L of ethylene glycosides, 4-8 μm of ol/L of Astragaloside IV, 2-3 μm of ol/L of Isorhamnetin.
Preferably, first culture medium includes DMEM/F12 culture medium and following final concentration containing 10% fetal calf serum
Component: 1.1 μm of ol/L of dexamethasone, insulin 6 μm of ol/L, Indomethacin 5nmol/L, 4.5 μm of ol/L of astaxanthin, hexichol second
1.5 μm of ol/L of alkene glycosides.
Preferably, second culture medium includes DMEM/F12 culture medium and following final concentration containing 10% fetal calf serum
Component: 1.1 μm of ol/L of dexamethasone, insulin 5 μm of ol/L, Indomethacin 5nmol/L, 2.4 μm of ol/L of astaxanthin, hexichol second
0.8 μm of ol/L of alkene glycosides, 5 μm of ol/L of Astragaloside IV, 2.5 μm of ol/L of Isorhamnetin.
A kind of adipogenic induction method of fat mesenchymal stem cell, by fat mesenchymal stem cell first described in the claim
The first culture medium in cultivated, then cultivated in second culture medium.
Preferably, fat mesenchymal stem cell is first cultivated 5-7 days in first culture medium, then described the
Continue to cultivate in two culture mediums.It is highly preferred that fat mesenchymal stem cell is first cultivated 6 in first culture medium
It, then continue culture 4 days in second culture medium.
Preferably, the fat mesenchymal stem cell is the fat mesenchymal stem cell in 3-6 generation.
Preferably, it is (1-2) × 10 that the fat mesenchymal stem cell, which is seeded to the inoculum density in the first culture medium,4
A/cm2。
Preferably, the culture is at 37 DEG C, the CO of 5% volume2Under the conditions of carry out.
Preferably, it when fat mesenchymal stem cell is cultivated in first culture medium, is replaced every 36-48h
Fresh culture when being cultivated in second culture medium, replaces fresh culture every 18-24h.
Beneficial effects of the present invention are as follows:
In culture medium raw material of the invention, dexamethasone can reduce the table of Adipocyte Differentiation inhibiting factor (pref21)
It reaches, promotes Adipocyte Differentiation；Insulin can be by adjusting the phosphorylation of cAMP response element binding protein (CREB) and turning
Record promotes transcription factor C/EBP expression, stem cell is promoted to be divided into fat cell；Indomethacin is the inhibitor of Cycloxygenase,
Osteoclast and osteoblast differentiation can be prevented, while promoting the differentiation to fat cell.On this basis, by being trained to first
It supports and adds suitable concentration astaxanthin in base, Stibene-glucoside is cooperated, and peroxide activator enzyme body vegetation is better played
The effect of activated receptor γ (PPAR- γ), further promotes the differentiation to fat cell；It is added into the second culture medium suitable dense
The Astragaloside IV of degree, Isorhamnetin are cooperated, and can more effectively be raised PPAR gamma collaboration and be swashed
The expression of the factor -1 (PGC-1 α) living further promotes cell death to activate egg by the synergistic effect of PGC-1 α and PPAR- γ
The transcription of Bai Kangti (CIDEC), to preferably inhibit fat point under the premise of partial fat Derived from Mesenchymal Stem Cells
Solution increases Fat Accumulation, promotes the further differentiation and the development of fat drips of fat mesenchymal stem cell.
The present invention breaks up fat mesenchymal stem cell using the first culture medium and the second culture medium combined induction, passes through culture
The adjustment of the ingredient of base and each constituent concentration, more effectively promote differentiation from fat mesenchymal stem cell to fat cell and
The development of fat drips, reach not only improve fat mesenchymal stem cell at rouge differentiation rate, but also shorten the effect of adipogenic induction time.
Specific embodiment
In the following, technical solution of the present invention is described in detail by specific embodiment.
Embodiment 1
Prepare the first culture medium and the second culture medium, in which:
First culture medium includes the component of the DMEM/F12 culture medium containing 10% fetal calf serum and following final concentration: ground plug
Rice 1 μm of ol/L of pine, insulin 5 μm of ol/L, Indomethacin 5nmol/L, 4 μm of ol/L of astaxanthin, 1 μm of ol/L of Stibene-glucoside；
Second culture medium includes the component of the DMEM/F12 culture medium containing 10% fetal calf serum and following final concentration: ground plug
Rice 1 μm of ol/L of pine, insulin 4 μm of ol/L, Indomethacin 5nmol/L, 2 μm of ol/L of astaxanthin, 0.6 μm of ol/L of Stibene-glucoside,
4 μm of ol/L of Astragaloside IV, 2 μm of ol/L of Isorhamnetin.
It is 1 × 10 that 3rd fat subsitutes mescenchymal stem cell, which is pressed inoculum density,4A/cm2It is seeded in the first culture medium,
37 DEG C, the CO of 5% volume2Under the conditions of carry out culture 6 days, then in the second culture medium, at 37 DEG C, the CO of 5% volume2Under the conditions of
Continue to cultivate.
Wherein, when being cultivated in the first culture medium, every 48h replace fresh culture, in the second culture medium into
When row culture, every replacing fresh culture for 24 hours.
Embodiment 2
Prepare the first culture medium and the second culture medium, in which:
First culture medium includes the component of the DMEM/F12 culture medium containing 10% fetal calf serum and following final concentration: ground plug
Rice 1.1 μm of ol/L of pine, insulin 6 μm of ol/L, Indomethacin 5nmol/L, astaxanthin 4.5 μm of ol/L, 1.5 μ of Stibene-glucoside
mol/L；
Second culture medium includes the component of the DMEM/F12 culture medium containing 10% fetal calf serum and following final concentration: ground plug
Rice 1.1 μm of ol/L of pine, insulin 5 μm of ol/L, Indomethacin 5nmol/L, astaxanthin 2.4 μm of ol/L, 0.8 μ of Stibene-glucoside
Mol/L, 5 μm of ol/L of Astragaloside IV, 2.5 μm of ol/L of Isorhamnetin.
It is 1 × 10 that 3rd fat subsitutes mescenchymal stem cell, which is pressed inoculum density,4A/cm2It is seeded in the first culture medium,
37 DEG C, the CO of 5% volume2Under the conditions of carry out culture 6 days, then in the second culture medium, at 37 DEG C, the CO of 5% volume2Under the conditions of
Continue to cultivate.
Wherein, when being cultivated in the first culture medium, every 48h replace fresh culture, in the second culture medium into
When row culture, every replacing fresh culture for 24 hours.
Embodiment 3
Prepare the first culture medium and the second culture medium, in which:
First culture medium includes the component of the DMEM/F12 culture medium containing 10% fetal calf serum and following final concentration: ground plug
Rice 1.2 μm of ol/L of pine, insulin 8 μm of ol/L, Indomethacin 6nmol/L, 6 μm of ol/L of astaxanthin, 2 μm of ol/L of Stibene-glucoside；
Second culture medium includes the component of the DMEM/F12 culture medium containing 10% fetal calf serum and following final concentration: ground plug
Rice 1.2 μm of ol/L of pine, insulin 6 μm of ol/L, Indomethacin 6nmol/L, astaxanthin 2.5 μm of ol/L, 1.2 μ of Stibene-glucoside
Mol/L, 8 μm of ol/L of Astragaloside IV, 3 μm of ol/L of Isorhamnetin.
It is 1 × 10 that 3rd fat subsitutes mescenchymal stem cell, which is pressed inoculum density,4A/cm2It is seeded in the first culture medium,
37 DEG C, the CO of 5% volume2Under the conditions of carry out culture 6 days, then in the second culture medium, at 37 DEG C, the CO of 5% volume2Under the conditions of
Continue to cultivate.
Wherein, when being cultivated in the first culture medium, every 48h replace fresh culture, in the second culture medium into
When row culture, every replacing fresh culture for 24 hours.
Comparative example 1
Prepare conventional adipogenic induction culture medium comprising the DMEM/F12 basal medium containing 10% fetal calf serum and with
The component of lower final concentration: 1 μm of ol/L of dexamethasone, 10 μm of ol/L of insulin, Indomethacin 5nmol/L, 3- isobutyl group -1- methyl
Xanthine 0.5mmol/L.
It is 1 × 10 that 3rd fat subsitutes mescenchymal stem cell, which is pressed inoculum density,4A/cm2It is seeded to and is trained in conventional adipogenic induction
Support base, at 37 DEG C, the CO of 5% volume2Under the conditions of cultivated.
Experimental example
(1) the primary separation of fat mesenchymal stem cell and passage
By the human fat tissue extracted under aseptic condition after PBS is rinsed, using 0.1%I collagen type enzyme at 37 DEG C
Digest 30min, the fat cell and supernatant of centrifugation removal floating, with 2 × 105/cm2Density be seeded in containing 10% tire ox blood
In clear DMEM/F12 culture medium, at 37 DEG C, the CO of 5% volume2Under the conditions of cultivate, fresh culture is replaced after 48h for the first time, is connect
Get off every 2-3d replacement fresh culture, when cell fusion degree 80-90%, passes in the ratio of 1:3.
(2) adipogenic induction
It will be by the fat mesenchymal stem cell that step (1) progress secondary culture obtains respectively by embodiment 1-3 and comparison
The method of example 1 carries out adipogenic induction.
(3) oil red O stain
Cultivating total number of days is 10d, measures each composition rouge differentiation rate, specific measuring method respectively are as follows: suction culture medium, 10%
Neutral formalin fixes 30min, distilled water flushing, and room temperature oil red O stain 20min discards extra dyestuff, under inverted microscope
Observation is taken pictures, and the staining positive cells number and total number of cells in 3 visuals field of recorded at random are calculated as rouge differentiation rate.Calculation formula
Are as follows: at rouge differentiation rate=(staining positive cells number/total number of cells) × 100%.The results are shown in Table 1:
Table 1 is at rouge differentiation rate
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example 1 | |
| At rouge differentiation rate (%) | 88.2 | 91.4 | 87.7 | 68.1% |
It can be seen that the present invention can make fat mesenchymal stem cell shorter compared with conventional adipogenic induction culture medium
In induction time, reaches higher at rouge differentiation rate, there is excellent adipogenic induction effect.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (9)
1. a kind of culture medium for fat mesenchymal stem cell adipogenic induction, which is characterized in that including the first culture medium and
Two culture mediums；
First culture medium includes the component of the DMEM/F12 culture medium containing 10% fetal calf serum and following final concentration: ground plug
Rice 1-1.2 μm of ol/L of pine, insulin 5-8 μm of ol/L, Indomethacin 5-6nmol/L, 4-6 μm of ol/L of astaxanthin, Stibene-glucoside
1-2μmol/L；
Second culture medium includes the component of the DMEM/F12 culture medium containing 10% fetal calf serum and following final concentration: ground plug
Rice 1-1.2 μm of ol/L of pine, insulin 4-6 μm of ol/L, Indomethacin 5-6nmol/L, 2-2.5 μm of ol/L of astaxanthin, talan
0.6-1.2 μm of ol/L of glycosides, 4-8 μm of ol/L of Astragaloside IV, 2-3 μm of ol/L of Isorhamnetin.
2. a kind of culture medium for fat mesenchymal stem cell adipogenic induction according to claim 1, which is characterized in that
First culture medium includes the component of the DMEM/F12 culture medium containing 10% fetal calf serum and following final concentration: dexamethasone
1.1 μm of ol/L, insulin 6 μm of ol/L, Indomethacin 5nmol/L, 4.5 μm of ol/L of astaxanthin, 1.5 μm of ol/L of Stibene-glucoside.
3. a kind of culture medium for fat mesenchymal stem cell adipogenic induction according to claim 1, which is characterized in that
Second culture medium includes the component of the DMEM/F12 culture medium containing 10% fetal calf serum and following final concentration: dexamethasone
1.1 μm of ol/L, insulin 5 μm of ol/L, Indomethacin 5nmol/L, 2.4 μm of ol/L of astaxanthin, 0.8 μm of ol/L of Stibene-glucoside,
5 μm of ol/L of Astragaloside IV, 2.5 μm of ol/L of Isorhamnetin.
4. a kind of adipogenic induction method of fat mesenchymal stem cell, which is characterized in that first weighing fat mesenchymal stem cell
Benefit requires to be cultivated in the first culture medium described in any one of 1-3, then of any of claims 1-3 second
It is cultivated in culture medium.
5. a kind of adipogenic induction method of fat mesenchymal stem cell according to claim 4, which is characterized in that will be fatty
Mescenchymal stem cell is first cultivated 5-7 days in first culture medium, then continues to train in second culture medium
It supports.
6. a kind of adipogenic induction method of fat mesenchymal stem cell according to claim 4 or 5, which is characterized in that institute
State the fat mesenchymal stem cell that fat mesenchymal stem cell is 3-6 generation.
7. a kind of adipogenic induction method of fat mesenchymal stem cell, feature according to any one of claim 4-6
It is, it is (1-2) × 10 that the fat mesenchymal stem cell, which is seeded to the inoculum density in the first culture medium,4A/cm2。
8. a kind of adipogenic induction method of fat mesenchymal stem cell, feature according to any one of claim 4-7
It is, the culture is at 37 DEG C, the CO of 5% volume2Under the conditions of carry out.
9. a kind of adipogenic induction method of fat mesenchymal stem cell, feature according to any one of claim 4-8
It is, when fat mesenchymal stem cell is cultivated in first culture medium, replaces fresh culture every 36-48h,
When being cultivated in second culture medium, fresh culture is replaced every 18-24h.
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