CN109161525A - Promote the method for Proliferation of Human Mesenchymal Stem Cells - Google Patents
Promote the method for Proliferation of Human Mesenchymal Stem Cells Download PDFInfo
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- CN109161525A CN109161525A CN201811164543.1A CN201811164543A CN109161525A CN 109161525 A CN109161525 A CN 109161525A CN 201811164543 A CN201811164543 A CN 201811164543A CN 109161525 A CN109161525 A CN 109161525A
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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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- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention discloses the methods for promoting Proliferation of Human Mesenchymal Stem Cells.Mesenchymal stem cell is the progenitor cells of osteoblast in marrow, is always a big hot spot of field of orthopaedics to the research of its osteogenic differentiation capability, and promotes it to the differentiation of skeletonization direction and one of the main method of anti-curing osteoporosis.Currently, a difficult point of mesenchymal stem cell application is how largely to obtain mesenchymal stem cell.Present invention discover that, α-himachalene, β-cedrene and different cedrone can effectively facilitate the proliferation of human marrow mesenchymal stem cell, and it will not influence its Multidirectional Differentiation ability, α-himachalene, β-cedrene and different cedrone may be used as the culture medium that additive preparation promotes Proliferation of Human Mesenchymal Stem Cells, and the drug for promoting Proliferation of Human Mesenchymal Stem Cells can be made.
Description
Technical field
The invention belongs to biological fields, are related to a kind of method for promoting Proliferation of Human Mesenchymal Stem Cells.
Background technique
Mesenchymal stem cell is the progenitor cells of osteoblast in marrow, always to the research of its osteogenic differentiation capability
It is a big hot spot of field of orthopaedics, and promotes it to the differentiation of skeletonization direction and one of the main method of anti-curing osteoporosis.
Currently, a difficult point of mesenchymal stem cell application is how largely to obtain mesenchymal stem cell.
α-himachalene, β-cedrene, γ-cedrene and different cedrone are compound isolated in deodar, chemistry knot
Structure is as follows.
Have not yet to see report of the above compound in terms of promoting Proliferation of Bone Mesenchymal Stem Cells.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for promoting Proliferation of Human Mesenchymal Stem Cells.
Above-mentioned purpose of the invention is achieved by following technical solution:
A method of promoting Proliferation of Human Mesenchymal Stem Cells, includes the following steps:
Acquire fresh bone marrow, heparin sodium it is anticoagulant after plus same amount of normal saline, Percoll cell separating liquid density gradient from
The heart takes intermediate mononuclear cell layer with the cleaning of low sugar DMEM culture medium, is inoculated in culture dish, after complete medium is added, is placed in
5%CO2It is cultivated in incubator.It changes liquid afterwards for 24 hours, abandons not adherent cell.Later every 2~3d changes liquid, grows to 90% to cell
Converge rear trypsin digestion secondary culture；Passage cell is taken, digestion is made cell suspension, is inoculated in culture bottle, is incubated at
In complete medium, 37 DEG C, 5%CO2After culture for 24 hours, it is changed to drug containing complete medium and continues to cultivate；
The drug containing complete medium contains the different cedrone of effective concentration.
Preferably, the complete medium is the DMEM/F12 culture medium containing 10%FBS.
Application of the different cedrone in the culture medium that preparation promotes Proliferation of Human Mesenchymal Stem Cells.
Application of the different cedrone in the drug that preparation promotes Proliferation of Human Mesenchymal Stem Cells.
The utility model has the advantages that
It is a discovery of the invention that α-himachalene, β-cedrene and different cedrone can effectively facilitate human marrow mesenchymal stem cell
Proliferation, and will not influence its Multidirectional Differentiation ability, α-himachalene, β-cedrene and different cedrone may be used as additive preparation
The drug for promoting Proliferation of Human Mesenchymal Stem Cells can be made in the culture medium for promoting Proliferation of Human Mesenchymal Stem Cells.
Detailed description of the invention
Fig. 1 is that each group 450nm measures absorbance；
Fig. 2 is Alizarin red staining result, in which: A is control group coloration result, and B is α-himachalene group coloration result, and C is
β-cedrene group coloration result, D are different cedrone group coloration result；
Fig. 3 is oil red O stain result, in which: A is control group coloration result, and B is α-himachalene group coloration result, and C is β-
Cedrene group coloration result, D are different cedrone group coloration result.
Specific embodiment
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but guarantor of the invention is not limited with this
Protect range.
One, experimental material
DMEM/F12 culture medium, low sugar DMEM culture medium, fetal calf serum are purchased from U.S. Gibco company.α-himachalene, β-snow
Loose alkene, γ-cedrene and the self-control of different cedrone or purchase, purity are not less than 98%.
Cell counting Kit (CCK-8) is purchased from Amada Co., Ltd. colleague chemistry institute.
Two, experimental method
1, the separation and culture of human marrow mesenchymal stem cell
Marrow 20mL in healthy volunteer's frontal bone is acquired, after heparin sodium is anticoagulant plus same amount of normal saline, Percoll cell divide
Chaotropic (1.073 × 10-3G/L) density gradient centrifugation takes intermediate mononuclear cell layer with low sugar DMEM culture medium cleaning 2 times, inoculation
Enter in 15cm culture dish, after complete medium (the DMEM/F12 culture medium containing 10%FBS) is added, is placed in 5%CO2In incubator
Culture.It changes liquid afterwards for 24 hours, abandons not adherent cell.Later every 2~3d changes liquid, after cell grow to 90% converge after trypsase
Had digestive transfer culture culture, is denoted as P1.Subsequent experimental is used for using P5 cell.
2, it is grouped and intervenes
The 5th generation human marrow mesenchyme stem cell is taken, is grouped intervention by following group:
Control group: complete medium culture is used；
α-himachalene group: the complete medium culture containing 15 μM of α-himachalenes is used；
β-cedrene group: it uses and contains 15 μM of β-cedrene complete medium cultures；
γ-cedrene group: it uses and contains 15 μM of γ-cedrene complete medium cultures；
Different cedrone group: the complete medium culture containing 15 μM of different cedrones is used.
3, cell Proliferation vitality test
The 5th generation human marrow mesenchyme stem cell is taken, cell suspension is made in digestion, with 4 × 105/ hole is inoculated in the culture of 96 holes
It on plate, is incubated in complete medium, 37 DEG C, 5%CO2After culture for 24 hours, culture medium is replaced according to above-mentioned grouping and interference method
Continue culture for 24 hours；After culture, the 10 μ L of CCK-8 solution in Cell counting Kit is added in every hole, in cell incubator
Continue after being incubated for 2h, measures absorbance in 450nm with microplate reader respectively, every group of experiment is repeated 3 times.
4, induction verifying human marrow mesenchymal stem cell osteogenic lipogenesis differentiation capability
The 5th generation human marrow mesenchyme stem cell is taken, cell suspension is made in digestion, with 2.5 × 105A/cm2Cell number inoculation
It in culture bottle, is incubated in complete medium, 37 DEG C, 5%CO2After culture for 24 hours, replaced according to above-mentioned grouping and interference method
Culture medium continues culture for 24 hours；After culture, culture medium washing is abandoned, cell suspension is made in digestion, with 8 × 103A/cm2Cell
Several plants are in 12 orifice plates；Skeletonization is added respectively, at rouge induction liquid, every 3d changes liquid, utilizes alizarin after cultivating 20d, 16d respectively
Red, oil red O stain dyeing.Specific steps are as follows: after human marrow mesenchymal stem cell is rinsed, 4 DEG C, 40g/L paraformaldehyde fixes
10min；Deionized water rinsing, dyes 30min with the alizarin red of 1mL, oil red O stain respectively at room temperature, thoroughly micro- after cleaning
It takes pictures under the microscope.Wherein: it is containing 10% fetal calf serum, 0.1 μM of dexamethasone, 10mM β-phosphoglycerol that Osteoblast Differentiation, which induces liquid,
The DMEM/F12 of sodium, 50 μM of ascorbic acid；It is containing 10% fetal calf serum, 0.1 μM of dexamethasone, 10 μM of pancreases at rouge induction liquid
The DMEM/F12 of island element, 200 μM of Indomethacins, 0.5mM isobutylmethylxanthine.
5, data processing
It is indicated using mean ± standard deviation, carries out t inspection with SPSS17.0 statistical package, P < 0.05 is to have conspicuousness
Difference.
Three, experimental result
1, α-himachalene, β-cedrene, γ-cedrene and different cedrone are to Proliferation of Human Mesenchymal Stem Cells ability
It influences
As a result as shown in table 1 and Fig. 1, it is seen then that α-himachalene, β-cedrene and different cedrone can remarkably promote people's bone marrow
The proliferation of mescenchymal stem cell, difference have statistical significance (P < 0.05)；Unobvious (the P > of γ-cedrene facilitation
0.05)。
1 each group 450nm of table measures absorbance (OD450)
| Group | OD450 |
| Control group | 0.4622±0.023 |
| α-himachalene group | 0.7525±0.027 |
| β-cedrene group | 0.7454±0.026 |
| γ-cedrene group | 0.4908±0.021 |
| Different cedrone group | 0.8117±0.024 |
2, the influence of α-himachalene, β-cedrene and different cedrone to human marrow mesenchymal stem cell differentiation capability
Experimental result is as shown in Figure 2,3, as a result as it can be seen that each group human marrow mesenchymal stem cell all have excellent skeletonization at
Rouge differentiation capability, α-himachalene, β-cedrene and different cedrone will not influence the differentiation capability of human marrow mesenchymal stem cell.
To sum up, α-himachalene, β-cedrene and different cedrone can effectively facilitate human marrow mesenchymal stem cell
Proliferation, and will not influence its Multidirectional Differentiation ability, α-himachalene, β-cedrene and different cedrone may be used as additive preparation and promote
Into the culture medium of Proliferation of Human Mesenchymal Stem Cells, the drug for promoting Proliferation of Human Mesenchymal Stem Cells can be made.
The effect of above-described embodiment is specifically to introduce essentiality content of the invention, but those skilled in the art should know
Protection scope of the present invention should not be confined to the specific embodiment by road.
Claims (4)
1. a kind of method for promoting Proliferation of Human Mesenchymal Stem Cells, includes the following steps:
Fresh bone marrow is acquired, after heparin sodium is anticoagulant plus same amount of normal saline, Percoll cell separating liquid density gradient centrifugation take
Intermediate mononuclear cell layer is inoculated in culture dish with the cleaning of low sugar DMEM culture medium, after complete medium is added, is placed in 5%CO2
It is cultivated in incubator.It changes liquid afterwards for 24 hours, abandons not adherent cell.Later every 2~3d changes liquid, after cell grow to 90% converge after
Trypsin digestion secondary culture；Passage cell is taken, digestion is made cell suspension, is inoculated in culture bottle, is incubated at complete training
It supports in base, 37 DEG C, 5%CO2After culture for 24 hours, it is changed to drug containing complete medium and continues to cultivate；
It is characterized by: the drug containing complete medium contains the different cedrone of effective concentration.
2. according to the method described in claim 1, it is characterized by: the complete medium is the DMEM/F12 containing 10%FBS
Culture medium.
3. application of the different cedrone in the culture medium that preparation promotes Proliferation of Human Mesenchymal Stem Cells.
4. application of the different cedrone in the drug that preparation promotes Proliferation of Human Mesenchymal Stem Cells.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113005079A (en) * | 2021-05-08 | 2021-06-22 | 河北驰熙科技发展有限公司 | Additive for human bone marrow mesenchymal stem cell in vitro amplification and amplification method |
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2018
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113005079A (en) * | 2021-05-08 | 2021-06-22 | 河北驰熙科技发展有限公司 | Additive for human bone marrow mesenchymal stem cell in vitro amplification and amplification method |
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Application publication date: 20190108 |