CN107574147A - A kind of mescenchymal stem cell Proliferation, Differentiation nutrient solution using atractylenolide as trophic factors - Google Patents
A kind of mescenchymal stem cell Proliferation, Differentiation nutrient solution using atractylenolide as trophic factors Download PDFInfo
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- CN107574147A CN107574147A CN201710981339.8A CN201710981339A CN107574147A CN 107574147 A CN107574147 A CN 107574147A CN 201710981339 A CN201710981339 A CN 201710981339A CN 107574147 A CN107574147 A CN 107574147A
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- 235000015097 nutrients Nutrition 0.000 title claims abstract description 18
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 17
- 230000004069 differentiation Effects 0.000 title claims abstract description 14
- 230000001228 trophic effect Effects 0.000 title claims abstract description 11
- 230000004663 cell proliferation Effects 0.000 title claims abstract description 7
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 22
- 210000004271 bone marrow stromal cell Anatomy 0.000 claims abstract description 17
- 230000004072 osteoblast differentiation Effects 0.000 claims abstract description 12
- 230000001939 inductive effect Effects 0.000 claims abstract description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 210000003716 mesoderm Anatomy 0.000 claims 1
- 210000000988 bone and bone Anatomy 0.000 abstract description 16
- 239000001963 growth medium Substances 0.000 abstract description 2
- 241000132012 Atractylodes Species 0.000 description 21
- 150000002596 lactones Chemical class 0.000 description 18
- ZTVSGQPHMUYCRS-SWLSCSKDSA-N (4as,8as)-3,8a-dimethyl-5-methylidene-4a,6,7,8-tetrahydro-4h-benzo[f][1]benzofuran-2-one Chemical compound C=C([C@@H]1C2)CCC[C@]1(C)C=C1C2=C(C)C(=O)O1 ZTVSGQPHMUYCRS-SWLSCSKDSA-N 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 12
- OQYBLUDOOFOBPO-UHFFFAOYSA-N Asterolide Natural products C1C2C(=C)CCCC2(C)CC2C1=C(C)C(=O)O2 OQYBLUDOOFOBPO-UHFFFAOYSA-N 0.000 description 10
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 5
- 229910052791 calcium Inorganic materials 0.000 description 5
- 239000011575 calcium Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 4
- 230000007547 defect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 2
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 2
- 230000002146 bilateral effect Effects 0.000 description 2
- 230000002308 calcification Effects 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 229930184727 ginkgolide Natural products 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 210000002758 humerus Anatomy 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000012109 statistical procedure Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000002303 tibia Anatomy 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of mescenchymal stem cell Proliferation, Differentiation nutrient solution using atractylenolide as trophic factors, nutrient solution nutrient solution based on DMEM/F12 culture medium solutions, the atractylenolide containing valid density in the basic culture solution；The mescenchymal stem cell is mesenchymal stem cells MSCs, and the differentiation refers to Osteoblast Differentiation, and the concentration of the atractylenolide is 5 60 μM.It is a discovery of the invention that atractylenolide can promote the propagation and Osteoblast Differentiation of mesenchymal stem cells MSCs, it can be used for the nutrient solution for being prepared into inducing bone mesenchymal stem cell Osteoblast Differentiation, to effectively improve mesenchymal stem cells differentiation skeletonization bone amount.
Description
Technical field
The invention belongs to stem cell field, is related to stem cells hyperplasia differentiation, and in particular to a kind of using atractylenolide as battalion
Support the mescenchymal stem cell Proliferation, Differentiation nutrient solution of the factor.
Background technology
The research of bone tissue engineer provides a new hope for treatment large segmental bone defect.Bone tissue engineer is by dry thin
Born of the same parents, trophic factors and the key element of timbering material 3 composition.Treatment of the good stem cell and trophic factors to Cranial defect is most important,
Therefore, propagation and the strong stem cell of differentiation capability are found, possesses the trophic factors for inducing strongly and promoting to breed and breaking up, reaches
It is that field of orthopaedics deepens continuously the focus of research to optimal bone defect healing effect.
Trophic factors can promote mescenchymal stem cell skeletonization, be a composition portion essential in bone tissue engineer
Point.Due to simple Derived from Mesenchymal Stem Cells skeletonization, bone amount is less, therefore the effect of trophic factors just seems more important.
Atractylenolide is a kind of compound in the bighead atractylodes rhizome, is main in the bighead atractylodes rhizome with atractylenolide Ⅰ and atractylodes lactone III
Ginkgolide Component.Its chemical structural formula is as follows, and atractylodes lactone III is the oxidation product of atractylenolide Ⅰ in source of students, and atractylenolide is
The dehydration product of atractylodes lactone III.Generally, it is considered that atractylenolide Ⅰ and the main active that atractylodes lactone III is the bighead atractylodes rhizome, are them
Anti-inflammatory and anticancer effective component (bibliography：Atractylodes lactone constituents and its Advance on Pharmacological Activities, China Dispensary 2012
The 39th phase of volume 23).
It is found by the applicant that atractylenolide have it is many it is special, different from the activity of atractylenolide Ⅰ and atractylodes lactone III, than
As atractylenolide can promote mesenchymal stem cells MSCs Osteoblast Differentiation.At present, there has been no the open atractylenolide of research
These activity.
The content of the invention
It is an object of the invention to provide a kind of mescenchymal stem cell Proliferation, Differentiation using atractylenolide as trophic factors
Nutrient solution.
Realize that above-mentioned purpose technical scheme of the present invention is as follows：
A kind of mescenchymal stem cell Proliferation, Differentiation nutrient solution using atractylenolide as trophic factors, cultivated with DMEM/F12
Nutrient solution based on based sols, the atractylenolide containing valid density in the basic culture solution.
Preferably, the mescenchymal stem cell is mesenchymal stem cells MSCs.
Preferably, the differentiation refers to Osteoblast Differentiation.
Preferably, the concentration of the atractylenolide is 5-60 μM.
Application of the atractylenolide in the reagent for preparing inducing mesenchymal stem cell Osteoblast Differentiation.
Preferably, the mescenchymal stem cell is mesenchymal stem cells MSCs.
The outstanding advantages of the present invention：
It is a discovery of the invention that atractylenolide can promote the propagation and Osteoblast Differentiation of mesenchymal stem cells MSCs, Ke Yiyong
In the nutrient solution for being prepared into inducing bone mesenchymal stem cell Osteoblast Differentiation, with effectively improve mesenchymal stem cells differentiation into
Bone bone amount.
Brief description of the drawings
Fig. 1 is that CCK8 methods detect influence of the atractylodes lactone to Proliferation of Bone Mesenchymal Stem Cells；
Fig. 2 is calcium scoring coloration result.
Embodiment
Just in conjunction with the embodiments specific below to introduce essentiality content of the invention, due to length reason, experimentation is retouched
Stating can not accomplish very in detail, and the part not being described in detail in every experiment is conventional behaviour well known to those skilled in the art
Make.
First, experiment material
Atractylenolide, atractylenolide Ⅰ and atractylodes lactone III are made by oneself, and purity is more than 98%.
DMEM/F12(1:1) culture medium is purchased from Hyclone companies as serum-free medium.
2nd, experimental method
1st, the separation and culture of mesenchymal stem cells MSCs (BMSCs)
Raw 14d healthy SD rat is taken out, is put to death using cervical dislocation and is soaked in 10min in 75% alcohol, sterile bar
Bilateral femur, shin bone and humerus are taken under part, is placed in the nutrient solution of serum-free, bilateral metaphysis is cut, exposes ossis,
2.5mL culture mediums are drawn with syringe and rinse ossis repeatedly, are collected flushing liquor and are placed in centrifuge tube, centrifuge 1200r/min,
5min, supernatant is abandoned, cell is resuspended with the complete culture solution containing 10% hyclone, in 5%CO2, cultivate in 37 DEG C of incubators,
Liquid is changed after 24h, removes non-attached cell, liquid is changed once per 2-3d, when cell attachment grows 80%-90%, 0.25% pancreatin disappears
Change and pass on, liquid is changed once every 2d, with propagating method to cell purification, cellular morphology is observed under inverted microscope.
2nd, influence of the atractylodes lactone to Proliferation of Bone Mesenchymal Stem Cells
Third generation BMSCs is taken with 1 × 103The concentration in/hole is inoculated in 96 orifice plates, be separately added into 40 μM atractylenolide,
Atractylenolide Ⅰ and atractylodes lactone III (also set up control group, control group does not add atractylenolide, atractylenolide Ⅰ or atractylodes lactone
III), 3 multiple holes of every group of setting, culture is terminated when cultivating 5d, 100 μ L serum-free mediums and 10 μ L CCK-8 are added in every hole
Liquid is detected, is placed in incubator after 2h, uses measure OD values under ELIASA 450nm wavelength.
3rd, calcium scoring dyes
Take the 3rd generation cell, in inoculated and cultured ware, 2.5 × 105/ ware.20 μM are separately added into after cell conforms to 80%
Atractylenolide, atractylenolide Ⅰ and atractylodes lactone III, cultivated eventually after inducing 21d, PBS is rinsed 3 times, 95% alcohol fixation 15min
Afterwards, 0.1% Alizarin red staining, 37 DEG C of incubation 1h, distilled water rinse 3 times.Observe and take a picture, count calcified area percentage
(%).
4th, statistical procedures
Statistical analysis is carried out with the statistical softwares of SPSS 19.0, data are represented using mean ± standard deviation, are compared between group and are adopted
With one-way analysis of variance, P ＜ 0.05 represent that difference is statistically significant.
3rd, experimental result
1st, BMSCs cultures and identification
Visible a small amount of star, spindle cell adherent growth after rat primary cell culture 24h, visible cell is in radiation after 4d
Shape arranges, in fusiformis.Visible cell grows arrangement closely in the form of sheets after culture 7d, is grown in swirling, and BMSCs cultures are normal.
2nd, influence of the atractylodes lactone to Proliferation of Bone Mesenchymal Stem Cells
After atractylenolide, atractylenolide Ⅰ and atractylodes lactone III cultivate 5d respectively, BMSCs is detected using CCK-8 kits
Cell quantity.As a result show：Atractylenolide and atractylenolide Ⅰ can promote BMSCs propagation, and OD values are significantly higher than control
Group, difference have statistical significance (P ＜ 0.05)；Atractylodes lactone III has slight inhibitory action to BMSCs propagation.
Each group OD values are as shown in table 1 and Fig. 1.
The CCK8 methods of table 1 detect influence of the atractylodes lactone to Proliferation of Bone Mesenchymal Stem Cells
| Control group | Atractylenolide group | Atractylenolide Ⅰ group | III group of atractylodes lactone | |
| OD values (450nm) | 0.83±0.08 | 1.39±0.12 | 1.17±0.11 | 0.75±0.09 |
3rd, calcium scoring coloration result
Calcification phenomenon occurs in known Gegenbaur's cell, can dye red by alizarin red agent, the cell of non-calcification will not be colored.
As it is clear from fig. 2 that control group, atractylenolide Ⅰ group, III group of atractylodes lactone are without obvious calcium scoring phenomenon (calcified area hundred
Point than ＜ 5%), and atractylenolide group can be observed the calcium scoring of large area, and calcified area percentage is up to more than 70%.
Above-described embodiment proves that atractylenolide can promote the propagation and Osteoblast Differentiation of mesenchymal stem cells MSCs, can
For being prepared into the nutrient solution of inducing bone mesenchymal stem cell Osteoblast Differentiation, to effectively improve mesenchymal stem cells MSCs point
It is melted into bone bone amount.
Above-described embodiment is the embodiment to essentiality content of the present invention, for preferably explaining the present invention, but this area skill
Art personnel are it is to be understood that protection scope of the present invention should not be confined to above-mentioned specific embodiment.
Claims (6)
- A kind of 1. mescenchymal stem cell Proliferation, Differentiation nutrient solution using atractylenolide as trophic factors, with DMEM/F12 culture mediums Nutrient solution based on solution, it is characterised in that：Atractylenolide containing valid density in the basic culture solution.
- 2. nutrient solution according to claim 1, it is characterised in that：The mescenchymal stem cell is that medulla mesenchyma is dry thin Born of the same parents.
- 3. nutrient solution according to claim 1, it is characterised in that：The differentiation refers to Osteoblast Differentiation.
- 4. nutrient solution according to claim 1, it is characterised in that：The concentration of the atractylenolide is 5-60 μM.
- 5. application of the atractylenolide in the reagent for preparing inducing mesenchymal stem cell Osteoblast Differentiation.
- 6. application according to claim 5, it is characterised in that：The mescenchymal stem cell is mesenchymal stem cells MSCs.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114525242A (en) * | 2022-03-15 | 2022-05-24 | 武汉百翼生物科技有限公司 | Method for inducing ipsc to differentiate into myocardial cells |
| CN116376815A (en) * | 2023-02-15 | 2023-07-04 | 山东科金生物发展有限公司 | Culture medium for promoting osteoblast differentiation of mesenchymal stem cells |
| CN118516302A (en) * | 2024-07-19 | 2024-08-20 | 广东医科大学附属医院 | Osteogenesis induction culture medium and osteogenesis induction differentiation method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114525242A (en) * | 2022-03-15 | 2022-05-24 | 武汉百翼生物科技有限公司 | Method for inducing ipsc to differentiate into myocardial cells |
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| CN116376815A (en) * | 2023-02-15 | 2023-07-04 | 山东科金生物发展有限公司 | Culture medium for promoting osteoblast differentiation of mesenchymal stem cells |
| CN116376815B (en) * | 2023-02-15 | 2024-05-31 | 山东科金生物发展有限公司 | Culture medium for promoting osteoblast differentiation of mesenchymal stem cells |
| CN118516302A (en) * | 2024-07-19 | 2024-08-20 | 广东医科大学附属医院 | Osteogenesis induction culture medium and osteogenesis induction differentiation method |
| CN118516302B (en) * | 2024-07-19 | 2024-10-22 | 广东医科大学附属医院 | Osteogenesis induction culture medium and osteogenesis induction differentiation method |
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Address after: 410000, No. 102, South Section of Dongwu Road, Changsha Economic and Technological Development Zone, Changsha City, Hunan Province, China. Zhongnan Yuanpin Stem Cell Technology Co.,Ltd. Patentee after: Yuanpin Cell Biotechnology Group Co.,Ltd. Country or region after: China Address before: 410100 dormitory building 98, Li Xiang Road, Quan Tang street, Changsha economic and Technological Development Zone, Hunan 205 Patentee before: HUNAN YUANPIN CELL BIOTECHNOLOGY Co.,Ltd. Country or region before: China |