CN109161524A - A method of promoting human adipose-derived stem cell proliferation - Google Patents
A method of promoting human adipose-derived stem cell proliferation Download PDFInfo
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- CN109161524A CN109161524A CN201811164272.XA CN201811164272A CN109161524A CN 109161524 A CN109161524 A CN 109161524A CN 201811164272 A CN201811164272 A CN 201811164272A CN 109161524 A CN109161524 A CN 109161524A
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention discloses a kind of methods of promotion human adipose-derived stem cell proliferation, it include: to take fresh human fat tissue, PBS buffer solution is rinsed, and is cut into fragment after removing adipose tissue film and blood vessel, digestion, complete medium is added after digestion, the filtering of 200 mesh filter screens removes indigested fibrous connective tissue, and fat cell and fat drips that supernatant removal suspends are abandoned in centrifugation, cell precipitation is resuspended in complete medium, erythrocyte cracked liquid is added, room temperature solution is educated after mixing, is centrifuged, is removed supernatant；Cell precipitation is resuspended in complete medium, is inoculated in culture bottle, and culture changes culture solution afterwards for 24 hours and removes non-attached cell, changes liquid within every 3 days later, digests after cell fusion, passes on；Passage cell is taken, digestion is made cell suspension, is inoculated in culture bottle, is incubated at after cultivating for 24 hours in complete medium, be changed to the complete medium containing hinesol or agalloch eaglewood loop coil alcohol and continue to cultivate.
Description
Technical field
The invention belongs to biological stem cell fields, are related to a kind of method of promotion human adipose-derived stem cell proliferation.
Background technique
Fat stem cell is that a kind of multipotential stem cell can break up shape under the conditions of specific in vivo and in vitro in adipose tissue
At various kinds of cell type, such as fat cell, osteoblast, cartilage cell, nerve cell, muscle cell.Currently, fat is dry thin
Born of the same parents assist autologous fat free grafting to have been found to can effectively improve the survival rate of transplant fat.Relative to bone marrow matrix source
Stem cell, fat stem cell are considered as the optimal selection of cell auxiliary fat transfer, because passing through liposuction
Obtain a large amount of fat stem cell.A kind of precursor of the fat stem cell as adipose tissue, participates in various adipose tissue weights
It builds, the tissue expander including reparation and mechanical force induction after growth and development, morbid oberity, damage or after anoxic.
During cell transplantation tissue reconstruction, need to provide enough cell origins for neoblastic formation.But
In most cases, the cell of transplanting cannot survive and be proliferated well, therefore, find suitable drug or growth factor etc. to promote
Stem cells hyperplasia into transplanting is most important for the effect for improving cellular replacement therapy.
Hinesol and agalloch eaglewood loop coil alcohol are a pair of of isomer, and only the chirality of individual carbon atoms is different；Thujopsis dolabrata ketenes
It is a kind of sesquiterpenoids with enone structure, chemical structure is as follows.
Have not yet to see the report that the pure and mild Thujopsis dolabrata ketenes of hinesol, agalloch eaglewood loop coil can promote fat stem cell to be proliferated.
Summary of the invention
The purpose of the present invention is to provide a kind of methods of promotion human adipose-derived stem cell proliferation.
Above-mentioned purpose of the invention is achieved by following technical solution:
A method of promoting human adipose-derived stem cell proliferation, includes the following steps:
Taking fresh human fat tissue, PBS buffer solution is rinsed, and it removes and is cut into fragment after adipose tissue film and blood vessel, 0.1%
Type I collagen enzyme and 37 DEG C of 0.25% pancreatin digestion, during which shake repeatedly, complete medium, 200 mesh filter screens are added after digestion
Filtering removes indigested fibrous connective tissue, and 1200 × g is centrifuged 5min, abandons fat cell and fat drips that supernatant removal suspends,
Cell precipitation is resuspended in complete medium, the erythrocyte cracked liquid of 6 times of volumes is added, room temperature solution educates 6min, 1200 × g after mixing
It is centrifuged 5min, removes supernatant；Cell precipitation is resuspended in complete medium, is inoculated in culture bottle, in 37 DEG C, 5%CO2In incubator
Culture changes culture solution afterwards for 24 hours and removes non-attached cell, changes within every 3 days later liquid, with 0.25% pancreatin and 1mmol/ after cell fusion
L EDTA37 DEG C digests 3~5min, and complete medium terminates digestion, passage；
Passage cell is taken, digestion is made cell suspension, is inoculated in culture bottle, is incubated in complete medium, and 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 hinesol or agalloch eaglewood loop coil alcohol of effective concentration.
Preferably, the complete medium is the DMEM in high glucose culture medium containing 10% fetal calf serum.
Preferably, it is passed in the ratio of 1:2.
The application of hinesol or agalloch eaglewood loop coil alcohol in the culture medium that preparation promotes human adipose-derived stem cell proliferation.
It is a discovery of the invention that hinesol, agalloch eaglewood loop coil alcohol can effectively facilitate the proliferation of human adipose-derived stem cell, and will not influence
Its Multidirectional Differentiation ability, hinesol, agalloch eaglewood loop coil alcohol may be used as the culture that additive preparation promotes human adipose-derived stem cell proliferation
Base.
Detailed description of the invention
Fig. 1 is that each group 450nm measures absorbance；
Fig. 2 is oil red O stain result, in which: A is control group coloration result, and B is hinesol group coloration result, and C is agalloch eaglewood
Loop coil alcohol group coloration result, D are Thujopsis dolabrata ketenes group coloration result；
Fig. 3 is Alizarin red staining result, in which: A is control group coloration result, and B is hinesol group coloration result, and C is heavy
Neptunea cumingi cyclic alcohol group coloration result, D are Thujopsis dolabrata ketenes 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
Fresh human fat tissue is derived from stomach fat liposuction patient, free from infection and endocrine system disease.
DMEM in high glucose culture medium, fetal calf serum are purchased from U.S. Gibco company.Hinesol, the pure and mild thujopsene of agalloch eaglewood loop coil
Ketone self-control or purchase, purity are greater 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 fat stem cell
Fresh human fat tissue is taken, PBS buffer solution is rinsed 2~3 times, is cut into 1mm after removing adipose tissue film and blood vessel3
The fragment of size, 37 DEG C of digestion 30min of 0.1% Type I collagen enzyme and 0.25% pancreatin, during which shakes 3 times, after digestion repeatedly
It is added complete medium (DMEM in high glucose containing 10% fetal calf serum), the filtering of 200 mesh filter screens removes indigested fibrous connective group
It knits, 1200 × g is centrifuged 5min, abandons fat cell and fat drips that supernatant removal suspends, and cell precipitation is resuspended in complete medium, adds
Enter the erythrocyte cracked liquid of 6 times of volumes, room temperature solution educates 6min after mixing, and 1200 × g is centrifuged 5min, removes supernatant；Cell precipitation weight
It is suspended from complete medium, is inoculated in culture bottle, in 37 DEG C, 5%CO2It is cultivated in incubator, changes culture solution removing afterwards for 24 hours and do not paste
Parietal cell, changes liquid for every 3 days later, complete with 3~5min of 0.25% pancreatin and 37 DEG C of 1mmol/L EDTA digestion after cell fusion
Full culture medium terminates digestion, passes in the ratio of 1:2, the 5th generation cell is taken to carry out subsequent experimental.
2, it is grouped and intervenes
The 5th generation human adipose-derived stem cell is taken, is grouped intervention by following group:
Control group: complete medium culture is used；
Hinesol group: the complete medium culture containing 10 μM of hinesols is used；
Agalloch eaglewood loop coil alcohol group: the complete medium culture containing 10 μM of agalloch eaglewood loop coil alcohol is used；
Thujopsis dolabrata ketenes group: the complete medium culture containing 10 μM of Thujopsis dolabrata ketenes is used.
3, cell Proliferation vitality test
The 5th generation human adipose-derived stem cell is taken, cell suspension is made in digestion, with 5 × 103/ hole is inoculated on 96 well culture plates, training
It supports in complete medium, 37 DEG C, 5%CO2After culture for 24 hours, continue to train according to above-mentioned grouping and interference method replacement culture medium
It supports for 24 hours；After culture, the 10 μ L of CCK-8 solution in Cell counting Kit is added in every hole, continues to incubate in cell incubator
After educating 2h, absorbance is measured in 450nm with microplate reader respectively, every group of experiment is repeated 3 times.
4, induction verifying human adipose-derived stem cell osteogenic lipogenesis differentiation capability
The 5th generation human adipose-derived stem cell is taken, cell suspension is made in digestion, with 2 × 105A/cm2Cell number is inoculated in culture bottle
In, it is incubated in complete medium, 37 DEG C, 5%CO2Culture for 24 hours after, according to above-mentioned grouping and interference method replacement culture medium after
Continuous culture is for 24 hours；After culture, culture medium washing is abandoned, cell suspension is made in digestion, with 5 × 103A/cm2Cell number plantation in
12 orifice plates；Rouge and Osteoblast Differentiation induction liquid are added into respectively, and every 3d changes liquid, utilizes oil red O stain after cultivating 16d and 20d respectively
And Alizarin red staining.Specific steps are as follows: after human adipose-derived stem cell is rinsed, 4 DEG C, the fixed 10min of 40g/L paraformaldehyde；Go from
Sub- water rinsing, dyes 30min with the oil red O or alizarin red of 1mL at room temperature, and thoroughly microscopically observation is taken pictures after cleaning.
It is containing 10% fetal calf serum, 0.1 μM of dexamethasone, 10 μM of insulin, 200 μM of indoles beauty at rouge induction liquid
Pungent, 0.5mM isobutylmethylxanthine DMEM in high glucose；It is containing 10% fetal calf serum, 0.1 μM of ground plug rice that Osteoblast Differentiation, which induces liquid,
Pine, 10mM sodium β-glycerophosphate, 50 μM of ascorbic acid DMEM.
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, influence of the pure and mild Thujopsis dolabrata ketenes of hinesol, agalloch eaglewood loop coil to human adipose-derived stem cell proliferative capacity
Experimental result is as shown in table 1 and Fig. 1, as a result as it can be seen that the pure and mild Thujopsis dolabrata ketenes of hinesol, agalloch eaglewood loop coil can be significant
Promote the proliferation of human adipose-derived stem cell, difference has statistical significance (P < 0.05).
1 each group 450nm of table measures absorbance (OD450)
2, influence of the pure and mild Thujopsis dolabrata ketenes of hinesol, agalloch eaglewood loop coil to human adipose-derived 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 adipose-derived stem cell all have it is excellent at rouge Osteoblast Differentiation
Ability, the pure and mild Thujopsis dolabrata ketenes of hinesol, agalloch eaglewood loop coil will not influence the differentiation capability of human adipose-derived stem cell.
To sum up, the pure and mild Thujopsis dolabrata ketenes of hinesol, agalloch eaglewood loop coil can effectively facilitate the proliferation of human adipose-derived stem cell,
And will not influence its Multidirectional Differentiation ability, the pure and mild Thujopsis dolabrata ketenes of hinesol, agalloch eaglewood loop coil may be used as additive preparation and promote
The culture medium of human adipose-derived stem cell proliferation.
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 human adipose-derived stem cell proliferation, includes the following steps:
Fresh human fat tissue is taken, PBS buffer solution is rinsed, and is cut into fragment, 0.1% I type after removing adipose tissue film and blood vessel
Clostridiopetidase A and 37 DEG C of 0.25% pancreatin digestion, during which shake repeatedly, complete medium, 200 mesh filter screen mistakes are added after digestion
It filters off and removes indigested fibrous connective tissue, 1200 × g is centrifuged 5min, abandons fat cell and fat drips that supernatant removal suspends, carefully
Born of the same parents' precipitating is resuspended in complete medium, and the erythrocyte cracked liquid of 6 times of volumes is added, and room temperature solution educates 6min after mixing, 1200 × g from
Heart 5min, removes supernatant；Cell precipitation is resuspended in complete medium, is inoculated in culture bottle, in 37 DEG C, 5%CO2It is trained in incubator
It supports, changes culture solution afterwards for 24 hours and remove non-attached cell, change within every 3 days later liquid, with 0.25% pancreatin and 1mmol/L after cell fusion
EDTA37 DEG C of 3~5min of digestion, complete medium terminate digestion, passage；
Passage cell is taken, digestion is made cell suspension, is inoculated in culture bottle, is incubated in complete medium, and 37 DEG C, 5%CO2
After 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 hinesol or agalloch eaglewood loop coil alcohol of effective concentration.
2. according to the method described in claim 1, it is characterized by: the complete medium is the height sugar containing 10% fetal calf serum
DMEM culture medium.
3. according to the method described in claim 1, it is characterized by: being passed in the ratio of 1:2.
4. the application of hinesol or agalloch eaglewood loop coil alcohol in the culture medium that preparation promotes human adipose-derived stem cell proliferation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114796277A (en) * | 2022-04-14 | 2022-07-29 | 高山艳 | Preparation method of proportional mixture of adipose-derived mesenchymal stem cells combined with nano-adipose |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114796277A (en) * | 2022-04-14 | 2022-07-29 | 高山艳 | Preparation method of proportional mixture of adipose-derived mesenchymal stem cells combined with nano-adipose |
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