CN114181897A - Construction method of anti-aging autologous stem cell model - Google Patents
Construction method of anti-aging autologous stem cell model Download PDFInfo
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- CN114181897A CN114181897A CN202111446770.5A CN202111446770A CN114181897A CN 114181897 A CN114181897 A CN 114181897A CN 202111446770 A CN202111446770 A CN 202111446770A CN 114181897 A CN114181897 A CN 114181897A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal 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
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
Abstract
The invention discloses a construction method of an anti-aging autologous stem cell model, which comprises the following steps: s1, obtaining stromal vascular components from adipose tissues, separating the stromal vascular components to obtain primary adipose-derived stem cells, and performing primary subculture on the primary adipose-derived stem cells to obtain adipose-derived stem cells; s2, culturing for 5-10 days by using a stem cell culture medium added with transferrin, albumin and oleic acid; s3, in the stem cell culture medium, the content of transferrin is 8-20 ng/mL, the content of albumin is 8-20 ng/mL, and the content of oleic acid is 25-60 ng/mL. The method establishes the in vitro aging cell model of the autologous stem cells, can shorten the molding time, has simple operation, is easy to popularize and has obvious aging effect.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a construction method of an anti-aging autologous stem cell model.
Background
Aging is the functional degenerative change of tissues, organs and cells of a human body along with the aging and environmental influence, and is characterized in that the functional activity of the body is progressively reduced, the adaptive capacity to the environment is gradually reduced, and the metabolism in the body is imbalanced, so that a series of aging symptoms such as pale complexion, loose skin, increased wrinkles, easy fatigue, reduced mental and physical labor capacity, reduced sexual function and the like are caused. Since aging is a deterioration of systemic tissue and organ functions throughout the body, strategies for aging resistance and reshaping should also consider improving functions from the whole level.
Stem cells are a type of pluripotent cells with the ability to self-replicate, and under certain conditions, can differentiate into a variety of functional cells. The stem cells can perform regenerative repair on aging degenerative changes of various tissues, organs and systems, normalize and youthful the structure and function of the cells, integrally regulate and control the body state, and are systemic, systemic and fundamental health care.
The adipose-derived stem cells (ADSCs) are stem cells with multidirectional differentiation potential, the ADSCs can be stably proliferated in vitro and have low apoptosis rate, have multidirectional differentiation potential in vivo and in vitro, can be differentiated into adipocytes, chondrocytes, myocytes, osteoblasts, nerve cells, glial cells and islet cells under the action of different induction factors, and can secrete various angiogenesis promoting factors and anti-apoptosis factors. The adipose-derived stem cells have wide sources, easily-obtained materials, small damage to organisms, large in vivo reserve and capability of obtaining a large amount of stem cells from a small amount of tissues, and are suitable for autologous transplantation and large-scale culture, so that the adipose-derived stem cells gradually become one of the research hotspots in recent years, and a new idea is provided for treating a series of diseases.
Disclosure of Invention
The invention mainly solves the technical problem of providing a construction method of an autologous stem cell model with anti-aging function, the method establishes the autologous stem cell in-vitro aging cell model, can shorten the molding time, is simple to operate, is easy to popularize and has obvious aging effect.
In order to solve the technical problems, the invention adopts a technical scheme that: a method for constructing an autologous stem cell model with anti-aging effect comprises the following steps:
s1, obtaining stromal vascular components from adipose tissues, separating the stromal vascular components to obtain primary adipose-derived stem cells, and performing primary subculture on the primary adipose-derived stem cells to obtain adipose-derived stem cells;
s2, culturing for 5-10 days by using a stem cell culture medium added with transferrin, albumin and oleic acid;
s3, in the stem cell culture medium, the content of transferrin is 8-20 ng/mL, the content of albumin is 8-20 ng/mL, and the content of oleic acid is 25-60 ng/mL.
Furthermore, in the stem cell culture medium, the content of transferrin is 9-12 ng/mL, the content of albumin is 9-12 ng/mL, and the content of oleic acid is 27-35 ng/mL.
Further, in the culture process, the stem cell culture medium is replaced every 2 to 3 days.
Further, the stem cell culture medium is a serum-free medium.
Further, the stem cell culture medium also contains streptomycin and penicillin.
The autologous stem cell model with anti-aging effect obtained by the construction method.
The autologous stem cell model with anti-aging function is used for screening anti-aging drugs.
The invention has the beneficial effects that: the method for establishing the in vitro senescence cell model of the autologous stem cell can shorten the molding time, is simple to operate, is easy to popularize and has obvious senescence effect. Moreover, the obtained aging cell model is not easy to die, can still survive for a long time, more than 3 days after the model is established, and can be used for screening anti-aging drugs.
Drawings
FIG. 1 is a 40-fold microscopic image of the adipose stem cells of the present invention cultured for 24 hours.
Detailed Description
The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention.
Example 1 Primary isolation and in vitro culture of adipose Stem cells
The primary isolation and in vitro culture of the adipose-derived stem cells are carried out according to the following steps:
1) cleaning the collected adipose tissue with normal saline, placing the adipose tissue into a centrifuge tube, and cutting with sharp-pointed scissors to 1-5mm3Size;
2) adding collagenase I solution with the concentration of 2.5mg/ml into adipose tissues, and carrying out water bath oscillation digestion at 37 ℃ for 1-2 h;
3) filtering with 30 μm filter screen, centrifuging for 10min at 200g, and removing supernatant;
4) adding 1ml LPBS into the precipitate, adding 3ml erythrocyte lysate, mixing, and standing at room temperature for 5 min; centrifuging at 3500rpm for 7min, and removing the supernatant;
5) adding 10ml PBS to resuspend the precipitate, centrifuging at 1500rpm for 5min, and discarding the supernatant;
6) 3mL of buffer was added to the pellet to resuspend the cells, dilute 10-fold, and sort the appropriate cells.
7) Subjecting the cells to line negative enrichment by immunomagnetic bead sorting, including CD5, CD45R (B220), CD11B, Anti-Gr-1(Ly-6G/C), 7-4, Ter-119; then, carrying out CD117(C-kit) positive screening on the obtained cells by using an immunomagnetic bead sorting method;
8) the resulting cells were diafiltered with a stemspan SFEM (stemcell #09600) medium +10ng/ml transferrin +10ng/ml albumin +30ng/ml oleic acid +100UI/ml penicillin, 100UI/ml streptomycin, 5% CO2Culturing in an incubator at 37 deg.C for 8 days;
during the culture period, the medium was changed every 2-3 days, followed by 1:3 passages. The obtained cells are the fat stem cell in vitro aging cell model. As seen from the inverted microscope, as shown in FIG. 1, the adipose-derived stem cells of the primary culture day 1 were uniformly suspended in the culture medium, and the cell morphology was circular and conformed to the basic morphological characteristics of the stem cells.
The adipose-derived stem cell aging model obtained after 8 days of culture has good cell aging effect, and can survive for a long time, generally at least 3-5 days. Therefore, the in vitro aging cell model can be used for screening anti-aging drugs.
Example 2 flow cytometry detection of the ratio of Lineage-c-kit + cells
Detecting the purity of the sorted cells:
(1) collection of freshly sorted Lin-c-kit + cells and 1X 10 of each Lin-c-kit + cells cultured for 8 days in example 16Centrifuging at 1500rpm for 5 min;
(2) washing cells once, adding CD117-PE10ul, and incubating for 15 minutes at 4 ℃ in a dark place;
(3) wash cells once, dissolve cell pellet to 200-. Flow cytometry (Becton DickinosoAccuriTMC 6).
Example 3 senescence-associated beta-galactosidase staining
The method comprises the following steps: collect 1X 10 of each group61ml of beta-galactosidase staining fixative was added to HSC and fixed for 15 minutes at room temperature. Washing with PBS, adding beta-galactosidase staining working solution, mixing, and keeping at 37 deg.C without CO2The cells were incubated overnight (16h) under the conditions, and the positive cell rate was calculated as the number of positive cells out of 400 cells counted at random. SA-beta-gal (senescence-associated beta-galactosidase) is a hallmark indicator of senescence. The cells positive for SA- β -gal staining had increased volume, the cytoplasm was blue, and the negative cells were not stained.
Example 4 cell cycle
(1) Collect 1X 10 of each group6HSC were washed with pre-chilled PBS and pre-chilled 4% paraformaldehyde fixed for 1 h.
(2) The cells were pelleted by centrifugation at about 1000g for 5 minutes.
(3) Cells were washed once and fixed overnight in 70% glacial ethanol. The cells were pelleted by centrifugation at about 1000g for 5 minutes.
(4) Washing cells once, adding propidium iodide staining solution, carrying out dark temperature bath at 37 ℃ for 30min, detecting DNA combined with propidium iodide fluorescence at the wavelength of 488nm in an excitation wavelength by a flow cytometer, and analyzing the periodic distribution of each group of cells by appropriate software.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent structural changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to other related technical fields, are included in the scope of the present invention.
Claims (7)
1. A construction method of an autologous stem cell model with anti-aging function is characterized by comprising the following steps: the method comprises the following steps:
s1, obtaining stromal vascular components from adipose tissues, separating the stromal vascular components to obtain primary adipose-derived stem cells, and performing primary subculture on the primary adipose-derived stem cells to obtain adipose-derived stem cells;
s2, culturing for 5-10 days by using a stem cell culture medium added with transferrin, albumin and oleic acid;
s3, in the stem cell culture medium, the content of transferrin is 8-20 ng/mL, the content of albumin is 8-20 ng/mL, and the content of oleic acid is 25-60 ng/mL.
2. The method for constructing the autologous stem cell model with anti-aging effect according to claim 1, wherein the method comprises the following steps: in the stem cell culture medium, the content of transferrin is 9-12 ng/mL, the content of albumin is 9-12 ng/mL, and the content of oleic acid is 27-35 ng/mL.
3. The method for constructing the autologous stem cell model with anti-aging effect according to claim 1, wherein the method comprises the following steps: and in the culture process, replacing the stem cell culture medium every 2-3 days.
4. The method for constructing the autologous stem cell model with anti-aging effect according to claim 1, wherein the method comprises the following steps: the stem cell culture medium is a serum-free culture medium.
5. The method for constructing the autologous stem cell model with anti-aging effect according to claim 1, wherein the method comprises the following steps: the stem cell culture medium also contains streptomycin and penicillin.
6. The autologous stem cell model with anti-aging effect obtained by the construction method according to any one of claims 1 to 5.
7. The autologous stem cell model with anti-aging effect according to claim 6 is used for screening anti-aging drugs.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115025122A (en) * | 2022-06-25 | 2022-09-09 | 广东旺合生物科技有限公司 | Application of autologous adipose-derived stem cells in preparation of anti-aging and anti-immunocompromised medicines or products |
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| CN105586311A (en) * | 2016-03-06 | 2016-05-18 | 李倩 | Culture medium for culturing human adipose-derived stem cells |
| CN106190967A (en) * | 2016-07-19 | 2016-12-07 | 安徽惠恩生物科技股份有限公司 | A kind of autologous fat derived stem cell is for the preparation method of beautifying and antisenility |
| CN106399236A (en) * | 2016-11-28 | 2017-02-15 | 广东万海细胞生物科技有限公司 | Culture method for promoting growth of adipose stem cells |
| CN107858330A (en) * | 2017-11-20 | 2018-03-30 | 上海中医药大学 | A kind of method for building up of the external senile cell model of mouse bone marrow cells hematopoietic stem/progenitor |
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- 2021-11-30 CN CN202111446770.5A patent/CN114181897A/en active Pending
Patent Citations (4)
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| CN105586311A (en) * | 2016-03-06 | 2016-05-18 | 李倩 | Culture medium for culturing human adipose-derived stem cells |
| CN106190967A (en) * | 2016-07-19 | 2016-12-07 | 安徽惠恩生物科技股份有限公司 | A kind of autologous fat derived stem cell is for the preparation method of beautifying and antisenility |
| CN106399236A (en) * | 2016-11-28 | 2017-02-15 | 广东万海细胞生物科技有限公司 | Culture method for promoting growth of adipose stem cells |
| CN107858330A (en) * | 2017-11-20 | 2018-03-30 | 上海中医药大学 | A kind of method for building up of the external senile cell model of mouse bone marrow cells hematopoietic stem/progenitor |
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| CN115025122A (en) * | 2022-06-25 | 2022-09-09 | 广东旺合生物科技有限公司 | Application of autologous adipose-derived stem cells in preparation of anti-aging and anti-immunocompromised medicines or products |
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