AU2020104108A4 - Method for in Vitro Construction of Cell Photoaging Model - Google Patents
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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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- G01—MEASURING; TESTING
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- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
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Abstract
The invention discloses a method for in vitro construction of a cell
photoaging model, belonging to the field of medicine and cell biology. The
specific steps include: obtaining the primary skin fibroblast from newborn mice,
and culturing them with DMEM+10% FBS in vitro; (2) carrying out passage
when mouse skin fibroblasts grow to 80%, and allowing the cells to grow
adherently for 24 hours; then conducting the first medium-wave ultraviolet
radiation, followed by 4 times of repeated irradiation; after the last irradiation for
24-72 hours, the mouse skin fibroblast photoaging model is obtained. In the
present invention, mouse-derived cells are used to obtain a large number of
primary cells at one time, which avoids the shortcomings of repeated freezing and
insufficient activity of the cell lines. In addition, the cell activity is good when
cultured in vitro and will not interfere with the senescence phenotype induced by
UVB within a limited number of generations; and the molding process takes a
short time, only 2 days; the effect is stable, and the senescence phenotype can be
observed after the last irradiation for 24 hours.
Description
Method for in Vitro Construction of Cell Photoaging Model
The invention belongs to the field of medicine and cell biology, in particular to a
method for in vitro construction of a cell photoaging model.
The aging of human body is divided into endogenous aging and exogenous
aging. The former is a natural procedural process, regulated by genes and influenced
by family genetics; the latter is mainly affected by external environmental factors and
lifestyle, in which the ultraviolet radiation in the sun is the main factor, so we call the
latter photoaging. In the process of photoaging, the ECM components of the skin
undergo significant changes, such as the degradation of collagen in the fibrous
scaffolds, the deposition of abnormal elastic fibers, the change of fibronectin (FN) in
the adhesive components and hyaluronan (HA) in the gelatinous matrix. The changes
in these ECM components will affect the normal function of the cells, which appear
as rough, thickening skin, large wrinkles and pigmented patches in appearance.
As one of the main components of human tissue, ECM plays an important role
in maintaining the morphological structure and functional integrity of cells. It is a
reticulate structure composed of large molecule such as protens and polysaccharides
distributed outside the cell, including three major categories, namely, fibrous
scaffolds (Type I collagen and elastin), adhesive components (non-collagen
glycoproteins) and gelatinous matrix (Glycosaminoglycans and proteoglycans). Type
I collagen (80%) is the most important filler, providing the skin with a full
appearance and proper stretchability; and elastin (4%) can keep the skin elastic. After
the action of exogenous aging-promoting factors, Type I collagenous fibers are
mostly degraded into amorphous fragments, and are reduced in contents. While
elastin tends to be denatured and deposited, and a large amount of denatured elastin
tissue can be observed in the photoaged skin and deposited in the dermis. Due to the
lack of ordered structure and the absence of skin elasticity, the skin is generally
characterized by sagging and rough texture. Fibroblast (FB), as the main cell
component in the dermis, has the functions of secreting ECM and regulating local
microenvironment. It not only synthesizes matrix components such as collagen fiber
and elastic fiber, but also secretes cytokines and matrix metalloproteinase (MMPs)
and participates in the remodeling of ECM. Therefore, more and more attention has
been paid to the role of FB in the aging process. Under physiological conditions,
fibroblasts are embedded in the ECM network structure, forming multicellular
complexes with keratinocytes, endothelial cells and macrophages. They respond to
cytokines by secreting matrix components and MMPs, leaving ECM in dynamic
equilibrium. With the growth of age, this equilibrium is broken. The senescent
fibroblasts undergo changes in morphology and metabolism, and are characterized by
weakened response to cytokines, increased levels of intracellular stress, and
decreased ability to secrete matrix components, while producing more MMPs in
ECM to participate in the degradation of ECM.
Ultraviolet exposure accounts for 80% among exogenous aging-promoting
factors. The ultraviolet in sunlight can be divided into three types, namely, long-wave
(UVA), medium-wave (UVB), and short-wave (UVC) ultraviolet light. Among them,
with a wavelength of 320~275 nm, UVB is the most active part of ultraviolet
biological effect, and has very strong erythema reaction, so that it can penetrate the
epidermis, reach the dermis, and cause the senescence of dermal fibroblasts. The in
vitro experiments have confirmed that UVB can be absorbed by pigment cells and
photosensitizers to produce Reactive Oxygen Species (ROS), which will damage
DNA and activate a series of signal pathways related to cell proliferation,
differentiation and senescence.
In order to study the mechanism of photoaging induced by UVB, a plurality of
authors used the method of culturing FB in vitro and irradiating it with UVB to
simulate ultraviolet exposure in daily life. Straface et al. cultured human WI-38
fibroblast cell lines in vitro, and used the single UVB exposure method to induce the
photoaging model. However, according to previous reports, the single irradiation
dose is difficult to control, and greatly depends on the cell state. When the dose is too
high, the cells will start the process of apoptosis and will not show senescence; when
the dose is too low, the cells can recover to normal state by their own repair
mechanism. Similar problems have arisen in our exploration, that is, it is difficult to
obtain a stable senescence phenotype through single irradiation. In the meantime,
photoaging is caused by long-term ultraviolet exposure, which is different from the
effect obtained by single irradiation. Single irradiation tends to cause acute injury,
just like the skin erythema effect observed clinically, and will not cause photoaging.
The main point of the so-called photoaging is the cumulative effect, that is, the
long-term and repeated exposure to UVB. Based on this idea, Debacq-Chainlaux et al.
successfully induced a plurality of senescence phenotypes by repeatedly exposing the
human AG04431 fibroblast cell lines to UVB. The reason why this method is
difficult to popularize is that first, cell lines tend to fluctuate in activity during
long-term transportation, and repeated freezing and resuscitation will also change cell
activity; second, these cell lines belong to limited cell lines, and are prone to
shortening telomeres during repeated passages, and they will appear senescence
phenotype along with passage, thus interfering with the identification of
ultraviolet-inducedsenescencephenotype.
In order to solve the existing problems, the invention provides an in vitro
construction method of a cell photoaging model.
In order to achieve the above objective, the present invention provides the
following scheme:
The invention provides an in vitro construction method of a cell photoaging
model, which is characterized in that, it comprises the following steps:
(1) The primary skin fibroblast cells are obtained from the newborn mice and
cultured with DMEM+10% fetal bovine serum in vitro;
(2) The passage is carried out when the mouse skin fibroblasts grow to 80%, and
after the adherent growth of the cells for 24 hours, the medium-wave ultraviolet
irradiation is repeated 4 times, and then the mouse skin fibroblasts photoaging model
is obtained after the last irradiation for 24-72 hours.
Further, the newborn mice in Step (1) are C57BL/6 mice born 18-24 hours.
Further, the passage number of mouse skin fibroblasts in Step (2) is 1-3
generations.
Further, the irradiation is repeated once every 12 hours in Step (2).
Further, in Step (2), the culture solution containing 1% FBS is used for culture
after the first 3 irradiation, and the mouse fibroblasts after the last irradiation are
cultured in a petri dish containing DMEM+10% FBS.
Further, in Step (2), the irradiation method is to suck off the cell culture solution
and cover it with a thin layer of colorless buffer solution, and place it 30cm below the
UVB lamp, in which the UVB lamp emits medium-wavelength ultraviolet light with
an emission peak value of 311 nm.
Further, the irradiation dose is 120 mJ/cm2 in Step (2).
Further, in Step (2), the irradiation is started when the degree of fusion of mouse
skin fibroblasts is greater than or equal to 50%.
The invention discloses the following technical effects:
The mouse-derived cells are used to obtain a large number of primary cells at
one time, which avoids the shortcomings of repeated freezing and insufficient activity
of the cell lines. The cell activity is good when cultured in vitro and will not interfere
with the UVB-induced senescence phenotype within a limited passage number; the
molding process takes a short time, only 2 days; the effect is stable, and the
senescence phenotype can be observed after the last irradiation for 24-72 hours.
Figure 1 is a quantitative study of the mRNA expression of ECM-associated proteins after exposure to 120 mJ/cm 2 of UVB for 4 times.
The embodiments of the present invention are further illustrated below in
combination with the appended figures, which shall not be considered as a limitation
of the present invention but shall be construed as a more detailed description of
certain aspects, characteristics and embodiments of the present invention.
It shall be understood that the terms described herein are for the purpose of
describing particular embodiments only and are not intended to limit the present
invention. In addition, for the numerical range in the present invention, it should be
understood that each intermediate value between the upper limit and the lower limit
of the range is also specifically disclosed. Each smaller range between intermediate
values within any stated value or stated range and any other stated value or
intermediate values within the stated range is also included within the present
invention. The upper and lower limits of these smaller ranges may be independently
included within or excluded from the range.
Unless otherwise stated, all technical and scientific terms used herein have the
same meaning as commonly understood by those skilled in the art in the field of the
present invention. Although the present invention describes only preferred methods
and materials, any methods and materials similar or equivalent to those described
herein may be used in the practice or testing of the present invention. All documents
mentioned in this specification are incorporated by reference for the purpose of
disclosing and describing methods and/or materials related to the documents. The contents of this specification shall prevail in the case of any conflict with any incorporated document.
Various modifications and variations can be made in the specific embodiments
of the present specification without departing from the scope or spirit of the invention,
which will be apparent to those skilled in the art. Other embodiments derived from
the specification of the invention will be apparent to those skilled in the art. The
specification and embodiments of the present application are only exemplary.
As used herein, terms "comprising", "including", "having", "containing" and the
like are all open terms, which mean including but not limited to.
EMBODIMENT 1
Isolation and Culture of Mouse dermal fibroblasts (MDFs)
The cells were derived from C57BL/6 inbred mice born 18-24 hours, and the
mice with red skin and normal peristalsis should be selected.
(1) The mouse was placed in a petri dish on ice, and the killed mouse was
immersed in iodine for 2 minutes. The steps were repeated twice. The suckling mouse
was immersed in demonized water to remove the iodophor, and then immersed in 70%
ethanol for 2 minutes. The steps were repeated twice. Thereafter, the petri dish was
placed on the ice.
(2) The tweezers and scissors were used to cut off the limbs and tail of the
suckling mouse, and then the skin along the direction of the spine was cut slowly and
carefully with scissors along the tail gap, with attention not to cut too hard to cause
the massive bleeding of the internal organs. After cutting to the head, the two sides of
the skin were torn along the opposite direction respectively with tweezers, and some of the adhered tissues were cut away with scissors, and finally the whole skin was torn off and the skin containing the beard on the skin was removed.
(3)The peeled skin was placed in a petri dish on ice, and then the subcutaneous
connective tissue and blood vessels were slowly removed with tweezers. Thereafter,
the skin was placed in a pre-cooled PBS (with double antibodies, penicillin and
streptomycin) solution for 30 minutes, then the skin slice was placed in another
sterile petri dish, with the dermis facing down, spreading out as much as possible.
The pre-cooled 0.1% of pancreatic enzyme solution was added carefully add slowly
to the petri dish to make the skin float over the pancreatin solution, and after covering
it with a lid, the petri dish was transferred to a 4-degree refrigerator for digestion
overnight.
(4) The epidermal tissue was peeled off from the skin with tweezers, and the
remaining dermal tissue was cut into small pieces of 1 cmx1 cm with ophthalmic
scissors on a super-clean bench. The dermal tissue was sucked into a conical flask
containing a stirrer with a straw, and then placed on the ice and digested for 20
minutes after adding 0.1% of Type I collagenase, then the conical flash was shake
gently every 5 minutes.
(5) The conical flash was placed in a constant temperature (37 °C or so)
magnetic stirrer for 30 minutes, then the cell supernatant was collected and
transferred to a 15ml centrifuge tube, and centrifuged to discard the supernatant.
Thereafter, the cell was resuspended with a fresh medium and then collected to a new
ml centrifuge tube.
(6) The 5ml of 0.06 pancreatin solution was added to the tissue blocks in the conical flask, and then the solution was digested on a magnetic stirrer for 5 minutes.
Thereafter, the supernatant was placed on ice and removed. These steps were
repeated 3 times.
(7) About 3ml of DMEM medium containing 10% FBS was added to the
supernatant containing the pancreatin solution to stop the digestion, and after the
centrifugation at 1500rpm at room temperature for 10 minutes, the cell precipitation
was resuspended with 1-2ml of DMEM medium containing 10% FBS and recollected
into a new 15ml centrifuge tube.
(8) The above Step (5), (6) and (7) were repeated until the skin tissue
disappeared.
(9) All cells collected by resuspension was centrifuged at 1500 rpm for 8
minutes.
(10) After the supernatant was removed and the cell precipitation was
resuspended with fresh medium, the cells were inoculated into T25 culture flask and
cultured in a cell incubator at 37 °C for 1 hour.
(11) After the differential velocity adherent, the cells in the supernatant were
discarded, and the DMEM medium containing 10% FBS was added to continue the
culture. The passage was carried out when the mouse skin fibroblasts grew to 80%,
and after the adherent growth of the cells for 24 hours, the first irradiation was started.
The 40 pm filter mesh can be used to remove impurities during the passage.
Preferably, the passage 1-3 was used to build the model.
EMBODIMENT 2
UVB irradiation
(1) Preparation of UVB lamps: aseptic disinfection and ultraviolet sterilization
was carried out to the ultraviolet lamps capable of emitting medium-wave ultraviolet
(280-320nm).
(2) The measurement of UVB irradiation dose: the two sides of the lamp was
installed with a centrifuge tube holder respectively with the distance of 30cm away
from the plane of the ultra-clean bench. After adjusting the height of the ultraviolet
lamp, the light source was turned on to keep the output light intensity stable, and the
dose of UVB (the unit displayed on the instrument is mJ/cm 2 -s) was measured by
Lutron UV light meter (Lutron, Taiwan), and then the required irradiation time was
calculated according to the measured data (the irradiation time = 120/UVB measured
dose (with the calculated value in seconds).
(3) UVB irradiation: when the fusion degree of mouse skin fibroblasts with
passage 1 to 3 prepared in Embodiment 1 was 50%, the culture solution was removed,
a thin layer of PBS was covered, the lid was opened and then the fibroblasts were
placed directly below the UVB lamp for the first irradiation with the irradiation dose
of 120 mJ/cm 2 . After the irradiation, PBS was removed, and DEME culture solution
was added to continue the culture, that is, irradiated once 12 hours, a total of 4 times.
After each irradiation, the culture medium containing 1% FBS was used to continue
the culture; and the DMEM+10% FBS culture was carried out after the last
irradiation.
(4) Senescence phenotype detection: The senescence phenotype can be detected
after the last irradiation for 24-72 hours.
(a) The toxic effects of UVB on cells were dose-dependent
After the irradiation of MDF with different doses, the proliferation rate slowed
down with the increase of irradiation dose, and the cell activity was significantly
different from that of the control group at 120 mJ/cm2 . When the dose reached 150
2 mJ/cm or above, the number of apoptotic cells observed increased and the cell
density decreased obviously. Thus, we will use 120 mJ/cm 2 as the dose of induction
model.
(b) The UVB irradiation raised the expression level of senescence-related
proteins p53 and p21.
Both P53 and p21 are proteins that play an important role in the process of cell
cycle regulation. Because senescence itself means growth arrest, it also inevitably
leads to cell cycle arrest, so in the process of senescence, p53, p21 and other cell
cycle regulatory proteins will appear abnormal changes, which can be used as
molecular markers of senescence. In order to verify whether UVB-induced dermal
fibroblasts have senescence or not, it is found by detecting changes in the protein
content of p53 and p21 that the protein levels of p53 and p21 increased in a
dose-dependent manner with the increase of UVB irradiation dose.
(c) Secretion and degradation of ECM: As shown in Figure 1, the real-time
quantitative PCR confirmed that the mRNA expression of type I collagen and elastin
decreased significantly, while MMP-1 that degraded ECM increased by fold.
Through the above example, it can be confirmed that after 4 times of repeated
UVB irradiation with a dose of120mJ/cm 2 , the cells may have stable senescence
phenotype, including cell cycle arrest and decreased secretion function, etc., which is
consistent with the corresponding changes of fibroblasts during photoaging. The above results indicate that repeated subcellular toxicity dose of UVB irradiation can effectively avoid the shortcomings of single irradiation, such as cell death caused by too high dose, or too low dose, and the cells can repair themselves and return to their normal state. At the same time, using MDFs can also avoid the decrease of cell activity and difficulty to purchase during the transportation and frozen storage of limited cell lines. MDFs are easy to obtain, easy to amplify in vitro, and only take 7 hours to obtain primary cells. The model induced by this method can express a series of senescence phenotypes stably, and the induction time is short, only 2 days, which can accelerate the experiment process greatly.
The invention provides a feasible technical system for applying the cell
photoaging model. Using the convenient method provided by this technique can
effectively construct a cell photoaging model in vitro. The model provided by the
present invention can also be used to study the mechanism of photoaging induced by
UVB, such as how UVB produces ROS, which signal pathway causes the final
senescence phenotype; and it can also be used to test the effectiveness of
anti-photoaging drugs.
The above-described embodiments merely describes the preferred embodiments
of the present invention and are not intended to limit the scope of the present
invention, and various modifications and improvements thereof made by those of
ordinary skilled in the art without departing from the spirit of the design of the
present invention shall fall within the scope of protection defined by the claims.
Claims (8)
1. A method for in vitro construction of a cell photoaging model, which is
characterized in that it comprises the following steps:
(1) The primary skin fibroblast cells are obtained from the newborn mice and
cultured with DMEM+10% fetal bovine serum in vitro;
(2) The passage is carried out when the mouse skin fibroblasts grow to 80%, and
after the adherent growth of the cells for 24 hours, the medium-wave ultraviolet
irradiation is repeated 4 times, and then the mouse skin fibroblast photoaging model
is obtained after the last irradiation for 24-72 hours.
2. The in vitro construction method described in Claim 1, which is characterized
in that the newborn mice in Step (1) are C57BL/6 mice born 18-24 hours.
3. The in vitro construction method described in Claim 1, which is characterized
in that the passage number of mouse skin fibroblasts in Step (2) is 1-3 passages.
4. The in vitro construction method described in Claim 1, which is characterized
in that the irradiation is repeated once every 12 hours in Step (2).
5. The method of in vitro construction according to Claim 1 or 4, which is
characterized in that the culture solution containing 1% FBS is used for culture after
the first three irradiation in Step (2); and the mouse fibroblasts after the last
irradiation are cultured in a petri dish containing DMEM+10% FBS.
6. The in vitro construction method as described in Claim 1, which is
characterized in that, in Step (2), the irradiation method is to suck off the cell culture
solution and cover it with a thin layer of colorless buffer, and place it 30cm below the
UVB lamp, wherein the UVB lamp emits medium-wavelength ultraviolet light with an emission peak of 311 nm.
7. The in vitro construction method described in Claim 1, which is characterized
in that, in Step (2), the irradiation dose is 120 mJ/cm2
.
8. The in vitro construction method described in Claim 1, which is characterized
in that, in Step (2), the irradiation is started when the degree of fusion of mouse skin
fibroblasts is greater than or equal to 50%.
-1/1-
Figure 1
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Cited By (1)
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| WO2023221853A1 (en) * | 2022-05-18 | 2023-11-23 | 上海珈凯生物股份有限公司 | Method for constructing senescent cell and method for evaluating anti-senescence efficacy |
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| WO2023221853A1 (en) * | 2022-05-18 | 2023-11-23 | 上海珈凯生物股份有限公司 | Method for constructing senescent cell and method for evaluating anti-senescence efficacy |
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