WO2023221853A1 - 一种衰老细胞的构建方法及评价抗衰老功效的方法 - Google Patents

一种衰老细胞的构建方法及评价抗衰老功效的方法 Download PDF

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WO2023221853A1
WO2023221853A1 PCT/CN2023/093502 CN2023093502W WO2023221853A1 WO 2023221853 A1 WO2023221853 A1 WO 2023221853A1 CN 2023093502 W CN2023093502 W CN 2023093502W WO 2023221853 A1 WO2023221853 A1 WO 2023221853A1
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cells
uva
generation
senescent
culture
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French (fr)
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周利丹
卢伊娜
高雅
熊玥
施雪梅
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上海珈凯生物股份有限公司
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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
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0626—Melanocytes
    • 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
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0629—Keratinocytes; Whole skin
    • 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
    • C12N5/0656—Adult fibroblasts
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5038—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving detection of metabolites per se

Definitions

  • the invention relates to the technical field of cell biology, and specifically to a method for constructing senescent cells and a method for evaluating anti-aging efficacy.
  • UVR ultraviolet radiation
  • Cellular senescence is a prerequisite and final driver of the physiological aging process and is essentially invisible during much of its progression.
  • Cellular senescence is a cellular state stimulated by stress signals and exists in specific physiological processes, linking physiological and stress signals to tissue homeostasis and tissue health. It can be initiated by various cellular stresses, such as DNA damage, oncogene activation, oxidative stress or exogenous toxicant exposure.
  • Cell senescence is not the same as cell death. Aged cells still maintain metabolic activity for a period of time and will also undergo some obvious changes. They have four typical characteristics: cell cycle arrest, aging-related secretory phenotype, macromolecule damage and metabolic disorder. .
  • the evaluation methods used for skin aging mainly include clinical evaluation, recombinant three-dimensional dermal epidermal skin model evaluation, and routine in vitro cell evaluation.
  • the advantage of clinical evaluation is that it is closest to the real effect.
  • the disadvantages are as follows: 1) It requires proprietary clinical instrument evaluation equipment such as VISIA skin detector, skin microscope and active skin surface analysis system Visioscan VC98, skin ultrasonic detector, and skin elasticity detector. , Antera 3D skin imaging measuring instrument and other equipment; these instruments and equipment are generally expensive and require dedicated personnel to operate; 2) At least three testing personnel are required, one of whom is a clinician; 3) The number of effective subjects is required to be at least 30.
  • the clinical evaluation experiment period is relatively long, and it generally takes about 6 months from confirmation to final results; 5) It cannot be conducted through throughput, and generally only one sample can be tested at a time, which is not suitable for screening or cosmetic raw material testing; 6) The cost of testing is relatively expensive, and generally the cost of a sample may be more than 100,000 yuan; 7) The clinical evaluation error is large , only when the sample effect is extremely strong can relatively clear results be obtained; 8) Clinical evaluation safety risks are high.
  • the advantage of the evaluation of the recombinant three-dimensional dermis-epidermis skin model is that it is close to the clinical effect, but its disadvantages are as follows: 1) The cost of testing is relatively high, generally a sample costs at least 30,000 yuan; 2) The experimental cycle is long, and general laboratories do not have recombination The three-dimensional dermal epidermal skin model needs to be purchased, and it usually takes at least 3 months from confirmation to data result acquisition; 3) cannot be used for high-throughput screening; 4) the aging period is not clear, and the most vigorous period of the recombinant three-dimensional dermal epidermal skin model is often It is used to evaluate the anti-aging effect of aging products; and the anti-aging effect of the product cannot be subdivided; 5) Since the current commercialized recombinant dermal epidermal skin model is less stable, the cost of trial and error is relatively high.
  • the advantages of conventional in vitro cell evaluation methods are as follows: 1) High-throughput screening is possible; 2) The detection cycle is short, generally 7 tasks are required from confirmation to obtaining data results, and skilled operation may require 5 working days; 3) Few detection personnel are required : Generally, one sample requires only one professional tester; 4) The test cost is low; generally the test cost for 10 samples is about 10,000 yuan; 5) The error is minimal; 6) The test results are stable, the same processing method, different personnel The test results are stable.
  • the purpose of the present invention is to provide a method for constructing senescent cells and a method for evaluating anti-aging efficacy.
  • the present invention provides a method for constructing exogenous senescent cells, including the following steps:
  • A1 Inoculate the primary cells in complete culture medium and culture them for one week. During this period, the cells are changed twice, and the cells are irradiated with ultraviolet light during each medium change to obtain UVA-P1 generation cells;
  • UVA-P1 generation cells for the primary cells in step A1 and perform the same treatment to obtain UVA-P2 generation cells;
  • step A3 Repeat step A2 to obtain the corresponding UVA-P3 ⁇ UVA-Pn generation cells; among them, n in UVA-Pn It is a positive integer from 4 to 20;
  • UVA-P1 ⁇ UVA-Pn generation cells obtained in the previous steps, and use the UVA-P3 ⁇ UVA-P4 generation cells as early senescent cells, UVA -P5 ⁇ UVA-P7 generation cells were used as mid-stage senescent cells, and ⁇ UVA-P8 generation cells were used as late-stage senescent cells.
  • the primary cells are selected from any one of human primary fibroblasts, human primary keratinocytes, and human primary melanocytes;
  • the seeding density of the primary cells is 1.2 ⁇ 10 5 to 6 ⁇ 10 5 /dish;
  • the culture conditions were 37°C and 5% CO2 .
  • the specific steps for changing the medium are: discard the old culture medium, wash it once with PBS buffer, then add PBS buffer, use a UVA irradiator to irradiate the cell culture dish closely for 10 minutes, and irradiate.
  • the irradiation dose is 14.4J; after the irradiation, discard the PBS buffer, add fresh complete culture medium, and continue culturing;
  • the medium replacement was performed on the third and fifth days of culture.
  • step A4 the COL I content, MMP-1 content, and cell number of the early senescent cells, mid-term senescent cells, and late senescent cells are as shown in the following table:
  • the present invention provides a method for evaluating the anti-aging efficacy of skin, which includes the following steps:
  • UVA-P3 ⁇ UVA-P4 generation cells prepared by the above method are evaluated in the early clinical aging population of 25 to 29 years old, and the UVA-P5 ⁇ UVA-P7 generation cells are evaluated in the clinical mid-stage aging population of 30 to 45 years old.
  • ⁇ UVA- P8 generation cells are evaluated in clinically advanced aging people aged 46 years and above;
  • step S4 Detect the cells cultured in step S3, and use the cells cultured without the sample to be tested as a control group to evaluate the anti-aging effect of the sample to be tested.
  • the seeding density of the cells is 5 ⁇ 10 4 to 6 ⁇ 10 5 /well;
  • the cell culture plate is any one of a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, and a 96-well plate;
  • steps S2 and S3 the culture conditions are both culture at 37°C and 5% CO2 .
  • the detection indicators are cell cycle, DNA amount, and ATP;
  • the detection indicators are COL I, MMP-1/-3, ATP, SA- ⁇ -gel, Hes-1, 8-OHdG, GLS1, and ⁇ H2AX;
  • the detection indicators are cell number, COL I, ⁇ H2AX, MMP-3, Hes-1, and 8-OHdG.
  • the present invention provides a method for evaluating the anti-aging effect of a test substance on senescent cells at different stages, including the following steps:
  • step a2 Substitute P1 generation cells for the primary cells in step a1 and perform the same treatment to obtain P2 generation cells;
  • step a2 Repeat step a2 to obtain the corresponding P3 ⁇ Pn generation cells; where n is a positive integer from 11 to 20;
  • step b Combine the P3 to P4 generation cells, P5 to P7 generation cells, P8 to P10 generation cells, ⁇ P11 generation cells prepared in step a and the UVA-P3 to UVA-P4 generation cells, UVA-P5 to UVA prepared in claim 1 -P7 generation cells and ⁇ UVA-P8 generation cells were seeded on cell culture plates and cultured for 24 hours;
  • step d Test the cells cultured in step c, and use the cells cultured without the sample to be tested as a control group to evaluate the anti-aging effect of the sample to be tested on each cell.
  • the seeding density of the primary cells is 1.2 ⁇ 10 5 to 6 ⁇ 10 5 /dish;
  • the specific steps of changing the medium are: discard the old medium, wash it once with PBS buffer, then add fresh complete medium, and continue culturing; the medium change is performed on the third and fifth days of culture;
  • step a4 the COL I content, MMP-1 content, and cell number of the early senescent cells, mid-term senescent cells, and late senescent cells are as shown in the following table:
  • step b the seeding density of the cells is 5 ⁇ 10 4 to 6 ⁇ 10 5 /well;
  • the cell culture plate is any one of a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, and a 96-well plate;
  • the culture conditions are both culture at 37°C and 5% CO2 .
  • the detection indicators are cell cycle, DNA amount, and ATP;
  • the detection indicators are COL I, MMP-1/-3, ATP, SA- ⁇ -gel, Hes-1, 8-OHdG, GLS1, and ⁇ H2AX;
  • the detection indicators are cell number, COL I, ⁇ H2AX, MMP-3, Hes-1, and 8-OHdG;
  • the detection indicators are cell cycle, DNA amount, and ATP;
  • the detection indicators are cell cycle, DNA amount, and ATP;
  • the detection indicators are COL I, MMP-1/-3, ATP, SA- ⁇ -gel Hes-1, 8-OHdG, GLS1, and ⁇ H2AX;
  • the detection indicators are cell number, COL I, ⁇ H2AX, MMP-3, Hes-1, and 8-OHdG.
  • the present invention has the following beneficial effects:
  • the present invention simulates exogenous senescent (photoaging) cells by simultaneously using passage and direct ultraviolet (UVA) irradiation, and by detecting indicators of each senescent cell, the constructed senescent cells are divided into early senescent cells, Mid-stage senescent cells, late-stage senescent cells, and by correlating senescent cells in each stage with the skin aging stages of people of different ages, thereby establishing the corresponding relationship between the generation number of senescent cells and the age of the population, thereby using the constructed senescent cells in different stages as clinical aging In vitro replacement cells come Carry out corresponding anti-aging evaluation. This provides an objective basis for the evaluation of the effects of active ingredients such as cosmetics on early, middle and late aging wrinkles, etc., and provides an innovative, accurate and alternative clinical cytology method for testing skin aging.
  • UVA direct ultraviolet
  • the anti-aging efficacy evaluation results using the senescent cell model constructed in the present invention have a strong correlation with the clinical evaluation results and have fewer false positive results. Compared with the clinical evaluation method, the test cost is lower, the repeatability is high, and the test results are low. The advantages of short cycle and high screening throughput.
  • the present invention further constructs exogenous (photoaging) senescent cells and endogenous (natural senescence) senescent cells at different stages, and can accurately evaluate the effect of the test substance on endogenous or exogenous senescent cells at different stages. function, providing an experimental basis for the precise research and development of anti-aging products.
  • Figure 1 is the detection results of endogenous senescent cells and exogenous senescent cells of different generations constructed in Examples 1 and 2; wherein, Figure 1A is the COL I content result; Figure 1B is the MMP-1 content result; Figure 1C is the cell Quantitative results; the Aging group represents endogenous senescent cells, and the UVA-Aging group represents exogenous senescent cells;
  • Figure 2 shows the pro-proliferation effect of senescent cells at different stages in Example 3;
  • Figure 3 shows the results of using a skin ultrasound instrument to detect changes in the hyperechoic areas of the dermis in people of different age groups in Example 4;
  • Figure 4 is the evaluation results of mitochondrial membrane integrity of senescent cell models at different stages in Example 4.
  • Figure 5 shows the SA- ⁇ -SA evaluation results of senescent cell models at different stages in Example 4.
  • Figure 6 shows the results of changes in the hyperechoic area of the dermis in the mid-term clinical skin aging model using the Biomenta sample with a concentration of 3% in Example 4.
  • Fibroblasts are the most abundant cell type in the dermis and contribute to skin aging directly or indirectly through interactions with other cells. They are also cells frequently used to study skin aging in vitro. Human fibroblasts are cells with strong functional activities. The cells and nuclei are large and clear in outline. The nuclei are large and obvious. The cytoplasm is weakly basophilic and has obvious protein synthesis and secretion activities.
  • the present invention establishes an in vitro human primary fibroblast aging model by studying the commonalities between natural aging and photoaging of human primary fibroblasts in terms of cell cycle, aging-related secretory phenotypes, macromolecule damage, metabolic disorders, etc. .
  • Fibroblast senescence is closely related to the occurrence and development of skin aging. Fibroblasts are the most important cells that constitute the dermal layer of the skin. Their dysfunction is closely related to most skin phenotypic changes, such as wrinkle formation. Therefore, dermal fibroblasts have become the first choice for studying endogenous and exogenous aging at the cellular level. Model. The primary human fibroblasts used in the following examples were isolated from human foreskin tissue in our laboratory. Donor information: Male, 20 years old. Cell identification is based on functional expression of fibroblast collagen/MMP-1/MMP-3 and transcriptome gene comparison. Cell number G2018001FF.
  • the complete culture medium for fibroblasts used in the following examples is: a mixed solution containing 10% newborn calf serum, 1% penicillin, streptomycin and amphotericin B, and the balance is DMEM basic culture medium.
  • This embodiment provides a method for constructing an endogenous aging (natural aging) cell model. The specific steps are as follows:
  • P0 generation cells were seeded in a 10cm cell culture dish at a density of 6 ⁇ 10 5 /dish and cultured, labeled as P1 generation cells; the medium used was complete fibroblast medium.
  • steps 2)-4) on the P1 generation cells obtained after the aforementioned subculture to obtain P2 generation cells, and repeat this to obtain P3-P14 generation cells.
  • P1-P14 generation cells were seeded on a 96-well cell culture plate at 2 ⁇ 10 4 /well, and cultured in a 37°C, 5% CO 2 incubator for 24 hours.
  • the culture medium used was the aforementioned fibroblasts. Complete culture medium. After culture, the cells obtained were subjected to medium replacement, specifically: after discarding the culture medium, add fresh fibroblast complete culture medium to the culture plate, and culture it in a 37°C, 5% CO2 incubator for 48 hours.
  • step 6 Based on the test results in step 6), divide the aforementioned P1-P14 generation cells into growth phase cells (P3-P4 generation cells), early senescent cells (P5-P7 generation cells), and mid-stage cells according to the range shown in Table 1. Senescent cells (P8-P10 generation cells), late senescent cells (P10-P14 generation cells).
  • This embodiment provides a method for constructing an exogenous aging (photoaging) cell model. The specific steps are as follows:
  • P0 generation cells Take human primary fibroblasts numbered G2018001FF (hereinafter referred to as P0 generation cells);
  • UVA-P1 generation cells were seeded in a 10cm cell culture dish at a density of 6 ⁇ 10 5 /dish and cultured, labeled as UVA-P1 generation cells; the culture medium used was fibroblast complete culture medium, which contained 10% newborn calf serum, 1% penicillin/streptomycin/amphotericin mixed solution, and the remainder is DMEM basic medium.
  • the obtained UVA-P1 generation cells were cultured and passaged in steps 2)-4) to obtain UVA-P2 generation cells, and repeated to obtain UVA-P3 to UVA-P14 generation cells.
  • UVA-P1 Inoculate UVA-P1 to UVA-P14 generation cells at 2 ⁇ 10 4 /well on a 96-well cell culture plate, and culture them in a 37°C, 5% CO 2 incubator for 24 hours.
  • the culture medium used is as mentioned above. of fibroblast complete culture medium. After culture, the cells obtained are subjected to medium replacement, specifically: after discarding the culture medium, add fresh fibroblast complete culture medium to the culture plate. Cultivate in a 37°C, 5% CO2 incubator for 48h.
  • step 6 Based on the test results of step 6), divide the aforementioned UVA-P1 ⁇ UVA-P14 generation cells into early senescent cells (UVA-P3 ⁇ UVA-P4 generation cells) and mid-stage senescent cells according to the range shown in Table 2 (UVA-P5 ⁇ UVA-P7 generation cells), late senescent cells (UVA-P8 ⁇ UVA-P14).
  • This embodiment provides a method for evaluating the anti-aging effect of a test substance on senescent cells at different stages.
  • the test sample is VC, which includes the following steps:
  • Example 1 Take the P4 generation cells (growth phase cells), P7 generation cells (early senescent cells), P9 generation cells (middle senescent cells), P12 generation cells (late senescent cells) prepared in Example 1, and the UVA prepared in Example 2 -P4 generation cells (early senescent cells), UVA-P7 generation cells (middle senescent cells), UVA-P9 generation cells (late senescent cells), inoculate each at 2 ⁇ 10 4 /well (or 0.8 ⁇ 10 5 /dish) Cultivate on a 96-well cell culture plate (or 60mm cell culture dish) in a 37°C, 5% CO2 incubator for 24 hours.
  • the culture medium used was fibroblast complete culture medium.
  • step 2) After the culture in step 1), discard the old culture medium, add fresh detection medium without test samples to the culture plate as a control group, and add fresh detection medium containing test samples (VC at a concentration of 10 ⁇ g/mL).
  • the culture medium was used as the experimental group and continued to be cultured for 48 hours in a 37°C, 5% CO2 incubator.
  • the detection medium contains 1% newborn calf serum, 1% penicillin/streptomycin/amphotericin B mixed solution, and the balance is DMEM basic medium.
  • step 2) After the culture in step 2), detect the corresponding indicators of the entry and exit of senescent cells at each stage.
  • the specific detection indicators of each cell are shown in Table 3.
  • COL I and MMP-1/-3 Elisa kits can be used to complete the detection according to the standard operating procedures of the Elisa kits.
  • Cells seeded in 96-well plates can be tested for ATP according to the standard operating procedures of the ATP kit.
  • the specific MitoBright LT fluorescent probe can be used to detect mitochondrial membrane integrity according to the standard operating procedure of the kit.
  • the DNA amount can be detected according to the standard operating procedure of the Hoechest33342 kit.
  • METTL3, GLS1, and 8-OHdG can be detected according to the standard operating procedure of immunofluorescence.
  • SA- ⁇ -gel detection can be carried out according to the standard procedure of SA- ⁇ -gel kit; cells seeded in 60mm cell culture dishes can be carried out according to the standard operating procedure of flow cytometry detection of cell cycle. Cell cycle detection.
  • VC has a significant pro-proliferation effect on cells in the growth phase, but the pro-proliferation effect on early senescent cells is poor, and it has basically no effect on mid-term senescent cells and late senescent cells. Proliferation effect.
  • exogenous aging photosenescence
  • VC has a more obvious pro-proliferation effect on early senescent cells, but has a poor pro-proliferation effect on mid-term senescent cells, and has no pro-proliferative effect on late senescent cells.
  • Figure 2 shows the ATP detection results of P4 generation cells, P7 generation cells, UVA-P4 generation cells and UVA-P7 generation cells with or without VC.
  • test sample is Biomenta (produced by Shanghai Jiakai Biotechnology Co., Ltd., and is Bifidobacterium adolescentis obtained using the method in the patent document CN114292773A strain lysate), including the following steps:
  • Example 1 Use the P7 generation cells (early senescent cells) and P9 generation cells (mid-term senescent cells) prepared in Example 1, and the UVA-P4 generation cells (early senescent cells) and UVA-P7 generation cells (mid-term senescent cells) prepared in Example 2. cells), inoculate 2 ⁇ 10 4 /well (or 0.8 ⁇ 10 5 /dish) on a 96-well cell culture plate (or 60mm cell culture dish), and culture in a 37°C, 5% CO 2 incubator for 24 hours. The culture medium used was fibroblast complete culture medium.
  • Biomenta at a volume concentration of 0.625% to 5% has a certain inhibitory effect on mid-term senescent cells, but Biomenta has no obvious effect on early-stage senescent cells, and the effect of Biomenta on mid-term exogenous senescent (photoaging) cells is better than that of mid-term endogenous senescent cells. The effect is more obvious on senescent (naturally aging) cells.
  • test method was approved by the ethics committee and the volunteers signed an informed consent form.
  • UVA-P3 ⁇ UVA-P4 generation cells obtained by the method of Example 2 can be used as in vitro replacement cells for early clinical aging (26-30 years old) for early aging evaluation; UVA-P5 ⁇ UVA- P7 generation cells can be used as in vitro replacement cells for clinical mid-term aging (31 to 45 years old) for mid-term aging evaluation; UVA-P8 or greater than UVA-P8 generation cells can be used as an in vitro replacement for clinical late-stage aging (46 years old or older) Cells were evaluated for late senescence.

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Abstract

公开了一种衰老细胞的构建方法及评价抗衰老功效的方法，该衰老细胞的构建方法包括将原代细胞接种培养和传代培养，并在接种培养和传代培养中进行紫外光照射，得到UVA-P1～UVA-Pn代细胞，并根据各代细胞的COL I含量、MMP-1含量、细胞数量检测结果，将各代细胞分为早期衰老细胞、中期衰老细胞和晚期衰老细胞，可用于评价皮肤抗衰老功效。通过构建的各时期衰老细胞与不同年龄人群所处的皮肤衰老时期进行关联，建立衰老细胞代数与人群年龄的对应关系，构建的不同时期衰老细胞可作为临床衰老的体外替代细胞来进行相应的抗衰老评价。

Description

一种衰老细胞的构建方法及评价抗衰老功效的方法 技术领域
本发明涉及细胞生物学技术领域，具体涉及到一种衰老细胞的构建方法及评价抗衰老功效的方法。
背景技术
每个人从出生起就经历衰老，皮肤衰老是机体衰老最明显和最直观的表现，可以作为预期寿命和健康的预测指标。与人体其他器官不同，皮肤衰老不仅不可避免地受到内在因素的影响，而且还受到各种外在环境因素的影响，加速衰老，尤其是紫外线辐射(UVR)。其中，皮肤受到紫外线辐射、吸烟、风吹、日晒及有害化学物质等环境因素导致的皮肤衰老称为外源性衰老，其中由于日光中紫外线辐射是环境因素中导致皮肤衰老的主要因素，所以外源性衰老又称为光老化。内源性衰老和外源性衰老均可导致皮肤结构完整性降低、功能丧失和外观的累积变化，如皱纹增多、松弛、弹力纤维增生、毛细血管扩张和皮肤色素沉着异常。目前，虽然这两个皮肤衰老过程不仅均是高度复杂和尚未完全理解的，而且均是缺乏通用的生物标志物来明确检测和评估的，但是目前研究均认为皱纹是皮肤衰老的外在表现，内部细胞衰老才是根源。
细胞衰老是生理衰老过程的先决条件和最终驱动力，在其大部分进展过程中基本上是看不见的。细胞衰老是一种由压力信号刺激产生，存在于特定生理过程的细胞状态，将生理和应激信号与组织内稳态和组织健康联系起来。可由各种不同的细胞应激出发，如DNA损伤、癌基因激活、氧化应激或外源性毒物暴露等。细胞衰老不等同于细胞死亡，衰老的细胞在一段时间内仍保持代谢活性，也会出现一些明显的变化，具有典型的细胞周期停滞、衰老相关分泌表型、大分子损伤及代谢紊乱四个特征。
目前对于皮肤衰老使用的评价方法主要有临床评价、重组三维真皮表皮皮肤模型评价、常规体外细胞评价。
临床评价的优点是最接近于真实效果，缺点如下：1)需要专有的临床仪器评价设备如VISIA皮肤检测仪、皮肤显微镜及活性皮肤表面分析系统Visioscan VC98、皮肤超声检测仪、皮肤弹性检测仪、Antera 3D皮肤成像测量仪等设备；这些仪器设备一般价格昂贵且需要专有人员操作；2)需要的检测人员至少有三名，其中一位为临床医生；3) 需要有效受试者人数至少30例，受试者难招募，而且不同的化妆品针对的衰老表现可能不同；4)临床评价实验周期比较长，一般从确定进行到最终结果获得需要6个月左右；5)不能通量进行，一般一次只能测试一个样品，不适合筛选或化妆品原料测试；6)检测费用比昂贵，一般一个样品的费用可能在10万元人民币以上；7)临床评价误差很大，样品效果极强时才能获得比较明确结果；8)临床评价安全风险大。
重组三维真皮表皮皮肤模型评价优点是与临床效果比较接近，但其缺点如下：1)检测费用比较高，一般一个样品的费用至少为3万元人民币；2)实验周期长，一般实验室无重组三维真表皮皮肤模型，需要购买，一般从确定进行到数据结果获得至少3个月；3)不能用于高通量筛选；4)衰老时期不明确，常将重组三维真表皮皮肤模型最旺盛时期用于评价衰老产品的抗衰效果；且不能将产品抗衰效果细分；5)由于目前商品化重组真表皮皮肤模型稳定性的较少，因此试错成本比较高。
常规体外细胞评价方法优点如下：1)可高通量筛选；2)检测周期短，一般从确定进行到数据结果获得需要7个工作，熟练操作可能需要5个工作日；3)需要检测人员少：一般一个样品需要一个专业检测人员即可；4)检测费用低；一般10个样品的检测费用约1万元人民币；5)误差最小；6)检测结果稳定，同样的处理方式，不同的人员检测，结果稳定。但目前的体外细胞评价方法仍存在下述缺点：1)与临床评价结果相关度相对低；2)衰老时期不明确，可能将年轻细胞用于评价衰老产品的抗衰老；3)无明确细分自然老化和光衰老体外细胞评价方法，同样一个样品，采用不同处理方式评价效果可能不一样。
发明内容
针对现有技术的不足，本发明的目的是提供一种衰老细胞的构建方法及评价抗衰老功效的方法。
本发明的目的是通过以下技术方案实现的：
第一方面，本发明提供了一种外源性衰老细胞的构建方法，包括以下步骤：
A1、将原代细胞接种于完全培养基中培养一周，期间对细胞进行2次换液，且每次换液过程中对细胞进行紫外光照射，得UVA-P1代细胞；
A2、将UVA-P1代细胞代替步骤A1的原代细胞进行相同的处理，得UVA-P2代细胞；
A3、重复步骤A2，得到相应的UVA-P3～UVA-Pn代细胞；其中，UVA-Pn中的n 为4～20的正整数；
A4、将前述步骤获得的UVA-P1～UVA-Pn代细胞进行COL I含量、MMP-1含量、细胞数量检测，并根据检测结果将UVA-P3～UVA-P4代细胞作为早期衰老细胞、UVA-P5～UVA-P7代细胞作为中期衰老细胞、≧UVA-P8代细胞作为晚期衰老细胞。
优选地，步骤A1中，所述原代细胞选自人原代成纤维细胞、人原代角质形成细胞、人原代黑色素细胞中的任一种；
所述原代细胞的接种密度为1.2×105～6×105/皿；
所述培养的条件为在37℃、5％CO2条件下培养。
优选地，步骤A1中，所述换液的具体步骤为：弃旧培养基，使用PBS缓冲液清洗一遍，然后再加入PBS缓冲液，使用UVA辐照仪紧贴细胞培养皿进行照射10min，辐照剂量为14.4J；照射结束后，弃PBS缓冲液，加入新鲜完全培养基，继续培养；
所述换液在培养的第三天和第五天进行。
优选地，步骤A4中，所述早期衰老细胞、中期衰老细胞、晚期衰老细胞的COL I含量、MMP-1含量、细胞数量如下表所示：
第二方面，本发明提供了一种评价皮肤抗衰老功效的方法，包括以下步骤：
S1、以前述方法制备的UVA-P3～UVA-P4代细胞评价25～29岁的临床早期衰老人群、UVA-P5～UVA-P7代细胞评价30～45岁的临床中期衰老人群、≧UVA-P8代细胞评价46岁及以上的临床晚期衰老人群；
S2、将选取的细胞接种于细胞培养板上培养24h；
S3、弃旧培养基，向细胞培养板中加入含或不含待测样品的新鲜完全培养基，继续培养24-96h；
S4、对步骤S3培养后的细胞进行检测，以不含待测样品培养的细胞作为对照组，评价待测样品的抗衰老功效。
优选地，步骤S2中，所述细胞的接种密度为5×104～6×105/孔；
所述细胞培养板为6孔板、12孔板、24孔板、48孔板、96孔板中的任一种；
步骤S2和S3中，所述培养条件均为37℃、5％CO2下培养。
优选地，步骤S4中，当培养的细胞为UVA-P3～UVA-P4代细胞时，检测指标为细胞周期、DNA量、ATP；
当培养的细胞为UVA-P5～UVA-P7代细胞时，检测指标为COL I、MMP-1/-3、ATP、SA-β-gel、Hes-1、8-OHdG、GLS1、γH2AX；
当培养的细胞为≧UVA-P8代细胞时，检测指标为细胞数量、COL I、γH2AX、MMP-3、Hes-1、8-OHdG。
在本发明前期的实验中发现，基于不同衰老时期细胞采用不同的特定检测指标，可使检测结果更准确。
第三方面，本发明提供了一种评价待测物对不同时期衰老细胞的抗衰老功效的方法，包括以下步骤：
a.构建内源性衰老细胞
a1、将原代细胞接种于完全培养基中培养一周，期间对细胞进行2次换液，得P1代细胞；
a2、将P1代细胞代替步骤a1的原代细胞进行相同的处理，得P2代细胞；
a3、重复步骤a2，得到相应的P3～Pn代细胞；其中，n为11～20的正整数；
a4、将前述步骤获得的P1～P n代细胞进行COL I含量、MMP-1、细胞数量检测，并根据检测结果将P3～P4代细胞作为生长期细胞、P5～P7代细胞作为早期衰老细胞、P8～P10代细胞作为中期衰老细胞、≧P11代细胞作为晚期衰老细胞；
b.将步骤a制备的P3～P4代细胞、P5～P7代细胞、P8～P10代细胞、≧P11代细胞和权利要求1制备的UVA-P3～UVA-P4代细胞、UVA-P5～UVA-P7代细胞、≧UVA-P8代细胞分别接种于细胞培养板上培养24h；
c.弃旧培养基，向细胞培养板中加入含或不含待测样品的新鲜完全培养基，继续培养24-96h；
d.对步骤c培养后的细胞进行检测，以不含待测样品培养的细胞作为对照组，评价待测样品对各细胞的抗衰老功效。
优选地，步骤a1中，所述原代细胞的接种密度为1.2×105～6×105/皿；
所述换液的具体步骤为：弃旧培养基，使用PBS缓冲液清洗一遍，然后加入新鲜完全培养基，继续培养；所述换液在培养的第三天和第五天进行；
步骤a4中，所述早期衰老细胞、中期衰老细胞、晚期衰老细胞的COL I含量、MMP-1含量、细胞数量如下表所示：
步骤b中，所述细胞的接种密度为5×104～6×105/孔；
所述细胞培养板为6孔板、12孔板、24孔板、48孔板、96孔板中的任一种；
步骤b和c中，所述培养条件均为37℃、5％CO2下培养。
优选地，步骤d中，当培养的细胞为UVA-P3～UVA-P4代细胞时，检测指标为细胞周期、DNA量、ATP；
当培养的细胞为UVA-P5～UVA-P7代细胞时，检测指标为COL I、MMP-1/-3、ATP、SA-β-gel、Hes-1、8-OHdG、GLS1、γH2AX；
当培养的细胞为≧UVA-P8代细胞时，检测指标为细胞数量、COL I、γH2AX、MMP-3、Hes-1、8-OHdG；
当培养的细胞为P3～P4代细胞时，检测指标为细胞周期、DNA量、ATP；
当培养的细胞为P5～P7代细胞时，检测指标为细胞周期、DNA量、ATP；
当培养的细胞为P8～P10代细胞时，检测指标为COL I、MMP-1/-3、ATP、SA-β-gel Hes-1、8-OHdG、GLS1、γH2AX；
当培养的细胞为≧P11代细胞时，检测指标为细胞数量、COL I、γH2AX、MMP-3、Hes-1、8-OHdG。
与现有技术相比，本发明具有如下的有益效果：
1.本发明通过采用传代和紫外线(UVA)直接照射同时发生的方式模拟外源性衰老(光老化)细胞，并通过对各衰老细胞进行指标检测，将构建的衰老细胞分为早期衰老细胞、中期衰老细胞、晚期衰老细胞，并通过各时期衰老细胞与不同年龄人群所处的皮肤衰老时期进行关联，从而建立衰老细胞代数与人群年龄的对应关系，从而将构建的不同时期衰老细胞作为临床衰老的体外替代细胞来 进行相应的抗衰老评价。由此为化妆品等活性成分对早、中、晚期皱纹等衰老功效的评价提供了客观依据，为皮肤衰老的测试提供了一种创新的、精准的、可替代临床的细胞学方法。
2.采用本发明构建的衰老细胞模型进行抗衰老功效评价的结果与临床评价结果的关联性强、假阳性结果少，且相比临床评价的方法具有测试成本较低、可重复性高、测试周期短、筛选通量高的优点。
3.本发明进一步通过构建的不同时期的外源性(光老化)衰老细胞和内源性(自然衰老)衰老细胞，可精准评价待测物对不同时期内源性或外源性衰老细胞的作用，为抗衰老功效产品的精准研发提供了实验基础。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为实施例1和2构建的不同代数内源性衰老细胞和外源性衰老细胞的检测结果；其中，图1A为COL I含量结果；图1B为MMP-1含量结果；图1C为细胞数量结果；其中的Aging组代表内源性衰老细胞，UVA-Aging组代表外源性衰老细胞；
图2为实施例3中不同时期衰老细胞的促增殖效果；
图3为实施例4中采用皮肤超声仪检测不同年龄段人群的真皮层高回声区变化结果；
图4为实施例4中的不同时期衰老细胞模型的线粒体膜完整性评价结果；
图5为实施例4中的不同时期衰老细胞模型的SA-β-SA评价结果；
图6为实施例4中的采用3％浓度的Biomenta样品在临床中期皮肤衰老模型的真皮层高回声区变化结果。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这些都属于本发明的保护范围。
本发明中的词语“优选的”、“优选地”、“更优选的”等是指，在某些情况下可 提供某些有益效果的本发明实施方案。然而，在相同的情况下或其他情况下，其他实施方案也可能是优选的。此外，对一个或多个优选实施方案的表述并不暗示其他实施方案不可用，也并非旨在将其他实施方案排除在本发明的范围之外。
应当理解，除了在任何操作实例中，或者以其他方式指出的情况下，表示例如说明书和权利要求中使用的成分的量的所有数字应被理解为在所有情况下被术语“约”修饰。因此，除非相反指出，否则在以下说明书和所附权利要求中阐述的数值参数是根据本发明所要获得的期望性能而变化的近似值。至少并不是试图将等同原则的适用限制在权利要求的范围内，每个数值参数至少应该根据报告的有效数字的个数并通过应用普通舍入技术来解释。
尽管阐述本发明的广泛范围的数值范围和参数是近似值，但是具体实例中列出的数值尽可能精确地报告。然而，任何数值固有地包含由其各自测试测量中发现的标准偏差必然产生的某些误差。
成纤维细胞是真皮中最丰富的细胞类型，直接或间接通过与其他细胞的相互作用导致皮肤老化，也是体外研究皮肤衰老经常使用的细胞。人成纤维细胞是功能活动旺盛的细胞，细胞和细胞核较大，轮廓清楚，核大而明显，细胞质弱嗜碱性，具有明显的蛋白质合成和分泌活动。本发明通过对人原代成纤维细胞在自然衰老和光衰老在细胞周期、衰老相关分泌表型、大分子损伤、代谢紊乱等方面的共同点研究来建立一种体外人原代成纤维细胞衰老模型。同时，通过一些细胞衰老的指标和人临床衰老指标进行对比建立体外人原代成纤维细胞衰老与临床衰老的关联。通过该方法提高体外评价的结果更接近于临床评价结果，为化妆品等活性成分对衰老的评价提供客观的依据。
成纤维细胞衰老与皮肤衰老的发生发展密切相关。成纤维细胞是构成皮肤真皮层最主要的细胞，其功能障碍和大多数皮肤表型变化，如皱纹形成密切相关，因此，真皮成纤维细胞成为细胞水平研究内源性和外源性衰老的首选模型。以下实施例中使用的人原代成纤维细胞是由本实验室从人包皮组织分离获得，供体信息：男，20岁。细胞鉴定依据成纤维细胞胶原蛋白/MMP-1/MMP-3等功能变现以及转录组基因比对。细胞编号G2018001FF。
以下实施例采用的成纤维细胞完全培养基为：包含10％新生牛血清、1％的青霉素、链霉素和两性霉素B三种的混合溶液、余量为DMEM基础培养基。
实施例1
本实施例提供了一种内源性衰老(自然衰老)细胞模型的构建方法，具体步骤如下：
1)取编号为G2018001FF分离的人原代成纤维细胞按接种密度为7.5×105/T175瓶接种于含成纤维细胞完全培养基的T175瓶中培养7天，所得细胞标记为P0代细胞；
2)第一天，按6×105/皿的密度将P0代细胞接种于10cm细胞培养皿中培养，标记为P1代细胞；所使用培养基为成纤维细胞完全培养基。
3)第三天和第五天，对P1代细胞进行换液，具体操作为：弃旧培养基，使用PBS缓冲液(磷酸盐缓冲液)洗一遍，加入新鲜的成纤维细胞完全培养基，放置37℃、5％CO2条件下进行传代培养。
4)第八天，将P1代细胞进行细胞消化和计数，得到P1代细胞及其细胞数量；所述细胞消化、计数具体操作为：弃旧培养基，使用PBS缓冲液洗两遍，每皿加入2mL0.05％胰酶消化液，37℃消化3min，弃胰酶消化液，再每皿加入5mL完全培养基终止消化，轻轻吹打细胞，得到细胞悬液，直接使用细胞计数仪测量8次，取平均值的细胞数量(见表1)。
5)第八天，将前述传代培养后得到的P1代细胞进行步骤2)-4)的操作，得到P2代细胞，并以此重复，得到P3-P14代细胞。
6)将P1-P14代细胞按2×104/孔各接种于96孔细胞培养板上，于37℃、5％CO2培养箱中培养24h，所使用的培养基为前述的成纤维细胞完全培养基。培养后所得细胞进行换液处理，具体为：弃培养液后，向培养板中加入新鲜成纤维细胞完全培养基，于37℃、5％CO2培养箱中培养48h。培养结束后，收集各培养上清，采用CollagenⅠ(COLⅠ)、MMP-1Elisa试剂盒，按照Elisa试剂盒标准操作流程完成检测，得到各代细胞中COL I含量结果(如图1A所示的Aging组)，MMP-1结果(如图1B所示的Aging组)各代细胞根据步骤4)的方法进行计数，得细胞数量(如图1C所示的Aging组)。
7)根据步骤6)的各检测结果，将前述P1-P14代细胞按照表1所示的范围进行划分为生长期细胞(P3-P4代细胞)早期衰老细胞(P5-P7代细胞)、中期衰老细胞(P8-P10代细胞)、晚期衰老细胞(P10-P14代细胞)。
表1

实施例2
本实施例提供了一种外源性衰老(光衰老)细胞模型的构建方法，具体步骤如下：
1)取编号为G2018001FF人原代成纤维细胞(以下称为P0代细胞)；
2)第一天，按6×105/皿的密度将P0代细胞接种于10cm细胞培养皿中培养，标记为UVA-P1代细胞；所使用培养基为成纤维细胞完全培养基，其包含10％新生牛血清、1％青霉素/链霉素/两性霉素混合溶液、余量为DMEM基础培养基。
3)第三天和第五天，对UVA-P1代细胞进行换液，具体操作为：弃旧培养基，使用PBS缓冲液洗一遍，每皿加入5mL PBS缓冲液，使用UVA辐照仪器中央部位紧贴细胞培养皿，照射10min(辐照剂量为14.4J)。然后弃PBS缓冲液，加入新鲜的成纤维细胞完全培养基，放置37℃、5％CO2条件下进行传代培养。
4)第八天，将UVA-P1代细胞进行细胞消化和计数，得到UVA-P1代细胞及其细胞数量；所述细胞消化、计数具体操作为：弃旧培养基，使用PBS缓冲液洗两遍，每皿加入2mL0.05％胰酶消化液，37℃消化3min，弃胰酶消化液，再每皿加入5mL完全培养基终止消化，轻轻吹打细胞，得到细胞悬液，直接使用细胞计数仪测量8次，取平均值的细胞数量(见表2)。
5)第八天，将得到的UVA-P1代细胞进行步骤2)-4)的培养和传代，得到UVA-P2代细胞，并以此重复，得到UVA-P3～UVA-P14代细胞。
6)将UVA-P1～UVA-P14代细胞按2×104/孔各接种于96孔细胞培养板上，于37℃、5％CO2培养箱中培养24h，所使用的培养基为前述的成纤维细胞完全培养基。培养后所得细胞进行换液处理，具体为：弃培养液后，向培养板中加入新鲜成纤维细胞完全培养基。于37℃、5％CO2培养箱中培养48h。培养结束后，收集各培养上清，采用CollagenⅠ(COLⅠ)、MMP-1Elisa试剂盒，按照Elisa试剂盒标准操作流程完成检测，得到各代细胞中COL I含量结果(如图1A所示的UVA-Aging组)，MMP-1结果(如图1B所示的UVA-Aging组)各代细胞根据步骤4)的方法进行计数，得细胞数量(如图1C所示的UVA-Aging组)。
7)根据步骤6)的各检测结果，将前述UVA-P1～UVA-P14代细胞按照表2所示的范围进行划分为早期衰老细胞(UVA-P3～UVA-P4代细胞)、中期衰老细胞(UVA-P5～UVA-P7代细胞)、晚期衰老细胞(UVA-P8～UVA-P14)。
表2
实施例3
本实施例提供了一种评价待测物对不同时期衰老细胞的抗衰老功效的方法，测试样品为VC,包括以下步骤：
1)取实施例1制备的P4代细胞(生长期细胞)、P7代细胞(早期衰老细胞)、P9代细胞(中期衰老细胞)、P12代细胞(晚期衰老细胞)、实施例2制备的UVA-P4代细胞(早期衰老细胞)、UVA-P7代细胞(中期衰老细胞)、UVA-P9代细胞(晚期衰老细胞)，按2×104/孔(或0.8×105/皿)各接种于96孔细胞培养板(或60mm细胞培养皿)上，于37℃、5％CO2培养箱中培养24h。所使用的培养基为成纤维细胞完全培养基。
2)步骤1)的培养结束后，弃旧培养液，向培养板中加入不含测试样品的新鲜检测用培养基作为对照组，加入含有测试样品(10μg/mL浓度的VC)的新鲜检测用培养基作为实验组，于37℃、5％CO2培养箱中继续培养48h。所述检测用培养基包含1％新生牛血清、1％青霉素/链霉素/两性霉素B混合溶液、余量为DMEM基础培养基。
3)步骤2)的培养结束后，对各时期衰老细胞进出相应指标的检测，具体各细胞检测指标如表3所示。使用收集的上清，可采用COL I、MMP-1/-3Elisa试剂盒，按照Elisa试剂盒标准操作流程完成检测，接种于96孔板中的细胞可按照ATP试剂盒标准操作流程进行ATP检测，可使用特异性MitoBright LT荧光探针按照该试剂盒标准操作流程进行线粒体膜完整性检测，可按照Hoechest33342试剂盒标准流程进行DNA量检测，可按照免疫荧光标准操作流程进行METTL3、GLS1、8-OHdG、γH2AX、Hes-1检测，可按按照SA-β-gel试剂盒标准流程进行SA-β-gel检测；接种于60mm细胞培养皿中的细胞可按照流式细胞术检测细胞周期标准操作流程进行细胞周期检测。
表3

检测结果显示，对于内源性衰老(自然衰老)，VC对于生长期的细胞有较明显的促增殖作用，而对于早期衰老细胞的促增殖效果较差，对于中期衰老细胞和晚期衰老细胞基本无促增殖效果。对于外源性衰老(光衰老)，VC对于早期衰老细胞有较明显的促增殖作用，而对于中期衰老细胞的促增殖效果较差，对于晚期衰老细胞无促增殖效果。图2为P4代细胞、P7代细胞、UVA-P4代细胞和UVA-P7代细胞加或不加VC的ATP检测结果。
实施例4
本实施例提供了一种评价待测物对不同时期衰老细胞的抗衰老功效的方法，测试样品为Biomenta(上海珈凯生物科技有限公司生产，为采用专利文献CN114292773A中方法获得的青春双歧杆菌菌株的溶胞产物)，包括以下步骤：
1)采用实施例1制备的P7代细胞(早期衰老细胞)和P9代细胞(中期衰老细胞)、实施例2制备的UVA-P4代细胞(早期衰老细胞)和UVA-P7代细胞(中期衰老细胞)，按2×104/孔(或0.8×105/皿)各接种于96孔细胞培养板(或60mm细胞培养皿)上，于37℃、5％CO2培养箱中培养24h。所使用的培养基为成纤维细胞完全培养基。
2)24h后，弃旧培养基，向培养板中加入含或不含有Biomenta的新鲜检测用培养基，于37℃、5％CO2培养箱中继续培养48h。培养结束后，按照线粒体检测试剂盒标准流程进行线粒体膜完整性检测以评价线粒体功能。按照SA-β-SA测试剂盒标准流程进行SA-β-SA检测以评价细胞衰老。线粒体检测部分结果(P7代细胞和UVA-P7代细胞)如图4所示，0.078％～5％体积浓度的Biomenta对早期衰老细胞和中期衰老细胞均有一定的抑制作用。其中Biomenta对中期衰老细胞的抑制作用大于早期衰老细胞，而且Biomenta对中期外源性衰老(光老化)细胞的效果比对中期内源性衰老(自然衰老)细胞的效果更明显。SA-β-SA检测部分结果(P7代细胞和UVA-P7代细胞)如图5所示， 0.625％～5％体积浓度的Biomenta对中期衰老细胞有一定的抑制作用，但是Biomenta对早期衰老细胞无明显作用，且Biomenta对中期外源性衰老(光老化)细胞的效果比对中期内源性衰老(自然衰老)细胞的效果更明显。
3)将Biomenta进行抗衰临床效果的检测，具体操作如下：
3.1试验方法经伦理委员会审批，志愿者签署知情同意书
3.2志愿者年龄范围18岁～65岁
3.3通过皮肤超声仪收集志愿者的皮肤数据，并根据检测不同年龄人群的真皮层高回声区(胶原)密度变化情况(如图3所示)，将临床皮肤划分为早期衰老、中期衰老、晚期衰老，并与细胞衰老的早期、中期、晚期细胞代数建立一一对应关系，如表3所示。
表3
3.4以25～55岁的志愿者为测试对象，按照临床评测标准流程，使用皮肤超声仪(为丹麦DermaLab Combo皮肤超声检测仪)对志愿者涂抹3％浓度的Biomenta样品前(O d)和连续涂抹3％浓度的Biomenta样品56天(56d)后进行真皮层高回声区密度(胶原)检测。结果如图6所示，3％浓度的Biomenta对中期衰老皮肤(30-45岁的志愿者)具有最明显的缓解作用。与步骤2)的中期外源性衰老(光老化)细胞结果一致。
本实施例的结果说明，采用实施例2的方法获得的UVA-P3\UVA-P4代细胞可作为临床早期衰老(26～30岁)的体外替代细胞进行早期衰老评价；UVA-P5～UVA-P7代细胞可作为临床中期衰老(31～45岁)的体外替代细胞进行中期衰老评价；UVA-P8或大于UVA-P8代细胞可作为临床晚期衰老(46岁或高于46岁)的体外替代细胞进行晚期衰老评价。
本发明具体应用途径很多，以上所述仅是本发明的优选实施方式。应当指出，以上实施例仅用于说明本发明，而并不用于限制本发明的保护范围。对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进，这些改进也应视 为本发明的保护范围。

Claims (10)

  1. 一种外源性衰老细胞的构建方法，其特征在于，包括以下步骤：
    A1、将原代细胞接种于完全培养基中培养一周，期间对细胞进行2次换液，且每次换液过程中对细胞进行紫外光照射，得UVA-P1代细胞；
    A2、将UVA-P1代细胞代替步骤A1的原代细胞进行相同的处理，得UVA-P2代细胞；
    A3、重复步骤A2，得到相应的UVA-P3～UVA-Pn代细胞；其中，UVA-Pn中的n为4～20的正整数；
    A4、将前述步骤获得的UVA-P1～UVA-Pn代细胞进行COL I含量、MMP-1含量、细胞数量检测，并根据检测结果将UVA-P3～UVA-P4代细胞作为早期衰老细胞、UVA-P5～UVA-P7代细胞作为中期衰老细胞、≧UVA-P8代细胞作为晚期衰老细胞。
  2. 根据权利要求1所述的外源性衰老细胞的构建方法，其特征在于，步骤A1中，所述原代细胞选自人原代成纤维细胞、人原代角质形成细胞、人原代黑色素细胞中的任一种；
    所述原代细胞的接种密度为1.2×105～6×105/皿；
    所述培养的条件为在37℃、5％CO2条件下培养。
  3. 根据权利要求1所述的外源性衰老细胞的构建方法，其特征在于，步骤A1中，所述换液的具体步骤为：弃旧培养基，使用PBS缓冲液清洗一遍，然后再加入PBS缓冲液，使用UVA辐照仪紧贴细胞培养皿进行照射10min，辐照剂量为14.4J；照射结束后，弃PBS缓冲液，加入新鲜完全培养基，继续培养；
    所述换液在培养的第三天和第五天进行。
  4. 根据权利要求1所述的外源性衰老细胞的构建方法，其特征在于，步骤A4中，所述早期衰老细胞、中期衰老细胞、晚期衰老细胞的COL I含量、MMP-1含量、细胞数量如下表所示：

  5. 一种评价皮肤抗衰老功效的方法，其特征在于，包括以下步骤：
    S1、以权利要求1-4任一项方法制备的UVA-P3～UVA-P4代细胞评价25～29岁的临床早期衰老人群、UVA-P5～UVA-P7代细胞评价30～45岁的临床中期衰老人群、≧UVA-P8代细胞评价46岁及以上的临床晚期衰老人群；
    S2、将选取的细胞接种于细胞培养板上培养24h；
    S3、弃旧培养基，向细胞培养板中加入含或不含待测样品的新鲜完全培养基，继续培养24-96h；
    S4、对步骤S3培养后的细胞进行检测，以不含待测样品培养的细胞作为对照组，评价待测样品的抗衰老功效。
  6. 根据权利要求5所述的评价皮肤抗衰老功效的方法，其特征在于，步骤S2中，所述细胞的接种密度为5×104～6×105/孔；
    所述细胞培养板为6孔板、12孔板、24孔板、48孔板、96孔板中的任一种；
    步骤S2和S3中，所述培养条件均为37℃、5％CO2下培养。
  7. 根据权利要求5所述的评价皮肤抗衰老功效的方法，其特征在于，步骤S4中，当培养的细胞为UVA-P3～UVA-P4代细胞时，检测指标为细胞周期、DNA量、ATP；
    当培养的细胞为UVA-P5～UVA-P7代细胞时，检测指标为COL I、MMP-1/-3、ATP、SA-β-gel、Hes-1、8-OHdG、GLS1、γH2AX；
    当培养的细胞为≧UVA-P8代细胞时，检测指标为细胞数量、COL I、γH2AX、MMP-3、Hes-1、8-OHdG。
  8. 一种评价待测物对不同时期衰老细胞的抗衰老功效的方法，其特征在于，包括以下步骤：
    a.构建内源性衰老细胞
    a1、将原代细胞接种于完全培养基中培养一周，期间对细胞进行2次换液，得P1代细胞；
    a2、将P1代细胞代替步骤a1的原代细胞进行相同的处理，得P2代细胞；
    a3、重复步骤a2，得到相应的P3～Pn代细胞；其中，n为11～20的正整数；
    a4、将前述步骤获得的P1～Pn代细胞进行COL I含量、MMP-1、细胞数量检测，并根据检测结果将P3～P4代细胞作为生长期细胞、P5～P7代细胞作为早期衰老细胞、P8～P10代细胞作为中期衰老细胞、≧P11代细胞作为晚期衰老细胞；
    b.将步骤a制备的P3～P4代细胞、P5～P7代细胞、P8～P10代细胞、≧P11代细胞和权利要求1制备的UVA-P3～UVA-P4代细胞、UVA-P5～UVA-P7代细胞、≧UVA-P8代细胞分别接种于细胞培养板上培养24h；
    c.弃旧培养基，向细胞培养板中加入含或不含待测样品的新鲜完全培养基，继续培养24-96h；
    d.对步骤c培养后的细胞进行检测，以不含待测样品培养的细胞作为对照组，评价待测样品对各细胞的抗衰老功效。
  9. 根据权利要求8所述的评价待测物对不同时期衰老细胞的抗衰老功效的方法，其特征在于，
    步骤a1中，所述原代细胞的接种密度为1.2×105～6×105/皿；
    所述换液的具体步骤为：弃旧培养基，使用PBS缓冲液清洗一遍，然后加入新鲜完全培养基，继续培养；所述换液在培养的第三天和第五天进行；
    步骤b中，所述细胞的接种密度为5×104～6×105/孔；
    所述细胞培养板为6孔板、12孔板、24孔板、48孔板、96孔板中的任一种；
    步骤b和c中，所述培养条件均为37℃、5％CO2下培养。
  10. 根据权利要求8所述的评价待测物对不同时期衰老细胞的抗衰老功效的方法，其特征在于，步骤d中，当培养的细胞为UVA-P3～UVA-P4代细胞时，检测指标为细胞周期、DNA量、ATP；
    当培养的细胞为UVA-P5～UVA-P7代细胞时，检测指标为COL I、MMP-1/-3、ATP、SA-β-gel、Hes-1、8-OHdG、GLS1、γH2AX；
    当培养的细胞为≧UVA-P8代细胞时，检测指标为细胞数量、COL I、γH2AX、MMP-3、Hes-1、8-OHdG；
    当培养的细胞为P3～P4代细胞时，检测指标为细胞周期、DNA量、ATP；
    当培养的细胞为P5～P7代细胞时，检测指标为细胞周期、DNA量、ATP；
    当培养的细胞为P8～P10代细胞时，检测指标为COL I、MMP-1/-3、ATP、SA-β-gel Hes-1、8-OHdG、GLS1、γH2AX；
    当培养的细胞为≧P11代细胞时，检测指标为细胞数量、COL I、γH2AX、MMP-3、Hes-1、8-OHdG。
PCT/CN2023/093502 2022-05-18 2023-05-11 一种衰老细胞的构建方法及评价抗衰老功效的方法 WO2023221853A1 (zh)

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