Construction method of double-layer cell model for evaluating 222 nm ultraviolet damage and application thereof

Abstract

The application discloses a method for constructing a double-layer cell model for evaluating 222nm ultraviolet damage and application thereof, and belongs to the technical field of cell models. The method comprises the following steps: inoculating cells on the bottom of a culture plate as a lower cell layer; inoculating cells in a culture plate coated with a temperature-sensitive medium at the same time, and culturing to form an upper cell sheet; transferring the upper cell sheet to above the lower cell layer, and stacking to form a double-layer cell model; using a 222nm deep ultraviolet light source with or without a filter to irradiate the model at a set dose; and detecting the activity of the upper cell sheet and the lower cell layer respectively by using an MTT method, and calculating the relative survival rate. The model simulates the layered structure of biological tissues by physical stacking, and uses the 222nm deep ultraviolet light source with or without a filter to irradiate, so that the penetration biological effects of different wave bands of ultraviolet on cells and the protective effect of the filter can be systematically and directly evaluated.

Description

Technical Field

[0001] This invention belongs to the field of cell model technology, specifically relating to a method for constructing a bilayer cell model for evaluating 222nm ultraviolet damage and its application. Background Technology

[0002] Ultraviolet (UV) radiation disinfection technology is a physical method that uses ultraviolet light to inactivate microorganisms. It is currently widely used in drinking water treatment, air purification, food processing, and medical disinfection. The core principle of this disinfection technology is to use UV light of a specific wavelength (usually in the 200-280nm band) to interfere with the replication of microorganisms' DNA and RNA, and to destroy protein activity to prevent replication and reproduction, thereby inactivating pathogenic microorganisms.

[0003] With the continuous development of ultraviolet (UV) disinfection technology, the requirements for disinfection effectiveness should not only focus on efficiency, but also on ensuring human safety while achieving highly efficient disinfection. Traditional UV disinfection (such as 254nm) can effectively kill pathogens, but its high-energy photons can penetrate human cells and damage the skin and eyes, requiring its use in unoccupied environments. In recent years, with advancements in UV disinfection technology, 222nm UV light has gained widespread attention due to its safety and effectiveness. 222nm UV light not only effectively kills bacteria and viruses, but also has extremely weak penetrating power in the air, thus enabling real-time disinfection in occupied environments, allowing for "human-machine coexistence." It is particularly suitable for high-contact locations such as hospitals, schools, and airports, significantly reducing the risk of airborne diseases.

[0004] In recent years, KrCl excimer lamps with a wavelength of 222nm have been used as ultraviolet light sources. The 222nm ultraviolet light they produce has a good effect on inactivating and inhibiting the growth of pathogens such as bacteria and viruses. However, it should be noted that in addition to emitting ultraviolet light with a main wavelength of 222nm, KrCl excimer lamps also produce some other wavelengths of ultraviolet light with lower intensity, which can cause some harm to the human body. Therefore, filters are needed to remove these stray waves to reduce the damage to the human body.

[0005] Currently, 3D cell structures based on scaffold-free self-assembling cell sheet technology are used in drug screening, organoid construction, and disease modeling, overcoming the technical challenge of low survival rates in single-cell transplantation while avoiding the potential risks of traditional scaffold materials. Traditional monolayer cell models have a limited structure and struggle to realistically simulate the penetration characteristics and tissue damage effects of 222nm ultraviolet radiation. Therefore, constructing bilayer cell models can, to some extent, compensate for the low simulation fidelity and significant discrepancies between evaluation results and in vivo findings in monolayer models, providing a more reliable in vitro research system for evaluating their biosafety and damage mechanisms.

[0006] 222nm ultraviolet light generated by KrCl excimer lamps shows good potential for pathogen inactivation, but its emission spectrum contains stray wavelengths, requiring filters to reduce potential biological risks. Traditional animal models are time-consuming and have low throughput, while cell models have the advantages of clear mechanisms and high efficiency, allowing direct evaluation of ultraviolet radiation-induced cell damage and the protective effect of test substances through cell detection. To understand the difference in cell damage caused by 222nm deep ultraviolet light with and without filters and to provide a theoretical basis for protecting against ultraviolet skin damage, it is urgent to establish a model evaluation method for ultraviolet radiation-induced cell damage. Summary of the Invention

[0007] The purpose of this invention is to provide a method for constructing a bilayer cell model for evaluating 222nm ultraviolet damage and its application, so as to provide a certain theoretical basis for understanding the difference in cell damage caused by 222nm deep ultraviolet light source with and without filter and for protecting against ultraviolet skin damage.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] The method for constructing a bilayer cell model for evaluating 222nm ultraviolet damage includes the following steps:

[0010] Step 1: Prepare a single-cell suspension using culture medium containing 10% fetal bovine serum, and culture it in DMEM high-glucose medium in an incubator for routine incubation. After several passages, during the logarithmic growth phase, digest the cells with 0.25% trypsin digestion solution, passage again, and then culture at 3.0 × 10⁻⁶ cells / year. 5 ~5.0×10 5 Cells were seeded at a density of 1 cell / mL in 24-well plates containing temperature-sensitive medium, with each well containing 1 mL. After seeding, the 24-well plates were incubated in an incubator until the logarithmic growth phase, yielding a single cell sheet.

[0011] Step 2: Using a pipette, remove the cell sheets and stack them in another 24-well plate containing a cell layer, forming a two-layer cell model. Divide the different 24-well plates into a blank control group and experimental groups with different UV irradiation doses.

[0012] Step 3: Place the 24-well plate in a biosafety cabinet equipped with a 222nm UV lamp. Preheat the cabinet for 15 minutes before irradiating the cells to stabilize the emitted light intensity. Place the cell-inoculated 24-well plate under the UV lamp. By controlling the irradiation time, different irradiation doses can be achieved to obtain cell models treated with different irradiation doses.

[0013] Step 4: After irradiation, UV irradiation damage indicators of the upper and lower cells are detected simultaneously, and the relative cell viability is determined using the MTT staining method.

[0014] As an improvement of the present invention, the cells are any one or a combination of at least two of the following: mouse fibroblast 3T3 cells, human immortalized epidermal keratinocytes HaCaT cells, human dermal fibroblasts HSF cells, human skin fibroblasts HDF cells and BJ cells, and human foreskin fibroblasts HFF cells.

[0015] As an improvement of the present invention, the temperature-sensitive medium in the 24-well plate is one of poly(N-isopropylacrylamide)PNIPAM and poly(N-isovinylcaprolactam)PNVCL. As an improvement of the present invention, the complete culture medium consists of DMEM medium, 10% fetal bovine serum (FBS) and 1% streptomycin-penicillin (P / S).

[0016] As an improvement of the present invention, in step one, the cells of the upper cell sheet may be the same as or different from the cells of the lower cell layer.

[0017] As an improvement of the present invention, the cell culture process in step one is as follows: take the frozen cells, place them in a 37°C water bath to thaw, then transfer them to a centrifuge tube containing complete culture medium, centrifuge, resuspend the precipitate, transfer them to a cell culture dish, and place them in a cell culture incubator for culture.

[0018] As an improvement of the present invention, the cell culture environment in the cell culture chamber in step one is set to 37°C, 5% CO2 and pH 7.2-7.4.

[0019] As an improvement of the present invention, the cell density in step one is 4.0 × 10⁻⁶. 5 per mL.

[0020] As an improvement of the present invention, the 222nm deep ultraviolet light fixture in step three is divided into two types: one with a filter and one without a filter.

[0021] As an improvement of the present invention, the 222nm deep ultraviolet irradiation intensity in step three is set to 0, 7.5, 15, or 30 mJ / cm. 2 .

[0022] As an improvement to this invention, the MTT staining method in step four for detecting relative cell viability works on the principle that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple formazan crystals, which are then deposited in the cells. Dead cells lack this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and its absorbance is measured at a wavelength of 490 nm using an ELISA reader. Within a certain cell number range, the amount of MTT crystals formed is directly proportional to the cell number. The number of live cells is determined based on the measured absorbance value (OD value); the higher the OD value, the stronger the cell activity (or, if measuring drug toxicity, the lower the drug toxicity).

[0023] As an improvement of the present invention, the specific method for detecting the relative cell viability using MTT assay in step four is as follows:

[0024] Step 1: After UV irradiation, aspirate the culture medium from each well, wash with PBS, add 1 mL of fresh culture medium, and continue culturing at 37°C, 5% CO2, and saturated humidity for 1-2 days.

[0025] Step 2: Four hours before the measurement, discard the original culture medium in each well, add a dilution of MTT:DMEM at a volume ratio of 1:9, and continue to incubate under the same conditions.

[0026] Step 3: During the assay, discard the culture medium from each well, add DMSO solution to each well, and shake for 10 minutes to allow the color to develop.

[0027] Step 4: Select a wavelength of 490nm, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well and calculate the viability.

[0028] The beneficial effects of this invention are as follows:

[0029] This study aims to establish a novel cell model of skin UV damage to evaluate the differences in the biological effects of 222nm ultraviolet radiation with and without filters on cells. This model requires no experimental animals, is low-cost, and highly sensitive. It allows for the molecular-level analysis of the mechanisms of UV-induced skin damage and provides an efficient platform for the screening and validation of UV-protective drugs. Compared to traditional animal models, which are time-consuming and have limited throughput, this cell system offers advantages such as a clearly defined mechanism and ease of operation. Attached Figure Description

[0030] Figure 1 The cell viability of a bilayer cell model after irradiation with 222 nm deep ultraviolet light without a filter is shown.

[0031] Figure 2 The cell viability of a bilayer cell model irradiated with 222nm deep ultraviolet light with a filter is shown. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. However, the following embodiments are only for explaining the present invention, and the scope of protection of the present invention should include all the contents of the claims. Moreover, through the description of the following embodiments, those skilled in the art can fully implement all the contents of the claims of the present invention.

[0033] Example

[0034] (1) Cell Culture and Sublayer Preparation: After resuscitation, mouse fibroblasts were placed in DMEM high-glucose complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and cultured routinely in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. When the cell confluence reached 80%-90%, the cells were passaged by digestion with 0.25% trypsin. The cell density was adjusted to 4.0 × 10⁶ cells / year. 5 Cells / mL were seeded into a standard 24-well plate (1 mL per well) and incubated overnight to form a dense lower cell layer.

[0035] (2) Preparation of upper cell sheet: 3T3 cells in the logarithmic growth phase were taken and cultured at the same density (4.0 × 10⁻⁶). 5 Cells (1 mL / well) were seeded into 24-well plates pre-coated with a thermosensitive polymer (such as PNIPAM or PNVCL). The cells were cultured until they fused to form a continuous, dense monolayer.

[0036] (3) Assembly of the bilayer model: Discard the culture medium in the well plate of step (2) and wash gently twice with sterile PBS. Gently peel off the edge with the tip of a pipette and peel off the entire upper cell sheet completely using the temperature response characteristics. Carefully transfer it to the surface of the 24-well plate containing the lower cell layer in step (1), let it settle naturally and adhere to the lower cell layer, and co-culture for 2 hours to construct a bilayer cell model.

[0037] (4) Ultraviolet irradiation treatment: The assembled bilayer cell model was placed in a biosafety cabinet, and deep ultraviolet excimer lamps (150W) with a wavelength of 222nm were used as the irradiation source, with and without filters respectively. The lamps were preheated for 15 minutes before irradiation to ensure stable light intensity. The experiment was divided into four groups: control group (0mJ / cm²) 2 Low-dose group (7.5 mJ / cm) 2 ), medium dose group (15mJ / cm) 2 ) and high-dose group (30mJ / cm 2 Radiation dose can be precisely controlled by adjusting the irradiation time.

[0038] ;

[0039] Radiation dose, Irradiation time, Irradiance.

[0040] (5) MTT assay for cell viability: After irradiation, the plate was returned to the incubator for 24 hours. Then, MTT reagent dilution was added to each well, and incubation continued for 4 hours. The supernatant was carefully aspirated, and an equal volume of DMSO was added to each well. The plate was shaken slowly on a shaker for 10 minutes to fully dissolve the formazan crystals. The absorbance (OD) of each well was measured at 490 nm using a microplate reader. The relative cell viability was calculated using the following formula:

[0041] ;

[0042] Cell viability: relative cell survival rate, OD experiment OD experimental group, OD blank : OD blank group, OD control OD control group.

[0043] The experiment was independently repeated three times (n=3), and the data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using t-tests with SPSS software, and a p-value < 0.05 was considered statistically significant.

[0044] The proportions of the main wavelength band (200~230nm) and clutter wavelength band (235~320nm) of the 222nm ultraviolet light source with and without filters were detected using a CCD spectrometer.

[0045] Table 1: Spectral band ratio of 222nm excimer lamp (without filters)

[0046]

[0047] Table 2: Spectral band ratios of 222nm excimer lamp (with filters)

[0048]

[0049] The proportions of the main and stray bands of the 222nm deep ultraviolet light source were measured using a spectrometer. The results, shown in Tables 1 and 2, indicate that after adding the filter, the relative spectral power of the 200-230nm main band significantly increased from 87.9% to 98.6%, while the total proportion of the 235-320nm stray band (including 235-280nm and 280-320nm) decreased sharply from 9.2% to 0.6%. This data demonstrates that the filter effectively suppresses stray ultraviolet radiation, concentrating the effective radiation of the light source at the bactericidal peak near 222nm, greatly improving the purity and targeting of the light source, thereby significantly reducing the risk of photochemical and biological damage to the human body and cells caused by stray radiation.

[0050] Within an irradiation distance of 0–250 cm, the irradiance of an ultraviolet light source with a filter was measured using an ultraviolet power meter, and the radiation dose under these conditions was calculated.

[0051] Table 3: Irradiance test results of 222nm deep ultraviolet light source at different heights with and without filter

[0052]

[0053] The irradiance of a 222nm deep ultraviolet light source at different distances was measured using an ultraviolet power meter. The results are shown in Table 3. The addition of a filter has a highly significant attenuation effect on the radiation intensity of the 222nm deep ultraviolet light source. At 0cm vertically below the light source, the light intensity with the filter added is 1376μW / cm². 2 (4890μW / cm) only without a filter 2 The radiation intensity with and without filters was approximately 28.1% lower than that without filters at all test heights (0–250 cm). This attenuation decreased synchronously with increasing distance, consistent with the physical diffusion law of ultraviolet radiation. This confirms that filters not only remove stray light but also significantly reduce the output dose of the dominant wavelength ultraviolet light, further verifying their crucial role in improving the safety of light source use at the physical level.

[0054] The damaging effects of 222nm deep ultraviolet light with and without filters on a bilayer cell model were evaluated using MTT staining. The results are as follows: Figure 1 and Figure 2As shown, 222nm deep ultraviolet light significantly inhibited the survival rate of the upper cell layer in the bilayer cell model in a dose-dependent manner, while having a smaller effect on the lower cell layer. Crucially, after irradiation with a filter, the survival rates of both the upper and lower cell layers remained at a high level without a significant dose-dependent decrease. These results indicate that the filter can effectively shield harmful stray light and significantly reduce the penetrating and killing effect of 222nm deep ultraviolet light on cells, confirming its role in improving ultraviolet biosafety.

[0055] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for constructing a bilayer cell model for evaluating 222nm ultraviolet damage, characterized in that, Includes the following steps: Step 1: Seed cells at the bottom of the culture plate as the lower layer; at the same time, seed cells at the bottom of the culture plate containing a temperature-sensitive medium as the upper cell sheet; Step 2: Stack the cell sheet obtained in Step 1 with the culture plate inoculated with the lower layer of cells to construct a two-layer cell model; Step 3: Irradiate the bilayer cell model using a 222nm deep ultraviolet light source with or without a filter; Step 4: The activity of cells after irradiation was detected by the MTT assay, and the relative cell viability was calculated to evaluate the differences in cell damage under different ultraviolet conditions.

2. The method according to claim 1, characterized in that, The cells mentioned in step one are selected from any one or a combination of at least two of the following: mouse fibroblast 3T3 cells, human immortalized epidermal keratinocytes HaCaT cells, human dermal fibroblasts HSF cells, human skin fibroblasts HDF cells, BJ cells, and human foreskin fibroblasts HFF cells.

3. The method according to claim 1, characterized in that, In step one, the cells in the upper cell sheet may be the same as or different from the cells in the lower cell layer.

4. The method according to claim 1, characterized in that, The temperature-sensitive medium mentioned in step one is poly(N-isopropylacrylamide)PNIPAM or poly(N-isovinylcaprolactam)PNVCL.

5. The method according to claim 1, characterized in that, In step three, the ultraviolet light irradiation dose is set to 0 mJ / cm. 2 7.5 mJ / cm 2 15mJ / cm 2 and 30mJ / cm 2 At least one of them.

6. The method according to claim 1, characterized in that, In step three, the filter is used to filter out stray ultraviolet rays in the 235-320nm band, thereby increasing the spectral power ratio of the 200-230nm main band to over 98%.

7. The method according to claim 1, characterized in that, In step four, the specific steps of the MTT method include: continuing to culture cells after irradiation, adding MTT diluent for incubation, then adding DMSO to dissolve formazan crystals, measuring the absorbance value at a wavelength of 490 nm, and calculating the survival rate according to the following formula: relative cell survival rate (%) = (OD test group - OD blank group) / (OD control group - OD blank group) × 100%.

8. A bilayer cell model constructed by the method of any one of claims 1-7.

9. The application of the double-layer cell model of claim 8 in evaluating the protective effect of a filter against cell-penetrating damage caused by 222nm ultraviolet radiation.

10. The application according to claim 9, characterized in that, The application is to evaluate the effect of filters on reducing ultraviolet cell damage by comparing the survival rate difference between the upper and lower cells in the bilayer cell model after irradiation with or without filters using a 222nm deep ultraviolet light source.

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