CN115039738A - Application of photonic crystal film in alleviating phototoxicity of nematodes - Google Patents
Application of photonic crystal film in alleviating phototoxicity of nematodes Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
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- Life Sciences & Earth Sciences (AREA)
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses an application of a photonic crystal film in alleviating phototoxicity of nematodes. The nematode is caenorhabditis elegans. The nematode diet is uracil-deficient Escherichia coli (E.coli) OP 50. The illumination mode of the nematodes is as follows: placing the NGM plate which is seeded with bacteria and contains nematodes in an incubator, keeping the temperature at 20 ℃, and illuminating with a full-spectrum light source simulating sunlight; the experiment comprises a control group, an illumination group, a photonic crystal film and an illumination group, wherein the control group is cultured under the dark condition at the temperature of 20 ℃; the light group was maintained for a period of 8 hours light/16 hours dark; the photonic crystal film and the illumination group are directly placed above the NGM board, and the illumination time of the photonic crystal film and the illumination group is kept consistent. The invention proves that the photonic crystal film can slow down the poisoning effect of illumination on nematodes to a certain extent, and provides a theoretical basis for potential application of the photonic crystal film in the aspects of photopoisoning resistance and aging resistance.
Description
Technical Field
The invention relates to the technical field of new application of photonic crystal films, in particular to application of a photonic crystal film in alleviating phototoxicity of nematodes, belonging to new application of a novel material in biology.
Background
Light is the most important energy source in nature, and is divided into ultraviolet light, visible light and infrared light. As a conservative environmental clue, light mediates the physiological functions of the organism and also causes various toxic effects due to long-term and excessive light exposure. Ultraviolet and short-wave blue light have well-known toxic effects on organisms. Ultraviolet radiation is an environmental carcinogen, and acute ultraviolet radiation induces DNA damage, promotes cellular aging, accelerates skin aging, and is carcinogenic. Visible short-wave blue light also showed a toxic effect leading to senescence, and adult drosophila in the 12-hour blue LED light exposure and 12-hour dark cycle showed symptoms of accelerated senescence compared to drosophila raised under constant dark conditions. Visible light also produces oxidative stress in the organism, leading to shortened nematode life and altered nematode development. While an increase in the level of mitochondrial-derived ROS is considered to be one of the possible mechanisms of phototoxic action. Visible light, short-wave visible light blue light and UVA radiation can induce the generation of reactive oxygen species ROS, cause lipid peroxidation and DNA damage, and further mediate cell death. Therefore, the light toxicity is effectively slowed down, and the method plays an important role in maintaining the normal physiological function of the organism.
The Photonic Crystal (PC) is an ordered microstructure, has a photonic band gap, can effectively reflect incident light with specific energy, and is particularly suitable for constructing a light control carrier to promote multiple absorption and utilization of the incident light. The Bragg diffraction is generated by the periodic arrangement of the substances of the photonic crystal, so that the light propagation performance can be regulated and controlled, and the light effect can be effectively improved and enhanced. Caenorhabditis elegans (c. elegans for short), a model organism. Because its genome is highly homologous to the human genome, it has been widely used in genetics, developmental biology, neurobiology, and aging research. In the text, the photonic crystal film is used for regulating and controlling the light intensity, so that the toxic action of light is effectively slowed down, the shortening of the service life of the nematode and the increase of ROS level caused by illumination are reversed, and the biological application field of the photonic crystal film is widened.
Disclosure of Invention
Aiming at the photonic crystal film, the invention provides the application of the photonic crystal film in the alleviation of the phototoxicity of the nematodes and researches the possible mechanism that the photonic crystal film can alleviate the phototoxicity effect of the nematodes.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: the invention relates to an application of a photonic crystal film in alleviating phototoxicity of nematodes.
Further, the nematode is caenorhabditis elegans.
Further, the nematode feed is uracil-deficient escherichia coli (e.coli) OP 50.
Furthermore, the light pattern of the nematodes is: placing the nematode-containing NGM plate with the well-sowed bacteria in an incubator, keeping the temperature at 20 ℃, and illuminating with a full-spectrum plant filling lamp simulating sunlight; photonic crystal thin film + illumination set: a photonic crystal film is placed directly over the NGM slab.
Further, OP50 medium was prepared: lysis Broth solid medium Lysogeny Broth, LB being the main food source for OP50 survival, 1000ml of LB solid medium was prepared: the following reagents were weighed into a 1L Erlenmeyer flask: 10g of trypsin, 10g of NaCl, 5g of yeast extract and 15g of agar (OP50 liquid culture medium does not need to be added), using deionized water to fix the volume to 1L, and performing autoclaving to obtain OP50 culture medium;
synchronizing nematodes: culturing the oviposition nematodes capable of laying eggs and developed to day 1 in a culture medium overnight, removing the female nematodes after 10h to obtain the nematodes in the same period, and culturing in an incubator at 20 ℃ until the nematodes are developed to young adult period for transfer.
Further, the environmental condition settings are: after the nematodes are synchronized, the nematodes are cultured in nematode culture media until adults grow, and after exposure treatment of each group, the adults are cultured in a constant-temperature incubator at 20 ℃.
Further, the photonic crystal film and the illumination group: illumination time: the photonic crystal film + light set was placed directly over the NGM slab with a 8 hour light/16 hour dark cycle per day.
Has the advantages that: the method has simple steps and good effect, and the experiment identifies that the photonic crystal film can slow down the poisoning effect of illumination on the nematode to a certain extent, and the ROS experiment can preliminarily determine the possible mechanism of the photonic crystal film for slowing down the poisoning effect of the nematode.
Drawings
FIG. 1 is a diagram of an experimental apparatus for administration of caenorhabditis elegans according to the present invention.
FIG. 2 is an SEM image of a photonic crystal film used in the present invention.
FIG. 3 is a table of the average lifetime of three experimental groups and a lifetime experimental chart of the present invention after irradiation with caenorhabditis elegans, and it is found that the lifetimes of the photonic crystal film and the irradiation group are higher than those of the direct irradiation group.
FIG. 4 is a graph of the in vivo ROS content of the direct illuminants and photonic crystal films of the invention and illuminants after illumination by C.elegans, showing that the photonic crystal films can reduce ROS levels in nematodes compared to the direct illuminants.
FIG. 5 is a graph of the in vivo ROS content of the direct light and photonic crystal film + light of the present invention after illumination with C.elegans, showing that the photonic crystal film can reduce ROS levels in the nematode relative to the direct light.
Detailed Description
In order to facilitate an understanding of the invention, the invention will now be described more fully hereinafter with reference to the accompanying drawings, in which several embodiments of the invention are shown, but which may be embodied in different forms and not limited to the embodiments described herein, but which are provided so as to provide a more thorough and complete disclosure of the invention.
Example 1
The invention relates to an application of a photonic crystal film in alleviating phototoxicity of nematodes.
The nematode is caenorhabditis elegans.
The nematode feed is uracil-deficient E.coli (E.coli) OP 50.
The illumination mode of the nematodes is as follows: placing the NGM plate which is seeded with bacteria and contains nematodes in an incubator, keeping the temperature at 20 ℃, and illuminating with a full-spectrum plant filling lamp simulating sunlight; the experiment comprises a control group, an illumination group and a photonic crystal film and illumination group, wherein the control group is always cultured under the dark condition at the temperature of 20 ℃; the light groups were maintained for a 8 hour light/16 hour dark cycle per day; and the photonic crystal film and the illumination group are used for directly placing the photonic crystal film above the NGM board, and the illumination time of the photonic crystal film and the illumination group is kept consistent.
Preparation of OP50 medium: lysis Broth solid medium Lysogeny Broth, LB is the main food source for the survival of OP50, 1000ml of LB solid medium was prepared: the following reagents were weighed into a 1L Erlenmeyer flask: 10g of trypsin, 10g of NaCl, 5g of yeast extract and 15g of agar (OP50 liquid culture medium does not need to be added), using deionized water to fix the volume to 1L, and performing autoclaving to obtain an OP50 culture medium;
synchronizing nematodes: placing the oviposition nematodes which have developed to day 1 in a culture medium for culturing overnight, removing the mother nematodes after 10 hours to obtain nematodes in the same period, and culturing in an incubator at 20 ℃ until the nematodes develop to young adult for transfer.
Setting environmental conditions: after the nematodes are synchronized, the nematodes are cultured in nematode culture media until adults grow, and after exposure treatment of each group, the adults are cultured in a constant-temperature incubator at 20 ℃.
The nematode experiments were divided into the following three groups: a first group: black and dark groups; second group: a direct illumination group; third group: a photonic crystal film and an illumination unit. Culturing in dark at 20 deg.C; the light groups were maintained for a period of 8 hours light/16 hours dark; the photonic crystal film and the illumination group are directly placed above the NGM board, and the illumination time of the photonic crystal film and the illumination group is kept consistent.
Test example 1
Experimental materials
Reagent: sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, cholesterol, agar powder, tryptone, yeast powder, peptone, 5-Fluorodeoxyuridine (FUDR) (Sigma).
Experimental and insect strains: uracil-deficient escherichia coli (e.coli) OP 50; wild type strain N2.
An experimental instrument:
AL 104-IC-electronic balance (mettler-toledo instruments), SMZ-168-dissecting microscope (Motic), DK-420-electric heating constant temperature water tank (shanghai forest communication laboratory instruments has (harbin eastern union), ZXSD-81430-biochemical culture (shanghai intelligent honest analytical instruments ltd.), D-1-automatic steam sterilizer (beijing fuenkomao limited), SM206-SOLAR power meter (synneu instrument ltd.), fluorescent microscope (nipwise shun yu instruments ltd.).
The nematode culture method comprises the following steps:
freshly prepared NGM medium containing OP50 was removed in a clean room sterile environment and placed in a clean room to cool for 5 minutes. Several nematodes of the L1 or L2 stage were picked up by a dissecting microscope into NGM medium, and the medium was incubated in an incubator at 20 ℃.
Setting environmental conditions: after the nematodes are synchronized, the nematodes are cultured in a nematode culture medium until adult nematodes are obtained, and the adult nematodes are cultured in a constant-temperature incubator at 20 ℃.
The illumination method of the nematode comprises the following steps: the photonic crystal film is arranged on the nematode culture medium at 0.5-0.7W/m 2 The nematode culture medium was exposed to 8 hours/16 hours daily light under light conditions.
Test example 1
The experimental device is shown in figure 1, the photonic crystal film is arranged on a nematode culture medium, and a light source vertically irradiates right above the culture medium.
Test example 2
As shown in fig. 2, fig. 2 is an SEM image of a photonic crystal thin film used in the present invention.
0.66g AA+110g H 2 O is in N 2 Reacting at 70 deg.C and 300rpm for 30min, adding 2.4g St to the reactant, reacting for 20min, adding 0.09g KPS and 3g H 2 Carrying out O reaction for 5-6h to obtain soap-free PS emulsion; centrifuging the obtained emulsion, washing off surfactant, diluting the 10% solid content emulsion with isovolumetric ethanol at equal ratio, performing hydrophilic treatment on membrane-extracted glass, inclining the membrane-extracted glass sheet on toilet paper, and drying with water to obtain the experimental photonic crystal film, wherein the experimental photonic crystal film is a double-layer photonic crystalA film. The photonic crystal thin film used in the experiment has a double-layer structure as shown by a cross-sectional Scanning Electron Microscope (SEM).
Test example 3
Nematode longevity experiment:
the synchronized nematodes were transferred to the medium supplemented with FUDR as described above, ensuring that 30 nematodes were transferred per medium.
Culturing until adult, transferring nematode to new culture dish with corresponding FUDR every 2-3 days to prevent culture medium contamination. Death data were not taken into account when drilling to the bottom of the culture dish, climbing the wall of the dish, dying by dehydration, no care for loss, etc., but they were recorded as experimental subjects and statistically analyzed.
Analysis of Life test statistical analysis was performed using an on-line application (OASIS; sbi. postech. ac. kr/OASIS), and the above test was independently repeated three times. As shown in FIG. 3, FIG. 3 is a table of the average lifetime of three experimental groups after irradiation with C.elegans of the present invention and a lifetime test chart, and it is found that the lifetimes of the photonic crystal film and the irradiated group are higher than those of the direct irradiated group.
Test example 4
ROS assay for nematodes:
when C.elegans developed to young adult, it was exposed to light. At least 20 C.elegans were taken from each group and treated with NGM plates. After 2 days of illumination, the sections were stained with 50. mu.M DCFH-DA solution, anesthetized with 1mM levamisole, and sectioned with 2% agarose, viewed under a fluorescent microscope, and the images were processed using Image J software. The experiment is divided into an illumination group and a photonic crystal film + illumination group. As shown in FIG. 4, FIG. 4 is a graph of the in vivo ROS content of the direct light group and the photonic crystal film with the light group after the C.elegans of the present invention was given light, and it was found that the photonic crystal film can lower the ROS level in the nematode worm as compared with the direct light group.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. An application of a photonic crystal film in alleviating the phototoxicity of nematodes.
2. Use according to claim 1, characterized in that: the nematode is caenorhabditis elegans.
3. Use according to claim 1, characterized in that: the nematode diet is uracil-deficient Escherichia coli (E.coli) OP 50.
4. Use according to claim 1, characterized in that: the illumination mode of the nematodes is as follows: placing the NGM plate which is seeded with bacteria and contains nematodes in an incubator, keeping the temperature at 20 ℃, and illuminating with a full-spectrum light source simulating sunlight; photonic crystal thin film + illumination set: a photonic crystal film is placed directly over the NGM slab.
5. Use according to claim 4, characterized in that: preparation of OP50 medium: lysis Broth solid medium Lysogeny Broth, LB being the main food source for OP50 survival, 1000ml of LB solid medium was prepared: the following reagents were weighed into a 1L Erlenmeyer flask: 10g of trypsin, 10g of NaCl, 5g of yeast extract and 15g of agar, diluting the mixture to 1L with deionized water, and sterilizing the mixture under high pressure to obtain an OP50 culture medium;
synchronizing nematodes: culturing the oviposition nematodes capable of laying eggs and developed to day 1 in a culture medium overnight, removing the female nematodes after 10h to obtain the nematodes in the same period, and culturing in an incubator at 20 ℃ until the nematodes are developed to young adult period for transfer.
6. Use according to claim 5, characterized in that: setting environmental conditions: after the nematodes are synchronized, culturing the nematodes in a nematode culture medium until the nematodes become adults, and culturing the nematodes in a constant-temperature incubator at 20 ℃.
7. Use according to claim 6, characterized in that: photonic crystal thin film + illumination set: illumination time: the photonic crystal film + light set was placed directly over the NGM slab with a 8 hour light/16 hour dark cycle per day.
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| CN113116871A (en) * | 2021-05-06 | 2021-07-16 | 南京大学 | Application of stilbene compounds in anti-aging small molecule drugs |
| US20210349324A1 (en) * | 2020-05-08 | 2021-11-11 | The Regents Of The University Of California | Multi-lens system for imaging in low light conditions and method |
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