Culture method and verification method of model for evaluating circadian rhythmicity

Abstract

The invention provides a culture method and a verification method of a model for evaluating circadian rhythmicity, and belongs to the field of disorder model establishment and active substance function evaluation. By culturing the nematodes with normal circadian rhythm and the nematodes with disordered circadian rhythm, the model for evaluating circadian rhythm is established, the method is simple and easy to control, the influence of environmental factors on circadian rhythm can be simulated more accurately, and a foundation is laid for establishing a stable and repeatable circadian rhythm disorder evaluation model. Whether the circadian rhythmicity of the nematodes is normal or not is effectively verified through the following method: manufacturing a test board, and placing the nematodes with normal circadian rhythmicity or the nematodes with disordered circadian rhythmicity in a test area; dropwise adding an inducer with a fixed volume at a fixed position, and covering the test plate; counting the number of nematodes in different areas of the plurality of test boards at fixed time intervals; and calculating a chemotaxis index, and carrying out circadian rhythm verification.

Description

Technical Field

[0001] The present application belongs to the technical field of circadian rhythm disorder model evaluation, and specifically relates to a cultivation method and a verification method of a model for evaluating circadian rhythmicity. Background Art

[0002] Circadian rhythm is an internal time regulation mechanism evolved by organisms to adapt to the periodic changes in the environment caused by the rotation of the earth. Almost all life forms, from simple single-celled cyanobacteria to complex mammals, including humans, exhibit this rhythmic characteristic.

[0003] Circadian rhythm affects individual physiological functions, such as sleep-wake cycles, cognitive abilities, and metabolic processes, by regulating a series of gene expression patterns. When external environmental factors (such as shift work, irregular lifestyles, or prolonged exposure to blue light from electronic screens) cause the internal biological clock to lose synchronization with external light conditions, circadian rhythm disorder (CRD) will occur. CRD not only interferes with normal physiological functions, but may also further lead to the occurrence and development of a variety of chronic health conditions such as obesity and neurodegenerative diseases.

[0004] Some researchers have used gene knockout technology to construct rhythm gene knockout mice (such as Bmal1 and Clock knockout mice), and obtained homozygous genotypes through breeding to simulate the model of circadian rhythm disorder. However, this method has problems such as long culture cycle, complex operation, high mortality rate of homozygous mice and high economic cost, which makes it difficult to meet the needs of efficient and economical research and development.

[0005] Therefore, there is an urgent need to obtain a method that can quickly, simply and scientifically evaluate circadian rhythmicity. Summary of the invention

[0006] The purpose of this application is to overcome the defects existing in the related art and provide a method for culturing a model for evaluating circadian rhythmicity, so as to obtain a substance that can quickly, easily and scientifically evaluate circadian rhythmicity and obtain verification.

[0007] In one aspect, the present application provides a method for culturing a model for evaluating circadian rhythmicity, comprising the following steps: a step of culturing nematodes with normal circadian rhythmicity and a step of culturing nematodes with disordered circadian rhythmicity;

[0008] The step of culturing nematodes with normal circadian rhythm comprises:

[0009] The eggs were obtained by simultaneous incubation, and the first-day larvae were obtained 12 hours later and recorded as L1 stage nematodes;

[0010] Primary entrainment: After 12 hours of constant low-temperature culture and then 12 hours of constant high-temperature culture, the first entrainment of nematodes was obtained;

[0011] Secondary entrainment: the first entrained nematodes were cultured in the dark for 12 hours, and then cultured in 400 lux for 12 hours to obtain the second entrained nematodes;

[0012] Three times of entrainment: the second entrainment nematodes are first cultured at 0% humidity for 12 hours, and then cultured at 20% humidity for 12 hours to obtain nematodes No. 1;

[0013] Placing the nematode No. 1 in an incubator at 20° C., 0 lux, and 0% humidity for 24 hours to obtain the nematode No. 2;

[0014] Four times of entrainment: the No. 2 nematode is first cultured in a constant low temperature, constant dark environment, and 0% humidity for 12 hours, and then cultured in a constant high temperature, constant light, and constant humidity for 12 hours;

[0015] The four entrainments were repeated twice to obtain the nematodes with normal circadian rhythm;

[0016] The step of culturing nematodes with disordered circadian rhythms comprises:

[0017] Repeat the steps of culturing nematodes with circadian rhythms,

[0018] The obtained nematodes with circadian rhythm are first exposed to 600 lux light and 14°C low temperature environment for 6 hours, then exposed to 600 lux light and 16°C high temperature stimulation for 6 hours, then exposed to 0 lux dark conditions and 16°C high temperature stimulation for 6 hours, and then exposed to 0 lux dark conditions and 14°C low temperature stimulation for 6 hours to obtain the nematodes with disordered circadian rhythm.

[0019] In an optional embodiment, the constant low temperature range is 13.5-17.0° C., the constant high temperature range is 18.5-22.5° C., the constant illumination range is 380-550 lux, and the constant humidity range is 0-5.4%.

[0020] Furthermore, the constant low temperature range is 15.5-16.5° C., the constant high temperature range is 19.5-20.5° C., the constant illumination range is 400-450 lux, and the constant humidity range is 0-1.0%.

[0021] In another aspect, a method for verifying whether the circadian rhythm of a nematode is normal is provided, comprising:

[0022] A test plate is prepared, and the nematodes with normal circadian rhythm or the nematodes with disordered circadian rhythm are placed in a test area;

[0023] Add a fixed volume of inducer at a fixed position and cover the test plate;

[0024] At fixed intervals, count the number of nematodes in different areas of multiple test plates;

[0025] The chemotaxis index was calculated and the circadian rhythm was verified.

[0026] In an optional embodiment, the nematode activity area d1 in the test plate is a circle with a radius ranging from 0.45 to 0.75 cm, and the test distance d2 between the inducer and the activity area is 0.30 to 1.5 cm.

[0027] Furthermore, the nematode activity area d1 in the test plate is a circle with a radius ranging from 0.50 to 0.60 cm, and the test distance d2 between the inducer and the activity area is 0.35 to 0.45 cm.

[0028] In an optional embodiment, the inducer type is 1-octanol, and one of methanol, ethanol, and n-hexane, wherein the proportion of 1-octanol is 1 to 5.5%, the inducer volume is 1 to 3 ul, and the reaction time is 10 to 60 min.

[0029] In an optional embodiment, the inducer type is 1-octanol, and any two of methanol, ethanol, and n-hexane in a volume ratio of 1:1, wherein the proportion of 1-octanol is 1 to 5.5%, the inducer volume is 1 to 3 ul, and the reaction time is 10 to 60 min.

[0030] In an optional embodiment, the inducer is a mixture of 1-octanol and ethanol, wherein the proportion of 1-octanol is 3.2-3.8% and the rest is ethanol; the volume of the inducer is 1-1.5 ul, and the reaction time is 25-35 min.

[0031] A method as described in any of the above, wherein the nematode is Caenorhabditis elegans.

[0032] This application has at least the following beneficial effects:

[0033] The present application provides a method for cultivating a model for evaluating circadian rhythmicity by establishing standardized temperature, light and humidity control. The method is simple and easy to control, and can more accurately simulate the impact of environmental factors on circadian rhythmicity, laying the foundation for establishing a stable and repeatable circadian rhythm disorder evaluation model.

[0034] This application effectively verifies whether the circadian rhythm of the cultured nematodes is normal by designing a variety of experimental parameter combinations and inducers. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0036] Figure 1 This is a schematic diagram of the approximate distribution of the test plates used in Examples 1 to 10 and Comparative Examples 1 to 11 of the present application; DETAILED DESCRIPTION

[0037] In order to make the above-mentioned objects, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below in conjunction with specific embodiments.

[0038] As a classic model organism, Caenorhabditis elegans is widely used in research fields such as toxicology, drug screening and disease models due to its short life cycle, clear structure and simple operation. The circadian rhythm-related genes of Caenorhabditis elegans are somewhat conservative with those of higher animals, especially the genes and signaling pathways that regulate metabolism, stress response, neurotransmission and circadian rhythm. In addition, nematodes have a short developmental cycle and a small size, making the experimental cycle easier to control and low-cost, which is conducive to large-scale screening. Compared with cell culture, nematodes can adapt to drugs or compounds of different polarities, and various candidate substances can be added to both liquid and solid culture media for high-throughput screening. However, there is still a lack of an evaluation system that uses the Caenorhabditis elegans model to systematically evaluate the relationship between active substances and circadian rhythm disorders.

[0039] Based on the above analysis, the present application proposes a model culture method for evaluating circadian rhythmicity and a verification method thereof. In the following description, many specific details are set forth to facilitate a full understanding of the present application, but the present application can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0040] The reagents involved in this application can all be purchased commercially.

[0041] As shown in Table 1, the eggs and L1 stage nematodes obtained by synchronization in Examples 1 to 12 and Comparative Examples 1 to 18 of the present application were obtained by the following method:

[0042] Wash the adult worms on the plate with M9 buffer, wash each plate twice, collect them in a 15mL centrifuge tube, and wash them three times with M9 buffer; remove the upper supernatant, add 12mL lysis buffer, and lyse for 9 minutes. Centrifuge at 3500rpm for 1 minute, discard the supernatant, add 12-14mL M9 buffer, wash with M9, repeat three times, and finally add 1mL M9 buffer, incubate in a 20℃ shaker for 12h, and obtain L1 stage Caenorhabditis elegans.

[0043] The preparation method of M9 buffer is as follows: Na2HPO4 6 g, KH2PO4 3 g, NaCl 5 g, 1 M MgSO4 1 mL, add water to make up to 1 L, and sterilize with high-pressure steam at 121°C for 20 minutes.

[0044] The models for evaluating circadian rhythmicity provided in Examples 1 to 12 of the present application all adopt the following culture method:

[0045] The steps to cultivate C. elegans with normal circadian rhythms include:

[0046] The above method is used to obtain eggs and L1 stage nematodes in synchronization;

[0047] Primary entrainment: After 12 hours of constant low-temperature culture and then 12 hours of constant high-temperature culture, the first entrainment of nematodes was obtained;

[0048] Secondary entrainment: The first entrained nematodes were cultured in the dark for 12 hours, and then cultured in 400 lux for 12 hours to obtain the second entrained nematodes;

[0049] Three times of entrainment: the second entrainment nematodes were first cultured at 0% humidity for 12 hours, and then cultured at 20% humidity for 12 hours to obtain nematodes No. 1;

[0050] Place nematode No. 1 in an incubator at 20°C, 0 lux, and 0% humidity for 24 hours to obtain nematode No. 2;

[0051] Four times of entrainment: nematodes No. 2 were first cultured in a constant low temperature, constant dark environment, and 0% humidity for 12 hours, and then cultured in a constant high temperature, constant light, and constant humidity for 12 hours;

[0052] The four entrainments were repeated twice to obtain nematodes with normal circadian rhythms;

[0053] The steps for culturing nematodes with disrupted circadian rhythms include:

[0054] Repeat the above steps to cultivate nematodes with circadian rhythms.

[0055] The obtained nematodes with circadian rhythm were first exposed to 600 lux light and 14°C low temperature environment for 6 hours, then exposed to 600 lux light and 16°C high temperature stimulation for 6 hours, then exposed to 0 lux dark conditions and 16°C high temperature stimulation for 6 hours, and then exposed to 0 lux dark conditions and 14°C low temperature stimulation for 6 hours to obtain nematodes with disordered circadian rhythm.

[0056] Please refer to Table 1 for specific experimental parameter settings.

[0057] Similarly, comparative examples 1 to 18 also adopt the same culture method as specific embodiments 1 to 12, the difference lies in the specific experimental conditions, please refer to Table 1 for details.

[0058]

[0059]

[0060]

[0061] Table 1

[0062] Note: In Table 1, d1 is the radius of the nematode activity area in the test plate described in W1; d2 is the distance between the inducer and the activity area in W2.

[0063] The calculation method of the chemotaxis index described in Examples 1 to 12 and Comparative Examples 1 to 18 of the present application is as follows:

[0064] See also Figure 1 The nematode activity area is divided into two parts, namely the proximal end and the distal end. The proximal end refers to the section close to the test point, while the distal end refers to the section far away from the test point. First, the number of all nematodes in the activity area is counted under a microscope. After the test period is over, the number of nematodes in the proximal and distal areas is counted respectively. The chemotaxis index is calculated using the following formula:

[0065] Chemotaxis index = (number of distal nematodes - number of proximal nematodes) / total number of nematodes;

[0066] Among them, the nematodes obtained in Examples 1 to 10 are nematodes with circadian rhythms, while Examples 11 to 12 are nematodes without circadian rhythms. Compared with the culture method of Examples 1 to 10, Examples 11 to 12 have the following additional steps: first, 600 lux light exposure and 14°C low temperature environment for 6 hours, then 600 lux light exposure and 16°C high temperature stimulation for 6 hours, then 0 lux dark conditions and 16°C high temperature stimulation for 6 hours, and then 0 lux dark conditions and 14°C low temperature stimulation for 6 hours to obtain the nematodes with disordered circadian rhythms.

[0067] Another specific implementation of the present application: a verification method for the model of circadian rhythm used in Examples 1 to 12 and Comparative Examples 1 to 18, that is, verifying whether the circadian rhythm of nematodes is consistent with the expectations of the model establishment. Using CircWave software, the specific operation is to enter the chemotaxis index of nematodes at 6 time points for a total of 24 hours, 4 parallels for each point, and take the p value according to the analysis results. If the p value is <0.05, it is considered to have rhythmicity, please refer to Tables 1 and 2.

[0068] Table 2

[0069]

[0070]

[0071] According to the results of the chemotaxis experiment, it can be seen that the calculation results of the rhythmicity test of Examples 1 to 10 are all less than 0.05, indicating that by cultivating nematodes under the conditions of Examples 1 to 10, nematodes with significant circadian rhythms can be obtained. In addition, the p-value results of Examples 11 to 12 are all greater than 0.05. The method of inducing nematodes with circadian rhythm disorders provided by the present invention can induce rhythm disorders in nematodes with circadian rhythms in a short period of time, and obtain nematodes with circadian rhythm disorders. The p-values ​​of all the nematodes in Comparative Examples 1 to 18 are greater than 0.05, indicating that it is impossible to induce the establishment of nematodes with normal rhythms under the above conditions.

[0072] Therefore, the model culture method for evaluating circadian rhythm provided in the present application is effective, and nematodes with normal circadian rhythm and nematodes with disordered circadian rhythm were successfully cultured, laying the foundation for establishing a stable and repeatable circadian rhythm disorder evaluation model.

[0073] The embodiments provided above are not intended to limit the scope of the present application, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present application in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present application.

Claims

1. A method for culturing a model for evaluating circadian rhythmicity, characterized in that: The model includes nematodes with normal circadian rhythm and nematodes with disordered circadian rhythm; the method includes the following steps: a step of culturing the nematodes with normal circadian rhythm and a step of culturing the nematodes with disordered circadian rhythm; The step of culturing nematodes with normal circadian rhythm comprises: The eggs were obtained by simultaneous incubation, and the first-day larvae were obtained 12 hours later and recorded as L1 stage nematodes; Primary entrainment: After 12 hours of constant low temperature culture and then 12 hours of constant high temperature culture, the first entrainment of nematodes was obtained; Secondary entrainment: the first entrained nematodes were cultured in the dark for 12 hours, and then cultured in 400 lux for 12 hours to obtain the second entrained nematodes; Three times of entrainment: the second entrainment nematodes are first cultured at 0% humidity for 12 hours, and then cultured at 20% humidity for 12 hours to obtain nematodes No. 1; Placing the nematode No. 1 in an incubator at 20° C., 0 lux, and 0% humidity for 24 hours to obtain the nematode No. 2; Four times of entrainment: the No. 2 nematode is first cultured in a constant low temperature, constant dark environment, and 0% humidity for 12 hours, and then cultured in a constant high temperature, constant light, and constant humidity for 12 hours; The four entrainments were repeated twice to obtain the nematodes with normal circadian rhythm; The step of culturing nematodes with disordered circadian rhythm comprises: Repeat the steps of culturing nematodes with normal circadian rhythms, The obtained nematodes with normal circadian rhythm were first exposed to 600 lux light and 14°C low temperature environment for 6 hours, then exposed to 600 lux light and 16°C high temperature stimulation for 6 hours, then exposed to 0 lux dark conditions and 16°C high temperature stimulation for 6 hours, and then exposed to 0 lux dark conditions and 14°C low temperature stimulation for 6 hours to obtain the nematodes with disordered circadian rhythm.

2. The method according to claim 1, characterized in that The constant low temperature range is 13.5-17.0° C., the constant high temperature range is 18.5-22.5° C., the constant illumination range is 380-550 lux, and the constant humidity range is 0-5.4%.

3. The method according to claim 2, characterized in that The constant low temperature range is 15.5-16.5° C., the constant high temperature range is 19.5-20.5° C., the constant illumination range is 400-450 lux, and the constant humidity range is 0-1.0%.

4. A method for verifying whether the circadian rhythm of a nematode is normal, characterized in that: include, A test plate is prepared, and the nematodes with normal circadian rhythm or the nematodes with disordered circadian rhythm are placed in a test area; Add a fixed volume of inducer at a fixed position and cover the test plate; At fixed intervals, count the number of nematodes in different areas of multiple test plates; The chemotaxis index was calculated and the circadian rhythm was verified.

5. The method according to claim 4, characterized in that The nematode activity area d1 in the test plate is a circle with a radius ranging from 0.45 to 0.75 cm, and the test distance d2 between the inducer and the activity area is 0.30 to 1.5 cm.

6. The method according to claim 5, characterized in that The nematode activity area d1 in the test plate is a circle with a radius ranging from 0.50 to 0.60 cm, and the test distance d2 between the inducer and the activity area is 0.35 to 0.45 cm.

7. The method according to claim 4, characterized in that The inducer type is 1-octanol, and one of methanol, ethanol, and n-hexane, wherein the proportion of 1-octanol is 1-5.5%, the inducer volume is 1-3ul, and the reaction time is 10-60min.

8. The method according to claim 4, characterized in that The inducer types are 1-octanol and any two of methanol, ethanol and n-hexane in a volume ratio of 1:1, wherein the proportion of 1-octanol is 1-5.5%, the inducer volume is 1-3ul, and the reaction time is 10-60min.

9. The method according to claim 7, characterized in that The inducer is a mixture of 1-octanol and ethanol, wherein the proportion of the 1-octanol is 3.2-3.8% and the rest is ethanol; the volume of the inducer is 1-1.5ul, and the reaction time is 25-35min.

10. The method according to any one of claims 1 to 9, characterized in that: The nematode is Caenorhabditis elegans.

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