A culture method and a verification method for a model for evaluating circadian rhythmicity

Abstract

The application provides a culture method and a verification method for evaluating a model of circadian rhythm, and belongs to the field of disorder model establishment and active substance function evaluation. By culturing nematodes with normal circadian rhythm and nematodes with circadian rhythm disorder, a model for evaluating circadian rhythm is established. The method is simple and easy to control, can more accurately simulate the influence of environmental factors on circadian rhythm, and lays a foundation for establishing a stable and repeatable evaluation model of circadian rhythm disorder. Whether the circadian rhythm of the nematodes is normal or not is effectively verified by the following method: a test plate is prepared, the nematodes with normal circadian rhythm or the nematodes with circadian rhythm disorder are placed in a test area; a fixed volume of an inducer is added at a fixed position, and the test plate is covered; nematode numbers in different areas of multiple test plates are counted at intervals of a fixed time; a chemotaxis index is calculated to verify the circadian rhythm.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of circadian rhythm disorder model evaluation, and particularly relates to a culture method and a verification method for evaluating a model of circadian rhythmicity. BACKGROUND

[0002] Circadian rhythm is an internal time regulation mechanism that organisms have evolved to adapt to the periodic changes in the environment caused by the rotation of the earth. From simple unicellular cyanobacteria to complex mammals, including humans, almost all forms of life exhibit this rhythmic feature.

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

[0004] Some researchers have constructed rhythm gene knockout mice (such as Bmal1 and Clock knockout mice) using gene knockout technology, and obtained homozygous genotypes by breeding to simulate a model of circadian rhythm disorder. However, this method has the problems of long culture period, complex operation, high mortality of homozygous mice, and high economic cost, which cannot meet the efficient and economic research and development needs.

[0005] Therefore, there is an urgent need for a method that can quickly, simply and scientifically evaluate circadian rhythmicity. SUMMARY

[0006] The purpose of the present application is to overcome the defects in the related art, provide a culture method for evaluating a model of circadian rhythmicity, obtain a substance that can quickly, simply and scientifically evaluate circadian rhythmicity, and verify it.

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

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

[0009] Synchronize the eggs, obtain the first day of larvae 12h later, and record them as L1 stage nematodes;

[0010] Primary entrainment: the first entrainment nematodes are obtained after 12h constant low temperature culture and 12h constant high temperature culture;

[0011] Secondary entrainment: the second entrainment nematodes are obtained after 12h darkness culture and 12h, 400lux culture of the first entrainment nematodes;

[0012] Tertiary entrainment: the first nematodes are obtained after 12h, 0% humidity culture and 12h, 20% humidity culture of the second entrainment nematodes;

[0013] The first nematodes are placed in a 20℃, 0lux, 0% humidity incubator for 24h to obtain the second nematodes;

[0014] Quaternary entrainment: the second nematodes are cultured for 12h in constant low temperature, constant darkness and 0% humidity, and then cultured for 12h in constant high temperature, constant light and constant humidity;

[0015] The quaternary entrainment is repeated twice to obtain the nematodes with normal circadian rhythm;

[0016] The step of culturing the nematodes with disturbed circadian rhythm comprises:

[0017] The step of culturing the nematodes with disturbed circadian rhythm comprises:

[0018] The nematodes with circadian rhythm are obtained, and then cultured for 6h in 600lux light and 14℃ low temperature, then cultured for 6h in 600lux light and 16℃ high temperature, then cultured for 6h in 0lux darkness and 16℃ high temperature, then cultured for 6h in 0lux darkness and 14℃ low temperature to obtain the nematodes with disturbed circadian rhythm.

[0019] In optional embodiments, the constant low temperature ranges from 13.5 to 17.0℃, the constant high temperature ranges from 18.5 to 22.5℃, the constant light ranges from 380 to 550lux, and the constant humidity ranges from 0 to 5.4%.

[0020] Further, the constant low temperature ranges from 15.5 to 16.5℃, the constant high temperature ranges from 19.5 to 20.5℃, the constant light ranges from 400 to 450lux, and the constant humidity ranges from 0 to 1.0%.

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

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

[0023] In the fixed position, drop a fixed volume of inducer, cover the test plate;

[0024] Interval fixed time, respectively, statistics of multiple test plate different area of nematode number;

[0025] Calculate the chemotaxis index, and verify the circadian rhythm.

[0026] In an alternative embodiment, the nematode activity area d1 in the test plate is a circle with a radius range of 0.45-0.75 cm, and the test distance d2 between the inducer and the activity area is 0.30-1.5 cm.

[0027] Further, the nematode activity area d1 in the test plate is a circle with a radius range of 0.50-0.60 cm, and the test distance d2 between the inducer and the activity area is 0.35-0.45 cm.

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

[0029] In an alternative 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 1-1.5ul, and the reaction time is 25-35 min.

[0030] In an alternative 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 1-1.5ul, and the reaction time is 25-35 min.

[0031] The method as claimed in any of the preceding embodiments, wherein the nematode is Caenorhabditis elegans.

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

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

[0034] The present application effectively verifies whether the cultured nematode circadian rhythm is normal by designing various experimental parameter combinations and inducers. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor. Among them:

[0036] Figure 1 The approximate distribution of the test plates used in Examples 1-10 and Comparative Examples 1-11 of the present application is shown in the following table: DETAILED DESCRIPTION

[0037] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below.

[0038] Caenorhabditis elegans, as a classic model organism, is widely used in toxicology, drug screening and disease modeling research fields due to its short life cycle, clear structure and simple operation. The circadian rhythm-related genes of Caenorhabditis elegans have certain conservation with higher animals, especially the genes and signaling pathways that regulate metabolism, stress response, neurotransmission and circadian rhythm. In addition, the development cycle of nematodes is short, the volume is small, the experimental period is easy to control and the cost is low, which is helpful for large-scale screening. Compared with cell culture, nematodes can adapt to different polarity drugs or compounds, whether it is liquid culture medium or solid culture medium, various candidate substances can be added for high-throughput screening. However, there is still a lack of evaluation system for evaluating the relationship between active substances and circadian rhythm disorders using Caenorhabditis elegans model.

[0039] Based on the above analysis, the present application proposes a model culture method for evaluating circadian rhythm and a verification method thereof. In the following description, many specific details are set forth in order to provide a thorough 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 extensions without departing from the connotation of the present application, therefore the present application is not limited by the specific embodiments disclosed below.

[0040] The reagents involved in the present application can be obtained by market purchase.

[0041] As shown in Table 1, the synchronized hatching of eggs and L1 nematodes used in Examples 1-12 and Comparative Examples 1-18 of the present application were obtained by the following method:

[0042] Wash the adult worms off the plates with M9 buffer, twice per plate, collect in a 15 mL centrifuge tube, and wash 3 times with M9 buffer; remove the supernatant and add 12 mL lysis buffer and incubate for 9 minutes. Centrifuge at 3500 rpm for 1 minute, remove the supernatant and add 12-14 mL M9 buffer and wash 3 times with M9 buffer, and finally add 1 mL M9 buffer and incubate at 20°C for 12 hours to obtain L1 stage C. elegans.

[0043] The M9 buffer is prepared as follows: Na2HPO46 g, KH2PO43 g, NaCl 5 g, 1 M MgSO41 mL, and water to make 1 L, and sterilized by high-pressure steam at 121°C for 20 minutes.

[0044] The model for evaluating circadian rhythmicity provided in Examples 1-12 of the present application uses the following culture method:

[0045] The steps for culturing C. elegans with normal circadian rhythmicity include:

[0046] The synchronized eggs and L1 stage C. elegans are obtained using the above method.

[0047] First entrainment: the first entrained C. elegans are obtained by first culturing at a constant low temperature for 12 hours, and then culturing at a constant high temperature for 12 hours.

[0048] Second entrainment: the second entrained C. elegans are obtained by first culturing the first entrained C. elegans in the dark for 12 hours, and then culturing at 400 lux for 12 hours.

[0049] Third entrainment: the first number of C. elegans are obtained by first culturing the second entrained C. elegans at 0% humidity for 12 hours, and then culturing at 20% humidity for 12 hours.

[0050] The first number of C. elegans are placed in a 20°C incubator at 0 lux and 0% humidity for 24 hours to obtain the second number of C. elegans.

[0051] Fourth entrainment: the second number of C. elegans are first cultured at a constant low temperature, constant darkness, and 0% humidity for 12 hours, and then cultured at a constant high temperature, constant light, and constant humidity for 12 hours.

[0052] The fourth entrainment is repeated twice to obtain C. elegans with normal circadian rhythmicity.

[0053] The steps for culturing C. elegans with abnormal circadian rhythmicity include:

[0054] The steps for culturing C. elegans with circadian rhythmicity are repeated,

[0055] The obtained nematodes with circadian rhythm are first subjected to 600 lux light for 6 hours at 14°C low temperature, then subjected to 600 lux light for 6 hours at 16°C high temperature, then subjected to 0 lux dark condition for 6 hours at 16°C high temperature, then subjected to 0 lux dark condition for 6 hours at 14°C low temperature, to obtain nematodes with disturbed circadian rhythm.

[0056] The specific experimental parameter settings are shown in Table 1.

[0057] Similarly, the comparative examples 1-18 are also subjected to the same culture method as the specific examples 1-12, with the difference being the specific experimental condition settings, which are shown in Table 1.

[0058]

[0059]

[0060]

[0061] Table 1

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

[0063] The calculation method of the chemotaxis index in the specific examples 1-12 and the comparative examples 1-18 is as follows:

[0064] Referring to 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 part close to the test point, and the distal end refers to the part away from the test point. First, count the number of all nematodes in the activity area under a microscope. After the test period ends, count the number of nematodes in the proximal end and the distal end, respectively. The chemotaxis index is calculated using the following formula:

[0065] Chemotaxis index = (number of nematodes in the distal end - number of nematodes in the proximal end) / total number of nematodes.

[0066] Among them, the nematodes obtained in examples 1-10 are nematodes with circadian rhythm, and examples 11-12 are nematodes without circadian rhythm. The culture method of examples 11-12 is as follows: first subjected to 600 lux light for 6 hours at 14°C low temperature, then subjected to 600 lux light for 6 hours at 16°C high temperature, then subjected to 0 lux dark condition for 6 hours at 16°C high temperature, then subjected to 0 lux dark condition for 6 hours at 14°C low temperature, to obtain the nematodes with disturbed circadian rhythm.

[0067] Another embodiment of the present application is a method for verifying the model of circadian rhythm, i.e. verifying whether the circadian rhythm of the nematode is consistent with the expectation of the model. The CircWave software is used, and the operation is as follows: the chemotaxis index of the nematode at 6 time points for 24 hours is input, 4 parallels for each point, and the p value is taken according to the analysis result. If the p value is less than 0.05, it is considered to have rhythm. Please refer to Tables 1 and 2.

[0068] Table 2

[0069]

[0070]

[0071] According to the results of the chemotaxis experiment, the rhythm test calculation results of Examples 1-10 are all less than 0.05, indicating that the nematodes cultured under the conditions of Examples 1-10 can obtain nematodes with significant circadian rhythm. In addition, the p value results of Examples 11-12 are all greater than 0.05, and the method for inducing nematodes with circadian rhythm disorder provided by the present application can induce nematodes with circadian rhythm in a short time to obtain nematodes with circadian rhythm disorder. The p values of all the nematodes of Comparative Examples 1-18 are greater than 0.05, indicating that it is not possible to induce the establishment of nematodes with normal rhythm under the above conditions.

[0072] Therefore, the model culture method for evaluating circadian rhythm provided by the present application is effective, and nematodes with normal circadian rhythm and nematodes with circadian rhythm disorder are successfully cultured, which lays a foundation for establishing a stable and repeatable evaluation model of circadian rhythm disorder.

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

Claims

1. A culture method for evaluating a model of circadian rhythmicity, characterized by, The model comprises normal circadian rhythm nematodes and abnormal circadian rhythm nematodes, and the nematodes are Caenorhabditis elegans; The method comprises the following steps: a step of culturing the normal circadian rhythm nematodes and a step of culturing the abnormal circadian rhythm nematodes; The step of culturing the normal circadian rhythm nematodes comprises: The eggs are obtained synchronously, and the first-day larvae are obtained after 12 hours, and are recorded as L1 nematodes; First entrainment: the first entrainment nematodes are obtained after 12 hours of constant low-temperature culture and 12 hours of constant high-temperature culture; Second entrainment: the second entrainment nematodes are obtained after 12 hours of dark culture and 12 hours of 400 lux culture of the first entrainment nematodes; Third entrainment: the first nematodes are obtained after 12 hours of 0% humidity culture and 12 hours of 20% humidity culture of the second entrainment nematodes; The first nematodes are placed in a culture box with the conditions of 20°C, 0 lux and 0% humidity for 24 hours to obtain second nematodes; Fourth entrainment: the second nematodes are obtained after 12 hours of constant low-temperature, constant dark environment and 0% humidity culture and 12 hours of constant high-temperature, constant light and constant humidity culture; The fourth entrainment is repeated twice to obtain the normal circadian rhythm nematodes; The step of culturing the abnormal circadian rhythm nematodes comprises: The step of culturing the normal circadian rhythm nematodes is repeated, The normal circadian rhythm nematodes are obtained, and after 6 hours of 600 lux light and 14°C low-temperature environment, 6 hours of 600 lux light and 16°C high-temperature stimulation, 6 hours of 0 lux dark condition and 16°C high-temperature stimulation, and 6 hours of 0 lux dark condition and 14°C low-temperature stimulation, the abnormal circadian rhythm nematodes are obtained; The constant low-temperature ranges from 13.5 to 17.0°C, the constant high-temperature ranges from 18.5 to 22.5°C, the constant light ranges from 380 to 550 lux, and the constant humidity ranges from 0 to 5.4%.

2. The method of claim 1, wherein, The constant low-temperature ranges from 15.5 to 16.5°C, the constant high-temperature ranges from 19.5 to 20.5°C, the constant light ranges from 400 to 450 lux, and the constant humidity ranges from 0 to 1.0%.

3. A method for verifying whether the circadian rhythmicity of the nematode according to claim 1 or 2 is normal, characterized in that, Comprise, Test plates are prepared, and the normal circadian rhythm nematodes or the abnormal circadian rhythm nematodes are placed in test areas; A fixed volume of an inducer is added dropwise at a fixed position, and the test plate is covered; The number of nematodes in different areas of multiple test plates is counted at intervals; The chemotaxis index is calculated to verify the circadian rhythm; The active area d1 of the nematodes 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 active area is 0.30 to 1.5 cm; The inducer is 1-octanol, and one of methanol, ethanol and n-hexane, wherein the volume ratio of 1-octanol in the inducer is 1 to 5.5%.

4. The method of claim 3, wherein, The active area d1 of the nematode in the test plate is a circle with a radius of 0.50-0.60 cm, and the test distance d2 between the inducer and the active area is 0.35-0.45 cm.

5. The method of claim 3, wherein, The volume of the inducer is 1-3 μl, and the reaction time is 10-60 min.

6. The method of claim 3, wherein, The inducer is 1-octanol, and any two of methanol, ethanol, and n-hexane in a volume ratio of 1:1, wherein the volume ratio of the 1-octanol is 1-5.5%, the volume of the inducer is 1-3 μl, and the reaction time is 10-60 min.

7. The method of claim 5, wherein, The inducer is a mixture of 1-octanol and ethanol, wherein the volume ratio of the 1-octanol is 3.2-3.8%, and the rest is ethanol; the volume of the inducer is 1-1.5 μl, and the reaction time is 25-35 min.

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