Diurnal rhythm phase measurement method and diurnal simulation device for isolated culture cells
By simulating the circadian light-dark cycle cycle on ex vivo cultured cells and combining advanced analysis techniques, the problem that the existing technology cannot study the circadian rhythm of ex vivo cells is solved, and accurate measurement and verification of cell circadian rhythm parameters are achieved.
Patent Information
- Application Number
- CN202411882562.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-04-29
AI Technical Summary
The prior art cannot effectively study the circadian phase, period and amplitude of ex vivo cultured cells, especially it is impossible to determine whether these cells have independent circadian rhythms and whether they are subject to photoreceptors or central control.
A circadian rhythm phase measurement method for ex vivo cultured cells was used to culture cells in the incubator through a device that simulated the circadian light-dark cycle cycle, and combined with qRT-PCR and cosine statistical analysis, gene expression data were fitted to determine rhythm parameters.
Successfully studying circadian rhythm phase, period and amplitude on ex vivo cultured cells verified that cells have independent circadian rhythms under the lack of central control, and provided important molecular biological data on the biological clock and related diseases by regulating the moving rhythm phase of the light and dark cycles.
Smart Images
Figure CN119899893A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a circadian rhythm phase measurement method for in vitro cultured cells and a circadian simulation device, and belongs to the field of in vitro cell circadian rhythm research. Background Art
[0002] At present, the research methods for the circadian rhythm phase and clock genes of organisms can only be conducted on living bodies due to equipment and conditions limitations. However, more and more evidence shows that peripheral tissues and cells can have independent circadian rhythm phase cycles and amplitudes without central control. Therefore, current technology is not convincing for studying whether the circadian rhythm phase, cycle, and amplitude of in vitro cultured cells are photosensitive or controlled by the central nervous system, and this problem needs to be solved urgently. Summary of the invention
[0003] The present invention provides a circadian rhythm phase measurement method for in vitro cultured cells and a circadian simulation device, so as to solve the problem that traditional methods cannot study the circadian rhythm phase cycle and amplitude of in vitro cultured cells.
[0004] In order to solve the above problems, a method for measuring the circadian rhythm phase of in vitro cultured cells is proposed, and the specific steps are as follows:
[0005] (1) Extract and purify target cells through primary cell extraction technology.
[0006] (2) Using cell culture technology, the target cells are grown adherently in a suitable vessel in an incubator. When the cell confluence reaches 75%-85%, the cells are subcultured until the number of cells meets the number of groups required for the study.
[0007] (3) placing a device for simulating day and night lighting in an incubator, placing cultured cells in the device, and simulating the day and night light and dark cycle through the device;
[0008] (4) Cell collection and processing: After culturing for 3-5 days under a simulated light-dark cycle, cells were collected at different time points, centrifuged and total RNA was extracted. This step is to capture the gene expression of cells at different time points.
[0009] (5) qRT-PCR: Gene expression is measured using fluorescence quantitative PCR technology. The CT value (threshold cycle) is an important parameter for measuring gene expression levels. Genes with high expression levels have low CT values, and genes with low expression levels have high CT values.
[0010] (6) Cosine statistical analysis and Cosinor software fitting: Cosinor software and cosine analysis function model were used to fit the data to obtain the rhythm, phase, amplitude and median of gene expression.
[0011] In the aforementioned method, the target cells are epidermal cells of human or animal bodies;
[0012] In the above method, the target cells are primary human keratinocytes, and the culture environment in the incubator is 5% CO 2 air, a constant culture environment of 36.8°C;
[0013] In the above step (4), after culturing for 3-5 days in the environment simulating the light-dark cycle, the target cells develop a circadian rhythm consistent with the light-dark cycle. In the last 24 hours of the protocol, multiple groups of cells are collected by trypsin digestion at different time points, centrifuged at 1000 rap / min for 5 minutes, and the supernatant is discarded to use the cell pellet for the final measurement of the circadian rhythm phase, period, and amplitude;
[0014] In the above step (4), in the last 24 hours of the protocol, at 8:00, 13:00, 18:00, 23:00, 4:00, and 9:00, a total of 6 time points, the corresponding 6 groups of cells were collected by trypsin digestion;
[0015] In the above method, after collecting multiple groups of cells, it is first necessary to extract the total RNA in the cells. RNA is the direct product of gene expression, and the total RNA contains all types of RNA in the cells (mRNA, rRNA, tRNA, etc.); use an RNA extraction kit (such as TRIzol, RNeasy, etc.) according to the manufacturer's instructions. These kits usually contain lysis buffer, separation buffer and washing buffer, which can extract and purify RNA from cells; during the extraction process, it is necessary to avoid degradation by RNases, so the operation must be carried out under sterile conditions, using pre-treated RNase-free instruments and reagents.
[0016] In the aforementioned method, qRT-PCR (quantitative reverse transcription polymerase chain reaction):
[0017] Reverse transcription of the extracted total RNA into cDNA requires a reverse transcriptase (such as M-MLV reverse transcriptase) and specific primers;
[0018] After reverse transcription, qRT-PCR is used to quantify the target gene. qRT-PCR is a technique for real-time monitoring of DNA amplification, which uses fluorescent dyes (such as SYBR Green) or fluorescent probes (such as TaqMan probes) to detect fluorescent signals during the amplification process.
[0019] In qRT-PCR, target gene-specific primers and fluorescently labeled probes or dyes are added, and the target gene is amplified through set thermal cycling conditions (denaturation, annealing, extension);
[0020] As the PCR cycles proceed, the fluorescence signal increases with the exponential increase of the DNA template. The threshold value of the fluorescence signal (CT value) is the cycle number when the number of PCR cycles reaches the set threshold, which is inversely proportional to the initial amount of template DNA.
[0021] In the above method, the changes in periodic data are described by fitting a cosine curve to determine the phase, amplitude, median and peak period of the rhythm. Using the Cosinor software, the CT value of each group of cells is input, and the software will perform curve fitting based on the cosine analysis function model:
[0022] The cosine analysis function model is:
[0023] F(χ)=M+Acos[2π / T(χ-φ)]
[0024] M: Median line, which represents the middle line of cyclical fluctuations.
[0025] A: Amplitude, which indicates the size of periodic fluctuations.
[0026] T: Period, usually 24 hours, indicating the period of the circadian rhythm.
[0027] χ: time point.
[0028] φ: Phase, indicating the starting point of the periodic fluctuation.
[0029] Through fitting, the rhythmic parameters of gene expression can be obtained, including phase, amplitude, median and peak period (the time point with the highest gene expression). These two steps combined can accurately analyze the circadian changes in gene expression and provide important molecular biology data for the study of biological clocks and related diseases.
[0030] The device for simulating day and night lighting described in the above measurement method includes a box, one side of the box is provided with an openable and closable door, full-spectrum LED lamp beads are fixed in the box, the outer cover of the full-spectrum LED lamp beads is provided with a uniform light film, the full-spectrum LED lamp beads are externally connected to a power supply and the control end is placed outside the box.
[0031] In the above device, the box and the door are both made of black acrylic plates, which are connected by hinges, and the full-spectrum LED lamp beads are fixed to the upper end of the box through an aluminum lamp plate.
[0032] The existing research methods for the phase of animal circadian rhythm and related genes can only be studied in vivo due to equipment and condition limitations. Generally, in order to establish circadian rhythm for animals, animals are placed in a dark environment for a certain period of time in advance to make the animals lose the rules of day and night, and then artificially irradiated with light simulating natural light, wherein the lights are turned on for 12 hours to simulate daytime and turned off for 12 hours to simulate nighttime (different starting points can be selected according to one's own research), so that rhythm is formed or rhythm phase is shifted. After acclimation for 1-2 weeks according to this rule, genes and proteins of animals are extracted to observe the relationship between rhythm changes and light. The present invention provides a research method for the phase, period and amplitude of circadian rhythm of in vitro cultured cells, which is studied on in vitro cultured cells. The circadian rhythm was studied to verify that cells cultured in vitro may have photosensitive proteins or genes that can regulate their own gene expression and control the phase of the circadian rhythm by sensing the light / dark environment; this broke the limitation that circadian rhythm could only be created by light / dark environment intervention in living animals in the past; at the same time, a device that can simulate day and night light in a dark incubator was designed, which can successfully provide a day and night light environment simulation for cells cultured in vitro, and verify the circadian rhythm phase, cycle, amplitude, etc. by detecting the circadian rhythm genes of cells, and can also move the circadian rhythm phase by adjusting the light time in the device. The device designed to simulate the day and night environment and can be placed in the incubator can allow cells to be placed in 5% CO through an external control terminal or remote control. 2 The cells can grow normally in a constant culture environment of 36.8℃ while achieving light-dark cycle, which is closer to the physiological state. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 It is a schematic diagram of the structure of a device for simulating day and night lighting;
[0034] Figure 2 Artificially changing the day and night time causes the phase shift of the clock gene rhythm of human primary keratinocytes; among them: a (LD). 08:00-20:00 simulates daytime (with light), 20:00-08:00 simulates nighttime (dark room); b (DL). 08:00-20:00 simulates nighttime (dark room), 20:00-08:00 simulates daytime (with light); c (DD). 24 hours in a dark environment (no light); The curve in the figure is a fitting curve, and the points in the figure are data values, which means that the value of each measurement displayed on the coordinates is the point in the figure, and then these points are input into the software to fit the curve in the figure. DETAILED DESCRIPTION
[0035] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0036] Example
[0037] See attached Figure 1 This embodiment improves the measurement of the circadian rhythm phase of in vitro cultured cells. The specific improvement is to propose a method specifically for measuring the circadian rhythm phase of in vitro cultured cells. The specific steps are as follows:
[0038] (1) Extracting and purifying human primary keratinocytes using primary cell extraction technology;
[0039] (2) Using cell culture technology, primary human keratinocytes were grown in a suitable vessel in an incubator with a culture environment of 5% CO 2 of air and a constant culture environment of 36.8°C. When the cell confluence is 75%-85%, the cells are subcultured until the number of cells meets the number of groups required for the research. The device for simulating day and night lighting used includes a box 1 made of a black acrylic board. The box 1 is a bottomless structure. A door body 2 that can be opened and closed is provided on one side of the box 1. The box 1 and the door body 2 are connected by a hinge. The door body 2 is also a black acrylic board. The full-spectrum LED lamp beads 3 are fixed to the upper end of the box 1 through an aluminum lamp board 5. The outer cover of the full-spectrum LED lamp beads 3 is provided with a uniform light film 4. The full-spectrum LED lamp beads 3 are externally connected to a power supply 6 and the control end is placed outside the box 1. The power supply 6 is a 5V constant voltage 120W high-power power charger. The connection method between the full-spectrum LED lamp beads 3 and the power supply 6 is a USB connector connection.
[0040] (3) placing a device for simulating day and night lighting in an incubator, placing cultured cells in the device, and simulating the day and night light and dark cycle through the device;
[0041] (4) Cell collection and processing: After culturing for 3-5 days under a simulated light-dark cycle, the cells will develop a circadian rhythm consistent with the light-dark cycle. In the last 24 hours of the protocol, at 8:00, 13:00, 18:00, 23:00, 4:00, and 9:00, the corresponding 6 groups of cells were collected using the trypsin digestion method. The cells were centrifuged at 1000 rap / min for 5 minutes, and the supernatant was discarded. The cell pellet was used for the final measurement of the circadian rhythm phase, period, and amplitude. After collecting the 6 groups of cells, the total RNA of the cells must be extracted first. RNA is the direct product of gene expression. The total RNA contains all types of RNA in the cell (mRNA, rRNA, tRNA, etc.). Use an RNA extraction kit (such as TRIzol, RNeasy, etc.) according to the manufacturer's instructions. These kits usually contain lysis buffer, separation buffer, and washing buffer, which can extract and purify RNA from cells. During the extraction process, RNase degradation must be avoided, so the operation must be performed under sterile conditions, using pre-treated RNase-free instruments and reagents.
[0042] (5) qRT-PCR (quantitative reverse transcription polymerase chain reaction):
[0043] Reverse transcription of the extracted total RNA into cDNA requires a reverse transcriptase (such as M-MLV reverse transcriptase) and specific primers;
[0044] After reverse transcription, qRT-PCR is used to quantify the target gene. qRT-PCR is a technique for real-time monitoring of DNA amplification, which uses fluorescent dyes (such as SYBR Green) or fluorescent probes (such as TaqMan probes) to detect fluorescent signals during the amplification process.
[0045] In qRT-PCR, target gene-specific primers and fluorescently labeled probes or dyes are added, and the target gene is amplified through set thermal cycling conditions (denaturation, annealing, extension);
[0046] As the PCR cycles proceed, the fluorescence signal increases with the exponential increase of the DNA template. The threshold value of the fluorescence signal (CT value) is the cycle number when the number of PCR cycles reaches the set threshold, which is inversely proportional to the initial amount of template DNA.
[0047] (6) Cosine statistical analysis and Cosinor software fitting: Cosinor software and cosine analysis function model were used to fit the data to obtain the rhythm, phase, amplitude and median of gene expression;
[0048] The changes in periodic data are described by fitting a cosine curve to determine the phase, amplitude, median, and peak period of the rhythm. Using the Cosinor software, input the CT value of each group of cells, and the software will perform curve fitting based on the cosine analysis function model:
[0049] The cosine analysis function model is:
[0050] F(χ)=M+Acos[2π / T(χ-φ)]
[0051] M: Median line, which represents the middle line of cyclical fluctuations.
[0052] A: Amplitude, which indicates the size of periodic fluctuations.
[0053] T: Period, usually 24 hours, indicating the period of the circadian rhythm.
[0054] χ: time point.
[0055] φ: Phase, indicating the starting point of the periodic fluctuation.
[0056] Through fitting, the rhythmic parameters of gene expression can be obtained, including phase, amplitude, median and peak period (the time point with the highest gene expression). These two steps combined can accurately analyze the circadian changes in gene expression and provide important molecular biology data for the study of biological clocks and related diseases.
[0057] Comparative experimental example:
[0058] Example 1 was used as experimental group a. Based on the experimental process of Example 1, a group of experimental groups with a 12-hour time difference between the illumination time and experimental group a and other experimental conditions were the same as those of experimental group a were designed as control group b; a group of control group c was completely in a dark environment and other experimental conditions were the same as those of experimental group a. It was observed whether the circadian rhythm-related genes of the cells lost their rhythmicity, whether the phase shifted, and whether the period and amplitude changed when the day and night were reversed or the daytime was completely lost. The experimental results are shown in the attached Figure 2 and Table 1 below.
[0059] Table 1 Disruption of normal day and night changes the median, amplitude, and peak period of the cell CLOCK gene rhythm
[0060] Grouping Mesor Amplitude Acorphase a(LD) 61.68454 75.35896659 6.37374507 b(DL) 47.20464 38.94905303 19.77050842 c(DD) 67.11627 17.05395116 21.05952731
[0061] Conclusion: When the light and dark environment is changed, the expression of the clock gene (clock) changes. When the light is from 8:00 to 20:00 and the dark is from 20:00 to 8:00, the expression of the clock gene is the highest at 6:00 in the morning and the lowest at 18:00 in the afternoon (Figure a). When the dark is from 8:00 to 20:00 and the light is from 20:00 to 8:00, the expression of the clock gene is the lowest at 6:00 in the morning and the highest at 18:00 in the afternoon (Figure b). Comparison of the two groups revealed that the phase of the circadian rhythm changed. When the environment is dark for 24 hours, the amplitude of the clock gene is significantly reduced.
[0062] Through the above-described process, the study of circadian rhythm-related genes was completed. Through the analysis of the results, it can be concluded that: (1) Human primary keratinocytes cultured in vitro have independent circadian rhythm phase, period, and amplitude in the absence of central control; (2) The phase of the rhythm shifts due to the change of the light-dark cycle environment; (3) When the day and night are reversed or the daytime is completely lost, the circadian rhythm of the cells changes, the phase shifts, and the period and amplitude change, providing support for the research on the rhythmicity of human or animal epidermal cells.
[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for measuring the circadian rhythm phase of in vitro cultured cells, characterized in that: The specific steps are as follows: (1) Extracting and purifying target cells through primary cell extraction technology; (2) Using cell culture technology, the target cells are grown in a suitable vessel in an incubator and subcultured when the cell confluence reaches 75%-85% until the number of cells meets the number of groups required for the study; (3) placing a device for simulating day and night lighting in an incubator, placing cultured cells in the device, and simulating the day and night light and dark cycle through the device; (4) Cell collection and processing: After culturing for 3-5 days under a simulated light-dark cycle, cells were collected at different time points, centrifuged, and total RNA was extracted; (5) qRT-PCR: measuring gene expression by fluorescence quantitative PCR technology; (6) Cosine statistical analysis and Cosinor software fitting: Cosinor software and cosine analysis function model were used to fit the data to obtain the rhythm, phase, amplitude and median of gene expression.
2. The method for measuring the circadian rhythm phase of in vitro cultured cells according to claim 1, characterized in that: The target cells are epidermal cells of human or animal bodies.
3. The method for measuring the circadian rhythm phase of in vitro cultured cells according to claim 1, characterized in that: The target cells are primary human keratinocytes, and the culture environment in the incubator is a constant culture environment of 5% CO2 in air and 36.8°C.
4. The method for measuring the circadian rhythm phase of in vitro cultured cells according to claim 1, characterized in that: In the above step (4), after culturing for 3-5 days in the environment simulating the light-dark cycle, the target cells develop a circadian rhythm consistent with the light-dark cycle. In the last 24 hours of the program, multiple groups of cells are collected by trypsin digestion at different time points, centrifuged at 1000 rap / min for 5 minutes, and the supernatant is discarded to use the cell pellet for the final measurement of the circadian rhythm phase, period, and amplitude.
5. The method for measuring the circadian rhythm phase of in vitro cultured cells according to claim 4, characterized in that: In the above step (4), in the last 24 hours of the protocol, at 8:00, 13:00, 18:00, 23:00, 4:00, and 9:00, the corresponding 6 groups of cells were collected by trypsin digestion.
6. The method for measuring the circadian rhythm phase of in vitro cultured cells according to claim 4, characterized in that: After collecting multiple groups of cells, the first thing to do is to extract the total RNA in the cells. RNA is the direct product of gene expression, and total RNA contains all types of RNA in the cells. Use an RNA extraction kit according to the manufacturer's instructions. These kits usually contain lysis buffer, separation buffer, and washing buffer, which can extract and purify RNA from cells. During the extraction process, it is necessary to avoid degradation by RNases, so the operation must be performed under sterile conditions, using pre-treated RNase-free instruments and reagents.
7. The method for measuring the circadian rhythm phase of in vitro cultured cells according to claim 1, characterized in that: Reverse transcription of the extracted total RNA into cDNA requires reverse transcriptase and specific primers; After reverse transcription, qRT-PCR is used to quantify the target gene. In qRT-PCR, target gene-specific primers and fluorescently labeled probes or dyes are added to amplify the target gene under set thermal cycling conditions. As the PCR cycles proceed, the fluorescence signal increases with the exponential increase of the DNA template. The threshold of the fluorescence signal is the cycle number when the number of PCR cycles reaches the set threshold, which is inversely proportional to the initial amount of template DNA.
8. The method for measuring the circadian rhythm phase of in vitro cultured cells according to claim 1, characterized in that: The changes in periodic data are described by fitting a cosine curve to determine the phase, amplitude, median, and peak period of the rhythm. Using the Cosinor software, input the CT value of each group of cells, and the software will perform curve fitting based on the cosine analysis function model: The cosine analysis function model is: F(χ)=M+Acos[2π / T(χ-φ)] M: median line, indicating the median line of cyclical fluctuations; A: Amplitude, which indicates the size of periodic fluctuations; T: period, usually 24 hours, indicating the period of the circadian rhythm; χ: time point; φ: phase, indicating the starting point of the periodic fluctuation; Through fitting, the rhythmic parameters of gene expression, including phase, amplitude, median and peak period, are obtained to accurately analyze the circadian changes of gene expression.
9. A device for simulating day and night lighting for use in the measurement method according to any one of claims 1 to 8, characterized in that: The invention comprises a box (1), one side of the box (1) is provided with an openable and closable door body (2), a full-spectrum LED lamp bead (3) is fixed in the box (1), an outer cover of the full-spectrum LED lamp bead (3) is provided with a light-uniform film (4), and the full-spectrum LED lamp bead (3) is externally connected to a power supply and a control end is arranged outside the box (1).
10. The device according to claim 9, characterized in that: The box (1) and the door body (2) are both made of black acrylic plates and are connected by hinges. The full-spectrum LED lamp beads (3) are fixed to the upper end of the box (1) via an aluminum lamp plate (5).