Method for improving myoblast proliferation and differentiation and human muscle group strength based on specific magnetic field parameters

Abstract

The invention discloses a method for improving myoblast proliferation and differentiation and human muscle group strength based on specific magnetic field parameters, which comprises the following steps of: respectively dividing related magnetic field parameters including magnetic field intensity, frequency, pulse number, duty ratio and intervention time into four levels, carrying out alternating-direct arrangement on the related magnetic field parameters, and carrying out a magnetic field intervention experiment according to data arrangement in an alternating-direct table; the method comprises the following steps: culturing cells to a density of 40% by using DMEM high glucose and 10% fetal calf serum, carrying out magnetic field intervention, dividing the intervention time into four levels of 10 min, 15 min, 20 min and 25 min, placing the four groups of cells in an environment which is the same as the magnetic field intervention group for intervention, isolating a magnetic field, and respectively standing for 10 min, 15 min, 20 min and 25 min; after the magnetic intervention is finished, immediately replacing the culture solution with a CCK8 reagent, and after the operation is finished, immediately putting the cells back to the cell culture box to be continuously cultured for 8 hours; taking out the cells after 8 hours, equivalently sucking out the CCK8 reagent in each group of cells, placing the CCK8 reagent in a 96-hole cell culture plate, and performing absorbance analysis on the CCK8 reagent in each group of cells by using a microplate reader so as to compare the proliferation speed of each group of cells.

Description

Technical Field

[0001] The invention relates to a method for improving the proliferation and differentiation of myoblasts and the strength of human muscle groups based on specific magnetic field parameters. Background Art

[0002] The prior art only points out that low-frequency pulsed magnetic fields have a certain promoting effect on cell proliferation and differentiation. In theory, different types of cells have different requirements for internal calcium ion channels and calcium ion content inside and outside the cells, which makes them have specific magnetic field sensitivity effects; even if the same cell is subjected to different magnetic field parameters, the magnetic intervention effect it shows is different; under the same magnetic field parameters, there may be great differences in the intervention effect on different cells; and the prior art does not provide the difference between the cell intervention effect and the difference in the optimal selection under different magnetic field parameters. The present invention applies safe pulsed magnetic fields of different intensities, frequencies, durations, and duty cycles to myoblasts, and clarifies the magnetic field parameters that have a positive promoting effect and the best promoting effect on myoblasts by its effect on the proliferation and differentiation of myoblasts. . Usually, this type of magnetic therapy requires a higher frequency or intensity magnetic field to intervene in specific human tissue parts, and due to the spatial distribution properties of the magnetic field, it is inevitable to irradiate the surrounding tissues. For high-frequency and high-intensity magnetic fields, the main side effects caused by them are peripheral nerve stimulation (PNS) and tissue heating. For example, in the field of magnetic particle imaging, in order to avoid PNS and tissue heating effects around the human limbs, the applied magnetic field and frequency should not be greater than 20kHz and 3mTrms. On the other hand, in some magnetic intervention application scenarios, a magnetic field of a certain intensity and frequency needs to be applied in the entire space, but even if only the spatial magnetic field distribution slightly larger than the human body size (2m*0.7m*0.7m) is considered, in order to apply a uniform magnetic field in space, the power supply capacity and various losses of the magnetic field generating coil also make it extremely difficult to generate a high-intensity magnetic field. (Specific simulation data can be added, such as how much current generates how much magnetic field in how much space).

[0003] Therefore, considering the need to achieve the best magnetic field intervention effect in the actual application of the product, it is necessary to clearly select the best magnetic field parameters for different magnetic intervention sites and magnetic intervention purposes. (1) The competitive conflict between magnetic field intensity and magnetic field safety, and (2) the need for the use of large-scale spatial uniform magnetic fields, require a method and device for generating human body intervention magnetic fields that has little impact on non-irradiated areas and can generate a large range of spatial magnetic fields.

[0004] Furthermore, for application scenarios in which protein supplementation through transdermal administration is required in magnetic stimulation muscle enhancement technology based on TRPC1 ion channels, a method for local magnetic field enhancement in hydrogel materials is further provided.

[0005] The existing technology applies a magnetic field to the entire area of ​​the part to be irradiated, and the same magnetic field strength is applied to all muscles in the entire area, which lacks the enhancement of local magnetic field changes. If a high-intensity spatial magnetic field is used, it will affect the central nervous system of the human body, and it is difficult to achieve an enhanced effect if a weaker spatial magnetic field is used.

[0006] The existing technology only uses magnetic fields to stimulate the TRPC1 protein pathway to achieve an enhancement effect. For individuals with low TRPC1 protein expression, the irradiation enhancement period is long and the effect is poor.

[0007] The present invention aims at the above problems, makes up for the deficiencies of the prior art, and provides a method for promoting local muscle enhancement based on soft magnetic materials and superparamagnetic materials in combination with TPRC1 protein reagent, so as to solve the problems arising from the above background technology.

[0008] The beneficial effects of the present invention are: The present invention clarifies the relevant magnetic field parameters that can be used to promote the proliferation and differentiation of myoblasts, greatly improving the efficiency of magnetic intervention in promoting physiological activities such as related cell proliferation and differentiation; The article Magnetostimulation Limits in Magnetic Particle Imaging proposes that PNS (peripheral nerve stimulation) and SAR (tissue heating) can be effectively avoided in a magnetic field with an intensity lower than 6mT and a frequency lower than 25KHz, and the human body will not feel any irritation in a magnetic field within this intensity and frequency range. The magnetic field safety threshold literature (6mT, 25KHz) is cited within the safe magnetic field threshold range, and the alternating magnetic field intervention treatment method of the human body is used to achieve continuous enhancement of local areas under low background magnetic fields, thereby reducing the side effects of long-term magnetic field intervention on non-treatment areas. By using soft magnetic powder or superparamagnetic nanoparticles to enhance the magnetic field strength in the magnetic field, the magnetic field strength is enhanced in specific parts, achieving the effect of local muscle strength enhancement.

[0009] The magnetic field parameters are refined, and the best magnetic field schemes are screened out for different intervention sites and intervention cell types to make the effect of magnetic intervention more significant. The relevant parameters such as magnetic field intensity, frequency, number of pulses, duty cycle, intervention time, etc. are arranged, and the experimental data are analyzed using the Taguchi method to determine the noise resistance and sensitivity of the individual parameter levels. Based on the above effects, the excitation magnetic field intensity or frequency required for large-scale irradiation is reduced, so that the power supply and magnetic field generating system can work efficiently and continuously, realizing the application requirements of large-scale and high-safety magnetic field intervention in free space.

[0010] Furthermore, in combination with the use of TRPC1 reagents, in application scenarios where protein supplementation is required through transdermal administration in magnetic stimulation muscle group enhancement technologies similar to those based on TRPC1 ion channels, a local magnetic field enhancement method in a hydrogel material is further provided. This method can enhance the expression of TRPC1 protein in the muscle and enhance the efficiency of the muscle tissue's response to magnetic field stimulation by applying or injecting TRPC1 reagents to the user, while ensuring the enhancement of the local magnetic field, thereby achieving the purpose of improving the magnetic field stimulation effect and shortening the irradiation enhancement period. Summary of the invention

[0011] The present invention provides a method for improving the proliferation and differentiation of myoblasts and the strength of human muscle groups based on specific magnetic field parameters.

[0012] The present invention is characterized in that relevant magnetic field parameters (magnetic field strength, frequency, number of pulses, duty cycle, intervention time) are divided into four levels and arranged in AC and DC. The relevant magnetic field waveform, level standard and AC and DC table are as follows.

[0013] Table 1 Table 2 According to the data arrangement in the cross-correlation table, the magnetic field intervention experiment was carried out. The cells were cultured to a density of 40% using DMEM high glucose and 10% fetal bovine serum for magnetic field intervention. The intervention time was clearly divided into four levels of 10min, 15min, 20min, and 25min. The four groups of cells were placed in the same environment as the magnetic field intervention group, isolated from the magnetic field, and left to stand for 10min, 15min, 20min, and 25min respectively. Immediately after the magnetic intervention, the culture medium was replaced with CCK8 reagent, and the cells were immediately returned to the cell culture incubator for continued culture for 8h.

[0014] After 8 h, the cells were taken out, and an equal amount of CCK8 reagent in each group of cells was aspirated and placed in a 96-well cell culture plate. The absorbance of the CCK8 reagent in each group of cells was analyzed using an ELISA instrument to compare the proliferation rate of each group of cells.

[0015] Beneficial effects of the present invention: The magnetic intervention of the present invention has a certain promoting effect on the proliferation of C2C12 myoblasts, but the promoting effect is different under different parameters. The Taguchi method can accurately predict the influence of each parameter on the magnetic intervention effect, and select the parameter combination with better intervention effect, which greatly saves the manpower and material resources required for repeated experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the main effects plot of the means.

[0017] Figure 2 is the main effects plot of the signal-to-noise ratio.

[0018] Figure 3 It is a comparison chart of the best parameters among 16 sets of parameter combinations.

[0019] Figure 4 It is the waveform of the magnetic field generator input signal. DETAILED DESCRIPTION

[0020] In production practices such as developing new products or improving old products, many experiments are often required. Proper experimental arrangement will reduce the number of experiments, avoid blindness, and get good results. On the contrary, the workload will not necessarily lead to good results. British RA Fisher and others have applied experimental design technology to agricultural production since the 1920s. In the 1950s, statistician Genichi Taguchi completed the tabulation of orthogonal design. The experiments in the table are evenly distributed and all experiments are neatly comparable. Researchers represented by Professor Genichi Taguchi have made great contributions to the wide application of experimental design technology. Therefore, when dealing with multi-factor experiments, the simple, fast and highly accurate Taguchi orthogonal experimental method has been widely used.

[0021] In this experimental design, the relevant magnetic field parameters (magnetic field intensity, frequency, number of pulses, duty cycle, intervention time) are divided into 4 levels and arranged in AC and DC. The relevant magnetic field waveforms, level standards and AC and DC tables are as follows.

[0022] According to the data arrangement in the cross-correlation table, a magnetic field intervention experiment was carried out on C2C12 mouse myoblasts. The cells were cultured to a density of 40% using DMEM high glucose and 10% fetal bovine serum for magnetic field intervention. Since it was impossible to accurately provide the cells with a standard incubator environment (carbon dioxide concentration of 5%, ambient temperature of 37°C) during the intervention, the cell activity would change with the intervention time. In this experiment, the intervention time was clearly divided into four levels of 10min, 15min, 20min, and 25min. Therefore, four groups of blank controls were used. The four groups of cells were placed in the same environment as the magnetic field intervention group, isolated from the magnetic field, and left to stand for 10min, 15min, 20min, and 25min, respectively.

[0023] After the magnetic intervention, the culture medium was immediately replaced with CCK8 reagent (try to avoid light during the operation), and the cells were immediately returned to the cell culture incubator for further culture for 8 hours.

[0024] After 8 hours, the cells were taken out, and an equal amount (100ul) of CCK8 reagent in each group of cells was aspirated and placed in a 96-well cell culture plate. The absorbance of the CCK8 reagent in each group of cells was analyzed using an ELISA instrument to compare the proliferation rate of each group of cells. The experimental data are shown in the figure below.

[0025] After normalizing the experimental data, the Taguchi method was used to analyze them. The analysis results are shown in the figure, where A, B, C, and D correspond to the magnetic field intensity, frequency, number of pulses, and intervention time, respectively.

[0026] According to the main effect diagram of signal-to-noise ratio, B, C, and D have the greatest influence on the signal-to-noise ratio of the target characteristic, so they are called divergence factors. According to the main effect diagram of mean, A and D have a greater influence on the mean, so they are called position factors. Among them, A is both a divergence factor and a position factor, so it can be identified as a regulating factor.

[0027] According to Taguchi analysis prediction, the best parameters are A4B2C3D3, that is, the intensity is 6mT, the frequency is 3000Hz, the number of pulses is, and the intervention time is 15min. This parameter was used to perform magnetic intervention on C2C12 myoblasts again and compared with the best parameters in the 16 groups of parameter combinations.

[0028] According to the above experimental results, magnetic intervention has a certain promoting effect on the proliferation of C2C12 myoblasts, but the promoting effect is different under different parameters. The Taguchi method can accurately predict the influence of each parameter on the effect of magnetic intervention, and select the parameter combination with better intervention effect, which greatly saves the manpower and material resources required for repeated experiments.

Claims

1. A method for improving myoblast proliferation and differentiation and human muscle strength based on specific magnetic field parameters, characterized in that: The relevant magnetic field parameters: magnetic field intensity, frequency, number of pulses, duty cycle, and intervention time are divided into 4 levels, and then arranged in AC and DC. The relevant magnetic field waveforms, level standards, and AC and DC tables are as follows: Table 1 Table 2 According to the data arrangement in the cross-correlation table, the magnetic field intervention experiment was carried out. The cells were cultured to a density of 40% using DMEM high glucose and 10% fetal bovine serum, and the magnetic field intervention was carried out. The intervention time was clearly divided into four levels of 10min, 15min, 20min, and 25min. The four groups of cells were placed in the same environment as the magnetic field intervention group, isolated from the magnetic field, and left to stand for 10min, 15min, 20min, and 25min respectively. Immediately after the magnetic intervention, the culture medium was replaced with CCK8 reagent, and immediately after the operation, the cells were returned to the cell culture incubator and continued to be cultured for 8 h; After 8 h, the cells were taken out, and an equal amount of CCK8 reagent in each group of cells was aspirated and placed in a 96-well cell culture plate. The absorbance of the CCK8 reagent in each group of cells was analyzed using an ELISA instrument to compare the proliferation rate of each group of cells.

Images