Method and system for improving vascular smooth muscle cell proliferation based on specific magnetic field parameters

Abstract

According to the method and system for improving vascular smooth muscle cell proliferation based on the specific magnetic field parameters, experiments verify that the sensitivity of vascular smooth muscle cells to a magnetic field is similar to that of skeletal muscle cells, and in a certain magnetic field parameter range, permutation and combination of different magnetic field parameters have different influences on the intervention effect of the vascular smooth muscle cells; parameters such as magnetic field intensity, frequency, pulse number, duty ratio, intervention time and the like are divided into four levels and are arranged into 16 parameter combinations in an alternating-direct mode, magnetic intervention is carried out on cells under different magnetic field parameters, cell proliferation speeds and differentiation degrees under different magnetic field parameters are compared through a technical means, and the cell differentiation accuracy is improved. The anti-noise capability, the sensitivity and the like of each parameter on the magnetic field intervention effect are calculated by utilizing a related formula, so that the optimal magnetic field intervention scheme under different conditions is screened out.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell proliferation, and in particular relates to a method and system for improving the proliferation of vascular smooth muscle cells based on specific magnetic field parameters. Background Art

[0002] The existing technology is to intervene in skeletal muscle cells through magnetic fields, so as to achieve the purpose of promoting skeletal muscle cell proliferation. For example, in the invention patent applied for by our team in the early stage (a method based on low-frequency pulsed magnetic field to induce TRPC1 promotion and enhancement), a weak pulsed magnetic field with a special waveform, frequency, pulse time, waiting time, intervention duration, etc. is applied to the skeletal muscle tissue to activate the TRPC1 transient receptor potential ion channel, promote the influx of calcium ions in muscle cells and mitochondrial respiration, thereby achieving the purpose of promoting the growth and differentiation of muscle cells.

[0003] In the prior art, it is only pointed out that special low-frequency pulsed magnetic fields can promote the proliferation and differentiation of skeletal muscle cells to a certain extent. In theory, various types of cells have different requirements for internal calcium ion channels and intracellular and extracellular calcium ion content, which makes the magnetic field sensitivity of various types of cells different. When the same cell is subjected to magnetic fields with different parameter combinations (intensity, frequency, pulse time, rest time, intervention duration), the magnetic intervention effect it exhibits is also different; the objective shortcomings of the prior art are as follows: 1. It does not propose the intervention effect of the magnetic field on other types of cells; 2. The prior art does not provide the difference between the cell intervention effects and the difference in optimization selection under different magnetic field parameters. Summary of the invention

[0004] The technical solution adopted by the present invention is: the method and system for improving the proliferation of vascular smooth muscle cells based on specific magnetic field parameters, by applying safe pulsed magnetic fields of different intensities, different frequencies, different durations, different duty cycles, etc. to the vascular smooth muscle cells, and the effect on the proliferation and differentiation of vascular smooth muscle cells is used to clarify the magnetic field parameters that have a positive promoting effect and the best promoting effect on the related cells by magnetic field intervention.

[0005] Considering that in the actual application of the product, magnetic field intervention needs to be performed on a variety of cells to achieve the effect of promoting their proliferation, it is necessary to conduct magnetic field intervention experiments on different cells to determine the optimal magnetic field parameters for different types of cells.

[0006] Experiments have verified that the sensitivity of vascular smooth muscle cells to magnetic fields is similar to that of skeletal muscle cells, and within a certain range of magnetic field parameters, the permutations and combinations of different magnetic field parameters have different effects on their intervention effects.

[0007] Parameters such as magnetic field intensity, frequency, number of pulses, duty cycle, and intervention time are divided into four levels and arranged in 16 parameter combinations. Magnetic intervention is performed on cells under different magnetic field parameters. The cell proliferation rate and degree of differentiation under different magnetic field parameters are compared through technical means, and the relevant formulas are used to calculate the noise resistance and sensitivity of each parameter to the magnetic field intervention effect, thereby screening out the best magnetic field intervention plan under different situations.

[0008] Beneficial effects of the present invention:

[0009] 1. The present invention expands the application scope of low-frequency pulse magnetic induction technology, determines its specific parameters for application in vascular smooth muscle cells, and compares the effects under different parameters.

[0010] 2. Limit the safety of the magnetic field. When the intensity is lower than 6mT and the frequency is lower than 25KHz, PNS (peripheral nerve stimulation) and SAR (tissue heating) can be effectively avoided. Therefore, the human body will not feel any irritation under the magnetic field within this intensity and frequency range. The magnetic field intensity, frequency, number of pulses, duty cycle, intervention time and other related parameters are arranged according to the AC / DC table. The experimental data are analyzed using the Taguchi method to determine the noise resistance and sensitivity of the individual parameter levels.

[0011] 3. The optimal magnetic field to promote the proliferation and differentiation of vascular smooth muscle cells is proposed, which can be applied to postoperative vascular regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a diagram of the equipment used in the experiment of the present invention.

[0013] Figure 2 It is the magnetic field waveform, horizontal standard and alternating and direct table diagram of the present invention.

[0014] Figure 3 It is an experimental data diagram of the present invention comparing the proliferation rate of each group of cells.

[0015] Figure 4 It is the mean main effects plot of the present invention.

[0016] Figure 5 It is the main effect diagram of the signal-to-noise ratio of the present invention.

[0017] Figure 6 This is a comparison chart showing that the present invention again performs magnetic intervention on human umbilical vein endothelial cells and compares the results with the best parameters in 16 groups of parameter arrangements and combinations. DETAILED DESCRIPTION

[0018] Angiogenesis is a new vascular network formed by existing endothelial cells. It is a complex process regulated by multiple factors, including the interaction between endothelial cells and the surrounding extracellular matrix. This process is mainly the process of endothelial cell proliferation, migration and vascular lumen formation. In production practices such as developing new products or improving old products, many tests are often required. Proper experimental arrangements will reduce the number of tests, avoid blindness, and obtain good results. On the contrary, an increase in workload may not necessarily lead to good results.

[0019] In this experimental design, the BP4610 power amplifier is used to provide different output signals for the Helmholtz coil: the relevant magnetic field parameters (magnetic field strength, frequency, number of pulses, duty cycle, intervention time) are divided into 4 levels, and they are arranged in AC and DC. The relevant magnetic field waveforms, level standards and AC and DC tables are as follows.

[0020] According to the data arrangement in the cross table, a magnetic field intervention experiment was conducted on human umbilical vein endothelial cells. The SMC cell cryopreservation tube was taken out from the -80°C refrigerator and immediately placed in a 37°C water bath to melt it as quickly as possible (the best time for SMC cells is within 2 minutes). The cryopreservation tube was gently and slowly shaken during the water bath to thaw it as quickly as possible. After it was completely dissolved, it was quickly disinfected with an alcohol cotton ball. The cryopreservation solution in the cryopreservation tube was sucked into a 10ml centrifuge tube with a 1ml pipette tip pipette, and centrifuged at 800rpm / min for 5min. After centrifugation, the supernatant was discarded, 3ml of sterile PBS was added to the centrifuge tube for washing 3 times, and then transferred to a DMEM culture medium containing 10% FBS, and the cell suspension was blown and fully mixed with a pipette. When the cells were completely resuspended in the culture medium, the cell suspension was transferred to a 10cm culture dish, and finally the culture dish was placed in a 37°C, 5% CO2 incubator for static culture. Magnetic field intervention was carried out when the cells were cultured to a density of 40%. 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: 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] According to the main effect diagram of signal-to-noise ratio, A, B, and C 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 C 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.

[0025] According to Taguchi analysis prediction, the best parameters are A4B1C4D4, that is, the intensity is 6mT, the frequency is 2000Hz, the pulse time is 0.01, the rest time is 0.1, and the intervention time is 15min. This parameter is used to perform magnetic intervention on vascular smooth muscle cells again, and compared with the best parameters in the 16 sets of parameter combinations. The comparison chart is as follows.

[0026] According to the above experimental results, magnetic intervention has a certain promoting effect on the proliferation of vascular smooth muscle cells, 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. The improvement of the proliferation rate of vascular smooth muscle cells can greatly accelerate the healing and regeneration of blood vessels after surgery.

Claims

1. A method and system for increasing the proliferation of vascular smooth muscle cells based on specific magnetic field parameters, characterized in that: By applying safe pulsed magnetic fields of different intensities, frequencies, durations, and duty cycles to vascular smooth muscle cells, the effects on the proliferation and differentiation of vascular smooth muscle cells are used to identify the magnetic field parameters that have a positive and optimal promoting effect on the related cells.

2. The method and system for increasing the proliferation of vascular smooth muscle cells based on specific magnetic field parameters according to claim 1, characterized in that: Considering that in the actual application of the product, magnetic field intervention needs to be performed on a variety of cells to achieve the effect of promoting their proliferation, it is necessary to conduct magnetic field intervention experiments on different cells to determine the optimal magnetic field parameters for different types of cells.

3. The method and system for increasing the proliferation of vascular smooth muscle cells based on specific magnetic field parameters according to claim 1, characterized in that: Experiments have verified that the sensitivity of vascular smooth muscle cells to magnetic fields is similar to that of skeletal muscle cells, and within a certain range of magnetic field parameters, the permutations and combinations of different magnetic field parameters have different effects on their intervention effects.

4. The method and system for increasing the proliferation of vascular smooth muscle cells based on specific magnetic field parameters according to claim 3, characterized in that: Parameters such as magnetic field intensity, frequency, number of pulses, duty cycle, and intervention time are divided into four levels and arranged in 16 parameter combinations. Magnetic intervention is performed on cells under different magnetic field parameters. The cell proliferation rate and degree of differentiation under different magnetic field parameters are compared through technical means, and the relevant formulas are used to calculate the noise resistance and sensitivity of each parameter to the magnetic field intervention effect, thereby screening out the best magnetic field intervention plan under different situations.

Images