Method for improving human umbilical vein endothelial cell proliferation based on specific magnetic field parameters

Abstract

According to the method for improving human umbilical vein endothelial cell proliferation based on the specific magnetic field parameters, experiments verify that the sensitivity of human umbilical vein endothelial 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 human umbilical vein endothelial 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 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 invention belongs to the technical field of cell proliferation, and in particular relates to a method for increasing the proliferation of human umbilical vein endothelial cells based on specific magnetic field parameters. Background Art

[0002] There are many methods for growth or therapeutic intervention of human tissues based on magnetic fields, such as the invention patent applied for by our team in the early stage: the patent is a method based on low-frequency pulsed magnetic field to induce TRPC1 promotion and enhancement. This patent applies a weak pulsed magnetic field with a special frequency and waiting time to skeletal muscle tissue, activates TRPC1 transient receptor potential ion channels, promotes calcium ion influx and mitochondrial respiration in muscle cells, and promotes the growth and differentiation of muscle cells.

[0003] The area to be irradiated and enhanced is placed in a uniform magnetic field, and the TRPC1 protein is stimulated by a special low-frequency pulse magnetic field, thereby activating and strengthening the three ion pathways related to skeletal muscle metabolism, muscle fiber transformation and hypertrophy, thereby promoting muscle enhancement.

[0004] The prior art only points out that low-frequency pulsed magnetic fields have a certain promoting effect on the proliferation and differentiation of skeletal muscle cells. 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 exhibits is different; under the same magnetic field parameters, there may be great differences in the intervention effects on different cells; and the prior art does not provide the differences between the cell intervention effects under different magnetic field parameters and the differences in the optimal selection. The present invention applies safe pulsed magnetic fields of different intensities, frequencies, durations, and duty cycles to human umbilical vein endothelial cells, and clarifies the magnetic field parameters that have a positive promoting effect and the best promoting effect on the relevant cells by using its effect on the proliferation and differentiation of human umbilical vein endothelial cells.

[0005] Therefore, 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. Summary of the invention

[0006] The technical solution adopted by the present invention is: the method for increasing the proliferation of human umbilical vein endothelial cells based on specific magnetic field parameters, angiogenesis is a new vascular network formed by existing vascular endothelial cells, and is a complex process regulated by multiple factors, including the interaction between vascular endothelial cells and the surrounding extracellular matrix. This process is mainly the process of vascular endothelial cell proliferation, migration and vascular lumen formation.

[0007] The present invention verifies through experiments that the sensitivity of human umbilical vein endothelial 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. The present invention divides parameters such as magnetic field intensity, frequency, number of pulses, duty cycle, and intervention time into four levels, and arranges them into 16 parameter combinations in an alternating and direct manner. Under different magnetic field parameters, magnetic intervention is performed on cells. The cell proliferation rate and differentiation degree under different magnetic field parameters are compared through technical means, and the relevant formulas are used to deduce the noise resistance and sensitivity of each parameter to the magnetic field intervention effect, thereby screening out the best magnetic field intervention scheme under different conditions.

[0008] Beneficial effects of the present invention:

[0009] 1. The present invention expands the application scope of low-frequency pulsed magnetic induction technology, determines the specific parameters of its application in human umbilical vein endothelial cells, and compares the effects under different parameters;

[0010] 2. Magnetostimulation Limits in Magnetic Particle Imaging proposes that PNS (peripheral nerve stimulation) and SAR (tissue heating) can be effectively avoided under magnetic fields with an intensity lower than 6mT and a frequency lower than 25KHz, and the human body will not experience any irritation under magnetic fields within this intensity and frequency range. The relevant parameters such as magnetic field intensity, frequency, number of pulses, duty cycle, intervention time, etc. are arranged according to the AC / DC table, and the experimental data are analyzed using the Taguchi method to determine the noise immunity and sensitivity of the individual parameter levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is the magnetic field waveform, horizontal standard and alternating and direct table diagram of the present invention.

[0012] Figure 2 It is an experimental data diagram of the present invention comparing the proliferation rate of each group of cells.

[0013] Figure 3 It is the main effect diagram of the signal-to-noise ratio of the present invention.

[0014] Figure 4 It is the mean main effects plot of the present invention.

[0015] Figure 5 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

[0016] 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 were evenly distributed and all the experiments were 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.

[0017] In the present invention, the relevant magnetic field parameters (magnetic field intensity, frequency, number of pulses, duty cycle, intervention time) are divided into four levels, and the AC and DC are arranged. The relevant magnetic field waveform, level standard and AC and DC table are as follows: Figure 1 .

[0018] According to the data arrangement in the cross table, a magnetic field intervention experiment was conducted on human umbilical vein endothelial cells. The HUVEC 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 HUVEC cells is within 3 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 culture medium containing 10% FBSDMEM, 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.

[0019] 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.

[0020] 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 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 Figure 2 .

[0021] 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.

[0022] According to the main effect diagram of signal-to-noise ratio, A, C, and D have the greatest impact 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 impact 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.

[0023] According to Taguchi analysis and prediction, the best parameters are A4B3C4D4, that is, the intensity is 6mT, the frequency is 4000Hz, the number of pulses is, and the intervention time is 15min. This parameter was used to perform magnetic intervention on human umbilical vein endothelial cells again, and compared with the best parameters in the 16 groups of parameter combinations. The comparison chart is as follows Figure 5 .

[0024] According to the above experimental results, magnetic intervention has a certain promoting effect on the proliferation of human umbilical vein endothelial 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 screen out the parameter combination with better intervention effect, which greatly saves the manpower and material resources required for repeated experiments. The improvement of the growth rate of human umbilical vein endothelial cells can greatly accelerate the healing and regeneration of blood vessels after surgery.

Claims

1. A method for increasing the proliferation of human umbilical vein endothelial cells based on specific magnetic field parameters, characterized in that: Angiogenesis is the formation of a new vascular network by existing endothelial cells. It is a complex process regulated by multiple factors and involves the interaction between endothelial cells and the surrounding extracellular matrix. This process mainly involves the proliferation, migration and formation of vascular lumen of endothelial cells.

2. The method for increasing the proliferation of human umbilical vein endothelial cells based on specific magnetic field parameters according to claim 1, characterized in that: Experiments have verified that the sensitivity of human umbilical vein endothelial cells to magnetic fields is similar to that of skeletal muscle cells, and within a certain range of magnetic field parameters, the arrangements and combinations of different magnetic field parameters have different effects on their intervention effects. This patent divides parameters such as magnetic field intensity, frequency, number of pulses, duty cycle, and intervention time into four levels, and arranges them into 16 parameter combinations in alternating and direct current.

3. The method for increasing the proliferation of human umbilical vein endothelial cells based on specific magnetic field parameters according to claim 2, characterized in that: Magnetic intervention is performed on cells under different magnetic field parameters. The cell proliferation rate and differentiation degree under different magnetic field parameters are compared through technical means. The relevant formulas are used to calculate the noise resistance and sensitivity of each parameter to the magnetic field intervention effect, so as to screen out the best magnetic field intervention plan under different situations.

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