Magnetic activation preparation method and application of phagocytic anti-inflammatory phenotype microglial cells

Abstract

The invention relates to a magnetic activation preparation method and application of phagocytic anti-inflammatory phenotype microglial cells, and the method comprises the following steps: separating and culturing microglial cells, and after the cells are cultured to a preset fusion degree, stimulating the microglial cells by using a pulsed magnetic field to obtain high-phagocytic and high-anti-inflammatory phenotype microglial cells which are transplanted into a specific brain region, the pulse magnetic field is used for treating central nervous cell diseases, the stimulation frequency of the pulse magnetic field is 1-100 Hz, the number of stimulation pulses is larger than or equal to 500, and the number of stimulation days is larger than or equal to 1 day. The microglial cells obtained by adopting low-frequency pulse stimulation have stronger migration ability, can swallow more foreign matters or abnormal protein aggregation products, can secrete less proinflammatory factors, is beneficial to recovery of the microenvironment of the central nervous system, and can be used for improving the microenvironment of the central nervous system. The invention provides an efficient, accurate and economical new way for microglial cells to treat neuroinflammation regulation and control and abnormal protein aggregation related diseases, and solves the technical problems of large side effect, high cost, inaccurate target spot, difficulty in acting on deep microglial cells and the like in the prior art.

Description

Technical Field

[0001] The present invention relates to the cross - technical field of neuroscience and bioelectromagnetics, and in particular to a magnetic activation preparation method and application of phagocytic anti - inflammatory phenotype microglia. Background Art

[0002] Microglia originate from yolk - sac progenitor cells, mature in the central nervous system and perform macrophage functions, which are of great significance for regulating the body's immune function and maintaining neuronal homeostasis. However, in many central nervous system diseases (such as Alzheimer's disease, Parkinson's disease, stroke, amyotrophic lateral sclerosis, etc.), microglia will be abnormally activated. Activated microglia will secrete a large amount of inflammatory factors such as TNF - α and IL - 1β, triggering or exacerbating the inflammatory response in the brain, causing damage to neurons and disrupting the normal function of the nervous system. Therefore, it is necessary to inhibit the inflammation of microglia. In Alzheimer's disease, the phagocytic function of microglia declines, resulting in a weakened phagocytic ability for the Alzheimer's disease pathogenic factor Abeta, leading to the accumulation of pathological proteins and exacerbating the occurrence of Alzheimer's disease. Therefore, improving the phagocytic and anti - inflammatory functions of microglia can play a role in treating these neurodegenerative diseases. Currently, the intervention of microglia in the brain is mainly through drug treatment and gene editing methods, but there are still many problems that cannot be ignored. On the one hand, drug treatment is often accompanied by serious side effects, affecting the quality of life of patients; on the other hand, gene editing is not only expensive and unaffordable for ordinary patients, but also has a long action cycle and is difficult to bring obvious curative effects to patients in a short time.

[0003] Repetitive transcranial magnetic stimulation (rTMS) is a non - invasive neuromodulation technique based on the Faraday electromagnetic induction effect. By applying a pulsed magnetic field outside the skull, it can directly affect the activities of brain cells. In in vitro cell experiments, magnetic stimulation can accelerate the proliferation of various cells and enhance immune function; in in vivo experiments, weak magnetic fields can trigger various effects, such as promoting fracture healing, joint fusion, angiogenesis, and collagen production. In clinical applications, transcranial magnetic stimulation technology also has deficiencies such as inaccurate magnetic stimulation target points and difficulty in acting on deep brain regions, which limit the direct regulation of magnetic stimulation on microglia in the brain. Currently, there is a lack of relevant research and development cases on implanting in vivo after in vitro magnetic stimulation of microglia. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a magnetic activation preparation method and application of phagocytic anti - inflammatory phenotype microglia.

[0005] The present invention is realized by the following technical solutions:

[0006] A magnetic activation preparation method for phagocytic anti-inflammatory phenotype microglia, comprising the following steps:

[0007] S11. Obtain microglia differentiated from stem cells, and perform purification and culture;

[0008] S12. Perform pulsed magnetic stimulation on the purified and cultured microglia. The frequency of the pulsed magnetic stimulation is 1-100 Hz, the number of stimulation pulses ≥500, and the number of stimulation days ≥1 day to obtain phagocytic anti-inflammatory phenotype microglia.

[0009] According to the above technical solution, preferably, in step S11, the stem cells include embryonic stem cells, pluripotent induced stem cells or mesenchymal stem cells.

[0010] According to the above technical solution, preferably, in step S11, during the "stem cell induced differentiation", select mesenchymal stem cell medium as a suitable medium, with a cell density of 1×10 4 -1×10 5 cells / cm 2 , culture in an incubator at 37°C and 5% CO2, and select growth factors as inducers, with an induction time between 4 days and 1 week, and change the medium every 2-3 days.

[0011] According to the above technical solution, preferably, in step S11, the "purification" process includes:

[0012] Prepare the induced differentiated cells into a single cell suspension, add immunomagnetic beads, and incubate at 4°C for 30-60 minutes;

[0013] Add the single cell suspension to a magnetic field sorting column. Under the action of the magnetic field, the microglia combined with the magnetic beads are retained in the sorting column, while other cells flow out;

[0014] Remove the magnetic field, elute the microglia retained in the sorting column with a buffer solution, collect the eluate to obtain purified microglia.

[0015] According to the above technical solution, preferably, in step S11, when performing the "purification and culture", add 10-20% fetal bovine serum to the basal medium to provide growth factors and nutrients for the microglia, and add macrophage colony-stimulating factor with a concentration of 10-50 ng / mL to DMEM / F12 medium, and culture in an incubator at 37°C. The gas environment in the incubator is 5% CO2 and 95% air, and the humidity in the incubator is maintained at about 95%.

[0016] According to the above technical solution, preferably, in step S12, the frequency of the pulsed magnetic stimulation is 1-40 Hz.

[0017] The present application also discloses an application of phagocytic anti-inflammatory phenotype microglia. Based on the above magnetic activation preparation method of phagocytic anti-inflammatory phenotype microglia, the phagocytic anti-inflammatory phenotype microglia are used for transplantation into specific brain regions for the treatment of central nerve cell diseases. The specific brain regions include the hippocampus, prefrontal cortex, amygdala, and striatum related to the central nervous system. The central nerve cell diseases include Alzheimer's disease, Parkinson's disease, and cerebral ischemia related to abnormal microglia.

[0018] According to the above technical solution, preferably, the transplantation of the phagocytic anti-inflammatory phenotype microglia into specific brain regions includes the following steps:

[0019] S21. Digest and centrifuge the phagocytic anti-inflammatory phenotype microglia to obtain a precipitate containing microglia.

[0020] S22. Resuspend the precipitate containing microglia in a phosphate buffer solution to obtain high phagocytic anti-inflammatory phenotype microglia that can be used for injection into specific brain regions.

[0021] According to the above technical solution, preferably, step S21 includes the following steps:

[0022] Discard the culture supernatant, rinse the cells with PBS 1-2 times, add 1-2 ml of digestive solution to the culture flask. The digestive solution is 0.25% Trypsin - 0.53 mM EDTA, and place it in a 37°C incubator for digestion for 1-2 minutes.

[0023] Observe the cell digestion situation under a microscope. If most of the cells become round and detached, quickly take it back to the operating table, gently tap the culture flask a few times, then add more than 5 ml of complete medium containing 10% serum to terminate digestion, gently pipette the cells, and aspirate them after complete detachment.

[0024] Collect the digested cell suspension into a centrifuge tube, centrifuge it at 1000 RPM for 8-10 minutes, discard the supernatant, add 1-2 mL of culture medium and pipette evenly to obtain a precipitate containing microglia.

[0025] According to the above technical solution, preferably, in step S22, the volume of the phosphate buffer solution is 0.1-1000 μL, and the number of the high phagocytic anti-inflammatory phenotype microglia is 1×10 4 -1×10 7 cells.

[0026] The beneficial effects of the present invention are:

[0027] By deeply researching and optimizing the key parameters of magnetic stimulation, including magnetic field strength, frequency, pulse pattern, and action time, etc., and combining with a unique magnetic stimulation mode of action, the present invention realizes precise regulation of microglia. In addition, in the technical design, the particularity of microglia in deep brain regions is fully considered. Through in vitro magnetic stimulation and in situ implantation of microglia, it can effectively act on microglia in deep tissues, thus breaking through the limitation that traditional magnetic stimulation technology is difficult to regulate deep cells. Through this series of innovative improvements, the method of this application not only significantly reduces the side effects brought by traditional gene encoding or drug intervention, but also greatly reduces the operation cost, improves the feasibility and application scope of the technology, provides an efficient, precise and economical new way for the application of microglia in the regulation of neuroinflammation and the treatment of diseases related to abnormal protein aggregation, and effectively solves the long-term technical problems in the prior art such as large side effects, high cost, inaccurate targets, and difficulty in acting on deep microglia. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the flow chart of the method for magnetically activating microglia of the present invention.

[0029] Figure 2 A is the schematic principle diagram of the process of magnetically stimulating microglia of the present invention, Figure 2 B is the schematic diagram of the process of different magnetic stimulations of the present invention.

[0030] Figure 3 A is the effect of repetitive magnetic stimulation at different frequencies on the viability of BV2 cells in the present invention, Figure 3 B is to observe the cell morphology by immunofluorescence staining, the cell nucleus is stained with DAPI, scale bar = 50 μm, all values are expressed as mean ± standard error, n = 3 for each group, *p < 0.05, compared with the control group;

[0031] Figure 4 A is the representative result of the flow cytometry experiment, Figure 4 B is the schematic diagram showing that repetitive magnetic stimulation enhances the phagocytosis of cells.

[0032] Figure 5 A is the representative micrograph of the cell scratch experiment (scale bar = 500 μm), Figure 5 B is the schematic diagram of the effect of repetitive magnetic stimulation at different frequencies on the cell migration ability.

[0033] Figure 6 A is the mRNA level of IL-1β in cells, Figure 6 A is the mRNA level of TNF-α in cells, Figure 6 C is the protein expression of IL-1β in the cell supernatant, Figure 6D was the protein expression of TNF-α in the cell supernatant. The data were expressed as the mean ± standard error. n = 3 in each group, *p < 0.05, compared with the control group;

[0034] Figure 7 A were representative WB bands of iNOS, NF-κB p65, and p-NF-κB p65. Figure 7 B was the immunofluorescence staining of BV2 cells with anti-NF-κB p65 antibody and DAPI after repetitive transcranial magnetic stimulation. Scale bar = 20 μm. n = 3 in each group, *p < 0.05, compared with the control group. Detailed implementation manners

[0035] To enable those skilled in the art of the present technology to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and the best embodiments. Based on the embodiments in the invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the invention.

[0036] Example 1: The present application discloses a method for magnetically activating and preparing phagocytic anti-inflammatory phenotype microglia, including the following steps:

[0037] S11. Microglia obtained by inducing and differentiating stem cells were purified and cultured.

[0038] S12. The purified and cultured microglia were subjected to pulsed magnetic stimulation. The frequency of the pulsed magnetic stimulation was 1 - 100 Hz, the number of stimulation pulses was ≥500, and the number of stimulation days was ≥1 day to obtain phagocytic anti-inflammatory phenotype microglia.

[0039] Among them, in step S11, the stem cells include embryonic stem cells, pluripotent induced stem cells, or bone marrow mesenchymal stem cells. When inducing and differentiating stem cells, mesenchymal stem cell medium was selected as the appropriate medium, and its cell density was 1×10 4 -1×10 5 cells / cm 2 . They were cultured in an incubator at 37°C and 5% CO2, and growth factors were selected as inducers. The induction time was between 4 days and 1 week, and the medium was changed every 2 - 3 days.

[0040] Meanwhile, the purification process in step S11 includes: making the induced differentiated cells into a single cell suspension, adding immunomagnetic beads, incubating at 4°C for 30 - 60 minutes to allow the magnetic beads to fully bind to microglia; adding the single cell suspension to a magnetic field sorting column, under the action of the magnetic field, the microglia bound with the magnetic beads are retained in the sorting column while other cells flow out; removing the magnetic field, eluting the microglia retained in the sorting column with a buffer solution, collecting the eluate to obtain purified microglia.

[0041] When culturing the microglia induced and differentiated from stem cells in step S11, 10 - 20% fetal bovine serum is added to the basal medium to provide growth factors and nutrients for the microglia, and macrophage colony-stimulating factor (M-CSF) with a concentration of 10 - 50 ng / mL is added to the DMEM / F12 medium, and it is cultured in an incubator at 37°C. The gas environment in the incubator is 5% CO2 and 95% air, and the humidity in the incubator is maintained at about 95% to prevent evaporation of the medium moisture and maintain a stable environment for cell growth.

[0042] In addition, in step S12, magnetic stimulation is used to stimulate the cells to a preset cell fusion degree to obtain fused cells. In this example, the initial cell fusion degree of the cells ≤ 50%, and the preset cell fusion degree ≥ 80%. It is also worth noting that the frequency of pulsed magnetic stimulation is preferably but not limited to 1 - 40 Hz. Figure 4 、 6 respectively reflect the effects of different stimulation frequencies on the anti-inflammatory and phagocytic abilities of the induced microglia, and it is found that magnetic stimulation with frequencies of 20 Hz and 40 Hz may be able to cause microglia to exhibit the strongest phagocytic and anti-inflammatory abilities ( Figure 4 A and B illustrate that magnetic stimulation of microglia at 20 Hz and 40 Hz shows the strongest phagocytic ability. Figure 6 A and C illustrate that the inflammatory factors secreted by microglia after magnetic stimulation at 20 Hz and 40 Hz are reduced), and through subsequent p65 immunofluorescence staining and calcium ion concentration detection, it is found that the effects of 20 Hz and 40 Hz are related to their ability to increase the intracellular calcium ion concentration.

[0043] Example 2: The present application also discloses an application of phagocytic anti-inflammatory phenotype microglia. Based on the above magnetic activation preparation method of phagocytic anti-inflammatory phenotype microglia, the phagocytic anti-inflammatory phenotype microglia are used for transplantation into specific brain regions for the treatment of central nerve cell diseases. The specific brain regions include the hippocampus, prefrontal cortex, amygdala, and striatum related to the central nervous system, and the central nerve cell diseases include Alzheimer's disease, Parkinson's disease, and cerebral ischemia related to abnormal microglia.

[0044] According to the above technical solution, preferably, the transplantation of phagocytic anti-inflammatory phenotype microglia into a specific brain region includes the following steps:

[0045] S21. Perform multiple pulsed magnetic stimulations on the microglia in a sterile environment, digest and centrifuge the phagocytic anti-inflammatory phenotype microglia to obtain a precipitate containing microglia.

[0046] Specifically, first discard the culture supernatant, rinse the cells with PBS 1 - 2 times, add 1 - 2 ml of digestive solution to the culture flask. The digestive solution is 0.25% Trypsin - 0.53 mM EDTA, and place it in a 37°C incubator for digestion for 1 - 2 minutes; then observe the cell digestion under a microscope. If most of the cells become round and detached, quickly bring it back to the operating table, gently tap the culture flask a few times, and then add more than 5 ml of complete medium containing 10% serum to terminate digestion. Gently pipette the cells, and aspirate them after complete detachment; then collect the digested cell suspension into a centrifuge tube, centrifuge at 1000 RPM for 8 - 10 minutes, discard the supernatant, add 1 - 2 mL of culture medium and pipette evenly to obtain a precipitate containing microglia.

[0047] S22. Resuspend the precipitate containing microglia in phosphate buffer solution to obtain phagocytic anti-inflammatory phenotype microglia that can be used for injection into a specific brain region. Among them, in this example, the volume of phosphate buffer solution is preferably 0.1 - 1000 μL, and the number of phagocytic anti-inflammatory phenotype microglia is 1×10 4 -1×10 7 cells.

[0048] Example 3: Culture the induced and differentiated microglia in a medium containing 90% DMEM, 10% fetal bovine serum, and 1% penicillin / streptomycin, change the medium every 2 - 3 days, and digest and passage with 0.25% trypsin when the cell confluence reaches 80%. Using a magnetic stimulator, place the culture dish in the center of the stimulation coil, ensuring that the distance between the stimulation coil and the cells is 5 mm. Set different frequencies such as 0 Hz, 1 Hz, 5 Hz, 20 Hz, 40 Hz, stimulate twice a day, with an interval of 12 hours, and continuously stimulate for three days. The total number of stimulations at each frequency is 500, and the cells stay in the external environment for 500 s. Perform cell function detection on the obtained phagocytic anti-inflammatory phenotype microglia.

[0049] (1) Cell viability detection: Using the MTT method, add MTT solution with a final concentration of 0.5 mg / mL to each well after magnetic stimulation, incubate at 37°C for 4 h, read the OD value at 450 nm with an enzyme - linked immunosorbent assay (ELISA) reader, and calculate the cell survival rate according to the formula to judge the effect of magnetic stimulation on cell viability. After immunostaining with an anti - Iba - 1 (a specific microglia marker) antibody, the results all show that the stimulated microglia have smaller cell bodies and longer branches compared with the control group microglia (Figure 3 ) These results indicate that repetitive magnetic stimulation at different frequencies can benefit the morphological changes of BV2 cells without affecting their activity.

[0050] (2) Phagocytosis ability assessment: By flow cytometry, after ultrasonic treatment and vortex oscillation of PE-conjugated microspheres, add them to the wells at a ratio of cell:microsphere = 1:200, incubate at 37 °C for 1 h, and after operations such as washing with PBS, trypsin digestion, and termination of digestion, use a flow cytometer to analyze the proportion of microglia phagocytosing microspheres to measure the change in cell phagocytosis ability. After magnetic stimulation, compared with the control group, the phagocytosis ratio of stimulated microglia to PE-conjugated microspheres was significantly enhanced ( Figure 4 ). Repetitive magnetic stimulation at low and high frequencies can enhance the phagocytosis ability of microglia.

[0051] (3) Migration ability determination: Adopt the cell scratch assay. After magnetic stimulation, use a pipette tip to draw a straight line (15 - 20 μm) at the bottom of the culture dish, wash with PBS and then change to serum-free medium, sample and take pictures at 0 h and 24 h, and calculate the cell migration rate according to the formula to detect the effect of magnetic stimulation on cell migration ability. After magnetic stimulation, compared with the control group, the migration rate of stimulated microglia was significantly increased ( Figure 5 ). Repetitive magnetic stimulation at low and high frequencies can enhance the migration ability of microglia.

[0052] (4) Inflammatory factor analysis (mRNA level detection): Extract total RNA by the Trizol method, reverse transcribe it into cDNA using HiScript II Q RT SuperMix, and then perform qPCR detection using Taq Pro Universal SYBR qPCR Master Mix. Set the amplification parameters to cycle 40 times, analyze the relative mRNA expression levels of IL-1β and TNF-α relative to β-actin to determine the regulation of magnetic stimulation on the gene expression of inflammatory factors; Protein level detection: Collect the cell supernatant after magnetic stimulation by ELISA method, and read the OD value at 450 nm on an enzyme-linked immunosorbent assay analyzer to measure the protein concentrations of TNF-α and IL-1β, and evaluate the change in the expression of inflammatory factors at the protein level. Compared with the control group, magnetic stimulation at 5 Hz, 20 Hz, and 40 Hz significantly reduced the mRNA level of the pro-inflammatory cytokine IL-1β ( Figure 6 a), and magnetic stimulation at 5 Hz and 40 Hz significantly reduced the mRNA level of the pro-inflammatory cytokine TNF-α ( Figure 6 b). The ELISA results showed ( Figure 6 c, d) that compared with 0 Hz, magnetic stimulation at 5 Hz and 20 Hz significantly reduced the protein expression level of IL-1β, and other frequencies showed a decreasing trend; compared with the control group, magnetic stimulation at all frequencies showed a trend of decreasing the protein expression of TNF-α, but no significant difference was shown.

[0053] (5) Signal pathway exploration: Using immunofluorescence, after culturing and stimulating microglial cells, they were fixed with paraformaldehyde, permeabilized with Triton X-100, blocked with goat serum, and then incubated successively with anti-rabbit Iba-1, anti-rabbit NF-κB p65 primary antibodies and goat anti-rabbit IgG-AF594 secondary antibody. After DAPI staining the cell nuclei, the slides were sealed. The nuclear entry of NF-κB was observed with a confocal microscope. Using Western blotting, proteins were separated by SDS-PAGE and transferred to a PVDF membrane. After blocking with skim milk powder, they were incubated with primary antibodies such as iNOS, NFκB, p-NF-κB, β-actin at 4 °C overnight. After washing the membrane with TBS-T, they were incubated with the corresponding secondary antibody for 90 min. After washing, the protein bands were detected with enhanced chemiluminescence solution, and the signal gray values were obtained by analyzing the digital images with PS. Using β-actin as an internal reference, the effect of magnetic stimulation on the NF-κB signal pathway and the expression of related proteins was studied. Compared with the control group, the ratio of p-NF-κB p65 to NF-κB p65 and the protein expression of iNOS in microglial cells treated with magnetic stimulation were significantly down-regulated ( Figure 7 a). Observing the nuclear entry of NF-κB by immunofluorescence, compared with the control group, the nuclear entry ratio of NF-κB in stimulated microglial cells was significantly reduced ( Figure 7 b), further confirming that magnetic stimulation can reduce the expression of inflammatory factors in microglial cells. In summary, different frequencies of repetitive magnetic stimulation affect the activation of downstream iNOS by balancing the NF-κB signal pathway, and reduce the expression of inflammatory factors in microglial cells.

[0054] Example 4: Taking the clinical action on microglial cells in the mouse brain as an example, magnetically activated phagocytic anti-inflammatory phenotype microglial cells were prepared and injected into a specific brain region for transplantation, for the treatment of central nervous cell diseases, including the following steps:

[0055] S31. Extraction of primary microglial cells:

[0056] Magnetic-activated cell sorting (MACS): Newborn mice were deeply anesthetized by intraperitoneal injection of ketamine (10 mg / kg) and xylazine (1 mg / kg). The mouse brains were perfused with cold L15 medium and quickly chopped on ice. Subsequently, the mouse brains were dissociated with papain (16.5 U / mL) for 30 minutes at room temperature and then terminated with ovomucoid (2 mg / mL) in the medium. Next, myelin was removed from the single-cell suspension by Percol density gradient centrifugation (30% v / v). Then the cell pellet was resuspended in the medium containing 10% magnetic beads conjugated with anti-mouse CD11b antibody and incubated at 4 °C for 15 minutes. Then the cells were washed with L15 medium and resuspended in 0.01 M PBS containing 0.5% BSA. The flow cell suspension was passed through an LS column connected to a QuadroMACS separator, and the column was washed twice with 3 mL of 0.01 M PBS to enrich microglia (or mrBMT cells). Then the cells in the column were rinsed with 3 mL of 0.01 M PBS. To obtain purified CD11b-positive cells, the enrichment process was repeated twice to obtain primary microglia.

[0057] S32. Cell preparation and magnetic stimulation implementation:

[0058] An appropriate amount of microglia in the logarithmic growth phase was trypsinized and seeded in a culture dish, and cultured in an incubator at 37 °C and 5% CO2 until the confluence reached 80%.

[0059] According to the above magnetic stimulation parameter settings, the culture dish was placed in a magnetic stimulator for repeated magnetic stimulation at different frequencies. Two stimulation operations were performed regularly every day. During this period, the stability of the cell culture environment was closely observed to ensure that conditions such as temperature, humidity, and CO2 concentration met the requirements for cell growth, and microglia with a phagocytic anti-inflammatory phenotype were obtained.

[0060] S33. Injection of microglia:

[0061] S33-1. Cell preparation:

[0062] The cultured microglia were trypsinized, and the collected microglia were resuspended in PBS, and the cell concentration was adjusted to about 5×10 4 cells / injection.

[0063] S33-2. Surgical preparation:

[0064] The recipient mice were anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg / kg) and xylazine (10 mg / kg);

[0065] The mice were fixed on a stereotaxic apparatus, and the heads were disinfected and shaved.

[0066] S33-3. Injection process:

[0067] According to the experimental requirements, select the injection site (such as the hippocampal region, coordinates: AP: -2.0 mm, ML: +1.5 mm, DV: -1.5 mm);

[0068] Use a microsyringe (such as a Hamilton syringe) to slowly inject the microglia suspension into the target brain region. The injection speed is usually 0.1 - 0.2 μL / min to avoid tissue damage. After the injection is completed, keep the needle in place for 1 - 2 minutes to ensure complete cell diffusion, and then slowly withdraw the needle.

[0069] S33-4. Postoperative treatment:

[0070] After the injection, disinfect the wound of the mouse and give the mouse a pain reliever (such as meloxicam, 1 mg / kg) to relieve pain;

[0071] Within 1 - 2 days after the operation, give the pain reliever once a day, and at the same time monitor the recovery of the mouse.

[0072] Based on the above Examples 1 - 4, it can be seen that this application has the following technical effects:

[0073] This application clarifies that repetitive magnetic stimulation at different frequencies has no negative impact on the viability of microglia and can induce beneficial changes in cell morphology, providing a safety basis for the application of magnetic stimulation in microglia regulation and expanding the application potential of magnetic stimulation in the field of nerve cells;

[0074] This application successfully demonstrates that magnetic stimulation at all frequencies significantly enhances the phagocytic ability and migration rate of microglia, which is of great significance in the treatment of neuroinflammation, can promote the clearance and repair of pathogens and damaged tissues by microglia, and accelerate the recovery of nerve tissue;

[0075] This application accurately determines that magnetic stimulation at 5 Hz, 20 Hz, and 40 Hz can significantly reduce the level of IL-1β mRNA, magnetic stimulation at 5 Hz and 40 Hz can significantly reduce the level of TNF-α mRNA, and magnetic stimulation at 5 Hz and 20 Hz significantly reduces the level of IL-1β protein. Magnetic stimulation at all frequencies shows a tendency to reduce the expression of TNF-α protein, providing a frequency basis for targeted regulation of microglia inflammatory factor secretion and contributing to the development of precise magnetic stimulation treatment plans;

[0076] This application deeply reveals that magnetic stimulation at all frequencies reduces the expression of downstream iNOS protein and the phosphorylation degree of NF-κB p65 by balancing the NF-κB signaling pathway, and reduces the expression of inflammatory factors, clarifying the internal molecular mechanism of magnetic stimulation regulating microglia function, and providing a theoretical support for subsequent drug research and development or treatment method innovation based on this mechanism.

[0077] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing microglia with phagocytic anti-inflammatory phenotype by magnetic activation, characterized in that: The steps include: S11. Purify and culture microglia obtained by inducing differentiation of stem cells; S12. The purified and cultured microglia are subjected to pulsed magnetic stimulation, wherein the frequency of the pulsed magnetic stimulation is 1-100 Hz, the number of stimulation pulses is ≥500, and the number of stimulation days is ≥1 day, so as to obtain microglia with phagocytic anti-inflammatory phenotype.

2. The method for preparing a phagocytic anti-inflammatory phenotype microglia by magnetic activation according to claim 1, characterized in that: In step S11, the stem cells include embryonic stem cells, induced pluripotent stem cells or mesenchymal stem cells.

3. The method for preparing a phagocytic anti-inflammatory phenotype microglia by magnetic activation according to claim 2, characterized in that: In step S11, during the "stem cell differentiation induction", a mesenchymal stem cell culture medium is selected as a suitable culture medium, and the cell density is 1×10 4 -1×10 5 cells / cm 2 , cultured in an incubator at 37°C and 5% CO2, and growth factors were selected as inducers. The induction time was between 4 days and 1 week, and the culture medium was replaced every 2-3 days.

4. The method for preparing a phagocytic anti-inflammatory phenotype microglia by magnetic activation according to claim 3, characterized in that: In step S11, the "purification" process includes: The induced differentiated cells were made into a single cell suspension, immunomagnetic beads were added, and incubated at 4°C for 30-60 minutes; The single cell suspension is added to a magnetic field sorting column, and under the action of the magnetic field, microglial cells bound to the magnetic beads are retained in the sorting column, while other cells flow out; The magnetic field is removed, and the microglia retained in the separation column are eluted with a buffer solution, and the eluate is collected to obtain purified microglia.

5. The method for preparing a phagocytic anti-inflammatory phenotype microglia by magnetic activation according to claim 4, characterized in that: In step S11, during the "purification and culture", 10-20% fetal bovine serum is added to the basal culture medium to provide growth factors and nutrients for microglia, and 10-50 ng / mL macrophage colony stimulating factor is added to the DMEM / F12 culture medium. The cells were cultured in an incubator at 37°C. The gas environment in the incubator was 5% CO2 and 95% air. The humidity in the incubator was maintained at about 95%.

6. The method for preparing microglia with phagocytic anti-inflammatory phenotype by magnetic activation according to claim 1, characterized in that: In step S12, the frequency of the pulse magnetic stimulation is 1-40 Hz.

7. An application of phagocytic anti-inflammatory phenotype microglia, based on the magnetic activation preparation method of phagocytic anti-inflammatory phenotype microglia according to claims 1-6, characterized in that: The phagocytic anti-inflammatory phenotype microglia are used for transplantation into specific brain regions for the treatment of central nervous system diseases. The specific brain regions include the hippocampus, prefrontal cortex, amygdala, and striatum related to the central nervous system, and the central nervous system cell diseases include Alzheimer's disease, Parkinson's disease, and cerebral ischemia related to abnormal microglia.

8. The use of a phagocytic anti-inflammatory phenotype microglia according to claim 7, characterized in that: The method of transplanting the phagocytic anti-inflammatory phenotype microglia into a specific brain region comprises the following steps: S21. digesting and centrifuging the phagocytic anti-inflammatory phenotype microglia to obtain a precipitate containing microglia; S22. Resuspend the microglia-containing precipitate in a phosphate buffered solution to obtain microglia with a high phagocytic and anti-inflammatory phenotype that can be used for injection into specific brain regions.

9. The use of a phagocytic anti-inflammatory phenotype microglia according to claim 8, characterized in that: Step S21 includes the following steps: The culture supernatant was discarded, the cells were rinsed 1-2 times with PBS, 1-2 ml of digestion solution was added to the culture bottle, the digestion solution was 0.25% Trypsin-0.53 mM EDTA, and the cells were placed in a 37°C incubator for digestion for 1-2 minutes; Observe the cell digestion under a microscope. If most of the cells become round and fall off, quickly take them back to the operating table, tap the culture bottle a few times, add more than 5 ml of complete culture medium containing 10% serum to stop digestion, blow the cells gently, and aspirate them after they fall off completely; The digested cell suspension was collected into a centrifuge tube, centrifuged at 1000 RPM for 8-10 minutes, the supernatant was discarded, 1-2 mL of culture medium was added and blown evenly to obtain a precipitate containing microglia.

10. The use of a phagocytic anti-inflammatory phenotype microglia according to claim 8, characterized in that: In step S22, the volume of the phosphate buffer solution is 0.1-1000 μL, and the number of the highly phagocytic anti-inflammatory phenotype microglia is 1×10 4 -1×10 7 indivual.

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