CN112029725B - Method for promoting macrophage polarization to M1 type by utilizing piezoelectric effect and application - Google Patents
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Abstract
The invention relates to a method for promoting macrophage polarization to M1 type by utilizing piezoelectric effect and application thereof. The piezoelectric material is used as a cell culture substrate and is combined with ultrasonic stimulation, M1 type polarized macrophages are cultured in vitro, compared with a common biochemical method, the method does not need to introduce external PMAPs, and the commercialized piezoelectric material is utilized, so that the macrophages can be rapidly promoted to be polarized to the M1 type by applying the ultrasonic method; the invention has good effect of promoting macrophage polarization to M1 type by utilizing piezoelectric effect, and has clinical application potential of tumor immunotherapy and anti-infection therapy. The method of the invention can produce M1 polarized macrophage in batch to promote the application of piezoelectric material in immunotherapy.
Description
Technical Field
The invention relates to a method for promoting macrophage polarization to M1 type by utilizing piezoelectric effect and application, belonging to the technical field of biological materials.
Background
During inflammation, immune homeostasis changes dramatically as a result of microbial danger signals, collectively referred to as pathogen-associated molecular patterns (PAMPs), including microorganisms, Lipopolysaccharide (LPS), DNA, RNA, and the like. In the inflammatory microenvironment, PAMPs will guide the immune process, and immune cells can not only respond to PAMPs through pattern recognition receptors, but also coordinate or individually regulate immune cells through various microenvironment receptors sensing temperature, pH, osmotic pressure, oxygen, etc. Macrophages are the most abundant immune cells in the tumor microenvironment and play a great role in the occurrence and development of tumors, and are divided into an M1 subtype with tumor growth resistance and an M2 subtype with tumor growth promotion. The promotion of the differentiation of tumor-associated macrophages to M1 type is of great significance for the treatment of tumors.
Chinese patent document CN 109369795A (application No. 201811350918.3) discloses a protein PvEXP100 protein for regulating and controlling the immune function activity of macrophages, and the PvEXP100 protein acts on unactivated RAW264.7 macrophages to obviously enhance the capability of the RAW264.7 macrophages to secrete NO, IL-2, IL-6 and TNF-alpha. Chinese patent document CN 111518219a (application No. 202010383935.8) discloses a chimeric antigen receptor, the intracellular domain of which contains IFN-gamma receptor, the macrophage expressing the chimeric antigen receptor can keep M1 type state for a relatively long time, and the activity of macrophage M1 type after tumor cell antigen is combined with the chimeric antigen receptor is enhanced. Chinese patent document CN 108210482a (application No. 201611127159.5) discloses a miRNA-loaded composite nanoparticle, which includes a cationic targeting membrane material, an acid-sensitive membrane material, and at least one miRNA; the miRNA is selected from miRNA for inducing tumor-related macrophages to differentiate into M1 type macrophages, and the function of promoting the tumor-related macrophages to differentiate into M1 type macrophages is realized.
The development of new drugs for clinical treatment has been promoted by the development of a number of new drugs based on pattern recognition receptors and micro-environmental sensors to modulate immune cell responses. In addition to biological signals such as PAMPs, in recent years, some research has focused on the study of physical signals in modulating immune cell responses and playing an increasingly important role. However, little is known about the role of electrical stimulation in modulating macrophage immune responses. Under physiological or pathological conditions, cell death and tissue damage can cause fluctuations in the ionic current of the cellular microenvironment, thereby altering the potential of the microenvironment. Although electrical stimulation may be important in macrophage polarization, the regulatory mechanisms need to be further explored.
Implanted batteries, nano-generators or external electrical source stimulation are effective means of stimulating cells, and these methods have been applied to targeted modulation of cardiogenesis and neuronal cell function. However, they require invasive wires to connect external and complex devices for implantation into the body, which not only causes great inconvenience to the patient, but also may pose a risk of infection and the like. Therefore, the development of non-invasive electrical stimulation means for cells and thereby the regulation of macrophage polarization will have profound effects in the fields of tumor immunotherapy and anti-infection therapy.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for promoting the polarization of macrophages to M1 type by utilizing piezoelectric effect and application thereof, which adopts non-contact and non-invasive electrical stimulation to realize the accurate regulation and control of the immune response of the macrophages and avoids the damage of complicated operation to the macrophages and human bodies. The invention provides the method for promoting the polarization of macrophages to the M1 type direction by using various piezoelectric materials and generating an electric signal after the materials are deformed by applying ultrasonic waves.
The technical scheme of the invention is as follows:
a method for promoting macrophage polarization to M1 type by utilizing piezoelectric effect is carried out in vitro, piezoelectric material is used as a cell culture substrate, macrophage suspension is inoculated on the substrate, cells are cultured for 1-7 d, ultrasonic stimulation is applied in the culture process, and M1 type polarized macrophages are obtained through culture.
According to the invention, the piezoelectric material is preferably a material with piezoelectric effect, including but not limited to a beta-phase PVDF piezoelectric film polarized along the direction d33, a polarized lithium niobate wafer or a sheet-shaped bacterial cellulose hydrogel synthesized by acetobacter.
Preferably according to the invention, the macrophage is a human or animal macrophage, including but not limited to human monocytic leukemia cells (THP-1), mouse mononuclear macrophages (RAW264.7) or primary macrophages of mouse bone marrow origin.
According to a preferred embodiment of the present invention, the parameters of the ultrasound are: the frequency is 20 kHz-5 MHz, the power is 0-300W, the ultrasonic time is 0 min-30 min each time, and ultrasonic is performed for 1-10 times every day.
Further preferably, the parameters of the ultrasound are: the frequency is 20 kHz-1 MHz, the power is 10-200W, the ultrasonic time is 3 min-20 min each time, and the ultrasonic is performed for 1-7 times each day.
Further preferably, the parameters of the ultrasound are: the frequency is 80 kHz-1 MHz, the power is 20-100W, the ultrasonic time is 5 min-15 min each time, and the ultrasonic is performed for 1-5 times each day.
According to the invention, the method for promoting the macrophage polarization to M1 type by using the piezoelectric effect preferably comprises the following steps:
(1) the piezoelectric material is fully soaked and cleaned by deionized water and then cut into 0.5 multiplied by 0.5cm2～3×3cm2Small pieces with the thickness of 20 mu m-3 mm are reserved;
(2) placing the piezoelectric material obtained in the step (1) as a cell culture substrate in a cell culture pore plate or a cell culture dish, adding 75% alcohol to soak for 0.5-12 h, simultaneously irradiating with an ultraviolet lamp in an aseptic operation table for 30-120 min to sterilize, cleaning residual alcohol with PBS (phosphate buffer solution), soaking the piezoelectric material in a cell culture medium overnight, and cleaning with the PBS for 2-3 times for later use;
(3) digesting or mechanically beating the macrophages, dispersing the macrophages by using a cell culture medium to obtain macrophage suspension, wherein the volume of the macrophage suspension is 0.01-2 mL, and the number of the contained macrophages is 1 multiplied by 1032 to 4X 106Inoculating the macrophage suspension on the surface of a piezoelectric material in a cell culture pore plate or a cell culture dish, standing for 0-90 min, supplementing 0-2 mL of cell culture medium into the pore plate or the cell culture dish, culturing the cells for 1-7 days, applying ultrasonic stimulation with the frequency of 20 kHz-5 MHz and the power of 0-300W, carrying out ultrasonic stimulation for 0-30 min each time, carrying out ultrasonic stimulation for 1-10 times every day, and replacing the cell culture medium every 2-3 days to obtain M1 type polarized macrophages.
Preferably, according to the invention, the cell culture conditions in step (3) are 5% CO at 37 ℃2The humidity of the wet environment is saturated humidity.
According to the invention, the cell culture medium is preferably high-sugar DMEM medium added with 10% of newborn calf serum or fetal calf serum and 1% of double antibody, or 1640 medium added with 10% of newborn calf serum or fetal calf serum, 1% of double antibody and 0.05mM of beta-mercaptoethanol.
The invention utilizes the piezoelectric material with piezoelectric effect to prepare M1 type macrophage by adopting an in vitro polarization mode, can be applied to the immunotherapy of tumors, for example, the M1 type macrophage is obtained by carrying out in vitro polarization on the macrophage of the invention and then is input into the focus part of the tumor of the invention, thus not only the cellular immunotherapy can be carried out, but also the infection and rejection reaction can be reduced.
The application of the piezoelectric material with the piezoelectric effect in promoting the polarization of macrophages to M1 type is characterized in that the piezoelectric effect is generated by the piezoelectric material through applying ultrasonic stimulation, so that the polarization of macrophages to M1 type is promoted.
According to the invention, the piezoelectric material is preferably a material with piezoelectric effect, including but not limited to a beta-phase PVDF piezoelectric film polarized along the direction d33, a polarized lithium niobate wafer or a sheet-shaped bacterial cellulose hydrogel synthesized by acetobacter.
Preferably according to the invention, the macrophage is a human or animal macrophage, including but not limited to human monocytic leukemia cells (THP-1), mouse mononuclear macrophages (RAW264.7) or primary macrophages of mouse bone marrow origin.
According to a preferred embodiment of the present invention, the parameters of the ultrasound are: the frequency is 20 kHz-5 MHz, the power is 0-300W, the ultrasonic time is 0 min-30 min each time, and ultrasonic is performed for 1-10 times every day.
Further preferably, the parameters of the ultrasound are: the frequency is 20 kHz-1 MHz, the power is 20-200W, the ultrasonic time is 5 min-30 min each time, and the ultrasonic is carried out for 1-7 times each day.
The application of the invention can be used for preparing M1 type macrophages, and can also be used for placing piezoelectric materials with piezoelectric effect on the body surface or in vivo, and the effect of promoting the macrophages to polarize to M1 type is achieved by applying ultrasonic stimulation to the piezoelectric material part.
Use of a piezoelectric material having piezoelectric effect for the preparation of a device for promoting the polarization of macrophages to M1 type.
Preferably according to the invention, the device comprises a piezoelectric material having a piezoelectric effect and an ultrasonic emitter. The device can be used for immunotherapy of superficial body tumors.
Has the advantages that:
1. the invention discovers and discloses that the piezoelectric effect can promote macrophage polarization to M1 type for the first time, compared with the traditional invasive electric stimulation means, the piezoelectric material is combined with the ultrasound to trigger non-contact and non-invasive electric stimulation, the important function of the electric stimulation in the immune regulation is verified, and the tumor immune therapy and anti-infection therapy clinical application potential is realized. The invention takes the piezoelectric material as the cell culture substrate and combines ultrasonic stimulation to culture M1 polarized macrophage in vitro, which can be used for cellular immunotherapy and can reduce infection and rejection reaction.
2. Compared with a common biochemical method, the method for promoting the polarization of the macrophages to the M1 type does not need to introduce external PMAPs, and can quickly promote the polarization of the macrophages to the M1 type by applying an ultrasonic method by utilizing a commercial beta-phase PVDF piezoelectric film, a polarized lithium niobate wafer or a piezoelectric material such as a flaky bacterial cellulose hydrogel synthesized by acetobacter on the market; experiments prove that the effect of promoting the macrophage to be polarized to M1 type by utilizing the piezoelectric effect is good. The method for promoting macrophage polarization to M1 type by utilizing piezoelectric effect provides a quick and effective method for tumor immunotherapy and anti-infection therapy.
3. The piezoelectric material selected by the invention is representative, for example, a beta-phase PVDF piezoelectric film has been proved to have long-term safety when being implanted into a body, and the bacterial cellulose hydrogel is a well-known excellent biomedical material, is low in price and easy to produce, so that the piezoelectric material can be placed on the body surface or in the body, and the effect of promoting macrophage polarization to M1 type is achieved by applying ultrasonic stimulation to the piezoelectric material part. The method of the invention can produce M1 polarized macrophage in batch to promote the application of piezoelectric material in immunotherapy.
Drawings
FIG. 1 is a Scanning Electron Microscope (SEM) photograph of a beta-phase PVDF piezoelectric film polarized along the direction d 33;
FIG. 2 shows the piezoelectric signal results of a Piezoelectric Force Microscope (PFM) test on a beta-phase PVDF piezoelectric film polarized in the direction d33, wherein the left graph is a phase diagram and the right graph is an amplitude diagram;
FIG. 3 is a bar graph of the expression level of the M1 type marker TNF α of THP-1 cells under the conditions of cell slide and beta-phase PVDF piezoelectric film with or without ultrasound;
FIG. 4 is a bar graph of the expression levels of the M1-type marker TNF α in THP-1 cells under conditions of cell slide and alpha-phase PVDF piezoelectric-free thin film with or without ultrasound.
Detailed Description
The invention will be further illustrated with reference to specific examples, without limiting the scope of the invention thereto.
Meanwhile, the experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents, materials and equipment are commercially available, unless otherwise specified.
The cell culture media used in the following examples were: the high-glucose DMEM complete culture medium is prepared by adding 10% of newborn calf serum or fetal calf serum and 1% of double antibodies into the high-glucose DMEM culture medium, and the 1640 complete culture medium is prepared by adding 10% of newborn calf serum or fetal calf serum, 1% of double antibodies and 0.05mM beta-mercaptoethanol into the 1640 culture medium.
Example 1:
observing the morphological characteristics of the beta-phase PVDF piezoelectric film polarized along the d33 direction by adopting a scanning electron microscope, wherein the scanning electron microscope photo is shown in figure 1, and the surface of the film is smooth and flat; the piezoelectric response of the beta-phase PVDF film is verified by a piezoelectric force microscope, and the result is shown in FIG. 2, and a butterfly curve represents the piezoelectric response of the material.
The piezoelectric film is used as a cell culture substrate to promote macrophage polarization to M1 type, and the method comprises the following steps:
1) soaking and cleaning beta-phase PVDF piezoelectric film with deionized water, and cutting into 1 × 1cm2Small pieces with the thickness of 100 mu m are reserved;
2) placing the beta-phase PVDF piezoelectric film in a cell culture pore plate, adding 75% alcohol to soak for 2h, irradiating for 60min in an aseptic operation table for sterilization, and cleaning residual alcohol with PBS buffer solution; soaking the beta-phase PVDF piezoelectric film in a 1640 culture medium overnight, and then washing the beta-phase PVDF piezoelectric film for 2-3 times by using a PBS buffer solution for later use;
3) digesting the THP-1 cells, dispersing the cells by using a 1640 culture medium to obtain a cell suspension, inoculating the THP-1 cells on the piezoelectric film treated in the step 1), wherein the volume of the inoculated cell suspension is 0.2mL, and the number of the cells contained in the cell suspension is 2 multiplied by 106Standing for 20min, supplementing 1640 complete culture medium containing 0.05mM beta-mercaptoethanol into a pore plate, wherein the volume of the supplemented culture medium is 0.8mL, and adding 10nM phorbol ester to induce cell adherence;
4) the cells were incubated at 37 ℃ with 5% CO2Culturing in vitro under saturated humidity for 3 days, applying ultrasonic stimulation once a day, wherein the ultrasonic frequency is 80kHz, the power is 90W, and the ultrasonic stimulation time is 10min each time; and a control group was set under the same experimental conditions: cell climbing sheet group, cell climbing sheet ultrasonic group and beta-phase PVDF non-ultrasonic group.
Performing gene detection on the THP-1 cells cultured for 3 days under different conditions, and detecting the expression level of an M1 type macrophage marker TNF alpha, wherein the result is shown in figure 3, and the result shows that the expression level of the TNF alpha gene of the THP-1 cells cultured on the beta-phase PVDF piezoelectric film and applied with ultrasonic stimulation is remarkably improved compared with that of a control group, and the expression level is respectively 42.7 times that of a cell climbing group, 30.5 times that of a cell climbing ultrasonic group and 3.2 times that of a beta-phase PVDF non-ultrasonic group; it is demonstrated that the polarization of macrophages to M1 type can be significantly promoted by the beta-phase PVDF piezoelectric film polarized along the direction d33 in combination with ultrasonic stimulation.
Example 2:
the beta-phase PVDF piezoelectric film is used as a cell culture substrate to promote the polarization of macrophages to M1 type, and the steps are as follows:
1) soaking and cleaning beta-phase PVDF piezoelectric film in deionized water, and cutting into 1.5 × 1.5cm2Small pieces with the thickness of 200 mu m are reserved;
2) placing the beta-phase PVDF piezoelectric film in a cell culture pore plate, adding 75% alcohol to soak for 2h, irradiating for 60min in an aseptic operation table for sterilization, and cleaning residual alcohol with PBS buffer solution; soaking the beta-phase PVDF piezoelectric film in a high-glucose DMEM culture medium overnight, and then washing the beta-phase PVDF piezoelectric film for 2-3 times by using a PBS buffer solution for later use;
3) digesting RAW264.7 cells, dispersing the cells by using a high-glucose DMEM culture medium to obtain a cell suspension, inoculating the RAW264.7 cells on the piezoelectric film treated in the step 1), wherein the volume of the inoculated cell suspension is 0.2mL, and the number of the cells contained in the cell suspension is 2 multiplied by 106After standing for 20min, supplementing a high-glucose DMEM complete culture medium into the pore plate, wherein the volume of the culture medium required to be supplemented is 0.8 mL;
4) the cells were incubated at 37 ℃ with 5% CO2Culturing in vitro under saturated humidity environment for 1 day, applying ultrasonic stimulation once a day with ultrasonic frequency of 0.5MHz and power of 20W, and stimulating for 5min each time; and a control group was set under the same experimental conditions: cell climbing sheet group, cell climbing sheet ultrasonic group and beta-phase PVDF non-ultrasonic group.
The RAW264.7 cells cultured for 1 day under different conditions are subjected to gene detection, the expression level of the M1 type macrophage marker TNF alpha is detected, and the expression level of the M1 type macrophage marker TNF alpha gene of the RAW264.7 cells cultured on the beta-phase PVDF piezoelectric film and subjected to ultrasonic stimulation is found to be remarkably improved compared with that of a control group.
Example 3:
the beta-phase PVDF piezoelectric film is used as a cell culture substrate to promote the polarization of macrophages to M1 type, and the steps are as follows:
1) soaking and cleaning beta-phase PVDF piezoelectric film with deionized water, and cutting into 1 × 1cm2And small pieces with the thickness of 500 mu m are reserved;
2) placing the beta-phase PVDF piezoelectric film in a cell culture pore plate, adding 75% alcohol to soak for 2h, irradiating for 60min in an aseptic operation table for sterilization, and cleaning residual alcohol with PBS buffer solution; soaking the beta-phase PVDF piezoelectric film in a 1640 culture medium overnight, and then washing the beta-phase PVDF piezoelectric film for 2-3 times by using a PBS buffer solution for later use;
3) digesting the primary mouse macrophage, dispersing the primary mouse macrophage by using a 1640 culture medium to obtain a cell suspension, inoculating the primary mouse macrophage on the piezoelectric film treated in the step 1), wherein the volume of the inoculated cell suspension is 0.1mL, and the number of contained cells is 2 multiplied by 105After standing for 20min, supplementing 1640 complete culture medium into the pore plate, wherein the volume of the supplemented culture medium is 0.9 mL;
4) the cells were incubated at 37 ℃ with 5% CO2Culturing in vitro under saturated humidity environment for 4 days, applying ultrasonic stimulation once a day, wherein the ultrasonic frequency is 1MHz, the power is 20W, the ultrasonic stimulation time is 3min each time, and replacing the culture medium once every 2 days; and a control group was set under the same experimental conditions: cell climbing sheet group, cell climbing sheet ultrasonic group and beta-phase PVDF non-ultrasonic group.
The gene detection is carried out on the mouse primary macrophages cultured for 4 days under different conditions, the expression level of the M1 type macrophage marker TNF alpha is detected, and the expression level of the M1 type marker TNF alpha gene of the mouse primary macrophages cultured on the beta-phase PVDF piezoelectric film and subjected to ultrasonic stimulation is remarkably improved compared with that of a control group.
Example 4:
the polarized lithium niobate piezoelectric wafer is used as a cell culture substrate to promote the polarization of macrophages to M1 type, and the steps are as follows:
1) the lithium niobate piezoelectric wafer is fully soaked and cleaned by deionized water and then cut into 1.5 multiplied by 1.5cm2Small pieces with the thickness of 300 mu m are reserved;
2) placing a lithium niobate piezoelectric wafer in a cell culture pore plate, adding 75% alcohol to soak for 2h, irradiating for 60min in an aseptic operation table to sterilize, and cleaning residual alcohol with PBS buffer solution; soaking the lithium niobate piezoelectric film in a 1640 culture medium overnight, and then washing the film for 2-3 times by using a PBS buffer solution for later use;
3) digesting the THP-1 cells, dispersing the cells by using a 1640 culture medium to obtain a cell suspension, inoculating the THP-1 cells on the piezoelectric wafer treated in the step 1), wherein the volume of the inoculated cell suspension is 0.1mL, and the number of the cells contained in the cell suspension is 1 multiplied by 106Standing for 20min, supplementing 1640 complete culture medium containing 0.05mM beta-mercaptoethanol into a pore plate, wherein the volume of the supplemented culture medium is 0.9mL, and adding 20nM phorbol ester to induce cell adherence;
4) the cells were incubated at 37 ℃ with 5% CO2Culturing in vitro under saturated humidity environment for 3 days, applying ultrasonic stimulation once a day, wherein the ultrasonic frequency is 120kHz, the power is 90W, and the ultrasonic stimulation time is 15min each time; and a control group was set under the same experimental conditions: cell climbing sheet group, cell climbing sheet ultrasonic group and lithium niobate piezoelectric wafer ultrasonic-free group.
The THP-1 cells cultured for 3 days under different conditions are subjected to gene detection, the expression level of the M1 type macrophage marker TNF alpha is detected, and the expression level of the M1 type macrophage marker TNF alpha gene of the THP-1 cells cultured on the lithium niobate piezoelectric wafer and applied with ultrasonic stimulation is remarkably improved compared with that of a control group.
Example 5:
the sheet-shaped bacterial cellulose piezoelectric hydrogel synthesized by acetobacter is used as a cell culture substrate to promote the polarization of macrophages to M1 type, and the steps are as follows:
1) the sheet-shaped bacterial cellulose piezoelectric hydrogel is cut into 2 multiplied by 2cm after being fully soaked and cleaned by deionized water2Small pieces with the thickness of 1mm are reserved;
2) placing the sheet-shaped bacterial cellulose piezoelectric hydrogel in a cell culture pore plate, adding 75% alcohol, soaking for 2h, irradiating for 60min in an aseptic operation table for sterilization, and cleaning residual alcohol with PBS buffer solution; soaking the flaky bacterial cellulose piezoelectric hydrogel in a high-sugar DMEM culture medium overnight, and then washing the flaky bacterial cellulose piezoelectric hydrogel for 2-3 times by using PBS buffer solution for later use;
3) digesting RAW264.7 cells, dispersing the cells by using a high-glucose DMEM culture medium to obtain a cell suspension, and inoculating the RAW264.7 cells after the treatment of the step 1)The piezoelectric hydrogel of (1), the volume of the seeded cell suspension is 0.2mL, and the number of cells contained therein is 1X 106After standing for 20min, supplementing a high-sugar DMEM complete culture medium, wherein the volume of the supplemented culture medium is 0.8 mL;
4) the cells were incubated at 37 ℃ with 5% CO2Culturing in vitro under saturated humidity environment for 1 day, applying ultrasonic stimulation once a day with ultrasonic frequency of 1MHz and power of 20W, and each ultrasonic stimulation time of 3 min; and a control group was set under the same experimental conditions: cell climbing sheet group, cell climbing sheet ultrasonic group and piezoelectric hydrogel ultrasonic-free group.
The RAW264.7 cells cultured for 1 day under different conditions are subjected to gene detection, the expression level of the M1 type macrophage marker TNF alpha is detected, and the expression level of the M1 type marker TNF alpha gene of the RAW264.7 cells cultured on the piezoelectric hydrogel and subjected to ultrasonic stimulation is found to be remarkably improved compared with that of a control group.
Comparative example 1:
the alpha-phase PVDF non-piezoelectric film is used as a cell culture substrate to regulate and control the polarization of macrophages, and the steps are as follows:
1) fully soaking and cleaning the alpha-phase PVDF non-piezoelectric film by deionized water, and then shearing the film into 1 multiplied by 1cm2Small pieces with the thickness of 100 mu m are reserved;
2) placing the alpha-phase PVDF non-piezoelectric film in a cell culture pore plate, adding 75% alcohol to soak for 2h, irradiating for 60min in an aseptic operation table to sterilize, and cleaning the residual alcohol with PBS buffer solution; soaking the alpha-phase PVDF non-piezoelectric film in a 1640 culture medium overnight, and then washing the alpha-phase PVDF non-piezoelectric film for 2-3 times by using a PBS buffer solution for later use;
3) digesting the THP-1 cells, dispersing the cells by using a 1640 culture medium to obtain a cell suspension, inoculating the THP-1 cells on the non-piezoelectric film treated in the step 1), wherein the volume of the inoculated cell suspension is 0.2mL, and the number of the cells contained in the cell suspension is 2 multiplied by 106Standing for 20min, supplementing 1640 complete culture medium containing 0.05mM beta-mercaptoethanol into a pore plate, wherein the volume of the supplemented culture medium is 0.8mL, and adding 10nM phorbol ester to induce cell adherence;
4) the cells were placed at 37 deg.CContaining 5% CO2Culturing in vitro under saturated humidity for 3 days, applying ultrasonic stimulation once a day, wherein the ultrasonic frequency is 80kHz, the power is 90W, and the ultrasonic stimulation time is 10min each time; and a control group was set under the same experimental conditions: alpha phase PVDF no ultrasound group.
The expression level of TNF alpha as a M1 type macrophage marker was detected by performing gene detection on THP-1 cells cultured for 3 days under different conditions, and as a result, as shown in FIG. 4, it was found that the expression level of TNF alpha gene of THP-1 cells cultured in a non-piezoelectric film and subjected to ultrasonic stimulation was almost unchanged compared with the expression level of TNF alpha gene of a non-piezoelectric film control group to which no ultrasonic wave was applied, indicating that the material without piezoelectric response also did not have the effect of promoting macrophage M1 type polarization in combination with ultrasonic wave.
Claims (8)
1. A method for promoting macrophage polarization to M1 type by utilizing piezoelectric effect is characterized in that the polarization is carried out in vitro, a piezoelectric material is used as a cell culture substrate, macrophage suspension is inoculated on the substrate, cells are cultured for 1-7 d, ultrasonic stimulation is applied in the culture process, and M1 type polarized macrophages are obtained through culture;
the piezoelectric material is a beta-phase PVDF piezoelectric film polarized along the direction d33, a polarized lithium niobate wafer or a flaky bacterial cellulose hydrogel synthesized by acetobacter;
the size of the piezoelectric material is 0.5 multiplied by 0.5cm2~3×3 cm2The thickness is 20 mu m-3 mm;
the cell culture medium adopted by the cell culture is as follows: adding 10% fetal calf serum and 1% double antibody into a high-glucose DMEM complete culture medium; or 1640 complete culture medium, adding 10% fetal calf serum, 1% double antibody and 0.05mM beta-mercaptoethanol into the 1640 culture medium;
the cell culture conditions were 5% CO at 37 ℃2Culturing in a humid environment, wherein the humidity of the humid environment is a saturated humidity;
the ultrasonic parameters are that the frequency is 20 kHz-5 MHz, the power is 10-300W, the ultrasonic time is 3 min-30 min each time, and the ultrasonic is performed for 1-10 times every day.
2. The method of claim 1, wherein the macrophage is a human monocytic leukemia cell, a mouse mononuclear macrophage, or a mouse bone marrow-derived primary macrophage.
3. The method of claim 1, comprising the steps of:
(1) the piezoelectric material is fully soaked and cleaned by deionized water and then cut into 0.5 multiplied by 0.5cm2~3×3 cm2Small pieces with the thickness of 20 mu m-3 mm are reserved;
(2) placing the piezoelectric material obtained in the step (1) as a cell culture substrate in a cell culture pore plate or a cell culture dish, adding 75% alcohol to soak for 0.5-12 h, simultaneously irradiating with an ultraviolet lamp in an aseptic operation table for 30-120 min to sterilize, cleaning residual alcohol with PBS (phosphate buffer solution), soaking the piezoelectric material in a cell culture medium overnight, and cleaning with the PBS for 2-3 times for later use;
(3) digesting or mechanically beating the macrophages, dispersing the macrophages by using a cell culture medium to obtain macrophage suspension, wherein the volume of the macrophage suspension is 0.01-2 mL, and the number of the contained macrophages is 1 multiplied by 103Is 4 x 106Inoculating the macrophage suspension on the surface of a piezoelectric material in a cell culture pore plate or a cell culture dish, standing for 20-90 min, supplementing 0.8-2 mL of cell culture medium into the pore plate or the cell culture dish, culturing the cells for 1-7 days, applying ultrasonic stimulation with the frequency of 20 kHz-5 MHz and the power of 10-300W, wherein the ultrasonic stimulation time is 3 min-30 min each time, performing ultrasonic stimulation for 1-10 times every day, and replacing the cell culture medium every 2-3 days to obtain M1 type polarized macrophages.
4. The method of claim 3, wherein the cell culture conditions in step (3) are 5% CO at 37 ℃2The humidity of the wet environment is saturated humidity.
5. The application of the piezoelectric material with the piezoelectric effect in promoting the polarization of macrophages to M1 type is characterized in that the piezoelectric effect is generated by the piezoelectric material by applying ultrasonic stimulation, so that the polarization of macrophages to M1 type is promoted;
the piezoelectric material is a beta-phase PVDF piezoelectric film polarized along the direction d33, a polarized lithium niobate wafer or a flaky bacterial cellulose hydrogel synthesized by acetobacter;
the size of the piezoelectric material is 0.5 multiplied by 0.5cm2~3×3 cm2The thickness is 20 mu m-3 mm;
the cell culture medium adopted for polarizing the macrophages to M1 type is as follows: adding 10% fetal calf serum and 1% double antibody into a high-glucose DMEM complete culture medium; or 1640 complete culture medium, adding 10% fetal calf serum, 1% double antibody and 0.05mM beta-mercaptoethanol into the 1640 culture medium;
the macrophage is polarized to M1 type under the culture condition that the macrophage contains 5% CO at 37 DEG C2Culturing in a humid environment, wherein the humidity of the humid environment is a saturated humidity;
the ultrasonic parameters are that the frequency is 20 kHz-5 MHz, the power is 10-300W, the ultrasonic time is 3 min-30 min each time, and the ultrasonic is performed for 1-10 times every day.
6. The use of claim 5, wherein the macrophage comprises a human monocytic leukemia cell, a mouse mononuclear macrophage or a mouse bone marrow-derived primary macrophage.
7. The application of piezoelectric material with piezoelectric effect in preparing equipment for promoting macrophage polarization to M1 type;
the piezoelectric material is a beta-phase PVDF piezoelectric film polarized along the direction d33, a polarized lithium niobate wafer or a flaky bacterial cellulose hydrogel synthesized by acetobacter;
the size of the piezoelectric material is 0.5 multiplied by 0.5cm2~3×3 cm2The thickness is 20 mu m-3 mm;
the cell culture medium adopted for polarizing the macrophages to M1 type is as follows: adding 10% fetal calf serum and 1% double antibody into a high-glucose DMEM complete culture medium; or 1640 complete culture medium, adding 10% fetal calf serum, 1% double antibody and 0.05mM beta-mercaptoethanol into the 1640 culture medium;
the macrophage is polarized to M1 type under the culture condition that the macrophage contains 5% CO at 37 DEG C2The humidity of the wet environment is saturated humidity.
8. Use according to claim 7, wherein the device comprises a piezoelectric material having a piezoelectric effect and an ultrasonic emitter.
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