Method for promoting fusion of macrophages and increasing content of phosphatidic acid in macrophages and application of method

Abstract

The invention discloses a method for promoting fusion of macrophages and increasing the content of phosphatidic acid in the macrophages and application of the method, and belongs to the technical field of biomedicine. In order to provide a method for effectively treating vascular calcification, an in-vitro cell experiment proves that the combination of a sound-sensitive agent and low-frequency low-intensity ultrasound can promote M1 type macrophages to be fused into multinuclear osteoclast-like cells and can improve the content of PA in the M1 type macrophages; the sound-sensitive agent is combined with low-frequency low-intensity ultrasound, so that mouse aortic smooth muscle cell calcification can be relieved by increasing the PA content in M1 type macrophages; a mouse in-vivo experiment further proves that the content of PA in an early calcification region of the aorta root of the mouse can be increased by combining the sound-sensitive agent with low-frequency and low-intensity ultrasonic treatment, so that aortic atherosclerotic calcification of the mouse is relieved. The invention provides a new idea for the treatment of vascular calcification.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for promoting macrophage fusion and increasing the phosphatidic acid content in macrophages and its application. Background Art

[0002] Vascular calcification increases the risk of cardiovascular diseases and all-cause mortality, but there is no effective treatment yet. Vascular calcification is similar to bone formation and is a highly regulated process. Osteoclast-like cells differentiated from macrophages have the multinucleated morphology of osteoclasts and express osteoclast-specific markers such as tartrate-resistant acid phosphatase. In vitro experiments show that osteoclast-like cells have certain osteoclastic functions, but the osteoclast-like cells around calcified tissues are often immature and have low functions. Multinucleated osteoclast-like cells fused from macrophages exist in human atherosclerotic plaques. Osteoclast-like macrophages around calcification play a key role in reducing calcification.

[0003] PA, 1,2-diacylglycerol-3-phosphate, is an important class of membrane phospholipids that exist in almost all cell membranes. PA is also a signaling molecule that participates in the regulation of many cell signaling pathways and functions as well as membrane rearrangement. It can regulate processes such as cell proliferation, survival, cytoskeleton organization, vesicle trafficking, secretion, and responses to hormones and abiotic stresses. Previous studies have shown that PA is regulated by reactive oxygen species and can promote macrophage fusion and enhanced osteoclastic function, but the regulatory role of PA in vascular calcification has not been found yet.

[0004] Currently, there is no method to reverse vascular calcification. Cholesterol-lowering statins cannot reduce or reverse vascular calcification; anti-inflammatory treatment cannot reduce vascular calcification in animal experiments; calcification treatment acting on osteoclasts may have adverse effects on the skeletal system. Therefore, in-depth exploration of the regulatory method of PA in macrophages is of great significance for the prevention and treatment of vascular calcification. Summary of the Invention

[0005] In order to provide an effective method for treating vascular calcification, the present invention has confirmed through in vitro cell experiments that a photosensitizer combined with low-frequency and low-intensity ultrasound can promote the fusion of M1 macrophages into multinucleated osteoclast-like cells and can increase the content of PA in M1 macrophages. The photosensitizer combined with low-frequency and low-intensity ultrasound can reduce the calcification of mouse aortic smooth muscle cells by increasing the content of PA in M1 macrophages; through in vivo experiments in mice, it is further confirmed that treatment with the photosensitizer combined with low-frequency and low-intensity ultrasound can increase the content of PA in the early calcified area of the mouse aortic root, thereby reducing atherosclerotic calcification of the mouse aorta.

[0006] To solve the above technical problems and achieve the corresponding technical effects, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide a method for fusing macrophages into multinucleated osteoclast-like cells. The method is to incubate M1 macrophages with a photosensitizer. When the photosensitizer in the cells reaches an effective concentration, irradiate the M1 macrophages with low-frequency and low-intensity ultrasound. The ultrasound frequency of the low-frequency and low-intensity ultrasound is 0.5 MHz - 1.5 MHz, and the ultrasound intensity is 0.2 W / cm 2- 1.0 W / cm 2 .

[0008] In one embodiment of the present invention, the photosensitizer is an organic molecular photosensitizer, an inorganic nano-photosensitizer or an organic-inorganic hybrid nano-photosensitizer.

[0009] In one embodiment of the present invention, the photosensitizer is sodium hematoporphyrin, 5-aminolevulinic acid, curcumin or emodin.

[0010] In one embodiment of the present invention, the photosensitizer is sodium hematoporphyrin; the incubation concentration of sodium hematoporphyrin in the method is 0.1 - 1 μmol / L, and the incubation time is 4 h.

[0011] In one embodiment of the present invention, the irradiation time is 3 - 7 min.

[0012] The second object of the present invention is to provide multinucleated osteoclast-like cells obtained by the above method.

[0013] The third object of the present invention is to provide the application of the above multinucleated osteoclast-like cells in the preparation of drugs for preventing and / or treating vascular calcification.

[0014] The fourth object of the present invention is to provide a method for increasing the phosphatidic acid content in M1 macrophages. The method is to incubate M1 macrophages with a photosensitizer. When the photosensitizer in the cells reaches an effective concentration, irradiate the M1 macrophages with low-frequency and low-intensity ultrasound. The ultrasound frequency of the low-frequency and low-intensity ultrasound is 0.5 MHz - 1.5 MHz, and the ultrasound intensity is 0.2 W / cm 2- 1.0 W / cm 2 .

[0015] In one embodiment of the present invention, the photosensitizer is an organic molecular photosensitizer, an inorganic nano-photosensitizer or an organic-inorganic hybrid nano-photosensitizer.

[0016] In one embodiment of the present invention, the photosensitizer is sodium hematoporphyrin, 5-aminolevulinic acid, curcumin or emodin.

[0017] In one embodiment of the present invention, the photosensitizer is sodium hematoporphyrin; the incubation concentration of sodium hematoporphyrin in the method is 0.1 - 1 μmol / L, and the incubation time is 4 h.

[0018] In one embodiment of the present invention, the irradiation time is 3 - 7 min.

[0019] The fifth object of the present invention is to provide an M1 macrophage with increased phosphatidic acid content obtained by the above method.

[0020] The sixth object of the present invention is to provide the use of the M1 macrophage with increased phosphatidic acid content in the preparation of a drug for preventing and / or treating vascular calcification.

[0021] Advantages of the present invention:

[0022] By using a photosensitizer combined with low - frequency and low - intensity ultrasound to intervene in M1 macrophages, it is found that the combination of the photosensitizer and low - frequency and low - intensity ultrasound can significantly increase the PA level of M1 macrophages, and can promote the fusion of M1 macrophages and improve their osteoclast ability. In addition, by co - culturing calcified smooth muscle cells with M1 macrophages treated with a photosensitizer combined with low - frequency and low - intensity ultrasound, it is found that the treatment with a photosensitizer combined with low - frequency and low - intensity ultrasound can reduce the calcification of mouse aortic smooth muscle cells by increasing the PA content in M1 macrophages.

[0023] The present invention further takes the atherosclerotic calcification model of apolipoprotein E (APOE) gene - knockout mice as the research object, based on mass spectrometry spatial omics, conducts image segmentation of biological specimens, and combines tissue morphological characteristics to in - situ screen the atherosclerotic calcification area of APOE gene - knockout mice and detect the changes in lipid metabolites in the calcified area. It is found that PA increases in the early calcification area, and the treatment with a photosensitizer combined with low - frequency and low - intensity ultrasound can reduce atherosclerotic calcification of the mouse aorta, further confirming that PA in macrophages can be a potential target for the treatment of vascular calcification.

[0024] The present invention first provides a method for regulating PA in macrophages in atherosclerotic calcification areas by using a photosensitizer combined with low - frequency and low - intensity ultrasound. This method can achieve precise regulation of cellular PA, has the advantage of cell selectivity, and can perform differential regulation according to the absorption and metabolism laws of porphyrin drugs by different cells, providing a new strategy and method for regulating PA in specific cells and tissues in vivo. Moreover, since PA in macrophages can promote the fusion of macrophages into osteoclast - like cells and reduce vascular calcification, macrophage PA can be used as a candidate target for anti - vascular calcification and related diseases. The treatment with a photosensitizer combined with low - frequency and low - intensity ultrasound described in the present invention can treat vascular calcification, providing a theoretical and experimental basis for the prevention and treatment of vascular calcification and related diseases.

[0025] In addition, the PA regulation method also has potential application value. In vascular calcification, osteoclast-like macrophages with the ability to decompose calcification have the disadvantages of incomplete fusion and low osteoclastic function. PA can participate in the fusion and rearrangement of cell membranes, promote macrophage fusion and enhance its osteoclastic function. By regulating macrophage PA, we may be able to affect the osteoclastic ability of macrophages, thereby reducing vascular calcification and providing new ideas for the treatment of cardiovascular diseases. In addition, regulating PA also has a neuroregulatory function. Research shows that PA is involved in neurotransmitter transmission and can control the hormone secretion of neuroendocrine cells. Therefore, regulating PA in the nervous system can provide a new solution for nerve function regulation and disease treatment. In addition, PA plays an important role in the membrane fusion during spermatogenesis and fertilization. Regulating PA may help improve reproductive function and has positive significance for the treatment of diseases related to the reproductive system. PA is also involved in the regulation of the muscular system. Research shows that PA can activate the mammalian target of rapamycin (mTOR) through different mechanisms and promote muscle anabolism. Therefore, regulating PA may help maintain or restore skeletal muscle mass and treat aging-related diseases.

[0026] In summary, the method for regulating PA provided by the present invention is expected to play an active role in the treatment of cardiovascular system diseases, nervous system diseases, reproductive system diseases and muscular system diseases, and provide new potential methods for the treatment of related diseases. Description of the Drawings

[0027] Figure 1 It is a mass spectrometry spatial omics detection result diagram of the PA content in the early calcified area of atherosclerotic plaques in mice aorta; among them, Figure 1 A in it is the mass spectrometry imaging diagram of the PA content in the early calcified area of atherosclerotic plaques in mice aorta, a is the ApoE of the control group - / - Mass spectrometry imaging diagram of the PA content in the early calcified area of atherosclerotic plaques in mice aorta, b is the ApoE intervened by the combination of photosensitizer and low-frequency low-intensity ultrasound - / - Mass spectrometry imaging diagram of the PA content in the early calcified area of atherosclerotic plaques in mice aorta, Figure 1 B in it is the mass spectrometry imaging data of PA, Figure 1 C in it is the chemical structure diagram of PA;

[0028] Figure 2Result graph for measuring the PA content in macrophages by enzyme-linked immunosorbent assay; among them, M2 is the group of untreated M2 macrophages, M1 is untreated M1 macrophages, M1-T-3 is the group of M1 macrophages 3 hours after treatment with a photosensitizer combined with low-frequency and low-intensity ultrasound, M1-T-6 is M1 macrophages 6 hours after treatment with a photosensitizer combined with low-frequency and low-intensity ultrasound, M1-T-12 is M1 macrophages 12 hours after treatment with a photosensitizer combined with low-frequency and low-intensity ultrasound, M1-T-24 is M1 macrophages 24 hours after treatment with a photosensitizer combined with low-frequency and low-intensity ultrasound, and M1-T-NAC is M1 macrophages treated with a photosensitizer incubation, NAC incubation, and then low-frequency and low-intensity ultrasound; * represents P < 0.05, and **** represents P < 0.0001;

[0029] Figure 3 Scanning result graph of macrophages detected by atomic force microscopy; among them, M1 is untreated M1 macrophages, M1+Rankl is M1 macrophages after Rankl intervention, and M1+Rankl+SDT is M1 macrophages treated with a photosensitizer combined with low-frequency and low-intensity ultrasound and Rankl intervention;

[0030] Figure 4 Statistical result graph of macrophage diameter obtained from the scanning result graph of macrophages detected by atomic force microscopy; M1 is untreated M1 macrophages, M1+Rankl is M1 macrophages after Rankl intervention, and M1+Rankl+SDT is M1 macrophages treated with a photosensitizer combined with low-frequency and low-intensity ultrasound and Rankl intervention;

[0031] Figure 5 Alizarin red staining result graph of the direct co-culture system of macrophages and smooth muscle cells; among them, Figure 5 A in it is the observation graph of the alizarin red staining result of the direct co-culture system of macrophages and smooth muscle cells, Figure 5 B in it is obtained from Figure 5 The statistical result graph of the proportion of the calcified area obtained from A in Figure 5 C in it is obtained from Figure 5Statistical result graph of the average optical density obtained by A in [description]; SMC+M1 is the co-culture of calcified smooth muscle cells and untreated M1 macrophages, SMC+M1+SDT is the co-culture of calcified smooth muscle cells and M1 macrophages treated with a photosensitizer combined with low-frequency low-intensity ultrasound, SMC+M1+SDT+NAC is the co-culture of calcified smooth muscle cells and M1 macrophages treated with a photosensitizer combined with low-frequency low-intensity ultrasound and intervened with NAC, SMC+M1+PA is the co-culture of calcified smooth muscle cells and M1 macrophages intervened with PA; * indicates P<0.05, ** indicates P<0.01, ns indicates no statistical difference;

[0032] Figure 6 Graph of the observation results of immunofluorescence staining for early calcification of atherosclerotic plaques in mouse aorta; among them, Baseline10* / Baseline 40* is the baseline group magnified 10 / 40 times by the microscope objective lens, Control-15day 10* / Control-15day 40* is the control group 15 days after sham intervention magnified 10 / 40 times by the microscope objective lens, Control-30day 10* / Control-30day 40* is the control group 30 days after sham intervention magnified 10 / 40 times by the microscope objective lens, SDT-15day 10* / SDT-15day 40*: the treatment group 15 days after intervention with a photosensitizer combined with low-frequency low-intensity ultrasound magnified 10 / 40 times by the microscope objective lens; SDT-30day 10* / SDT-30day 40*: the treatment group 30 days after intervention with a photosensitizer combined with low-frequency low-intensity ultrasound magnified 10 / 40 times by the microscope objective lens;

[0033] Figure 7 Graph of the statistical results of the ratio of the immunofluorescence positive area of BMP2 to the plaque area and the average fluorescence intensity for early calcification of atherosclerotic plaques in mouse aorta; among them, Control-15 is the control group 15 days after sham intervention, SDT-15 is the treatment group 15 days after intervention with a photosensitizer combined with low-frequency low-intensity ultrasound, Control-30 is the control group 30 days after sham intervention, SDT-30 is the treatment group 30 days after intervention with a photosensitizer combined with low-frequency low-intensity ultrasound. Detailed implementation manners

[0034] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with specific implementation manners and the accompanying drawings of the specification. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents and instruments used are all conventional materials, reagents and instruments in the art unless otherwise specified, and those skilled in the art can obtain them through commercial channels.

[0035] Example 1: Sensitizer combined with low-frequency and low-intensity ultrasound to increase the PA content in calcified tissues

[0036] 1. Construction of an animal model of early calcification of atherosclerotic plaques in mice

[0037] The experimental animals used in this invention are 6-week-old apolipoprotein E gene knockout C57BL / 6 mice (i.e., ApoE - / - mice), with a body weight of 18 - 20 g, purchased from Qingzilan Technology Co., Ltd. Feed the ApoE - / - mice with an atherogenic diet (22% fat, 0.12% cholesterol) and raise them in a specific pathogen-free environment for 20 weeks to obtain an animal model of early calcification of atherosclerotic plaques in mice aorta.

[0038] 2. Intervention of the animal model of early calcification of atherosclerotic plaques in mice with a sensitizer combined with low-frequency and low-intensity ultrasound

[0039] Intravenously inject sodium huaporfin into the animal model of early calcification of atherosclerotic plaques in mice (the dosage of the sensitizer is 4 mg / kg based on the body weight of the mice). After 4 h, anesthetize the mice. Place a ultrasonic transducer with a diameter of 35 mm and a resonance frequency of 1.0 MHz under the precordial area of the mice through a degassed water column, with an ultrasonic intensity of 0.4 W / cm 2 ², and the treatment time is 15 min. Use the animal model of early calcification of atherosclerotic plaques in mice without the intervention of a sensitizer combined with low-frequency and low-intensity ultrasound as the control group.

[0040] 3. Identification of early calcification areas of atherosclerosis and detection of PA content

[0041] Based on mass spectrometry spatial omics and combined with tissue morphological characteristics, screen the early calcification areas of atherosclerosis at the molecular level. The specific method is as follows:

[0042] (1) Two weeks after the end of high-fat feeding, take samples of atherosclerotic plaques at the aortic root from the control group ApoE - / - mice and ApoE - / - mice intervened with a sensitizer combined with low-frequency and low-intensity ultrasound respectively. Immediately wash the plaque samples with ice-cold saline, freeze them in liquid nitrogen for 1 min, and store them at -80 °C. Cut the samples into fragments about 5 mm in size and embed them in sodium carboxymethylcellulose with a mass fraction of 2%. Cut frozen sections (14 μm / slice) with a cryostat and transfer the sections to glass slides coated with conductive indium tin oxide (ITO), and store them at -80 °C for spatial mass spectrometry imaging and histochemical staining. Before the experiment, place them in a desiccator and dry for 40 min.

[0043] (2) Perform spraying treatment on the matrix, and then use a mass spectrometry imaging instrument to conduct mass spectrometry imaging scanning experiments on the regions of interest. Important imaging conditions: positive ion mode, SmartBeam 3D laser (355 nm), scanning range of m / z 200 - 900 Da, and laser intensity of 40%.

[0044] (3) Perform segmentation processing on the mass spectrometry imaging data for picture data. The overall operation includes finding mass spectrometry peaks, peak alignment, and noise reduction, and use K-Means to perform picture segmentation processing on the aligned results. Among them, during the process of finding mass spectrometry peaks, 100 mass spectrometry peaks are found in each spectrum with a step of 16 spectra; peak alignment is calculated using the average spectrum of the complete mass spectrometry imaging dataset. After segmentation processing, combined with tissue morphology spectra, early calcified areas of the specimen are identified and PA content is detected.

[0045] The results are as Figure 1 shown. PA(25:3) m / z: 615.126 is distributed in the early calcified areas of the mouse aortic root and is significantly upregulated in the group intervened with the combination of photosensitizer and low-frequency low-intensity ultrasound, that is, compared with the control group of ApoE - / - mice, the combination of photosensitizer and low-frequency low-intensity ultrasound intervention increased the content of PA in the early calcified areas of the mouse aortic root.

[0046] Example 2: The combination of photosensitizer and low-frequency low-intensity ultrasound increases the expression level of PA in M1 macrophages

[0047] 1. Induction of M1 / M2 phenotype RAW264.7 macrophages

[0048] Culture RAW264.7 cells in a 37°C constant temperature incubator containing 5% carbon dioxide. Observe the cell status and change the culture medium every 24 to 48 hours. When necessary, passage at a ratio of 1:2 or 1:3 to maintain the good growth state of the cells. 12 hours before intervention, seed the cells at a density of 10 5 cells / mL in a 35 mm cell culture dish. When the cells are completely adherent and in good growth state, add different concentrations of stimulating factors. For the induction of M1 (pro-inflammatory type) macrophages, add 2.5 ng / mL interferon γ + 200 ng / mL lipopolysaccharide and act together for 12 h; for the induction of M2 (anti-inflammatory type) macrophages, add 10 ng / mL interleukin 4 and act for 12 h.

[0049] 2. Perform the treatment of the combination of photosensitizer and low-frequency low-intensity ultrasound on M1 / M2 phenotype RAW264.7 macrophages

[0050] The M1-phenotype RAW264.7 macrophages were respectively seeded on 35-mm culture dishes, incubated with sodium hematoporphyrin monomethyl ether at a final concentration of 0.8 μM for 4 h, and then the culture dishes were placed in a degassed water bath 30 cm away from a 35-mm-diameter ultrasonic transducer with a resonance frequency of 1.0 MHz, and treated with ultrasonic intensity of 0.2 W / cm 2 for 7 min. Macrophages were taken for detection at 3 h, 6 h, 12 h, and 24 h after the combination of the photosensitizer and low-frequency low-intensity ultrasound. M1-phenotype RAW264.7 macrophages and M2-phenotype RAW264.7 macrophages without the combination of the photosensitizer and low-frequency low-intensity ultrasound treatment were used as controls. To study whether the combination of the photosensitizer and low-frequency low-intensity ultrasound acts through reactive oxygen species, an intervention group of the combination of the photosensitizer and low-frequency low-intensity ultrasound + reactive oxygen species scavenger N-acetylcysteine (NAC) was set up. The treatment method of the combination of the photosensitizer and low-frequency low-intensity ultrasound + NAC was to seed M1-phenotype RAW264.7 macrophages on 35-mm culture dishes respectively, incubate with sodium hematoporphyrin monomethyl ether at a final concentration of 0.8 μM for 4 h, then add 5 mM NAC and incubate for 1 h, and place the culture dishes in a degassed water bath 30 cm away from a 35-mm-diameter ultrasonic transducer with a resonance frequency of 1.0 MHz, and treat with ultrasonic intensity of 0.2 W / cm 2 for 7 min. Macrophages were taken for detection 12 h after ultrasound.

[0051] 3. Enzyme-linked immunosorbent assay for macrophage PA

[0052] RAW264.7 macrophages were washed with 1× phosphate-buffered saline. Samples of RAW264.7 macrophages treated differently were repeated with 6 freeze-thaw cycles (≤ -20 °C). The samples were centrifuged at 3000×g for 10 min at 4 °C to obtain the supernatant, and the PA level in the supernatant was measured using a mouse PA detection kit.

[0053] The detection results of PA content are as Figure 2 shown. Compared with M1 macrophages, M2 macrophages have a higher basal level of PA, while the combination of the photosensitizer and low-frequency low-intensity ultrasound treatment can increase the PA content in M1 macrophages. The PA level of M1 macrophages at 3 h after the combination of the photosensitizer and low-frequency low-intensity ultrasound treatment increased significantly, and this effect could last for at least 24 h. In addition, the PA level of M1 macrophages in the group of the combination of the photosensitizer and low-frequency low-intensity ultrasound treatment + reactive oxygen species scavenger NAC decreased significantly compared with that of M1 macrophages 12 h after the combination of the photosensitizer and low-frequency low-intensity ultrasound treatment, indicating that the combination of the photosensitizer and low-frequency low-intensity ultrasound acts through reactive oxygen species.

[0054] Example 3: The combination of the photosensitizer and low-frequency low-intensity ultrasound promotes the fusion of M1 macrophages

[0055] The M1 macrophages induced in Example 2 and the M1 macrophages treated with a photosensitizer combined with low-frequency and low-intensity ultrasound (incubated with sodium huaporfin at a final concentration of 0.8 μM for 4 h, ultrasound frequency of 1.0 MHz, and ultrasound intensity of 0.2 W / cm 2 for 7 min.) were respectively made into cell suspensions, inoculated into cell dishes at a density of 6.25×10 3 cells / cm 2 . After 1 h of cell inoculation, the cells were stimulated with 30 ng / mL of the cytokine RANKL. The culture medium was changed every 3 days, and the cytokine RANKL was supplemented. After 5 days of induction, atomic force microscopy was performed for observation, and the M1 macrophages without any treatment were used as a control. The cell dishes were gently washed three times with physiological saline, air-dried naturally, and then the samples were immediately scanned using a commercial atomic force microscope and the cell diameters were statistically analyzed.

[0056] The results of atomic force microscopy detection are shown in Figure 3 and Figure 4 . It can be seen from Figure 3 and Figure 4 that compared with the M1 macrophages without any treatment and the M1 macrophages stimulated with RANKL, the diameters of the M1 macrophages treated with a photosensitizer combined with low-frequency and low-intensity ultrasound and stimulated with RANKL increased significantly, and the cell diameters increased from about 10 μm to more than 30 μm, indicating that macrophage fusion had occurred. It can be seen that the photosensitizer combined with low-frequency and low-intensity ultrasound technology can promote M1 macrophage fusion.

[0057] Example 4: Application of Activating PA Expression in Macrophages in Alleviating Atherosclerotic Calcification

[0058] 1. Construction of a direct co-culture system of macrophages and calcified smooth muscle cells

[0059] Mouse smooth muscle cells (SMCs) were inoculated in 35-mm cell culture dishes containing high-glucose DMEM medium with 10 mM β-glycerophosphate, 2.8 mM calcium chloride, 50 μg / mL ascorbic acid, and 10% fetal bovine serum at a density of 1.0×10 4 cells / cm 2 . The culture medium was changed every 2 days and cultured continuously for 4 days. On the fourth day, untreated M1 macrophages and the M1 macrophages treated differently in Example 2 (M1 macrophages treated with a photosensitizer combined with low-frequency and low-intensity ultrasound and M1 macrophages incubated with a photosensitizer for 4 h, then incubated with NAC for 1 h and then treated with low-frequency and low-intensity ultrasound) were added to the smooth muscle cells, and the detection was performed after 4 days of co-culture.

[0060] 2. Alizarin red staining

[0061] Add an appropriate amount of alizarin red S staining solution to the cell culture dish, incubate in an oven at 37 °C for 30 min, wash thoroughly with distilled water, and then observe and take pictures under an optical microscope.

[0062] The alizarin red staining results of the direct co-culture system of macrophages and smooth muscle cells are as Figure 5 shown. As Figure 5 can be seen, the combination of sonosensitizer and low-frequency low-intensity ultrasound treatment can reduce the calcification degree of the direct co-culture system of M1 macrophages and calcified smooth muscle through macrophage reactive oxygen species, and M1 macrophages treated with exogenous PA (100 nM) have an effect similar to that of the combination of sonosensitizer and low-frequency low-intensity ultrasound treatment. It can be seen that the combination of sonosensitizer and low-frequency low-intensity ultrasound treatment can reduce the calcification of mouse aortic smooth muscle cells through PA.

[0063] 3. Immunofluorescence staining of early calcification of mouse aortic plaques

[0064] Take the arterial frozen sections prepared in 3(1) of Example 1, and perform the following operations after rewarming:

[0065] (1) Fix with pre-cooled acetone for 15 min;

[0066] (2) Immerse in PBS buffer 3 times, 5 min each time;

[0067] (3) Block with ready-to-use goat serum at room temperature for 30 min;

[0068] (4) Place the slide in a wet box, add the mixed primary antibody prepared with 1% BSA, and incubate overnight at 4 °C;

[0069] (5) Take out the wet box the next day and rewarm at 37 °C;

[0070] (6) Wash with PBS buffer 3 times, 5 min each time;

[0071] (7) Add the fluorescent secondary antibody prepared with PBS buffer to the slide and incubate in the dark at 37 °C for 1 h;

[0072] (8) Wash with PBS buffer 3 times, 5 min each time;

[0073] (9) Counterstain the cell nucleus with DAPI for 10 min;

[0074] (10) Wash with PBS buffer 3 times, 5 min each time;

[0075] (11) Add an anti-fluorescence quenching mounting medium and cover with a clean coverslip;

[0076] (12) Perform image acquisition with a fluorescence microscope and analyze with Image-Pro Plus software.

[0077] The results are asFigure 6 and Figure 7 As shown in Figure 7 , compared with the control group, the ratio of the immunofluorescence positive area of calcification-related bone morphogenetic protein 2 (BMP2) to the plaque area and the mean fluorescence intensity in the 15-day and 30-day groups after the combination of the sonosensitizer and low-frequency low-intensity ultrasound treatment were significantly decreased. It can be seen that the combination of the sonosensitizer and low-frequency low-intensity ultrasound treatment can reduce aortic atherosclerosis calcification in mice.

[0078] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for fusing macrophages into multinuclear osteoclast-like cells, characterized in that: The method comprises incubating M1 macrophages with a sonosensitizer, and when the sonosensitizer in the cells reaches an effective concentration, irradiating the M1 macrophages with low-frequency and low-intensity ultrasound; the ultrasound frequency of the low-frequency and low-intensity ultrasound is 0.5 MHz-1.5 MHz, and the ultrasound intensity is 0.2 W / cm 2- 1.0W / cm 2 .

2. A method for increasing the phosphatidic acid content in M1 macrophages, characterized in that: The method comprises incubating M1 macrophages with a sonosensitizer, and when the sonosensitizer in the cells reaches an effective concentration, irradiating the M1 macrophages with low-frequency and low-intensity ultrasound; the ultrasound frequency of the low-frequency and low-intensity ultrasound is 0.5 MHz-1.5 MHz, and the ultrasound intensity is 0.2 W / cm 2- 1.0W / cm 2 .

3. The method according to claim 1 or 2, characterized in that: The sonosensitizer is an organic molecular sonosensitizer, an inorganic nanosonosensitizer or an organic-inorganic hybrid nanosonosensitizer.

4. The method according to claim 3, characterized in that The sonosensitizer is sodium porphyrin, 5-aminolevulinic acid, curcumin or rhamnosine.

5. The method according to claim 4, characterized in that The sonosensitizer is sodium porphyrin; in the method, the incubation concentration of sodium porphyrin is 0.1-1 μmol / L, and the incubation time is 4 hours.

6. The method according to claim 1 or 2, characterized in that: The irradiation time is 3-7 minutes.

7. A multinucleated osteoclast-like cell obtained by the method of claim 1.

8. Use of the multinuclear osteoclast-like cells according to claim 7 in the preparation of a medicament for preventing and / or treating vascular calcification.

9. An M1 macrophage with increased phosphatidic acid content obtained by the method of claim 2.

10. Use of the M1 macrophages with increased phosphatidic acid content according to claim 9 in the preparation of a medicament for preventing and / or treating vascular calcification.

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