CN110794143B

CN110794143B - Method for researching interaction between adjacent cells

Info

Publication number: CN110794143B
Application number: CN201910886668.3A
Authority: CN
Inventors: 彭海生; 杜智敏; 唐雪枫; 李明慧; 刘肖莹; 扈丹丹; 王娜; 廉明明; 张长梅; 唐淑坤
Original assignee: Harbin Medical University
Current assignee: Harbin Medical University
Priority date: 2019-09-19
Filing date: 2019-09-19
Publication date: 2023-01-13
Anticipated expiration: 2039-09-19
Also published as: CN110794143A

Abstract

The invention discloses a method for researching the interaction between adjacent cells. The invention utilizes the combination of nanotechnology and a chemical synthesis method to prepare the nano-particle sound-sensitive agent, and then the nano-particle sound-sensitive agent is incubated with a type of cells to enable the cells to take in the nano-particle sound-sensitive agent. Then under the action of an external magnetic field, cells phagocytosing the nanoparticle acoustic sensitizer and other cells are mixed to construct a micro-tissue 3D cell population, and then the level of Reactive Oxygen Species (ROS) in the cells is regulated and controlled by accurately regulating and controlling the sound intensity and the trigger time in vitro by using ultrasound, so that the in-vitro timing, quantitative and positioning regulation and control of cell damage are realized. In vitro cell experiments, proteomics researches and the like prove that a large amount of proteins between damaged cells and adjacent cells change. The method can explore and clarify the internal relation between targeting and adjacent cell competitive uptake and the generation and development process of clinical diseases, and can be used in the technical fields of cell communication, preparation design, medicine research and development and the like.

Description

Research method for interaction between adjacent cells

Technical Field

The invention relates to a research method for the interaction between adjacent cells, in particular to a research method for the interaction between adjacent cells, which can clarify the internal relation between targeting and the competitive uptake of adjacent cells, explore the occurrence and development processes of diseases and research the pathogenesis of diseases, provide thought and theoretical basis for the design and clinical treatment of a targeted preparation and is used for the design of the targeted preparation, the research and development of new drugs, the research on the pathogenesis of diseases and the clinical treatment application. The invention belongs to the technical field of medicines.

Background

The cells are basic units of various biological structures and physiological functions on the earth, about 40 to 60 trillion human cells are combined into tissues and form organs, organ synthesis systems and various organs and systems to construct a complex human body, so that the research on the cells is very important in the process of exploring human development and ensuring human health. However, a series of activities and reactions generated by the body are not independently completed by a single cell, but are completed by a plurality of cells which are mutually matched and mutually assisted by transmitting 'signals'. Therefore, how to transmit the "signal" through the interaction between cells, especially between two adjacent cells, will become the focus of future cell research.

The targeted delivery of nano-drugs is an important field of drug research, aiming at improving the enrichment degree of drugs at the focus and reducing the systemic distribution of the drugs, thereby realizing high-efficiency and low-toxicity treatment. Overcoming the natural biological barriers of the human body in a targeted delivery system for nano-drugs is one of the challenges currently encountered by researchers. The neighbor cells are important microenvironments that must be considered for targeted delivery of the nano-drug. Firstly, adjacent cells share and co-establish an extracellular matrix microenvironment; secondly, intercellular communication is carried out among nerve cells in the modes of electric signals, chemical signals and physical contact, so that the physiological behaviors of the nerve cells are influenced; again, the ability of competitive uptake of nano-drugs between neighboring cells is a determinant of targeting, and the outcome may be affected by cell damage. It can be seen that the neighbor cell role is very important. There has been no study on the competitive uptake of nano-drugs between adjacent cells in tissues and which connections between different cells associated with the microenvironment exist.

In clinical treatment, healthy cells and damaged cells coexist at a focus part, and how to treat the damaged cells is always an aim which is struggled by researchers on the premise of not damaging the normal healthy cells. However, it is impossible to precisely locate and regulate the damaged cells at the present stage, and it is difficult to observe the effect of the damaged cells on the neighboring cells, i.e., healthy cells, which is not favorable for the reason and spatial-temporal order of the biological events. Although the in vitro two-dimensional culture is easy to position and simple and controllable, the three-dimensional structure of the tissue is lost, so that the cell biological information is changed and the credibility is lost; tissue engineering techniques provide a three-dimensional structure of tissue, but add interference to the scaffold material. If the 3D micro-tissue which is not provided with the bracket material and is close to the human body can be constructed in vitro, the defects of uncontrollable in vivo, in vitro distortion and material interference can be made up.

The inventor successfully prepares the damaged cells by using a 3D micro-tissue precision damaged cell technology for the first time, and measures the change of the proteomics of the adjacent cells caused by the influence of the interaction between the cells. The method comprises the following four steps: firstly, preparing a nano particle sound-sensitive agent by combining a nanotechnology with a chemical synthesis method, and incubating the nano particle sound-sensitive agent with a type A cell to enable the cell to take in the nano particle sound-sensitive agent; secondly, randomly mixing A type cells phagocytizing the nanoparticle sound-sensitive agent with another type of cells (B type cells) to construct a micro-tissue 3D cell population under the action of a magnetic effect; thirdly, regulating the level of Reactive Oxygen Species (ROS) through ultrasonic intensity, triggering time and the like, so as to realize the regulation of A-type cell damage in vitro in a timing, quantitative and positioning manner; fourth, changes in the 3D cell population proteins were analyzed using iTRAQ proteomics techniques. Protein analysis found that abnormal protein expression associated with cellular uptake, intracellular trafficking and energy metabolism (mitochondrial proteins, ATP-binding proteins) occurred, and these abnormal proteins, including clathrin and non-clathrin, were shown to be associated with cellular uptake. Meanwhile, ROS causes the increase of Glut1 on the surface of the A-type cell, which indicates that the uptake of nanoparticles by the cell is increased. In addition, there are a number of other proteins that have changed and are in need of further study.

Inducing the damage of the A-type cells in the 3D cell population of the microtissue by timing, quantifying and positioning by utilizing a nanotechnology and a microtissue engineering technology, and observing the influence of the damage on the adjacent cells. The method can explore and clarify the internal relation between targeting and adjacent cell competitive uptake and the occurrence and development process of clinical diseases, research the pathogenesis of diseases, provide thinking and theoretical basis for the design and clinical treatment of targeted preparations, and can be used for targeted preparation design, new medicine research, disease pathogenesis research and clinical treatment application.

Disclosure of Invention

The invention aims to provide a method for researching the interaction between adjacent cells, which can explore and clarify the internal relation between targeting and adjacent cell competitive uptake and the occurrence and development process of diseases in clinic, research the pathogenesis of diseases, provide thought and theoretical basis for the design of targeting preparations and clinical treatment, and can be used for the design of targeting preparations, the research and development of new drugs, the research of pathogenesis of diseases and the application of clinical treatment.

In order to achieve the purpose, the invention adopts the following technical means:

the invention relates to a research method of the interaction between adjacent cells, which comprises the following steps:

(1) Preparing a nano particle sound sensitive agent;

(2) Incubating the prepared nano particle sound-sensitive agent and A-type cells to enable the A-type cells to take in the nano particle sound-sensitive agent;

(3) Mixing the A-type cells and the B-type cells which phagocytose the nano particle sound-sensitive agent, re-suspending the mixture by using a culture medium, and preparing a paramagnetic cell suspension in a paramagnetic environment;

(4) Under the action of an external magnetic field, A cells and B cells phagocytosing the nano particle sound-sensitive agent can construct a micro-tissue 3D cell population, and then the level of Reactive Oxygen Species (ROS) in the A cells is regulated and controlled by ultrasound to damage the A cells;

(5) Researching the interaction mode between the A-type cells and the B-type cells;

here, the a-type cells and the B-type cells are both biologically adjacent cells in space, and the term "spatially adjacent" refers to adjacent cells in a 3D structure.

Preferably, the nanoparticle sound-sensitive agent is an emodin nanosuspension and is prepared by the following steps: weighing emodin, polyvinylpyrrolidone and sodium dodecyl sulfate in a beaker, adding deionized water, and homogenizing at high speed; then, a high-pressure homogenizer is adopted for multiple cycles to obtain an emodin nano suspension, namely a nano particle sound-sensitive agent;

wherein, preferably, the emodin nanosuspension is prepared by the following steps: weighing emodin 300mg, polyvinylpyrrolidone 1.5g and sodium dodecyl sulfate 30mg in a beaker, adding 100ml deionized water, and homogenizing at high speed for 5 times, each time for 20 seconds; and circulating for 15 times by using a high-pressure homogenizer at 700bar to obtain the emodin nano suspension, namely the nano particle sound-sensitive agent.

Preferably, the nanoparticle sound-sensitive agent can be prepared by the following steps:

(1) Preparing gold nanoparticles: putting chloroauric acid into a three-necked flask, adding ultrapure water, ultrasonically mixing, heating in a water bath kettle under reflux, stirring and heating; adding sodium citrate trihydrate solution under the condition of stirring; continuously refluxing, heating and stirring; stirring and cooling at room temperature to obtain wine red gold nanoparticles;

(2) Preparation of curcumin @ gold nanoparticles: weighing curcumin, dissolving the curcumin in ethanol, dispersing gold particles in the ethanol solution in which the curcumin is dissolved, standing in a dark place, and taking supernatant to obtain curcumin @ gold nanoparticles;

(3) Preparation of sugar-modified nanocrypsin: weighing egg yolk lecithin, cholesterol and sugar-PEG 600-DSPE (polyethylene glycol-galactose), wherein the sugar is mannose, glucose, galactose and the like, preferably the sugar is glucose or mannose, dissolving the substances in ethanol, uniformly mixing, performing spin drying to form a film, adding curcumin @ gold nanoparticle solution to disperse the lipid film, performing spin drying again by using a rotary evaporator, adding physiological saline for ultrasonic hydration, filtering by a polycarbonate film after probe ultrasonic treatment, centrifuging, and taking the supernatant to obtain sugar-modified nano curcumin, namely the nano-particle sonosensitizer;

wherein, the preferable mol ratio of the yellow lecithin, the cholesterol and the sugar-PEG 600-DSPE is 49:50:1; the ultrasonic hydration refers to that ultrasonic treatment is carried out for 10 seconds, is suspended for 6 seconds and is carried out for 10 times.

protoporphyrin liposome: dissolving egg yolk phosphatidylcholine (EPC), cholesterol (CHO) and DSPE-PEG-2000 in absolute ethanol in a rotary evaporator flask, and forming a thin lipid film by evaporating the mixture under reduced pressure; hydrating the lipid film with a physiological saline solution containing protoporphyrin; the liposome solution is dispersed by ultrasonic treatment, and the protoporphyrin liposome, namely the nanoparticle sonosensitizer, is obtained by extruding the probe through a polycarbonate membrane after ultrasonic treatment.

Among them, preferred is egg yolk lecithin (EPC), cholesterol (CHO), DSPE-PEG-2000 in a molar ratio of 49:50:1.

wherein, preferably, the A-type cells include but are not limited to glial cells, glioma cells, fibroblasts, vascular smooth muscle cells, endothelial cells, bacteria and fungi;

preferably, the B-type cells include, but are not limited to, neuronal cells, glial cells, cardiac muscle cells, vascular smooth muscle cells, vascular endothelial cells, bacteria, and fungi.

Wherein, preferably, the paramagnetic environment is provided by a paramagnetic solvent.

Wherein, preferably, the paramagnetic solution is a gadolinium solvent, and more preferably, the gadolinium solvent is gadobutrol.

Among them, it is preferable to detect proteomic changes of damaged a-type cells and undamaged B-type cells by timing, quantifying, and localizing regulation and damage a-type cells in a microtissue 3D cell population.

Preferably, the method for researching the interaction between adjacent cells can be applied to the technical fields of cell communication, preparation design and medicine research and development.

Preferably, the method for researching the interaction between adjacent cells can be used for researching and clarifying the internal relation between targeting and the competitive uptake of adjacent cells.

Compared with the prior art, the invention has the beneficial effects that:

the invention constructs a micro-tissue 3D cell group in vitro by an external magnetic field, intervenes healthy cells to damage the cells in a timing, quantitative and positioning manner by utilizing a 3D micro-tissue precise cell damage technology, and monitors the change of biological information such as signal conduction between the damaged cells and adjacent cells, proteomics, genomics and the like. In vitro cell experiments, proteomics research and the like prove that the related proteins of the uptake capacity, the uptake route, the apoptosis, the energy metabolism and the intracellular transportation between damaged cells and adjacent cells are all changed. The invention can explore and clarify the internal relation of the competitive uptake of targeting and adjacent cells and the occurrence and development process of diseases, research the pathogenesis of diseases, provide thought and theoretical basis for the design of targeting preparation and clinical treatment, and can be used for the design of targeting preparation, the research and development of new drugs, the research of pathogenesis of diseases and the application of clinical treatment.

Drawings

FIG. 1 is a particle size potential diagram of emodin nanosuspension (Emo-ns).

FIG. 2 is a particle size potential diagram of protoporphyrin liposome (Pro-LIP).

FIG. 3 is a particle size distribution graph (A) during the curcumin @ gold particle construction process; electron micrographs (B) of gold nanoparticles; an electron microscope image (C) of the gold nanoparticles coated on the surface; particle size before and after curcumin coating and fluorescence map (D).

FIG. 4 is a UV full-wavelength scan of sugar-modified nanocrystallized curcumin (MAN-Cur @ Au), which proves that sugar-modified nanocrystallized curcumin is successfully constructed layer by layer.

FIG. 5 is a flow chart of three formulations; the three nano-particle sound-sensitive agents are proved to be all taken up by cells.

FIG. 6 is a flow fluorescence shift plot (A) and a flow fluorescence histogram (B) of the change in cell surface antigen GLUT-1; the low-frequency ultrasound has a promotion effect on Glut1 on the surface of a C6 cell triggered by Emo-ns, but the promotion effect is related to the time for culturing the cell after the ultrasound, the Glut1 expression is enhanced most obviously at 0h after the ultrasound, and the Glut1 expression has a descending trend after 4h, which is possibly related to ROS generated by activating the Emo-ns by the ultrasound, and the generation of the ROS is instantaneous, so that the Glut1 expression reaches a peak value after the ultrasound, but the ROS signal is gradually weakened along with the time extension, and the phenomenon of high Glut1 expression also disappears.

FIG. 7 is a graph of mitochondrial membrane potential points (A, B, C) and a fluorescence histogram (D); the fact that the mitochondrial membrane potential of the cells treated by Emo-ns tends to be reduced is shown, the phenomenon is enhanced after ultrasonic stimulation, and the Emo-ns and ultrasonic treatment have the effect of inducing the reduction of the mitochondrial function of the cells, namely, cell damage is generated.

FIG. 8 is a Western blot and histogram of the apoptotic protein caspase 3 (A, C) and caspase9 (B, D) in cardiomyocytes; the low-frequency ultrasound has an enhancement effect on the expression of the Pro-LIP-induced apoptosis-related protein caspase 3/9 in the cells, the enhancement effect is related to the fact that the low-frequency ultrasound can promote the cells to take in the Pro-LIP, and the high expression phenomenon of the caspase 3/9 in the cells after the sonodynamic therapy is the effect of the reduction of mitochondrial function caused by the fact that the cells generate a large amount of active oxygen after being treated by the Pro-LIP combined ultrasound.

FIG. 9 is a graph of the microtissue 3D cell population for the effect of ROS in Vascular Smooth Muscle Cells (VSMCs) on the neighbouring cells Vascular Endothelial Cells (VECs). The results show that no obvious DCF green fluorescence is seen in the VEC + SDT group, the VSMC + VEC + SDT group and the VSMC group which absorbs MAN-Cur @ Au + VEC, the DCF green fluorescence cells of the VSMC group which absorbs MAN-Cur @ Au + VEC + SDT group are obviously more than those of the VSMC group which absorbs MAN-Cur @ Au + VEC, and the analysis shows that the damaged VSMC with increased ROS transmits oxidative stress signals to VEC of adjacent cells, but the deep mechanism is not clear.

FIG. 10 is a diagram of a microtissue 3D cell population constructed from different cell types; indicating that any cell can successfully construct a layer-by-layer mixed and randomly mixed micro-tissue 3D cell population.

FIG. 11 is a bar graph of the change in protein from neighbor cell proteomics studies; the difference of the protein expression quantity and type among different groups is illustrated, and the obvious protein expression difference exists in the 3D mixed construction micro-tissue under the action of ROS.

FIG. 12 is a Venturi map of the protein of interest in neighbouring cells; therefore, the adjacent cells of the cell microenvironment have obvious influence on the proteomics of the brain glioma cells (C6) and the neuron cells (PC 12), and the types and the numbers of the cells are obviously changed.

FIG. 13 is a graph showing the bioinformatics analysis of the expression of the neighbor cell protein profile; indicating that the expression of protein mass spectrum is changed by the existence of primary myocardial cells.

FIG. 14 is a graph of the analysis of the network of related proteins in neighbor cells; as a result of the analysis, it can be seen that some proteins of Vascular Smooth Muscle (VSMC) and Vascular Endothelial Cells (VEC) have multiple properties, possibly acting on ATP binding, cellular trafficking, mitochondrial structure or function, and intracellular responsiveness.

Detailed Description

The advantages and features of the invention will become more apparent from the following further description of the invention with reference to specific examples. However, the examples are only for illustrating the present invention and do not set any limit to the scope of the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The materials used in the examples are as follows:

chlorauric acid chloride was purchased from McLin Biochemical technology, inc. (Shanghai, china)

Curcumin was purchased from Shifeng Biotechnology limited (Shanghai, china)

Trisodium citrate dihydrate, purchased from prometha Biotech, inc. (Shanghai, china)

PEG 2000 from Aladdin industries, inc. (Shanghai, china).

p-carboxyphenyl-alpha-D-acetylmannosamine (Ac) ₄ MAN) was obtained from Innochem, beijing, china.

Egg yolk phosphatidylcholine (EPC) and Cholesterol (CHO) were purchased from Bio Life Science & technology co., ltd (shanghai, china).

Rhodamine B (Rho) and 1,1 '-dioctadecyl-3, 3' -tetramethyl-Diiodocyanine Iodide (DiR) are available from HEDE Biotechnology Ltd, beijing, china.

anti-GLUT-1 antibodies were purchased from Immunoway (Beijing, china).

ROS detection kit purchased from Beibo reagent (Shanghai, china)

Mitochondrial membrane potential kit purchased from Biyuntian biotechnology (Shanghai, china)

CCK-8 kit purchased from Biyuntian biotechnology (Shanghai, china)

Healthy Wistar rats 200-250 g purchased from the laboratory animal institute of Harbin medical university (SCXK 2013-001)

EXAMPLE 1 preparation of emodin nanosuspensions (Emo-ns)

Emodin nanosuspension: weighing emodin 300mg, polyvinylpyrrolidone 1.5g and sodium dodecyl sulfate 30mg in a beaker, adding 100ml deionized water, and homogenizing at high speed for 5 times, each time for 20 seconds; and then the high-pressure homogenizer is adopted to circulate for 15 times at 700bar to obtain the emodin nano suspension, namely the nano particle sound-sensitive agent.

Example 2 preparation of sugar-modified Nanocrystallized curcumin (MAN-Cur @ Au)

(1) Preparing gold nanoparticles: adding 250 μ l chloroauric acid into a three-necked flask, adding 100ml ultrapure water, ultrasonically mixing, heating in a water bath under reflux, stirring, and heating; adding 926 μ l of sodium citrate trihydrate solution under stirring; continuously refluxing, heating and stirring for 15min; stirring and cooling at room temperature to obtain the wine red gold nanoparticles.

(2) Preparation of curcumin @ gold nanoparticles: weighing 19.2mg of curcumin, dissolving in 3ml of ethanol, dispersing 1ml of gold nanoparticles in the ethanol solution of the curcumin, standing for 24 hours in a dark place, and taking the supernatant to obtain the curcumin @ gold nanoparticle solution.

(3) Preparation of mannose-PEG 600-DSPE: prepared according to the method disclosed in the Chinese patent application with the application number of 201811062535.6 and the invention name of 'sugar-polyethylene glycol-DSPE coupling compound and the preparation method and application thereof'.

(3) Preparation of sugar-modified nanocrypsin: the molar ratio of the mixture is 49:50:1, weighing yolk lecithin, cholesterol and mannose-PEG 600-DSPE, dissolving 15mg of lipid material in ethanol, uniformly mixing, then carrying out spin-drying to form a lipid membrane, adding 1ml of curcumin @ gold nanoparticle solution to disperse the lipid membrane, using a rotary evaporator to spin-dry again, adding physiological saline for ultrasonic hydration, carrying out ultrasonic treatment for 10 seconds, pausing for 6 seconds, working for 10 times, filtering through a polycarbonate membrane, centrifuging, and taking supernatant to obtain sugar-modified nanocryation curcumin, namely the nanoparticle sonosensitizer.

EXAMPLE 3 preparation of protoporphyrin liposomes (Pro-LIP)

The molar ratio of the mixture is 49:50:1 weighing egg yolk lecithin (EPC), cholesterol (CHO), DSPE-PEG-2000, dissolving the above 15mg of lipid material in absolute ethanol in a rotary evaporation flask, and evaporating the mixture under reduced pressure to form a thin lipid film; hydrating the lipid membrane with 1ml of physiological saline solution containing 0.01mg of protoporphyrin; the liposome solution was dispersed by sonication (run for 10 seconds, pause for 6 seconds, work for 30 times) and extruded through a 100nm polycarbonate membrane to obtain protoporphyrin liposome, i.e., nanoparticle sonosensitizer.

Example 4 characterization of nanoparticle sonosensitizers

(1) DLS detection particle size distribution and Zeta potential: at room temperature, 1mL of deionized water was taken, 10. Mu.L each of the prepared Emo-ns (prepared in example 1) and Pro-LIP (prepared in example 3) was taken, the samples were mixed with deionized water and injected into a dedicated cuvette of a dynamic light scattering particle sizer, and the dynamic light scattering particle sizer was used to measure the average particle size and zeta potential of each liposome. Particle size potential diagrams of emodin nanosuspensions (Emo-ns) and protoporphyrin liposomes (Pro-LIP) are shown in FIGS. 1 and 2, respectively.

(2) TEM detection morphology: the prepared MAN-Cur @ Au (prepared in example 2) is diluted properly, a copper mesh is added dropwise and kept stand for 1min, phosphotungstic acid is negatively dyed as required, a proper scale is selected, and the particle morphology is observed and photographed by using a TEM. The particle size potential diagram in the preparation process of MAN-Cur @ Au is shown in figure 3, which indicates that the sugar modified nano curcumin (MAN-Cur @ Au) is successfully constructed, the fluorescence of particles is enhanced after the curcumin is coated, and the particle size is increased. An ultraviolet full-wavelength scanning graph of sugar-modified nano curcumin (MAN-Cur @ Au) is shown in FIG. 4, which proves that sugar-modified nano curcumin is successfully constructed layer by layer.

EXAMPLE 5 culture of Primary astrocytes, neuronal cells

(1) Culturing primary astrocytes: placing a mouse (purchased from department of animals of Harbin medical university, SCXK 2013-001) within 3 days of birth on ice for 2min, and spraying alcohol for disinfection; after the mice were treated with 75% ethanol and PBS sequentially, the mice were killed by dislocation, brains were taken and placed in precooled PBS (PBS placed on an ice plate); placing the rat brain on a moist gauze, and removing blood filaments in the rat brain by using forceps; shearing rat brain with scissors, adding pancreatin for digestion, transferring to a 50mL centrifuge tube, and placing in a cell culture box for 10min; after the pipetting is uniform, medium (containing FBS) is added to stop digestion. And (5) filtering by using a filter screen. 12000rpm 5min at 4 ℃ while ddH2O cleaned the PLL coated bottles 3 times; the PLL is recovered. Centrifuging, discarding the supernatant, adding culture medium, blowing uniformly, counting cells, and transferring to a cell culture bottle.

(2) Culturing primary neuron cells: pregnant mice were anesthetized with 0.2mL10% chloral hydrate (C57 BL/6J, pregnancy 16-17 days, purchased from department of animals of Harbin medical university, SCXK (Black) 2013-001), and fetal mice were removed and immersed in PBS. Fetal mice were washed 2 times with PBS and surface blood was removed. Removing fetal membrane to remove mucus (passing 75% alcohol), taking out fetal mouse, placing in DMEM, and cutting. Fetal rat brains were isolated with surgical instruments and placed in PBS and the rat cortex was isolated under a microscope. The separated cortical tissue was placed in DMEM. The cortex was washed once with PBS, the tissue was minced with scissors (no significant clumps), digested with 0.5% trypsin by pipetting, and digested to no significant clumps (digestion was stopped by adding 20% fbs medium). Digestion was stopped with media and filtered, 1500rpm, centrifuged for 5min. The supernatant is removed and 10mL of inoculation medium (neutral medium +0.5% GlutaMAX +2% B27supplement +1% PS) is added, counted. Six-hole plates were layered at 1 × 106/hole, and the liquid was changed after four hours. One day after plating culture cytarabine was added to inhibit glial cell growth (1.

Example 6 culturing of Primary fibroblasts, primary cardiomyocytes

(1) Culturing primary fibroblasts: taking a suckling mouse of a Wistar rat with the age of 1-3 days, placing the suckling mouse in 75% alcohol for a moment of disinfection, cutting open the chest by an ophthalmological scissors, taking out the heart of a young mouse, rapidly cleaning the heart by PBS (phosphate buffer solution) in a super clean workbench, removing blood coagulation and fiber tissues around the heart, placing the heart into serum-free DMEM, cutting the heart of the suckling mouse into tissue blocks with the size of about 1mm & lt 3 & gt, then transferring the tissue block solution into a 15mL centrifuge tube, sucking out the DMEM solution after the tissue blocks are settled at the bottom of the tube, adding pancreatin digestive juice with the volume twice of the tissue amount, slowly shaking and digesting for 1-3 minutes in a 37 ℃ water bath pot, and discarding the pancreatin turbid. Repeatedly digesting for 3-4 times, collecting enzyme solution, wherein the volume ratio of the enzyme solution to the culture solution is 1:1, stop digestion in a refrigerator at 4 ℃. The collected digest was passed through a 200 mesh sieve, and then the filtrate was centrifuged at 2500rpm for 3min. Removing supernatant, collecting cell precipitate, resuspending the cell precipitate with DMEM culture solution containing serum, blowing cells until the cells are dispersed, transferring the cell suspension into a disposable cell culture bottle, culturing in an incubator at 37 ℃ for 2 hours, and allowing fibroblasts to adhere to the wall.

(2) Culturing primary myocardial cells: after the fibroblasts are attached to the wall, the culture solution in the cell bottle is collected, the residual cell suspension is transferred to a new culture bottle, the myocardial cells are purified and cultured in an incubator at 37 ℃ for 48 hours for later use.

Example 7 culturing vascular smooth muscle cells

2-3 healthy rats were taken and 1mL heparin (1 KU/mL) was injected intraperitoneally, followed by waiting for 20-30 minutes. After anesthesia with chloral hydrate, lung tissue was removed and placed in HEPES buffer or PBS buffer. Lung tissue and buffer were transferred to a clean bench and pulmonary vessels were isolated under a microscope. After vessel separation, the vessels were cut open longitudinally and endothelial cells were removed by gentle swabbing with a sterile cotton swab. The adventitia is scraped gently using a blade or scissors to remove the fibrosis. The vessels were then cut into small pieces, transferred to collagenase II digests and treated for 30 minutes at 37 ℃. The treatment fluid was collected and the digest neutralized with serum-containing medium. Observing the obtained cells under microscope, repeating the digestion step until long spindle-shaped smooth muscle cells are obtained, neutralizing the digestion solution, centrifuging the cells, suspending the cells in a culture solution (containing double antibody), and placing the cells in a medium at 37 ℃ in CO ₂ Culturing in an incubator. After the liquid change on the next day, the liquid change is carried out every other day.

Example 8 modeling of cellular injury

(1) Cell inoculation: taking rat glioma cells (or fibroblasts or vascular smooth muscle cells) in the logarithmic growth phase, washing the rat glioma cells (or the fibroblasts or the vascular smooth muscle cells) for 3 times by using sterile PBS after high pressure, digesting the rat glioma cells by using trypsin, neutralizing pancreatin by using a high-sugar DMEM culture solution containing 10% serum, properly blowing and beating the cells to uniformly disperse the cells, collecting the cells into a 10EP tube, centrifuging the cells at 2000rpm for 3min, and removing a supernatant; the cells were resuspended using culture medium and inoculated into T25 cell culture flasks.

(2) Ultrasonic treatment: after the cells are attached to the wall for 24 hours, the nano particle sound-sensitive agent (Emo-ns, pro-LIP or MAN-Cur @ Au) is given at the concentration of 9 mug/mL, and the cells are subjected to ultrasonic treatment by using a sound power physiotherapy instrument after 4 hours of administration. After the medicine is taken and before the ultrasonic treatment, incubating and bathing the DCFH-DA fluorescent probe for 30min, detecting the intracellular fluorescence intensity by a flow cytometer, and judging the intracellular active oxygen yield.

Finally, the inventive examples determined that the ultrasound intensity was 0.4W/cm ² And establishing a cell damage model when the ultrasonic frequency is 1MHz and the ultrasonic time is 15min. However, the skilled person can establish a corresponding cell damage model by adjusting the time (dosage) for taking the nanoparticle sonosensitizer, the ultrasonic intensity and the ultrasonic time, and controlling the cell damage degree in a timing, quantitative and positioning manner according to actual needs.

Example 9 cellular uptake of nanoparticle sonosensitizers

(1) Taking cells, washing the cells for 3 times by using sterile PBS after high pressure, digesting the cells by using trypsin, neutralizing the trypsin by using a high-sugar DMEM culture solution containing 10% serum, properly blowing the cells to uniformly disperse the cells, collecting the cells into a 10mLEP tube, centrifuging the tubes at 2000rpm for 3min, and removing a supernatant; resuspending cells in culture medium, inoculating, and adjusting density to 7.0 × 10 ⁵ A hole. The experimental groups were: blank control group, emo-ns group, MAN-Cur @ Au group, and Pro-LIP group.

(2) According to the designed experimental group, a nanoparticle sonosensitizer (Emo-ns, pro-LIP or MAN-Cur @ Au) was administered at a concentration of 9. Mu.g/mL, the cells and the drug were incubated for 4 hours, and after incubation, the cells were trypsinized, centrifuged at 2000rpm X3 min, resuspended in PBS, collected, and examined. The flow cytometer detects the fluorescence intensity of the nanoparticles in the cells, uses a corresponding filter to detect the fluorescence intensity, and analyzes the data by using FlowJo 7.6 software.

FIG. 5 illustrates that all three nanoparticle sonosensitizers can be taken up by cells.

Example 10 detection of cell surface antigen GLUT-1

(1) Cell inoculation: taking brain glioma cells (C6 cells) in logarithmic growth phase, washing the cells for 3 times by using sterile PBS after high pressure, digesting the cells by using trypsin, neutralizing the pancreatin by using a high-sugar DMEM culture solution containing 10% serum, properly blowing and beating the cells to uniformly disperse the cells, collecting the cells into a 10mL EP tube, centrifuging the tubes at 2000rpm for 3min, and removing supernatant; resuspending the cells in culture medium, seeding at 7.0X 10 density ⁵ The cells are divided into a blank Control group (Control), an emodin nanosuspension group (Emo-ns), an emodin nanosuspension + ultrasonic group (Emo-ns-SDT-0 h), an emodin nanosuspension + ultrasonic post-incubation group (Emo-ns-SDT-4 h).

(2) Antibody incubation: according to the designed experimental time and grouping, the nano particle acoustic sensitivity agent (Emo-ns) is given at the concentration of 9 mug/mL, and the cells are subjected to ultrasonic treatment by using an acoustic power physiotherapy instrument; ultrasonic intensity of 0.4W/cm ² Ultrasonic frequency is 1MHz, and ultrasonic time is 15min. After ultrasonic treatment, digesting cells, preparing a cell suspension, carrying out primary antibody incubation at 4 ℃ overnight, and then incubating a fluorescent secondary antibody; gently wash 3 times with PBS and detect on flow cytometer.

FIG. 6 shows that low-frequency ultrasound promotes Glut1 on the surface of C6 cells induced by Emo-ns, but the promotion effect is related to the time for cell culture after the ultrasound, the expression of Glut1 is enhanced most obviously at 0h after the ultrasound, and the expression of Glut1 has a descending trend after 4h, which is probably related to the ROS generated by curcumin activated by the ultrasound, and the generation of ROS is transient, so that the expression of Glut1 reaches a peak value after the ultrasound, but as the time is prolonged, the ROS signal is gradually weakened, and the phenomenon of high expression of Glut1 also disappears.

Example 11 detection of mitochondrial Membrane potential in cells

(1) Taking brain glioma cells (C6 cells) in logarithmic growth phase, washing the cells for 3 times by using sterile PBS after high pressure, digesting the cells by using trypsin, neutralizing the pancreatin by using a high-sugar DMEM culture solution containing 10% serum, appropriately blowing and beating the cells to uniformly disperse the cells, collecting the cells into a 10mLEP tube, and centrifuging the tube at 2000rpm3min, discarding the supernatant; resuspending the cells in culture medium, seeding at 7.0X 10 density ⁵ A hole. The experimental groups are: blank control group, emo-ns + SDT group.

(2) According to the designed experimental grouping, the nano particle sound-sensitive agent (Emo-ns) is given at the concentration of 9 mug/mL, cells and the nano particle sound-sensitive agent are incubated for 4 hours, and after incubation, ultrasonic treatment is carried out, wherein the ultrasonic intensity is 0.4W/cm ² Ultrasonic frequency is 1MHz, and ultrasonic time is 15min. The cells of each group were collected and resuspended with 0.5mL of a pre-formulated JC-1 staining working solution, and 0.5mL of cell culture solution was added to each sample, mixed well and placed in CO ₂ Incubate for 30min in the incubator. Preparation of JC-1 Xbuffer can be performed during the incubation. After the incubation is finished, centrifuging to remove the cell culture solution and the fluorescent staining working solution, repeating the operation twice by using 1 Xbuffer solution at 4 ℃ and 1000RPM for 4min to fully wash the cells, finally resuspending the cells by using 1 Xbuffer solution, and detecting on a computer.

FIG. 7 shows that the mitochondrial membrane potential of cells treated by Emo-ns is decreased, and the phenomenon is enhanced after ultrasonic stimulation, which proves that Emo-ns-SDT has the effect of inducing and reducing the mitochondrial function of cells.

EXAMPLE 12 changes in the expression level of apoptotic protein caspase 3/9 in cells

(1) Taking primary fibroblasts, washing the primary fibroblasts for 3 times by using sterile PBS (phosphate buffer solution) after high pressure, digesting the primary fibroblasts by using trypsin, neutralizing the pancreatin by using a high-sugar DMEM (modified eagle medium) culture solution containing 10% serum, properly blowing and beating the cells to uniformly disperse the cells, collecting the cells into a 10mL EP (enhanced EP) tube, centrifuging the cells at 2000rpm for 3min, and removing a supernatant; resuspending the cells in culture medium, seeding at 7.0X 10 density ⁵ A hole. The experimental groups were: blank control group, pro-LIP + SDT group.

(2) Taking appropriate amount of Pro-LIP, dispersing in culture solution to prepare stock solution of 9 μ g/mL, and keeping out of the sun 4; after the cells adhere to the wall, the medicine is administered for 4 hours, and the ultrasonic intensity is 0.4W/cm ² Culturing for 24h at ultrasonic frequency of 1MHz for 15min; the culture medium containing Pro-LIP was discarded, washed 3 times with PBS, and the protein was cleaved with RIPA and collected. At 4 deg.C, 13500rpm × 20min, taking supernatant, adding loading buffer, vortexing, 100 deg.CBoiling for 5min, and storing the sample in a refrigerator at-80 ℃ for later use; spreading glue, loading, performing electrophoretic separation, transferring to membrane, dyeing with ponceau, incubating at 4 deg.C for one time, and standing overnight; the membrane was washed with TBST buffer, incubated with secondary antibody 1h, washed with TBST, and developed using ECL.

FIG. 8 illustrates the enhancement of Pro-LIP-induced expression of caspase 3/9, which is an apoptosis-related protein in cells, by low-frequency ultrasound, and is related to the enhancement of Pro-LIP uptake by cells, wherein the phenomenon of high expression of caspase 3/9 in cells after sonodynamic treatment is the effect of decreased mitochondrial function caused by the large amount of active oxygen produced inside the cells after Pro-LIP combined with ultrasound treatment.

Example 13 Effect of neighbor cells

(1) Respectively washing blood Vessel Smooth Muscle Cells (VSMC) and blood Vessel Endothelial Cells (VEC) for 3 times by using sterile PBS after high pressure, digesting by trypsin, neutralizing pancreatin by using high-sugar DMEM culture solution containing 10% serum, properly blowing and beating the cells to uniformly disperse the cells, collecting the cells into a 10mL EP tube, centrifuging at 2000rpm for 3min, and discarding supernatant; resuspending cells in culture medium, inoculating, and maintaining the density at 1.0 × 10 ⁷ A hole. The experimental groups were: VEC + SDT group (ES), VSMC + VEC + SDT group (MES), VSMC + MAN-Cur @ Au + VEC group (MCE), and VSMC + MAN-Cur @ Au + VEC + SDT group (MCES).

(2) After the cells were attached to the wall for 24h, the VSMC was dosed with MAN-Curr @ Au at a concentration of 9. Mu.g/mL for 4h according to the experimental grouping requirements. Staining the vascular endothelial cells with DAPI, incubating with a DCFH-DA probe, and digesting the two types of cells respectively; randomly mixing VSMC phagocytosing the nano-particle sonosensitizer with VEC cells, re-suspending with culture medium, adding gadobutrol into the cell suspension to prepare paramagnetic cell suspension, constructing a micro-tissue 3D cell group under the magnetic effect, performing ultrasonic treatment with the ultrasonic intensity of 0.4W/cm ² Ultrasonic frequency is 1MHz, and ultrasonic time is 15min. And (4) fluorescence imaging.

FIG. 9 shows that DCF green fluorescence is not evident in ES group, MES group and MCE, and DCF green fluorescence cells are evident in MCES group and MCE group, and analysis suggests that damaged vascular smooth muscle with increased ROS transmits oxidative stress signals to adjacent vascular endothelial cells, but the deep mechanism is not clear.

Example 14 microtissue 3D cell population construction and site-directed Induction of ROS injured cells

(1) Taking brain glioma cells (C6 cells) and normal neuron cells (PC 12 cells), respectively washing for 3 times by using sterile PBS after high pressure, digesting by using trypsin, neutralizing pancreatin by using a high-sugar DMEM culture solution containing 10% serum, properly blowing and beating the cells to uniformly disperse the cells, collecting the cells into a 10mL EP tube, centrifuging at 2000rpm for 3min, and removing supernatant; cells were resuspended using High DMEM10% serum medium and seeded into T25 cell flasks.

(2) Adding MAN-Curr @ Au into the C6 cells at the concentration of 9 mu g/mL after the C6 cells are attached to the wall for 24 hours, and incubating for 4 hours;

(3) Taking C6 cells for taking nano particle sonosensitizer MAN-Cur @ Au, digesting by trypsin, centrifuging at 2000rpm for 3min, and respectively re-suspending the cells in an EP tube by using a proper amount of High DMEM serum-free culture medium for later use; diO (1 mg/mL) was added to the C6 cell suspension to label the cell membrane, and the mixture was incubated at 37 ℃ for 5min.

(4) Random mixing: taking PC12 cells, adding a proper amount of DAPI (1 mg/mL) to mark cell nuclei, and incubating for 15-20 min at 37 ℃. Randomly mixing the C6 cells phagocytosing the nanoparticle sonosensitizer with the PC12 cells, re-suspending with a high-sugar serum-free culture medium, adding gadobutrol into the cell suspension to prepare paramagnetic cell suspension, and constructing a micro-tissue 3D cell population under an external magnetic field.

Mixing layer by layer: taking PC12 cells, adding a proper amount of DAPI (1 mg/mL) to mark cell nuclei, and incubating for 15-20 min at 37 ℃. Adding gadobutrol into the cell suspension to prepare paramagnetic cell suspension, constructing a PC12 cell 3D cell population under an external magnetic field, then adding a C6 cell suspension phagocytosed by the nanoparticle sonosensitizer, constructing a PC12 cell-C6 cell 3D cell population under the external magnetic field, and repeating the steps to construct a micro-tissue 3D cell population under the external magnetic field by a layer-by-layer mixing method.

(5) After the micro-tissue 3D cell population is established for 30 minutes, inducing the C6 cells which take the nano particle sonosensitizer to generate ROS to damage the C6 cells by using the ultrasonic effect, wherein the ultrasonic intensity is 0.4W/cm ² Ultrasonic frequency of 1MHz and ultrasonic time of 15min, and is used for observing the change of mixed protein of adjacent cells of 3D cell population of the micro-tissueAnd (4) making conditions.

According to the above steps, vascular Smooth Muscle Cell (VSMC) and Vascular Endothelial Cell (VEC) microtissue 3D cell populations and cardiomyocyte and fibroblast microtissue 3D cell populations were constructed, respectively.

Fig. 10 illustrates that different cells can successfully construct layer-by-layer mixed and randomly mixed micro-tissue 3D cell populations that can be used for subsequent studies.

Example 15 proteomics study

And (3) after the microtissue 3D cell group constructed by the C6 cells and the PC12 cells is subjected to ultrasonic treatment, the microtissue 3D cell group is blown away by a physical blowing method, and after the cells are collected, the cells are processed according to a protein extraction method, so that a research object is provided for iTRAQ protein analysis.

(1) Protein extraction and quantification

The cells were taken out and washed with PBS 1-3 times in a 1.5 mL centrifuge tube at 2000rpm 3min, added with 300. Mu.L of lysis buffer [8M urea (480 mg/mL) +10mM DTT (1.54 mg/mL) + ultrapure water ], sonicated with an ice probe 20 times in total, lysed sufficiently, centrifuged at 20000g for 10min, and the supernatant was aspirated and stored at-80 ℃. A small amount of the supernatant solution was taken and the protein concentration was determined by BCA method.

(2) Pancreatin digestive protein

Dithiothreitol was added to the protein solution to give a final concentration of 5mM, and the solution was reduced at 56 ℃ for 30min. Further iodoacetamide (final concentration: 10 mM) was added, and incubated at room temperature in the dark. Finally, the urea concentration of the sample was diluted to below 2M. The method comprises the following steps of 1: adding pancreatin in the mass ratio of 50 (pancreatin: protein), and performing enzymolysis at 37 ℃ overnight. Then, the ratio of 1: adding pancreatin in the mass ratio of 100 (pancreatin: protein), and continuing enzymolysis for 4h.

(3) iTRAQ marker

And (3) passing the peptide fragments through a column to remove salt, and freeze-drying the sample. Peptide fragments were dissolved with TEAB (0.5M) and labeled according to the iTRAQ kit protocol. Namely: dissolving and thawing the labeled reagent with isopropanol, mixing with the peptide segment, incubating at room temperature for 2h, mixing the labeled peptide segment, desalting, and lyophilizing.

(4) RP reverse phase separation

The peptide fragments were fractionated by high pH reverse phase HPLC using C18 as the chromatographic packing (manufactured by military institute). The operation is as follows: the peptide fragment gradient is 10%, 12.5%, 15%, 17.5%, 25% and 50%, the solution pH is 10, 80 μ L collects one component, and the subsequent operation is carried out after freeze-drying.

(5) Liquid chromatography-mass spectrometry

The peptide fragment was dissolved in mobile phase A (0.1% (v/v) formic acid aqueous solution) by liquid chromatography, and then separated by using EASY-nLC1000 ultra performance liquid system. The mobile phase A is an aqueous solution containing 0.1 percent of formic acid and 2 percent of acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. Gradient elution was carried out with a flow rate of 400nL/min. The ESI source was ionized and analyzed by QOxitve HF mass spectrometry. The peptide fragment parent ion and its secondary fragment were detected and analyzed using a high resolution Orbitrap. Data is collected using a data dependent scanning (DDA) program mode.

(6) Database search

Secondary mass spectral data were retrieved using Maxquant (v1.5.2.8). And (3) retrieval parameter setting: the database is SwissProt Rat, a reverse library is added to calculate the false positive rate (FDR) caused by random matching, and a common pollution library is added to the database to eliminate the influence of pollution proteins in the identification result. The quantitative method is set as iTRAQ-8plex, and the FDR of protein identification and PSM identification is set as 1%.

A total of 2587 proteins were identified in this study, and if normalized to 1.2-fold or greater up-regulation threshold and 0.83-fold or less down-regulation threshold, then the histone changes in the quantified proteins are shown in figure 11. FIG. 11 illustrates the differences of the amount and type of protein expression among different groups, which shows that the 3D mixed micro-tissue has significant protein expression differences under the action of ROS.

FIG. 12 is a Venturi plot of the relevant proteins in neighbouring cells; the method shows that the adjacent cells of the cell microenvironment have obvious influence on the proteomics of the brain glioma cells (C6) and the neuron cells (PC 12), and the types and the numbers of the cells are obviously changed.

Example 16 biological systems informatics analysis-protein expression profiling

After ultrasonic treatment, a micro-tissue 3D cell population constructed by primary fibroblasts and primary cardiomyocytes is blown away by a physical blowing method, the cells are collected and then processed according to a protein extraction method, and after analysis by iTRAQ protein, protein clustering analysis is performed.

Clustering analysis: applying R language, referring to heatmap R code, analyzing with file protein data csv, taking out protein with score value greater than threshold 5, applying heatmap function in R package gplots and ggplot2, finding out clustering relation, and forming cluster file.

Figure 13 illustrates that the presence of primary fibroblasts altered the expression of primary cardiomyocyte protein profiles.

Example 17 biological systems informatics analysis-network analysis

After the microtissue 3D cell group constructed by VSMC cells and VEC cells is subjected to ultrasound, the microtissue 3D cell group is blown away by a physical blowing method, the cells are collected and then processed according to a protein extraction method, and network analysis is performed after iTRAQ protein analysis.

Network analysis: firstly, differential protein classification is carried out on protein data, the protein data are arranged into a relation pair csv file, then R language is applied, a relation pair R code is referred, weight is calculated (weight algorithm: in the same function, each protein appears in several groups of experiments, namely the weight of the protein with the function), a 'relation _ weight.csv' file is obtained, and finally cytoscape software is used for drawing a network graph. Drawing a protein network relation graph of ATP, mitochondria, endocytosis and cell transportation, and being used for explaining the relation between energy metabolism and cell uptake and transportation; and drawing an ATP, mitochondria and apoptosis protein network relation graph to explain the relation between energy metabolism and apoptosis.

FIG. 14 results from network analysis show that some proteins have multiple properties and may function in ATP binding, cellular trafficking, mitochondrial structure or function, and endocytosis.

Claims

1. A method for studying the interaction between adjacent cells, comprising the steps of:

(1) Preparing a nano particle sound sensitive agent;

(2) Incubating the prepared nano particle sound-sensitive agent and A type cells to enable the A type cells to take in the nano particle sound-sensitive agent;

(4) Under the action of an external magnetic field, constructing a microtissue 3D cell group by using A cells and B cells which phagocytose nano particle sound-sensitive agents, and then regulating the level of Reactive Oxygen Species (ROS) in the A cells by using ultrasound to damage the A cells;

2. The method for studying the interaction between adjacent cells according to claim 1, wherein said nanoparticle sonosensitizer is an emodin nanosuspension, and is prepared by the steps of: weighing emodin, polyvinylpyrrolidone and sodium dodecyl sulfate in a beaker, adding deionized water, and homogenizing at high speed; and then the emodin nano suspension, namely the nano particle sound-sensitive agent, is obtained by multiple circulations of a high-pressure homogenizer.

3. The method for studying the interaction between adjacent cells according to claim 2, wherein said emodin nanosuspension is prepared by the steps of: weighing emodin 300mg, polyvinylpyrrolidone 1.5g and sodium dodecyl sulfate 30mg in a beaker, adding 100ml deionized water, and homogenizing at high speed for 5 times, each time for 20 seconds; and then the high-pressure homogenizer is adopted to circulate for 15 times at 700bar to obtain the emodin nano suspension, namely the nano particle sound-sensitive agent.

4. The method for studying the interaction between adjacent cells according to claim 1, wherein the nanoparticle sonosensitizer is prepared by the steps of:

(1) Preparing gold nanoparticles: putting chloroauric acid into a three-necked flask, adding ultrapure water, ultrasonically mixing, heating in a water bath kettle under reflux, stirring and heating; adding sodium citrate trihydrate solution under the stirring condition; continuously refluxing, heating and stirring; stirring and cooling at room temperature to obtain wine red gold nanoparticles;

(2) Preparation of curcumin @ gold nanoparticles: weighing curcumin, dissolving the curcumin in ethanol, dispersing the gold nanoparticles in ethanol solution in which the curcumin is dissolved, standing in a dark place, and taking supernatant to obtain curcumin @ gold nanoparticles;

(3) Preparation of sugar-modified nanocrypsin: weighing yolk lecithin, cholesterol and sugar-PEG 600-DSPE, wherein the sugar is mannose, glucose or galactose, dissolving the yolk lecithin, the cholesterol and the sugar-PEG 600-DSPE in ethanol, uniformly mixing, spin-drying to form a film, adding curcumin @ gold nanoparticle solution to disperse the lipid film, spin-drying again by using a rotary evaporator, adding physiological saline for ultrasonic hydration, filtering by a polycarbonate film after probe ultrasonic treatment, centrifuging, and taking the supernatant to obtain sugar-modified nano curcumin, namely the nano particle sound-sensitive agent.

5. The method of studying the interaction between adjacent cells according to claim 4, wherein said sugar is glucose or mannose

6. The method of claim 4, wherein the molar ratio of lecithin, cholesterol, and sugar-PEG 600-DSPE is 49:50:1; the ultrasonic hydration refers to that ultrasonic treatment is carried out for 10 seconds, is suspended for 6 seconds and is carried out for 10 times.

7. The method for studying the interaction between adjacent cells according to claim 1, wherein the nanoparticle sonosensitizer is prepared by the steps of:

protoporphyrin liposome: dissolving egg yolk phosphatidylcholine (EPC), cholesterol (CHO) and DSPE-PEG-2000 in absolute ethanol in a rotary evaporation bottle to obtain a mixture of egg yolk phosphatidylcholine (EPC), cholesterol (CHO) and DSPE-PEG-2000, and forming a thin lipid film by evaporating the mixture under reduced pressure; hydrating the lipid film with a physiological saline solution containing protoporphyrin; dispersing the liposome solution by ultrasonic treatment, and extruding the liposome solution through a polycarbonate membrane to obtain the protoporphyrin liposome, namely the nanoparticle sonosensitizer.

8. The method of studying o-cell interaction of claim 7, wherein the molar ratio of egg yolk lecithin (EPC), cholesterol (CHO), DSPE-PEG-2000 is 49:50:1.

9. the method of claim 1, wherein the type A cells include but are not limited to glial cells, glioma cells, fibroblasts, vascular smooth muscle cells, endothelial cells, bacteria, and fungi.

10. The method of claim 1, wherein the B-type cells include but are not limited to neuronal cells, glial cells, cardiac muscle cells, vascular smooth muscle cells, vascular endothelial cells, bacteria, and fungi.

11. The method of studying o-cell interaction of claim 1, wherein said paramagnetic environment is provided by a paramagnetic solution.

12. The method according to claim 11, wherein the paramagnetic solution is a gadolinium-based solvent.

13. A method according to claim 12, wherein the gadolinium solvent is gadobutrol.

14. The method of claim 1, wherein the proteomic changes of damaged a-type cells and undamaged B-type cells are detected in a microtissue 3D cell population by timing, quantitative and localized regulation and damage of a-type cells.

15. The method of any one of claims 1 to 14, wherein the method is used in the fields of cell communication, pharmaceutical design, or pharmaceutical development.

16. The method of any one of claims 1-14, wherein the method is used to explore and elucidate the intrinsic association of particle targeting with the competitive uptake of neighboring cells.